abstract from all papers on microbiology, serology and related sciences, published in netherlands...

PREFACE.

Sinc~ the occupation of" the Netherlands in May 1940 postal exchange with foreign countries, especially those overseas, has gradually decreased and broke down completely in 1941, thereby depriving us of the opportunity to keep in touch with our colleagues abroad by exchangeing periodicals and reprints of papers in our scientific field, as in every other branch of science.

Happily many of our colleagues after the war have been quick to provide us with their publications issued in the past few years and the Board of the Netherlan~ls Society of Microbiology is glad in its turn to seize the opportunity of reciprocity by collecting in one volume abstracts of all papers on research work in the field of microbiology, serology and related sciences from periodicals issued in the Netherlands during the years 1940--1945, both in dutch and foreign languages. In the medical field papers dealing mainly with pathology and epidemiology are omitted.

It is hoped that the exhange of papers from 1946 onwards will follow its normal course as in the years before the war, thereby reestablishing the many friendly .relations which, existed between dutch microbiologists and their ioreign colleagues.

The abstracts are arranged according to subjects. The main classification is:

General microbiology Biochemical investigations Serological investigations Medical bacteriology and serology Animal pathology Technical microbiology Mycology and plant pathology Soil bacteriology We owe a dept of gratitude to the staff of our periodical Antonie

van Leeuwenhoek for the considerable amount of work involved in preparing this issue.

The Netherlands Society of Microbiology,

JAN S~Irr, Chairman

IN MEMORIAM PROF. G. KAPSEI~BERG

November 9th 1942 G. KAPSENBERG, Professor in Hygiene and Medical Police at the University of Groningen passed away quite unexpectedly.

GERARDUS KAPSENBERG WaS borne April 24th 1883 at-Rotter- dam. After having passed the High School at The Hague he Studied medicine at the University of Leiden. On April 8th 1909 he received .his medical certificate. Already as a student, he showed his ability, e.g., by the treating of a subject proposed for prize competition by the Medical Faculty at Leiden. His paper bore the title: ,,An experimental and clinical investigation of the reaction of the organism on the products which the decay of the cells of its own organs give rise to". The Senate valued this investigation with the golden medal, accorded in a public meeting in. 1909.

The extensive scientific interests of KAPSENBERG were evidenced by his fulfilling the function of demonstrator in various branches, before as well as after having attained his medical certificate. First he was demonstrator to R. P. VAN CALCAR, Professor of Hygiene. Then during .two years he received a surgical training under the direction of Prof. J .A . KORTEWEG. In 1911 he returned to'Prof. VAN CALCAR for a short period and from Oct. 1st 1912 to Dec. 1st 1917 he was demonstrator and later head demonstrator to Dr.D. A. DE JONG, professor of Comparative Pathology. Moreover he prac- tised in surgery as well as in bacteriology and serology! .During the first world war KAPSENBERG occupied several other govern- mental functions, viz., surgeon in the military medical Service, adviser for hygiene of the city of Leiden, controlling medical officer of the food department and school doctor.

In 1919 he was appointed as Director of the newly estabLished service of Hygiene at Groningen which had been completely organised by him. And as the culmination of his scientific career after the death of ALEX. KLEIN he was appointed as Professor of the University" of Groningen in January 1936. On June 4th 1936 he held his inaugural address on ,,Microbiology and immunology as a science." . . . . . . .

The scientific work of KAPSENBERG as well bears evidence of his many sidedness. First of all his publications in the immunological field draw the attention, which bear chiefly on the relation of the antibodies of the serum globulines and which culminate in an interesting study on the formation of antibodies.

Next to this his work in the field of ba.cteriological diagnostics is of chief interest. As early as 1913 and 1919 he published some papers on the technique of the Wassermann reaction. In 1922 he thrice detected Microspomm pubescens ]lavescens thus far never detected in the Netherlands. Further he was the first in the Nether-

4: in memoriam Prof. G. Kapsenberg.

lands to isolate the bacillus of Cohen as the causal agent of meningitis (1924). A still rarer bacterium, viz., Listerdla monocytogenes, detected in 1926 in infections of rabbits, was isolated by KAPSEN- BERG for the first time from man, also in a case of meningitis. A detailed investigation of bacteriological diagnostics of diphtheria (1932), in which he improved the nutrient medium by the addition of egg's yolk and glucose, gNes evidence of his lasting interest in routine tests.

The epidemiology of infectious diseases as well has been enriched by his work. This is evidenced by a study of meningitis cerebro- spinalis (.1918), a report on the danger of contagion in tuberculosis composed together with G. J. HOET, by descriptions of minor epidemics of typhoid and paratyphoid fever at Groningen (1920, 1924) and by his observations of the plague flea Ceratophyllus /asciatus at Groningen (I 926). Even in the surgical field he worked scientifically. A paper on a hip trouble published in 1916 bears witness thereof.

In all of his work the technical side of our difficult branch of science had his special interest. He imagined continuously technical improvements or new apparatus, which he knew how to realize with great dexterity. His papers on methods of dialysis by means of amnion membranes, on a metal dop as a substitute for the cotton plug and on new apparatus for filtration, elution and extraction bear witness of the important results arrived at. KAPSENBERG was an excellent speaker and it was always a treat to follow his carefully prepared courses and lectures. But most of all he was a sincere and open-hearted man, kind-hearted and ready to help. Thus his sudden passing away means a great void for his many friends, co-workers and pupils. They will not forget what he did for them.

A. E. B.

IN MEMORIAM Dr H. A. DIDDENS

Many of her friends in Holland and also abroad will have heard with deep sorrow o.f the sudden death of MA~m~. DIDDENS during the war. She died the third of December 1944 at Amersfoort. She met with an accident, misled by the darkness and died immediately after .

She had a very active life and the work she had alreadY achieved offered so many expectations fo~ her future. I t seems incredible tha t such a valuable life has ended now for ever.

HERMANNA ANTONIA DIDDENS was born in February 8, 1902 at Winschoten, where she at tended the elementary school and Latin school. In 1921 she entered the University of Amsterdam as a s tudent in biology. She passed her final examination in general botany and zoology, entomology, phytopathology and microbiology in 1937 and .continued-to work on phytopathology at Professor WESTERDIJK'S Ins t i tu te at Baarn. She took her doctors degree on a thesis entitled: , ,Investigations on a flax disease caused by Pythium megalacanthum de Bary" at Amsterdam, in 1931.

She had interrupted her s tudy at Baarn, however, for about a year to work as a demonstrator in phytopathology at Professor SCHAFFm~r's Labora tory at Bonn.

When she still worked for her thesis she was, in April 1929 appointed as collaborator at the ,,Centraalbureau voor Schimmel- cultures" where she remained till September 1942. Her chief work at the , ,Bureau" was the control of the collection and the inden- tification of the numerous cultures sent to the ,,Bureau". Moreover she did the extensive correspondence. Besides this work she largerly contributed to a s tudy on.the systematics of the Mycotoruloideae. In 1932 she spent, in connection with this work some time at the Inst i tute of Parasitology of Dr. LANGERON at Paris.

She showed a deep interest in work going on in other laboratories and in methods used there. This interest connected with a great passion for travelling made her visit many laboratories and inst i tutes, during her numerous journeys in Germany, England,. France, Italy, Denmark.

In 1936 she a t tended the second- and in 1939 the third inter- national Congress for Microbiology resp. at London and at New York. At the lat ter she read a paper entitled: ,,Variations occuring in type culture collections". After the New York Congress she visited many laboratories and institutes in the eastern part of the U.S.A. She stayed for some time at the Inst i tute of Dr. WAKSMAN at New Brunswick (N.J.) and at Dr. WESTON'S Inst i tute at Cam- bridge (Mass.). The war menacing'her own country made her return to Holland at the beginning of 1940.

In 1942 ~he chan6ed her position at the ,,Cenfraalbureau

In memoriam Dr. H. A. Diddens.

for another, which offered her bet ter prospects. She, then, was appointed as a mycologist at the , ,Laboratory for Bulbresearch" at Lisse. Her work there, more in the field of phytopathology, had her highest interest. Unfor tunate ly war-conditions Very soon made scientific work almost impossible. To the ,,Centraalbureau voor Schimmelcultures" the departure of its able collaborator was a very great and deeply felt. loss.

She had been secretary of the Dutch Society for Plantpathology. During this period she published - - in '1941 - - together with T. A. C. SCHOEVERS and H. L. G. DE BRUYN a list of Dutch names for plant diseases in agricultural plants. Her other publications were mainly about the work going on at the , ,Centraalbureau" and several of them were published in this periodical.

She was a very talented scientist and an accurate researcher. But besides the deep interest she showed in her work, she also had an open eye for other things. She was very fond of sports, of music and other branches of art. She had all kind of favourJte pursuits e~g., she studied Russian for some time and had.made such progress in this language that she succeeded together with a fellow pupil to translate a russian scientific book.

As a schoolgirl she was president of the club of grammar-school pupils and as a s tudent she was member of the board of the corpo- ration of students.

She liked domestic comfort and was an excellent housewife, and the pleasant years we lived together in the little house ,,Madoera" at Baarn I will always remember as a good time. Her chief trait of character was her perfect honesty as well in scientific as in human respects. This together with her great modesty, her cheerfulness, her warm feeling towards others and her keen. sense of humour made the friendship-with her of so great a value.

Therefore the tragic 'end of her life will be both a loss to science and a grief to her many friends, who will always .remember her peculiar charm and vital personality.

J . L .

IN MEMORIAM PROF. Dr W. C. DE 6RAAFF

After a l o n g iUness passedaway on May 10, 1944 Dr W. C. DE GRAAFF, Professor in Pharmacography, Galenic Pharmacy and Applied BiOlogy at the University of Utrecht and Honorary Doctor of the University of Amsterdam..As intensive as his interest in many branches of science as extensive were his knowledge and the field he covered inhis teaching. In fact he taught pharmaCography as well as the science of foodstuffs, of medicinal, nutritional and toxic plants, the medical chemistry and applied microbiology..As to his lectures they were always clear and' captivating, enlivened by his humour and irony.

When merely his microbiological work will be mentioned here, this d o e s not mean that he would have been less active in other fields, far from it. I t may be stated, however, that microbiology stood first in his interest. This is evidenced by the large number (20) of theses on microbiological subjects which have been composed under his direction.

Next to the strictly scientific microbiology applied microbiology had his interest. The important work performed by him first as member and later as president o.f the Committee ex Art. 17 of the Wares Act needs mentioning. In this function he .did not keep to his official instruction, but, and this the more so when the bacteriological condition of. victuals or stimulants was in question, he arrived at his opinion by means of personal investigations, an opinion which generally would further-find its way into the Royal Warrants. Also the establishment of the Normal Leaf hl 1028 bearing on the bacteriological examination of drinking water is due to his initiative and has been accomplished under his direction after a period of about 9 years, wherein many investigations bearing on this matter had been carried out in his laboratory.

In the more strictly scientific field he became known by his investigations of the bacterial fermentation, where he a t t empted to attain insight in the fermentation of sugar by means of fermentation of simpler compounds. As micro-organisms he studied mainly coliform bacteria, which have always roused his special interest.

In his laboratory for the first time in the Netherlands the antigenic structure of bacteria has been investigated, viz., of various types of pneumobacilIi.

Already from his Leiden period dated his interest in the methodics of clinical, bacteriological research, which was evidenced in the book he wrote together with Prof. Dr El GORTER, ViZ., ,,~Klinisch¢ Diagnostiek"~ a widely valued laboratory manual,

In memoriam Prof. Dr W. C . de Grsaff.

Until his death he was president of the Committee for the examination of clinical analysts, instituted by the Chemical Society. He has always actedvigorously for a thorough training of these auxiliary laboratory workers.

IN,M, EMORIAM PROF. Dr P. C. FLU

On December t~9~ .1945 ~he .Uniyersity. of Leiden. as well as:, the Ins t i tu t ion for Tropical Medicine had to encash a heavy ~ s s : b y the,passing away, of PAUL CHI~ISTI~N FLU. :Not mere ly as-a ~¢ery ou ts tand ing scientist but as well as organiser .and leader, of this Laboratory he was to be valued greatly. He-was always re,ady for his: coworkers ,and if ~one had~ the privilege, of composing a thesis unde~ :his dir~gtion ~- one, rnigli~ be sure,,th~,t Professor: FLy. would assist: w i t h wbrd and .deed and: when neededwonld~ give: actual perso.n, al help. Also for: the lower staff working under his,direc.tioh the death of this beloved, chief, means a heavy loss. To his: ~ e a t simplicity aud to the interest :he. took,,iIi, t he i r difficulties it' is ~due tha t ;they not merely ~lose a Director but even more a counsellor upon ,whom -they knew, to ma,y~ depend,

Professor FLu was born irr. 1884 in Paramaribo~ where he was trained.aS a. West-Indiau physician. By his outstanding ,qualities he drew the a t tent ion in such measure ' tha t he was among those who. migh~t-finish theirs, studies in :the Netherlands. :At the age of ha rd ly22 he ,obtained, in 1906: a±" Utrec~t,-his ce~ificate as a ph,ysl, cian. After a period Of-one year in'which, he!was, d.emonstr~or-o1 Prof. SPRONCK he was appointed as a mili tary surgeon second:class in the a rmy of the Netherland East Indies. ,In order t o increase as much as.p'ossible his ,medical. knoWledge h e worked" first- some years a~ Paris and Hamburg. In~ the latte.r ,city he.,worked i n the, Ins t i tu t ion :for Naval ~and Tropical .Diseases,: where Dr YON :P~o, WAREK ;directed his studies, I ~ this! Ins t i tu i ions his first :publi'- ca t ions in the parasitological field: saw the light.

This period.of life c a m e t o a ~ close in 1~908 when: he. h a d t o ~o ,l]ow his dest, ina±ion for t,h'e~,Mflitary 'Hospital a t Paramaribo., He re he was put to work in the pathological laboratory.- An .order to..-iff; vestigatescientificaUy the ocemrrence :of" malaria i,I~ ;theu~pe~ 1Ands of Suriname followed i,n 1:94'0..Professor F l u teaC.hi~ag, meaBwhile in. the, Medical -S~hoot a s well, ~it, t~eeds no fur,ther! stress, t'hat-~in this period,,of. ~19081 t6 ; 19-1,0 ".~n, enormot~s a.motmt¢ of Work:,,was performed:: As-,the,-~eshlt thereof, he,:.wrbte s0me~,-v, ery :imp0~.a~t- communications whick ,were main ly laublished,ir~ fo~bign~periodicats. These,paperS, which ~ n p t i l l Imw,ha~,e:-~ot i~n,,theleast,10s~.in,vM, u~', ha.ve:.~liigMy qontr~buted: ~0:,the. drAwi~g?ofithe~ A.t, temiOri,,.o~ ~ s s turdy scientific worker. More and more he appeared.to be 6f~.gVe@t value::m:Vhe..furtl~e~i~ag~of.'tl~e.~l~lowtedge of .tropiCa, l~'~liS~a~,e. I t is h im tliat .~etheriand..East~and!-W.e~ Indies~mky-th , a ~ f w iikipogtai~t observations-~l~ieh :'l~,#e ,promoted-* ~public .he~J,fli~:in'.:,~: mar&ed degree,

10 In memoriam Prof, Dr P. C. l~lu.

On July '2~ 1911 he was appointed once more in the Netherland East Indies where a further development of his scientific aptitude was opened to him, when he had to replace the fourth medical officer in the medical laboratory at Weltevreden. In "1915 after the leaving of Dr G. GRUNS his appointment as Director followed. In the mean. t ime Prof. F L u taiight ' general l~ra~itology i~ the Indonesian Medical School, thus the amount of work performed here did no t remain under the level attained at in his West Indian period.

No~withstanding this he had always a ready ear for us younger physicians, who .gladly came to him with our problems. For ,many of us he has thus been of great value.

His gifts of leadership did not remain in the dark. In 192:1 the chair for Tropical Hygiene at Leiden was offered to him and June 1 I, 1921 he held here .his inaugural address on the,subject: The influence of factors outside the human organism, and the spreading of infectious diseases in the tropics.

Having initially worked in a fairly primitive laboratory on the Keizersgracht at.Leiden, it Was a great satisfaction for Professor FLu to be able to move over to th~ l~boratory on the Rapeffburg, arranged according_ to hi s directions. At the s ame time he was appointed as Director of this tasteful and well equipped Institution for Tropical Hygiene.

In his further scientific investigations he centred mainly on one subject , viz., the bacteriophages, following on a period of intimate co-operation with D'HI~RELLE.'In his research work Professor FLU assumed the help of many younger workers which gave rise to various Doctor's theses.

In the latter 25 years of his life he has kept to this subject nearly uninterruptedly. A short interruption of these activities occurred, when during 4 months he travelled for study in Suriname charged b y t h e ,,Vereeniging voor Tropische Hygiane".

Once again a change would be brought about in the branches of science taught by Professor FLU, when in 1936 as the ~esult o f a concentration of chairs he was charged with the teaching of Hygiene and Bacteriology.

Meanwhile many distinctions fell to his part. He was Knight of the Order. of Oranje Nassau met de Zwaarden and Commander in the order of the Nile, whilst the University of Utrecht conferred upon h im.a Honorary .Doctor's degree~ Besides.he was a corresponding member of the Royal Academy of. Science at Amsterdam and o f - the ,,Vereenig.',m,g "tot Bevordering der Geneeskundige Wetenschappen in India and finally of the ,,S0ci~te de Pathologic exotique" and of. t h e Belgian ,,Vereeniging vo0r Tropische Genees- kunde '~.

In 1938 Pto~essor F~.u was called u p o n as Rec tor Magnificus of the ~Univer~i.ty of-Leiden. Up-:till then the line of-hislife had .tended: upward. Alas, i n that same. year. 1938 he became a victim of his work as by a laboratory accident he incurred a severe in-

In memoriam Prof. Dr P. C. Flu. 11

fection. After. having got through a serious illness, it .initially appeared as if i t w o u l d turn out f0r:the good, but gradualiy it became apparent that his health had suffered greatly. Still Professor FLu untiredly kept on working at his favourite subject,:,the bacteriophage, wherein the problem of vaccination against plague.had taken up an important part.

But fate did not favour him any more. Heavy afflictions were bestowed'upon him, which finally were too great for his decreased corporal resistance. Still he kept on fighting' up till the m0me~t that the summarising of the knowledge of the bacteriophage driving the latter 25 years had been brought to a ~lose. This work will be published ere long.

Among his further larger publications we cite: The textbook of parasitary diseases and o~f Hygiene (3 volumes), Textbook of Troi)lcal Hygiene, his report of his visit of Suriname and the Chapter ,Die Pest" in MEI~SE~s Handbuch der" Tr0penkrankheiten and finally ,,Voordrachten over de Aetiologie en Prophylaxe van Infectie- ziekten", the re-edition of which will be issued ere long, whici~ has still.been supervised by Professor FLu. A.P.

6ENERAL MICROBIOLO6Y

Verzamelde Geschi-iften van M. W. BEIJERINCK. Zesde deel. Met registers bp al'le zes deelen benevens eene beschrijving -van zijn leven en beschouwingen over zijn work door G. W~N ITERS0~ Jr., L. E. DEN ~DOOREN DE ~ONG en A. J,. KLUYV~R. (Collected papers o:f M. W. BEIJERINCK. Sixt~l volume. W~th i~dices on the .sJ,x volumes and a description of his work by G. VAN ITERSON. Jr., L. E. DEN DOOREN DE ,JONG and A. J. KL~WER). Martinus Nijhoff, den Haag, 1940.

The sixth volume has been published.20 years after the-initial 5 volumes. Next to some scientific papers published by.B~IjE- glNCK after he had left the chair at Delft VolUme 6 contains 3 indices (Author Index; Index to organisms; Subject Index) which are a great help to the reader, who wants to get the most out of the wide variety of subjects masterfully treated by B~:IJERINCK. The chief value, however, lies in the three treatises in the English language, composed by his successors in the chair and by his last pupil and demonstrator, vA~ ITERSON hastreated the b0tanicalpart of the work of BEIJEmNCX, KLU~:VER the microbiological part. By these most competent workers in their' fields the work of BEIJERmCK has been discussed in the light of present-day knowledge and the products of B~IJ~RINCK'S creativ.e imagifiation can stand this light. DEN DOOREN WE JONG treats the scientist from the human side. He has composed a biography and has drawn a portrait of BEIJERINCK as a scientific worker. He has succeeded in drawing a portraff very true to life and although a fervent. admirer of BEIJ~RINCK he has been honest enough not to leave out the shade parts. In the chapter ,,BEIJEtUNCK at work" a very vivid picture is presented of the scientist, of his, sparkling vitality and his characteristic habits and sayings.

The sixth volume rounds up the impressive work of the five earlier very worthily.

L. E. DXN DOOI~N DE SONG, De voedingswaarde der bacteri6n in verband met hun pathogeniteit. (Nutritive conditions of bacteria in connection wfffa their pathogenicity). Chemisch Weekblad-29, 131, 1942. Cf. Over de eVolutie der bacter/~n in verband met hot ontstaan der pathogeniteit. (On the e~rolution of bact.eria ~ con: nection with the origin of the pathogenicity). Ned. T. voor Genees- kUnde 86, 734, 1942. Cf. also: Een en ander over bketerieele groei- s toffen . (Notes on bacterial growth substances): Vakblad v00r Biologen 28, 38, 1942.

The pathoeenicitv of bacteria, when seen from the standuokat'of the evolution~ary do~ctr/ne, is. ~n acquired property; oni~ deve]opod

14 General Microbiology.

after the existence of higher plants and animals had become possible and after bacteria had succeeded in conquering the natural resistance Of organisms. According to KNIGI~T five evolutionary stages can be noted :among bacteria: I. Autotrophic bacteria. 2. Heterothropic bacteria. 3. Bacteria requiring amino-acids as N-source. 4. Bacteria which need growth-substances in addition to the organic C- and N-compounds. 5. Bacteria which can develop exclusively in animal organisms. Among bacteria of group I and 2 no real pathogenic forms occur, most of them belonging to group 4. Transitions are found between the different stages. The synthetic ability decreases from ! to 5. The pathogenicity of bacteria is caused by their forming endo- or exo-toxines. The question arises why a saprophytic bacterium m a y have started to develop pathogenic properties. Comparing pathogenic bacteria With saprophytic bacteria o f the same gelius it becomes evident that - - w i t h o u t excepti6n - - the pathogenic put_much more complicated demands on nutrition. With the increasing of pathogenicity the bacteria have lost the ability of synthesising certain substances themselves. On the other hand, cultivation on artificial media may decrease the pathogenic ability and increase the enzymatic potencies. (E. tyt~hosa, M. tuberculosis}.

I t is nowsupposed that pathogenic bacteria have developed out of saprophytes becati~e through t h e stay in rich nutritive surroundings the synthetic ability is decreased. The bacteria are now forming toxic substances to attack living tissue in order by these means to get hold of the necessary nutritive substances.

P. C. FLU, Das Ultravirus als Krankheitsursache, seine Eigen: schaften und eine kritische Uebersicht fiber die Ansichten bezfig]ich seiner Art. (The ultravirus as a pathogene, its characteristics and a critical survey of the existing conceptions as to its nature). Acta Leidensia (Mededeelingen uit her Instituut voor Tropische Hygiene, Leiden) 15--16, 25, 1940--1941.

The author gives a clear survey of the characteristics of ultravirus (generally including phage also in this term) as far as they are founded on experiments. These well-f.ounded facts are: The whole group of agents which are embraced by the term .,,ultravirus" are not uniform. The smallest and most primitive among them, such as virus diseases of plants, the phages and viruses of many human and animaldiseases - - as far as they have been s tudied . - - consist of nucleoproteids, which, when purifi.ed as far as possible, sometimes have been obtained as paracrysials. The higher organised agents such aS the elementary hddies of vaccine and of vari01a moreover consist of a fat soluble substance and carbohydrat6s, i In- accordance w i t h their dimensions which oscillate between 10-- 150 p/~ they -foll0w,.-thelaws .of ,the: hydrophile ¢oltoidM-s~ate of matter,~ To, develop .theirs, ,f tractions .~ they need an equilib~iiun of

General Microbiology. 15

electrolytes in the same way as living pr0toplasm. Whenever this eqUilibrium is destroyed, they are rendered inactive or they are annihilitated. All these characteristics are characteristics of living matter.

The author discusses critically the various theories brought forward as to the nature of ultraviru$, a n d more in detail the theory of endogenous origin, and that of dead matter. The author claims that virus, phage grid ultravirus exist in the region on(the borderline of living and d~ad matter, and it seems to him wise to ackn6~vledge here the same mystery which is adherent to 'all living matter.

P. C. FLU, Wordenna contact met een bacteriophaag megatherium niet-lysogene stammen lysogeen, omdat z~ met den phaag worden geinfecteerd, of omdat zij tot phaagvorming worden ge- induceerd ? (Do non-Iy.sogene strains aftez contact with a bacteriophage megatherium ~'ow lysogene, because they~are infected with the phage or because they are induced into phage formatidn~ Acta Letdenma IMededeehngen mt het Instltuut voor Tropische Geneeskunde, Leiden) 15---16, 52, 1940--1941.

The lysogene Bacill.us m~gatherium ~38 which, in ordinary circumstances, does not produce linage, whe~ Cultivated in peptone-water, produces the samephage that dissolved it and rendered it resistdnt and l~'sogene. The probab'fl~ty that tl~is faculty is due to phages that pone{rated into i~s plasma and later were enclosed in the spores,: is greater than that it has asgumed the property by some mysterious induction.

S. J. C. DUNLOP~ Investigations on the occurrence of bacterlo- phage-fre e spores of Bacillus '/megaa~eriuraS99 of DEN DOOREN DE JoNG.. Antonie VSal Leeuwenhoek 7, 234; 1941.

It has been tried to i~elate from B. mega~herium, strain 899. of DEn DOOmZN DE Jo14G a spore~that dues no t contain phage, This has failed. Moreover it has been proved, that in case a suspension containing spores .is heated at 80 ° C., not all the spores are resistant to this temperature as one would suspect, but only about 1%. I t is .left opei~ whether phage-free spores may occur.

T. Y. I~1~o~a BoLTj~, Some experiments.wlth blown glasses. Antonie ~an:~Le~uwenhoek 7, 61, 194~1.

Experiments were carrie.d out: with blown glass spheres in order to ascertain whether the ;0bservatiohs reposed by vAN LSEUWSN~ HOEK might be made by means of these. Microphotographs made through such lenses show dear ly t h a t this is actually the case. VAN LEEUWENHOEK, however, mentions his use of ground lenses. At, low magnification these are certainly better than b!own ~ glasses,

16 General Micxobiology~

The possibility of VAS LEE~IWENHOEK,, having used coverslips iS discussed.,

A. E. B~u~x, MOthode simple pour la dessiccation dans' ie vide de cultureS, bact~fiennes. (Simple meth0d:for :fhe drjringin vacuo of bacterial cultures). A~tonie Van.Leeu.wen~oek- 1'9, :71, 1944---19~5.

The apparatus used is a modification o f the model of (:OOPER and GRABELL., A simple and rapid, method for the drying of bacterial cultures has b e e n arrived at.

P. . .H.H. DE BRUYZ¢, The cultivation of filterable viruses in vitro. An~onie van Leeuwenh0ek: 8, 19, 1942.

A review of the recent methods for:cultivation-of!viruses'on tissue cultures .and cell suspensions i~ presented. The aims.whiCh may be arrived at by these m a ~ s are discussed.

A. Bos, Her desinfecteerend vermogen van" kresyli~e op enkele bac~terie-soorfe'n. '(The disiflfecting action- of ~e:syHfie ofisom~ species.of bacteria): T4jdsctirifft:x.oor-Die~,gene.e.sku.nde-'~0, '55," 1.94"3;

The disinfecting .effect of kresyline upon::=Escheri~hia coti, Br¢~- celia abortus, Erysipdothrix r, husio~hiae,, S.~igdla equul~ Salmo- nella en~eri#idis var. dublin, staphylo~gccus aureus and S~reptOcoccu* pyosep~icus (syn. Str. pyoge~-e~~pffeared to be equal or bet ter than tha t of creolin, and surpassed t h a t o f carbolic acid. These bacteria are killed by a J % solution Within 2 ilfinnt~s~ I n fact the effect on the ge rms oLblack leg a n d ar~.thr.ax was bet ter than tha t of creolin or citopogeli, but distinctly less than tha t of carbolic acid. A 10 °/o solution :of" .kresyline..ld~ed Cl~stridiu,r, ohauvoei within 30 minutes, anthra, x.wit~hin 2 days .

C. F. VAN eYe-N, Eenige, onderz.oe~gen, omt~ent de bacte~e- d00dende werking ~" der' 'We'stingiabuse~-~erilamp~ ~Sotne in4c~s~i: gations, of ttie bactericidal-.action of the WestinghOuse-Sterilamp). Tijdschrift vOor Diergeneeskunde 67, 586, 1940.

Irradiation with the..'.Westinghouse.-$.ter~mp, ki.'lls a,: good .deal o f micrococci, B. coli and-Salmonellae in a - th in layer of agar. ~he"effect depends on the length'~of~;time oLicmdiation~,~the ~iix- imrature" and. the ,'distance: 'between-,l~mp ~an~d " the ~iay~r ~ 6f~ a ~ , ...

Biochemical .Investigations 1,7

BIOCHEMICAL INVESTI.GATION$

A. J-. Kxtrrv~.R and M. TH. J. CUST~,RS, The suitability of disaccharides a.s respiration and assimilation substrates of yeast~ which. do not ferment these sugars.. Antonie van Lceuwenhoek 6, 121, ~99~-~1940.

Yeast species claimed' as non,felanen.ting, disaccharides .were studied as to their ability to ferment maltose; lactose:or saccharbse: Non-maltose and non-lactose~fexaner~ting yeasts gave completely negati~;e resultS. In tests for the felanentat.ion of saccharose Torula monosa-was quite negative, in 7 other species, however, a weak yet Unmistakable fermentation could be observed. Heavy inoculations had to be applied and sometimes the time of observation had to be prolonged.

The assimilation was tested by means of: the auxanographic method of BEIJEmNCK and, by growth experiments in liquid media with quan$itative determination of. the disappearance o f sugar. The non-fermenting, species. Torulopsis.,datbeba, Torulo#sis .utilis,. Mycoca~d$~a parakmsei ,and. Bre#~omyces ~no.mal~s gave p0sffive results. Saceharomyces [~agilis, Tomla, cmmorSs .and Toruta monosa did not consume any maltose uhder anaerobic conditions~, The consumption': by ' Zygosa¢ch.aromybes Marxianus, " Saccharomyces exiguus and ~:Sa~charomyces. L~dwigii .was so low that it may .be practically neglected. Among the. non-lactose fermenting yeasts Blast6d-e~¢on i~terme~ium, consmned .a- ~considerabl6: amo.un.t of lactose, whilst &accharom~ces carlsbergenSis, and Saccharomyus ,erevisae.:d.id-~Iot. consume.any. ~ non-saccharose fermenting yeast species ,with .Oae .excePtion of Torula monosa-consumed saccharose ~uncler azlaerobie..conditions.

The sUi.t:~bfli~ty.-of,the disaccharides as a .respiration substra{e has been- tested by meansof t,ke. W A R B U R G ~ manometer,me,hod, D, is.c r'~min~tion between .respiration ~.o,t sugar, and of accompan~ng impurities .has ~een obt ~a~nedby ex.~en ~6hag-the experiment over a longer, period .o!; :,time~ All yeasts .whieh,:had ~hown posffive results ,ha, the fermen~tior~-test with, ima~C,6se~ were able to ,:~se i t :as a respiration' substrate %Novae .of~ the non-.lactose-fermel~thag yeast~ Could use !lac,tose as, ,such. The-~n~.~sacehar0se' termed, ring, yeasts with~-:the, exception of~,,Tor.,ul~t :~o~osa :could use ~,saccha~o~'e:, ~s a respiration,.substrate~.

Eor ,TorulopsSs:. dattil*a";i¢. ¢outd be proven,: that',an: aerol~ie, fer- • menia~on,:e~iSts :.a~on, g,w,i~.t~..the respi~ktiom ;::W~.,en ,the::,t~p~ion

aerobic :~ermentat'ion. Thus a hydr0iase. ~is!.p~esex~t,:'m'~his~;~e~:'~-t, Maltase wag deiectedl/as 'Well by meahs of the. method of, Wn~- S T ~ T T E R ' , " : S ~ H N E i D E R and "B:~v~x~ll4.::i~I6reoqer in:. M~ycoc~nd~d~

Some unexplained- anomalies, stfll:,~x/stin~ in. ,the. :behav/e.u,r of : , " - " " ' " ' ' - " ' ' ' "" : "" " ' " " ~ ~"" " " . . . . " " " F ' . . . . :" ~ ' ' "

18 Biochemical Investigations"

them. By cultivating Blastodendrion inte~medium on a lactose medium, respiration of lactose was obtained.

It is discussed that apparently hydrolases are partially or completely inactivated under anaerobic conditions. Probably it is the state of reduction which will entail this inactivation. Proteinases are activated b y reductiofl and it may be conceived that this increased proteolysis will affect the carbohydrases which contain proteins as main components.

M. TH. J. CUSTERS, Onderzoekingen over-her gistge$lacht Brettanomyces. (Investigations of the yeast genus Eretta~omyces). "~hesis, Delft, 1940.

17 strains of Brettanomyces yeasts were studied as to their morphological and physiological characters. Cells of a particular ,,ogive" shape frequently occur. All strains produce marked quantities of acid from glucose. Brettanomyces Kufferath et van Laer is to be maintained as a separate genus in the subfamily Mycotoruloideae. For the moment four species and two varieties should be distinguished within the genus.

Under anaerobic conditions ethyl alcohol and CO2 were the only dissimilatory products formed by Brettanomyces Cla~issenii. Under aerobic conditions Bre~tanomyces Clausenii a n d Brettanom~ces bruxdlensis produced a considerable amount of acetic acid besides ethyl alcohol and CO~. I t is deemed probable that the acetic acid is a product of the oxidation of the ethyl alcohol. Under similar conditions Saccharomyces cerevisiae-did not .produce any acetic. acid. I n manometric respiration experiments it was found that Brettanomyces Claussenii oxidises the alcohol to acetic acid only at pH = 6.40, whilst at pH = 4.35 and 3.77 this acetic acid is further oxidised to CO, and water.

Manometric experiments were made to study the influence of O8 on t h e fermentation of gl~lcose. An early decline is apparent and caused by the formation of acetic acid from the accumulated ethyl alcohol, The most remarkable result was that the aerobic fermentation surpassed the anaerobic ferrqentation. W;hen using cells of older cultures higher values of both aerobic and anaerobic fermentation were observed and these Cells also show a normal PASTEUI~-effect. Ceils derived.from anaerobic cultures were characterized by a Very low value of respiration, whilst the intensities of both aerobic and anaerobic felmentation were high. and almost equal. A discussion has been given o f the causes und~!yin:g the xaegative PASTeUR-effect.

E. VAN OLDEN, Manometric investigations on bacterial deni~ trification." Pr6¢. Kon. Ned. Akad. vaxi We~. 48,, 685,' I940.

The aim' of, the invest igst ion ',was the~ testing of, the possibflit,~'

Bioc-hemic~l-t~nves*ig~ion~. I9

O~ studying denitrification of ,;resting bacteria" by:means of the manometric method. It was found necessary to use ceils which had grown anaerobically in the presence .of nitrate._ Then in the manometric experiments an evolution of nffrogen could, be observed, of the same orde r of magnitude as that of: the aerobic gaseous metabolism. The rate of nitrogen production proved con- stant as long .as a sufficient am6unt of nitrate .was present. Thus no intermediate,siages,of the nitrate reduction were formed in any appreciable amounts. The total amount oLnitrogen evolved c~rresponded wffh the amount calculated when the nitrate added was completely converted. Thus the manometric method appears suitable for the study' of denitrification.

Certain bacteria appeared to. denit.rify almost equally well in the presence as-in the absence of organic substrates. SO these are able to oxidize reserve materiM.present in the cells at. the expense of. the ni t ra te .added. Th, is: process has been. termed ,endogenous denitrification".

The previous history of. the cells shows a decisive influence on their denitrifying activity, Recently arguments have been given in favour of the view that :.denitrification. is depending on a speciM ,,nitrate reductaseT'. This has tc~ be considered then.as an ,;ad~ptive enzyme".

H.. B~uwI~s, Sur les ferments prof~ol~iques du. glbeiO cholerae et du. Vibrio ~l Toe. (On the-proteolytic enz-ymes of. Vibrio ~holera, e and V~brio El. To 0. An.tonie ,van Lee~wenhoek 6~ 48, 1939-~1940; Cf. also: Acta Leidensfa l~--d6, 1~4, 1940---194L

Haemodigestion and t h e liquefying of gelatine are caused by separate enzymes. In 4 strains of V . El To t cultivated bn:agar the splitting up of gela.tine, estimated by .means: of, the Viscosimeter of OSTW.~LD,, and the haem0digestion weremore ir~tense, than in strains kept on white of egg. In the latter thehaem6digestive action had quite disappeared.

S. DX Boxi~; Nitrate assimilation, of Asp¢,,g~ltus ~ge~. ~an Tieghem. Pr6c. -I~0n. Ned: A~ad. V~r~-."~et. 4'3,. 715,. I'940~

Tke xylenol method for.detemair~g the ~nff~ate in fm~g~ and: their culture solutions yields satisfactory results. .~i~ate is r io t : : ac~u- iated: in As~ergillug~niger bu~ :is metabo,~ise.d~ af~er 'upta~ke, I,n."':the case ,o~.starvedfungous,:mats:,the pH doeslnot:, appe~r:t'O have mY effect.'upon: the assimilation ,o~ .n:£tm~e., In ~bw./~ag:: .ftmgi.~-~'~: ~ simflati0n.. ~f ni~tmte, is: st, r0ngest at. pH..4;, ~ao~espondmg ,Wi*-~ :the s~ongesi~el~vetopment, Ni, tm.te ass~,mrlk~ion~is--ha'cre~e6 by ~d~ti~fon of .glucose,

20 Biochemical Investigation~.

A..:GORT~-R, Amino acid breakdown by ,4spergillus niger, l~ro¢~ Kon. Ned. Akad. Van Wet.. 4~, 721, 1940.

The variousenzymes,hitherto: known for the amino acid breakdown ..play no. part in tlie. deami nation Of:amino acids .by Starved mats of. A spe'rgillus,niger. In ~th'~s Organism the oxidative deam'm_.- ati6n of £rnilfO acid~s.-proceedg'~b'est at 15H 2-.-4 of the surr~und~'g medium. It is cl0sOly .coupled With the ordinary cell respiiation, and is probabl~r Cau~d by ,,Unspecific" oxidation enzymes.

L. H. C. PERQUIN, On t~i e incidental occurrence of rod-shaped, dextran producing bacteri~ in a beet sugar factory. Autonie Van Leeuwenhoek 6, 227, ~1~39--1940.

From a ,,FroschlaiCh" formed hi a sugar-factory, the responsible organism was isolated. This org~mism could be identified with the dextran-forming, heidrofermentative, rod-shaped lactic acid bacterium, Betabacteriu~i vermi/orm¢, a bacterium first described by WARD as one of the constituents of the ,,ginger-beer plant" and r-ecognized by MAYER as being also one of the components of-the ,,tibi"-consortium. Thus i~c has, been s~hown that the , forma, ti0n of the .,,Froschlaich" in sugar-faCtories.is not always due to £euco- no~toc mesenterioides or.to Bac~l~s Vulgatus, but can also be formed by BaabacUrium vermi/orme.

A. hitherto unk, nown species oi the genus Stmptobaaerium was found to-be an. accompanying :organism. This rod-shaped, homo- fermentative, dext.r'an-f0rmlng bacterium has been- desoribed .,in detail as the species Streptobacterium dextranicum nov. spec.

F. A. M. ~. "$~zzs. V~ -*WAes~xRO~, Onderzoekingen over microben-amylasen. ?(I~@estigations of:microbial amylases). -Thesis D,elft, t9~1.

The aims of the investigations reported o~ in thts thesis consist-in: I o. the studying of the occurrence o~ a/nylolytic propertiesin vari:b~s yeast, species, incliid~tg .a' more de~tailed~ study of, tRe. arny~.d occurring in" s,, c ,rb yces//ragilis; :and..2_?. gaimng-~fi insigh£.:in the action' of thebacterial amylases which occur nowadays on the market and .~'thoro~h.~nveS~ga~cion .of.the products of,hydzoly, sis of .~sta~h: .produced~)by tiie:-lat~e~

The investigation a s : ~ t~he,,qccurvence ~of ~amy!ases ~in 64 yeast species belong'mgilto ~wide'ly , v , ~ i n g gen.era :has; s ~ itha.t.:by f~r th:e:~ea~ ,e~j~umbe_r of these ,possessed a~definite I.aculty.to a~z star~h: ,mO~e..~or. :~ss: .~ten~i~l~:Tkis . amyl~y~tic: ~adulty,~as~ ~ i~

In. th i s ~nnection the , ~ ~ p a ~ e d . t h e ~hrious :meth0:ds by_ me ,arts:-of, :whic . -h~t l ie :~f l~o~on of: .starch.might, be ascert~ec~

Biochemicsl ~nve~tg~fions~

qualitatively. I t could be proven that some species of H a ~ l a ~ndSaccharomyces i n laure:cultures could-:.~:ferment p ~ t .of the starch; ,wi~h the 'production of,-carbgn dioxide and alcohol. ~It appeared that the amylolytic properties of Sa¢ch'atomyees fragil~s ~ J0rgensen could be whol ly attributed: :to, ..phosphorylase: The preparation, of amylase derived from. ba~eria, s~ch..as :Super- clastase, Rapidase,. Biolase N:extr~ is discnssed~, All. these preparations .appeared to be free fre~ maltase, The cCCilrrence of glucose and maltose could be ascertained wi'tTt~ertainty as products ~f, hydrolysis induced by-Superciastase.-Moreover a polysaccharide fermentable, by Sacchaeor~yces cerevisiae could be isolated and i~nt if ied as a trisaccharide.

j. H. BEKIf~ZR, Les mutilats du bacille, charbonneux. (The mutilates of Bsc~llus snthmcis). Antonie van Leeuwenhoek 7~ 180, i9~1.

Fo~ 11 .strains q f Bacillus...anthraCis i t has been investigated whether, they were rehd~re.d asporogenous~by incubating at :42 ° C. Some strains never lose the i r sporogenous Character, 0ther:are asporogenous ..tot a period, but soon grow..sporogenous kgain and finally there ate .some strainS, which .remain for a lon{~: pei~odor perhaps permanently asporogenous. The asporogenous stTfains differ from .th.e. original sp.0rogenous st~in, by a ~ecreased-vii-ulence for the mouse, an: increased sensitivityfor the bacteriophage and loss of ..some biochemical properties,

I£. T. WI~mN~A, Bacillus agav-exede~,-a new s.!?ecies, decomposing agar: Ant0nie' van Leeuwenlioek 7i 121, 1941.

' In, stable ,mama,re,' leafm01d~and shill (especially .fihose with. a high content. of-orgar~ic mat ter! .agaX' de~i~posing .bac~: i ~are reIRtix~ely cornmon.-.,The' occurrence .of d,i~feren~ :t3)pes .of -these bacter~a"-~- Bac~Rus, wgar-exedens ~-~ ir~.materiaIS with .a higli"eonten~ :of organic matter ~sugge~ts: .the ~ 'prosenee:irL ~ e s e m~terials of ]~igherorganic compounds; especia~y apt fov;th¢ii-...nutr.ition..

.K.T. WmRmG,%,. The formation .ot ~¢etic acid. ,f~om, .carbon ~li0xide-and ~c~c~gen ~ b.y:~maer..obi¢ Sp.ore~ormin g>bacteriv~.~ ~toniV van Leeuwenhoek 7~ ,251,~ J94~.

Furtherexperimentson ananaerobic bacillus synthesising acetic aCi@~f~omCo~ amd' :H:~ are des~ribed.. ~ T.l~e ~t)~g~ii~S .m ~! q,d~s!ign~ ~ classilidd.,as- C~16si~idi~m aeaic,gm ~ov~spec: ~.:Acetf~ a.qi~ ~s ~so forme~from sugar. ,ftwassho .wn ~hat:at anymomeff~ ~e:n~ber of H~ moieculds ~sed. tOr',Sy~,thesis is'. ~Opo~On.~t~ ~fl~e~number of molecules present. Con.t~aaus~ prov'isio~l ~: .wit~:"g~ and CO~

2~2 Biochemical ~n~'es~iga~ions.

influenoes the rate of..~the, process I~vourab!y. In such'~conditions a culture may absorb as much as .4,5 liter ,of H~ a ., day at 30 ° C. The pH range of,C1, a~ti~.rn is betw.een 7:5,and ]0.5'~, the optimum being between. 8 and 9.

A growth promotingsubstance is present in alkaline mud extract. This ~ Substance can be concentrated by means of absorption or precipitation. Its nature is still unknown.

A. T~SM~N en A. C. BRANDWIJK, St0fwisselingsproeven met C.- diph~.keri~e: IV. (Experiments. on "the me~aboti.~m of C: dip1( tt~griae.. IV), Geneeskundige Gids 19, 240; 260, 1941.

The production, of diphtheria toxine cannot be induced under anaerobic conditions; either by culturing in the usual way or by preventing the formation of a pellicle by shaking the medium. When, ~ however, the organism is cultured in aeration:flasks, the dissimilation of the sugar takes a completely riormal course. The figures and curves bearing on the fermentation of'the sugar supplied, the course of the pH .and-thepfoduct~on of CO~, NH~ .and toxine agree completely with_those obtained in former experiments with ,,stagnant" cultures. Diphtheria toxine appears to:beable to stand a strong aeration:.with o,xygen. The experiments desCribed confirm the assumption ,that the toxine is an. exotoxine.

A. TASM~N e~ A. ~B. F. k~.. PONDMAN; Su~ la fermentation de la gl=co'se par CL ' .~=~. (Oh ~ie fermentation of glucose by CL' ~g~). . Antonic van Leeuwenhoek 7, 169, 19~1'.

I n contradiction with the investigations of BOORSMA, PR~-VOT and VEIL~-0N respectively P.R~V0T and K!RCHHEINER It has ~ot been possible t o fermen,t glucose,"-either, by three strains of CL ~et=~¢ or by a bacteriologically pure strain, isolated from the strain ,,L:~ ~h~eh has been made use drby.the: f0nner:investiga~ors. So it: has to be: admitted a tong:wi thKoi~ an d ~-IETSCH .anti,many other investigators that.it is absolu,~ely.: ~possible tb make CL4¢~n/ fer m__ ent .glucose. The gaseous~pro,d~cts,.c~bon dioxide-and.ammonia are..exclusively, du.e,, to,: :the -deComposi:ti6n-~of ~itrogen: :.products~ derived from .the pept~n~s:and:aminoac~ds,a~ded~to the,aut~ient medium. i t is very probable: that t he curious resutts presented by B0oRs~A, PR~VOT :and ygzL~,oN are-due to the use of a bacterio- 10gical]y impu~e;c-ultur~:, The f~rmation o~,te~ani¢, toxin~is certainly fa~o~ed:,by ~he' a~i~ig ~:~,f~.g!~se~te .'~ke~tefanie broth. The ~dause of this fa:vourable action ,is s:t~iti~"urle~piairie~l '.

The impuffty,-of~'~he ~ i n use~..is,- clisela]m~ed,

Biochemica l InveStigations. ~3

A. 'r~SMAN et A'. B,. F. A, PONDMAN, Sur la fermentat.ion de la gl,cose pax Cl. tetan%. '(On the fermentatidn of gluco.se by Cl. tetani). An~onie van Leeuwenhoek .7~ 242, 1941.

The authors keep to:their opinion as to the impurity of the strain such. as i t areived 'in their p0gsession.

E-C. WASSlNK .and A.-M~NTEN Some .observations on ¢be utilization of organic compounds by purple sulphu.r bacteria. Antonie van Leeuwenhoek-8, 155, i942.

Enrichment cnltures of purple s.u!phur bacteria from Delft mud were m.ade in inorganic :media. By .means of shake-cultures in agar with-iiiorgau.ic salts it was at tempted to isolate a strain,-the growth of ~vhich was fgvoured by orga~fic substances. This aim.could not be attained as a.l!strains isolated throve better in media containing sodium malate t h a n in-completely inorganic ones. So it may be concIuded that purple sulphur badteria, a~ least those isolate'd !rom ordinary mud, show a better develdpment under photo-heterotrophic than under pi~oto-autotrophic conditions.

A. MANTEN, The isolation 01 Chromatium Ohenii and its behaviour in di{ferent media. Antonie' van Lee~wenhoek 8, .1tt4, 1942.

An. ,enrichment. culture of Chroma~ium Okenii, ,obtained from m u d o:f a di tch. in Delft, was used for isolation, of ,this species: .The bacteria o f the strain~ isolated by making 5 successiveshake cu.ltu~es in an agar medium were, however, less than half the size of Chr. Okenii when cul.~ivated :~.'der the same :conditions as the. bacteria in , the original culture., By. v.ariation of the culture: conditions :f0r the pure,str.ain,-.-it appeured that in a mineral mediumcontaining fa.irly, high coricentrati0ns~of sodium thiosuIpha~e:and fairly:!o;w. concentr~ti0ns or sodium malate,:bacte~ia developed of. nearly the same size,!as.present in the .e~richment. cul. ture. This supports the view of WINOG~ADsKY that -media of this type offer the , most natural conditions ¢o purple gulphur bacteria.

M~R~x P. LOHNIs, Sind hatitbildende Helen bef~higrt elementaren Sticks~off. zu assimilieren?, '(.'A_re :pell~cle '.forming" 3/easts" abie to assimilate: elementary nitrogen?) )Lutonie van Leeuwenhoek 9, 13~, ~ ..i~9~;

It, was~ t r ied whether: :the, results, reported by,'Fm~I: as~ t,o~ the

,a~d: M~co~ie, r~r~a.~ni, could. ~ei r:epzvduced, ,~Altho:~.h,Tthe!.~s :am, ~

2~ Biochemical Investigations.

K. C. WINKLER, ,Iets over de stikstof, stofwisseling bij Bact. coli .(Aspects of the nitrogen metabolism of B. col 0. Chem. Week- b lad 40, 147, 1948.

The quotient, of the decrease of ammonium in the medium And the number of bacteria present is determined after various intervals in a medium containing ammonium sulphate as the sole source of nitrogen on which B. colt was cultivated. Along with the increase in age of the cultures the quotie6t decreases considerably (10C0 × or more).

As it is impossible that the bacteria would have contained initially a 10C0 fold amount of nitrogen, it is to be accepted that at the start a part of the ammonium is changed into aminer~nitrogen which is not taken up by the bacteria. This may lead to the supposition that the synthesis of .amino acids is an exogenic process located in the membrane of bactelia. Amino acids are probably produced already in the lag phase; perhaps a definite external concentration has to be reached before the synthesis of protein may occur.

J. D. TAK, On bacteria decomposing cholesterol. Antonie van Leeuwenhoek 8j 32, 1942.

I t has been definitely proved that in suitable enrichment media in which cholesterol is the only source of carbon this compound disappears with relatively great speed. Continuous shaking of these cultures greatly favours their development. From the e~richment cultures three Mycobaaeriu~n species have been isolated which proved "to be able to at tack this compound also in pure culture. One of these species which possesses this property in a very marked degree does not seem to have been described earlier. For this Species the name of Mycobacteriugn cholesterolicum has been proposed. Strair,s of Mycobaeteriuq~ phlei, M. lacticola, M. berolinense, M..salmog¢icolor and of M..rubru~n, taken from the collection of the Laboratory of Microbiology at Delft, were also able to decompose cholesterol.

A. J. KLUYVE'R and A. MANTEN, Some observations on the metabolism of bacteria oxidizing molecular hydrogen. Antonei ~an Leeuwenhoek 8~ 71, 1942.

Enrichment cultures for hydrogen oxidizing bacleria enabled the authors to isolate four different species having this faculty, one of which apparentlywas identical with Hydroge~omo~as [lava Niklewski. This strain proved to be suitable for mancme,ric experiments with resting bacteria. With suspensions of ~he~e bacteria manometric experiments regarding:th e. gaseous metabolism h a v e be~n. p~rfolaned with the ultimate aim of Contributing to the solution of , the problem o f chemo-autotrophic ca rbond iox ide

Biochemical Investiga~ons. $6

assimilation. It was ~ourid that the bacteria only brought ab~.t hydrogen oxidation, if they hacl also been grown under autotrophic conditions; heferotrophically grown bacteria did not oxidize molecular hydrogen, although they were able to respire normal}y with organic substrates. In contrast herewith the autotrophically grown, bacteria proved to be able to bring about both types of oxidation. Moreover: on addition of both hydrogen and lac, tate these substances proved to be oxidized simultaneously, the rate of each of these oxidations behlg unimpaired. Under certain conditions the tot,al gas consumption was even unmistakably enhanced. These .results seem to warrant the conclusion that hydrogen oxida"don asks for a special catalytic system independent of the catalysts active in the normal respiration process. Apparently the special system is only built up when the bacteria are growrl. ~ii~der autotrophic conditions and the faculty to s3mthesize this system i s gradually - - and after some time irreparably --- lost on cultivation under heterotrophic conditions.

Finally i t has been shown that bacteria, Which were active as f a r as hydrogen oxidation was concerned, were completely unable to bring about a reaction between hydrogen arid carbon dioxide. Herewith the theory regarding the nature of the metabolism of hydrogen oxidizing bacteria already advanced in 1906 by KASERER and still recently defended by BURK has been deTinitely refuted.

S. J. BUREMA and K. T. WIERINGA, Molydenum as a gro~ch factor of Arotobaaer'chroococcum. Anionic Van Leeuwenhoek 8~ 123, 1942.

Though .the discovery o f molybdenum as an element necessary for the growth of Azotobacter enables us to cultivate this organism in a purely synthetic medium,, free of combined nitrogen, the optimal medium for. its development is not known as yet. Evidence is given that organic matter of the soil is highly favourable. Molyb, denum acts as a reductor in the assimilation of atmospheric ni- trogom For thereduct ionof.ni t rate .ni t rogen less Mo is needed than for the "reduction of free nitrogen.

A. QuIsPEL, The lichenisation of aerophflic algae. Proc. Ned. Akad. van-Wet, 45; .276, 1942.

The fungal symbionts.in ~i:ehen.ized~algalcovers canbe c:ul.t~¥ated with more success-:than-.true lichen ~[ungi. Their gre~t s ~ l .arj'ty: ~;0 the la t ter makes it pmba.ble that..they are related to.certai~ h~:e lichen fungi and that ±his alga-fungus symbiosis is cbmpm'able to the tichen symbiosis, in consequence-they folTn an excellent object for the study of this symbiosis. The fungi are unable to fix atmospheric nitrogen. They cannot develop withouf aneurin, which they

26 Biochemical: Invesfigstlons.

obtain in nature:from, their .algal p~.rtner, ~I~, none :of .the c:ultures on ~ario.us media, .the preSenCe of lichenic acids., or ~imflar:pr.odu&s could, be .:detected.- :On -the- :contrary, i..t, appeared ,,that, the ~ga Apatoaoccus~is the producer of ~; rem.arkable:me.tabo!ic~.Prod:nct, ca!led ,apatococc~n, w i t h the te`n£a-tive, formula, ~3H~O,~N:. ,Some chemical properties of .,this substance` are. described. A relationshi~p with certain lichenic acids is suggested.

A. QtlISP~X, The-.mutual relat ions between .algae and fungl in lichens. Di~.s. Groningen, 194-3: ' Recueil des:-Trav: bot . ~ & r l . 40, 413, t9~t3.

The lichen-symbiosis was investigated by means of experiments with pure cultures of the components. As lichen-algae some Cys$#- coccus species were isolated, the only lichen-hmgus investigated was Xanthoriomyces ~arieti~iae. As an orientation; however, a great many experiments were performed with the fungi which are living in symbiosis with the aerial algae ~leurococcus and Apatoccus, as it appeared that these fungi are closely related to true lichen-fungi, whilst, their gvowth÷~velocity is .much better. I n consequence~ the'y are an excellerit object for the Study of lichen-symbiosis. As far as possible the results obtained with the investigation of these fungi were tested upon Xanthoriomyces.

It appeared that the fungi did not develop in synthetic culture ~olutions without the addition of certain nutrilites (aneurin, fl- alanin and other bios substances). The lichemalgae, can..provide the fungi with these nutrilites. These algae themselves were sti- mnlated by the addition of. aspara.gin, nicotinic acid and certain bios substances, when developing- m, .organic: culture solutions: In inorganic solutions a good develgpment could only be..obtained after the .addition of a smM1. amoun.t of-asc0r'bic :acid. I t is. very probable that the tic.hen=fungi, a r e able ,t6 st~imita:t.e the photosynthesis of the algae.by the production of ascorbic acid~ or a,related substance.

The fungi did .not produce lichenic acids, in cultures. :On-the other hand the alga Apatococcus.minor synthesizes,a, remarkable metabolic product, called apatococcin, which most probably is related with certain aliphatic' lichenic acids.

An investigation of the water-h0usehold Of some lichens showed that the protective influence of the .-fungus against a: desiccation of the algae is merely very small .and can be only perceived when the desiccation i s n o t t o o il~ense. ~he ~final;coiac'lusion.is ~hat~ the lichen-s~bi0sis ,may be regarded :as a ;,mutualistic symbiosis" in Wl~icl~ ti4eexcharige of nut~.tes' plays a~ important role.

Bic~hemlc~l ,$nves~iga~ions.

H. Boxrwx~s; tn-~estig~ibn, of ~he :symbioa~ Of, ,.4:l.n~s gl~t~osa, ..4lnus ~ncana~ mad: :H~'popl~a, .,,1,~m,ioid~s:-.. A~tonie' ~va~. Leetiwei~ hook. 9," 107, ,10~3.

The cult ivati0p:of the :symbiont:.,from: .the, various noct~.~.wa:s tried on man3r c~ture 5neclia, .These-experiments were. caged.out in aH:ii~onth§ of the yearl Next..t0. Bacillus subtilis an d af, ew b a c t ~ and. moulds .in morely, f~w cases .Acti.nomyces:de3retoped. Any Ac, tino, mvcetae :occuz~ing ~ve~e kept. in cultivati0n,and tested as. inocu!um. Never c,ould a.ny develo,l~ment.o£ nodules be iinduced~by.th..erm,-in synthetic.pure cultures 350 of which were .tested, InocU!atio~ ~ , h the strain Actinomycss ,aIni of. vol~ P~-OTH0 gave negative. re.s;ults. In sterfliz.ed .soil ~n .open. pots 2~6utof 100 uninocul~ed:.plants.bor, e noduleS, so the rare i~ositive restilts among the. inoculated tflant.s are deemed ~alueless. I,t is claimed: tha t the residts~ oi YON PLOTI~b arrived at by the .~amelmean'sand ~on fewer plantsd0 neffher a, dmit any valid concl.flsion, as to the identification of the endophyte of Alnus.

MARIE" P. L~-INIS, De symbiose va n Bacterium radieicola met vl~nderbloe~nigen. (The. symbiosis Of Bacterium radi~¢dla with leguminosae) Yakblad voorBiologen .25, 27, 19~4

The investigations on the symbiosis-of nodule organisms and their, hosts, carried out. after" the publication of the monograph of FRED,'"BALDWlI¢. and McC0v":in 1932 are reviewed.:"Stress' is lain o'ii the part' Of the host in the symbiotic .fixation" of nltt0gen.

P. B. I~OTTIER, Fluor0metrische en spec~ophotome,txische bepaling van ]actoflaviue in micro~orgauismen. (Fluordrbetrical,'afld spect-rophof0me~rical estimation 'o~ riboflavin'.in micf0.orgalfism~).. Tbesis~ Delft~ '19~2,

Riboflavin:is esiimated-nearly .exclusively by-.mea~s of 'three methods,,-: viz.,. ,biologiCally,- : speetrophotometrically:, and . fluoro= metrically..As, the. author ,is.of the. opiniqI~,that ~ it:has :y:et:icQ • be established that the biological method wfll:.cove~the total.amatm, t. of riboflavin; he has . . . . in. Collaboration with: othenmembers-o~.the Biophysical: .Research ~. Gronp,: in?Utrecht . . . . merel,.y, stnd~e6i.th,e physico-chemical,methods.

By means of measuring..:the absorption,spectra~:R"has-been established..fhat, a. sufficient .pufific~titm, of ,tie ~ex, tra¢t, oox]:d,,:not be realised,:..without,, considerable~:logses~of, rib0ftkvi~. ~Tht~s.-..the spectrophotometdeal method'.'.is inexact. ~Mere3yi~b.y :a ;ppt~g :an extrap0"l, ation -of,-..~he~ coinplete, ,".-a, bsoi~p~o~a; :.spectr~J~0~ ' .,)~,~xe! i io~ purified extrac-ts ~-,order to, determine:~h~.,absori~tio~,..o#fhei-lig~,{ by these iml~urifies ~. .it- has. bee~ possi, ble tO-..arri,*ze~t. app~Ximatel~ correct, results,

More.exa¢~.,ve~utts ,.,may. be. .~t.,tame& -'by ,.the._..flUorome~i~ metla0fl,

~8 Biochemical In~estlgatlo~'s.

In order-to,arrixre..at .this ~ it has, previously,~o ,be'made sure of~ .that ~ e~clusively the, fluorescence of. riboflavin 'is- .measured. A special screen for the elimination of fluorescenceof:anyimpurities

esent: has to be. applied.- Further the .decreasing influence on the br~seence caused by the ~ absdrption: of the incident light ii~ the

e~tracts and bY the presence bf! substances Which' will ext~ffigm'sh th~ fluorescence 'has t6 be. calctilate~.

A calibration diagram has been designed which permits ~he exact reading of the amount of ari3~ fluorescent substance in an ex~tra~t by ineans of the estimation 0f-the intensity Of fluorescence 0f two dilutidns of this extract.

The extraction of the' riboflavin from micro-organisms appeared t o be quantitative' merel3~ wheii Carried out by means ofheating in 'sal:furic acid Of a t least 3 %.

Some estimations have-showrt, that micro-organisms are richer in riboflavin than had ' been believed previously.

G. J. M, VAN DER KERK, Onderzoekingen o v e r de b i o l u m i n e s c e n t i e .der lich.tbacteri~m (Investigations 'of the bioluminescence of the lumin0us-bacteria). Thesis, Utrecht~ 1942. CA. also: A~ J~ ,KLuYv~R, G. J..M. VAN I),ER KERK: land A. VA~r DER BURG, The ,effect .of radiation on light emission by luminous bacteria, Proc. Ned.

Altad. v. ,wet. 45, 886~ 89t5, .1942.

The investigations made so ,~ar prove that in :all probability biolu}nesc~nce is in all ~ases,due io the oxidation b y m dlecular 6xygen '~- of 'a "thennostabile compou.ndof low moiecul~ weight: luciferin, through the: intermediary of an enzyme; luciferase. For luciferin it has been made acceptable that its.molecular structure inclUdes .:tW.O. oxidation possiNfi~ies! tiaereversibie,,,dark~!oxidation ~V.i~hi0xygen or.other inorganic 0Xi,'diser.t0 dehydro,lucifer~n is pro~ bably~ based on the presence of a polyphenoI grpuping (A~DzRSON), whereas in the I irreversible ,,luminescent oxidation with oxygen and i htci~emse, a-~CO~CHs0H~group, is oxidJsed: to :a~' GOOH- g.roltp:(CH~RAVORLySm~d.BALL~N,TINE ). Fresh~effortswetem'ade t o s~p~ate ,~he:lumino.us:,gystem fr0m-"the bacterial celll :,Action o~: tiqaid ~ air .off: Photobacterium_ . phOs~t~oreum "el, iel ;.,,~ro~tUe~:i ~',"cell ddsinteg~a'tion, bat nb e~a,eel l~lar l~,inescence~reselted:A~tempts toi: e)tte~ct ~ from,strongly: .!uminescen{: baexteria~ a s~lSs~a-nee. ~khidh would,be able to 'restore ~he larn'mous-eaPaeRy:of;~bacteria d ~ - ken,,'6d, .by-extlanstion',also, led -.to.~iiega .~ve,:resMts,

A~ ,gm~ent-s:.:h~d: been ~. Of.fared that a , . , f l ~ , enzyme might.,,co, operate; irt. ¢ehe :tlghgxea~t~ioa~",'!n~ order.~o ~.tes~.-~the ~posgi, bita'~,y ,that fi~.... ':b.,,.a,c,i.~i~-;l~eSeence.::.,zem~-,. ~apeei.a!~ .deriv..a~i~re.: of,,~l.~'~fla~

gblRea, ~mr~.,~/~: .~N'o@/¢or.e~n~:V'As~'to; its?.melNag--.poilat :and.~its

~ ! ) g~ tke! ie riboftkv.iri-tetraacet~te, a- ~resiilt ..wliieh~is"amfaveur..:-" a~bN .i~ e l ~ . .~ ~la~in ,h~g~h~ s i g . . ~ e k , ~ i i g v e s ~ g a ~ a ,seei~ed

BiochemicaL. Inws~ga~i~s. 29

to Q~er~l~tfle• prospect, an indirect metkod for Che ,iden~fiea~ia~ of one ,of the components: of ~tl~el luminous system was. :taken up,

I t had .-already been .proved b y ~V~N SCHO~N~B,U~G::and~ ~ DER BURG for luminous bacteria th~,.the,in~enSi~y of the ~em~t~ed light is, reversibly ~e~uced ~ y i ~ d i a t i ~ . i Ap~ en t !y : a.Ph~t~- chemical.conversio~ o~: ionei~o~ t.be c~mpO~er~ts ~ef.t~e. light, emi~tt'mg system occurs. This radiation effect, proved to b.e clearly dep-endeii~ on the wave-length, A s m d ~ of t~e radi~Ho~l eff~ctseemed,.th:ere- fore, ~o. open the possibility, of detergainingart~,daactiv.atiiS~a!.s~ec~ trum" which at ~ the: same:.time we~Id be the absorptipn.spe, ctAim of that component of the light emitting, systemwhich, has~absurbed the active radiation.

The question was exam~:ned in which way ~he req~cd: ab,- sorption-coefficient .for a de, finite w~ve-lengt-h wiU.depend on~.the exper'n-aen.,.tailydetermjnable quantitie:s~ ,v~., the.intensities; Y-o' ,aad: Y of the bacterial iight before aft& af~er .i~radi.atio~,::By cidcu-,~-~ h:ting, the"photOchemical action: ip.er incident q,,aantum, a.qnan~ity was obtained, defined as ,,specific photochemical :effect" ~Mch forms a direct measure for the required, absorption: co~fficien:t, The curve rePresenting-~the .reiafion between- the w~ve-te.~g~:hi::a~nd: t~e corresponding.specific, photochemical., .e:ffec¢ ~ the 4naeti.va:t4~.n spect~m~,.-::-.w.i,lI'~represent • at ~the •same -time. t~e'absorption~,ve in a relat.~ve' • measure~ofthe compound ~h, ich.has! absorbed: the. photochemically active radiation.

Homogenously grown plate .cultures were used, which were irradiated with the light Of a mercury lahip. ~ihe inte~sit:y bi the bacteria:H~gl~:t .before' and after irradiation' Co~ilc~.be estimated for a great number of wa~e:lengfhs b~megns "of the phofogtaph, ic method.

The photochemical inactivation spectrum of ,Ph. phost~horeum having- been: ~stimated, wi,thin: .~ide, bruits., the:,.pho~¢hem..~¢al actio~,:,p.r.o~e61 to. be i.s~tis,fa~t o ~ y .' .p, ropor.tion~ ..fo~.~-e~': ~ '~ , /~ f i~adlation...aaad :to.the ~a:diatidn.iritensit¥,. :$0 ' ~.:at:.~tie.. B ~ - Ros.~o,e. la:w., :,ap, p~ears t p , ~01d: ,f~qr the. ,pho.to.c~.~n. 1 ~ ~ ~ c ~ B ~ uader ~, exami~dti:6xl.. : The. .: ina¢~eation- s p e c ~ ~-sh:,~.~'~. "~n~]~,

~ad 320 mp, The r~e¢ogery of the. ligk~-i~tensiLy after irrndia~,o~ ~as S~u~ed.

I t aP.l~eaxed ~,flaat.~ *&ose:~p _,arts.;o!~ :the. ealmre wkieh .we~.q,=ad:i~ted witl~. ~ve!eng th s ~om 7:tkg xegion < 3¢0.:m~/~ skoveed,-~.~sec~4ary. ef%gt:i:af, tev ~n im.;~ial.: :m¢r~ase,-.'m. llgh, t..:in~ensity;~o a v~,~e e~r

that ~l~to4-~0'~m,',m~tes.~aft~r:,tke e ~ 6 , o £ ~ " g _ ~ e •

omy after- :this perio&- m~ abo~e;~enttonecV f f ee6n6~ ~ / r ~ e - comes mnnifes:t, From ~ i s :~ was~fer~ed thst" ~e::~i~te/~se!~,:.~h¢

30 Biochemlcai Investigation~.

intensity owing to irradiation is primarily due to the photochemical conversion of one and the same compound, and that consequently the inactivation spectrum actually represents the absorption spectrum of one single compound.

I t has been established that the presenceof oxygen is essential for the occurrence of the radiation effect on the light emission of Ph. phosphoreum.

I t is discussed whether a photo-sensitizer could have been responsible for the photo-inactivation observed. The action of riboflavin or a carotene a.s. such could be conclusively refuted; and it was considered less probable that a So far unknown- sensitizer would be involved. Various considerations led to the view that with great ,I~robability dehydro-lucif~rin has to be considered to be the photosensitive component: Oxygen will. be. essential for the photo-inactivation as oi~ly then a-Aarger quantity of dehydro- luciferin is present .in the cell.

Finally the author tried to an.swer the .question whether the absorption spectrum determined might contribute to the development-of a structural formula, of dehydroduciferin. Arguments could be given in favour of the view that dehydro-luciferin might be a derivative of 1,4-naphthoquinone, in which the CO-C!-I,OH- group is, a direct substitute of the quinone nucle~fs. For luciferin a corresponding naphthohydroquinone structure was deemed the most probable.

C. J.. P. SVRUIT and A. L. SCHUILI/~, On the influence ofnaph- thoquinones on the respiration and light emission of Photobacterium phosphoreum. Recueil" des travaux chimiques des Pays-Bas 64, 219, 1945.

By addition of naphthoquinone and some of i ts derivatives in low concentrationsto a suspension0fl Photobacterium pl~osphoreum; both •the light emission and t h e respiration of the bacterial suspension are inhibited, the, light emission much more strongly than the respiration. This inhibition is brought about by-fhe naphthoqtiinones .acting as hydrogen carriers, thus ~hiitifig the luciferin system •in the bacteria into a more oxidised stk~e. The hydrogenation of the naphthoquinones was shown to ]be-reversible and the naphthoquinones are dehydi'ogenated: b y a KCN sensitive ~atalvst. This result leads to a:-modificati0n O~ vAlq ScHo~IWEN- BUl~ff's scheme of respiratioia in :.lumfn0u's bacteria. Bacterial lueiferln .has a normal redox potential (E'0; pH = 7) of' the orde~ of ~ 5 0 inV. The inhibition, is nor. dete~niiaed, by-the concentration of naphthoquinone i n the medium in-'.which the bacter ia a~r6 sus-. Pended, bu.t by:the ratio of the-numbe# :0f bacter iat0 tile t imbe r - of: molecules Of na phttroquin0ne added, indicaiing that this" c6m-- pO,tlnff is practically ~omplet:dly absorbed by: the: cells.

Biochemfca~ ]~nves~igations.

]~. C. WAS$INK, Oft the ratio beL~veen-the 'hpfak~ of c~rbon dioxide and o.f the hydrogen donor in pu,ple salphur bacteria. Enzymologia 10, 257, 1942.

In suspensions Of' the purple sulphur bacterium, Chrosnatium. strata D, m phosphate buffer, pH 6.3, temperature 29 C., the ratio between the amount of carbon dioxide, and the a'frioutats of various hydrogen donors taken up in photosynthesis', Was detefrm'ned manometricalLy,

The ratio thiosulphate: C02 was measured by supplying limf~ed amounts of sodium thiosulphate frcm a 'sRle bulb~ and measuring subsequently the corresponding total am6unt of'C0~' assirniiated. Blanks receiving no dosage were run parallelly in each experiment. In these the gas uptake always was very low. "l he ratio tliiosult)hate'." C02 was 3.75, in good agreen{ent with the value pr6vioUsly fouhd by EYMERS and WASSINK following another method'.

In all cases studied the ratld's were practically irtdependent of the magnitude of the amounts supplied, thus the first step of the conversion seems to run to completion' before subsequent conver- sions play an appreciable rSle.

The rate of photosynthesis was always directly propoffiorial to the concentration of the supplied compounds as far as thi~ concen ,- tration was low.

E. KATZ, E. C. WASSlNX and R. DORR~STEIN, On some-mefJao- dical problems in the study of photosynthesis of uniceIlu,lar organisms. Enzymologia 10, 269, ~1942.

Chro~natium, strain D, again was used, The influence, of bacterial concentration in manometric measurements of photosynthesis was measured and discilssed. Both gas' exchange and fluorescence were considered. In the case of gas exchange the light gradient.in the suspension~is important for the shape of the curve showing, rate o~ photosynthesis versus incident light intensity. In ~conce~t~ated suspensions the average ligh,t'intensity is, lower and the gradiezi,i laxger. A photosynthesis irradiation curve for the average cog may be derived which shows a rather sharp transition between'hght.1/r~" tation and light saturation, The possible reasons causing a, transition r~'nge Are discussed. Concerning fluorescence the-~bsor,ptiort-,, of fluorescence l, ight within the suspen's~ion 'is reponsible, for the :ob ¢ served fact that the fl, uo~escence trah~ition, point~.~ntensi~y,¢iepends less upon concentf, a~iqn than that of gas exchange.:

In direct and atternatir~g current sod~-mn light no~.dif, feren~es 'of the' curves representing the rate of. photo, gyn~hesis ag~ins,t ~ligh~t intensi±y were fom~d.

Corrections for smal~ differerrces in, the~'bottom area 6f the,vessels used in the same~eries ate dig~cussed; t~he,~orrec*ions to be applie/l depend ,on thebacterial, concentrations: ~se&

32 Biochemical T~nves~Ige~don~.

E. C. WASSmK, E. KATZ and R. DORRESZEm, On photosynfJaes~s and fluorescence of bacterio-chrorophyll in Thiorhodaceae. Enzy- mologia 10,' 285, 1942.

Parallel measurements of gas exchange in photosynthesis, and of fluorescence of bacterio-chlorophyll were performed 'With suspensions of purp'le sulp hur bacteria, C ro ati. , s tun D, under a large variety of external conditions in order to investigate to which extent bacterio-chlorophyll is involved iri the ~chain of photo- synthet ic xeacfions.

The gas exchange shows the s~me relation to the incident in: tensity of light as in green, plant celis, indicating that also in the bacteria the process of ph0tosynthesis consists'of light-sensitive;~nd light,-insensitive links.

]?he curves of. fluorescence of bacterio:chlorophyll agafnst' the incident of intensity of light a~e bent curves, consisting of a9 ,,init.'ial" slope (at low incident intensities), a transition range, and a much steeper ,,final" slope (at high intensities). This indicktes • that the a cceptor of the excitatign, energy is present only in a limited amount. In a suspension to Which no hydrogen dongr .was added the transition between the initial and the final slope is found at a low mcadent mtenmty, upon supply of hydrogen donor it shifts tow~ds a higher intensity, so that the fluorescence-yield' at " idgh light intensities is decreased. It~ is .of special importahce that this decrease of fluorescence-yield is also observed if only a small rate of photosynthesis is allowed, "e.g., in .the absence of carbon dioxide. I t is tlms concluded that the hydrogen donor is primarily conne~ed with the transfer of light energy from bacterio-chlor0phyll to:~he energy acceptor. Above a certain concentrat, ion, of hydrogen donor fluorescence is not-influenced fur the~ at about the same concentration,also the rate,of photosynthesis-reaches its maximum. This leads to the conclusion that in the presence of an excess of hydrogen donor a certain syst.em l~ecomes'th~ limiting factor both for energy trs~usfer and for ,photosynthesis: .V~.,lth e-xces~ of hydrogen donor the energy transfer (s~nd/ed, by observ, i.ng flue~escence) and the ,rate ,o~ photosynthesis. :depend on temperature in the same: way..Thi:s indicates that:~he bruiting sysC.emmentioned is no t .the, sys tem:~ energy transfei itself, but an enzymatic ~reaht, ion', i~ which ~he hy~ogen .donor ~ mvol~ved..:This~mp, l~es,, f ~ t ~ m . ~e , 6 a t . t h ¢ hydr.og6n'elo~or does n6t,ocoupy t, he.,transf~r sys~tem as~sueh ..b~t~ gives.rise.~,o,the f~on4aation~ of ~he, energy :a.~ceptor ~bytke m e n t i o ~ reaction. This r e a s o n ~vas- found -to be~ mar, kelly: 8ensitiv~to, PH9 whiek~ sen6ffi~y',w~is ~ ~eren t ; , =fo~, y~iot~s, ~.ydr?g,en ~dga0~rs, In e~perim~m~sdn, wJ~ieh.h~d~og~n a~d~ t,h~os~lpha~te ,w.er.e s!m~lta,~e~- ously supp,h'ed, tl/e parts of "~he mentioned enz~a,tic:s~stem zea_e-

and\~hus'32'i~/~:in the reacfJ~ffwith hy6rogen. The uptake.or c~bon • d i ~ d e agai, nst'i,ncide~rlt ir/tenm~ty~l~vs ,an :$-~hape. Seine evidence

Biochemical ]Investigations. 3~

is presented in favour of the view that the ,,loss" at low intensities is only apparent, organic hydrogen acceptors which do not reflect in the manometric measurement, playing then a relatively more important r61e t h a n t h e y do at higher light intensities.

Carbon dioxide does not appear to interfere with the transfer of energy since fluorescence is not fundamentally influenced by with- drawal of COy Neither have cyanide and hydroxyl amine a significant influence upon fluorescence. I t is concluded that, besides the system reacting with the hydrogen donor, a second dark enzyme- system exists, at which carbon dioxide is converted, and which is sensitive to cyanide and to hydroxy] amine.

Sodium azlde and ethyl urethane also affect the latter system and moreover compete with ~he normal energy acceptor at the system of energy transfer.

I t follows from these observations that the ,,BLACKMAI~"-characteristics of the gas exchange-irradiation curves can be due to two different processes, viz~, 1) a process in which the hydrogen donor is converted into a substance involved in the transfer of energy; it is sensitive to temperature and pH, insensitive to cyanide, and precedes the transfer of light energy; 2) a process ill which carbon dioxide is involved,and obviously reduced; it is sensitive to cyanide and hydroxyl amine, and presumably also to temperature, and it 'follows the transfer of light energy.

The experimental results are in accordance with the assumption that the latter system is a dehydrogenase, the substrates of which are the hydrogen donors. I t is assumgd that kydrogen is transferred from these substances to a substance or to substances present at the ?~ransfer system, which thus become active as energy acceptors.

1"(. DORRXSTEIN, E. C. WASSINK and E. KATz, Theoretical con-. siderations concerning the relation be~veen photoaynthesis and fluorascence of bacterio-clilorophyll in purple sulphur bacteria, w~'th an outlook o~ the comparative physiology of photosynthesis. Enzy- mologia 10, 355, 1942o

I t was at tempted to construe a connection between the experimental results described in the preceding paper and the resuIts obtained earlier by Om~ST~I~, WASSlNK, and collaborator.s-for ChtorelZa.The scheme of photosynthesis to be developed for Chro- matium can'be denoted as an extension of the one proposed for Chlordla. The transfer of energy is preceded by a dark process in which the hydrogen donors react, and foilowe~by a dark process~ in which CO S or a derivative of it, is reduced.

A mathematical description of the picture has been given, ,whi.eh. also ,can be considered ,as an extension'of the formulation given'for, Chlordla-photosynthesis by, OR~ST~IN et al. Finally a general su~ey~ o f d a t a in connectionAvith' a discussion,, of -recent literature, ando a~ outlook on the comparative physiology of plioiosyfl.thesis h~s. been~ p~e~enAed,

34 Biochemical Investigatlon~.

E. C. WASSlNK et J . A.H. KERSTEN, Observations sur la p]aotb~ synth~se et la fluorescence chlorophyllienne des diatom~es. Enzy- mologia II, 282, 1943--1945.

Photosynthesis was studied with diatom-material collected in nature. I t consisted nearly wholly out of Nitzschia dissit~ata (Kiitz) Grun.; after purification by centrifugation i t could be preserved in an active state in the refrigerator for several weeks. Additional observations were made with uni-algal cultures obtained from this material; the species isolated was determined ~s Nitzschia spec. el. ovalis, and represents probably a culture form of the above mentioned one. Photosynthesis was measured with the Warburg- technique in Warburg~buffer No. 9 or in a modified Richter-solution in contact with air containing 5 % of' COy Fluorescence was measured with a photocell-amplifier-apparatus. Sodium light or yellow light, isolated by filters from an incandescent lamp, were used as light sources. Photosynthesis showed much the same characters as in Chlordla, remarkable were the large capacity of the BLACKMAN-System, e.g., at 6 ° C. high light intensities were still efficiently utilisable, and the strong respiratory intensity. Cyanide and ethyl ure thane acted much the same as in Chlordla..Respi- ration was also strongly sensitive to temperature. Aft.er correction for respiration photosynthesis in its light-limited region showed no significant temperature sensitivity. The quantum efficiency' O~/hv) in Warburg buffer was about 1/13 for the cells collected in nature; the fluorescence-yield about 0.25 %; the photosynthetic quotient (O~/COz) about 1.1 equal to the value found for Chlordla. Thus, fat formation does not seem to take place directly in photosynthesis, as was also found by. BARKER. Fluorescence showed a remarkable new phenomenon, wz., a considerable d e c r e a s e in yield beginning at about the light intensities at which photosynthesis is saturated. I t was considered of importance tha t this decrease is not found when photosynthesis is limited by COz-supply, and neither during the initial stages of an illumination. From observations with Chlordla, WASSINK and KATZ had previously concluded tha t in Chlordla the energy transfer is impeded by a reduced state of the cell. I t was now assumed tha t in the diatoms the reaching of light saturation under norrfial conditions is determined by the capacity of the Og-producing (OH-removing). system. Furthermore-i t seems likely tha t in the diatoms with their strong reopirafion part of the transfer system remains reduced as long as photosynthesis is not light-saturate~, so tha t the energy transfer then cannot reach its maximum value. This only becomes possible as soon as the OH-groups formed can no.longer be removed quickly catalytically, and can part ly be used for establishing a more oxidized state at the transfer system, enabling a better energy transfer,and, thus, causing a lower yield of fluorescence.

Measuring the accumulated productions of many separate ex- positions to light during only a few (5 or 10) seconds, the ,,induc- t ion" phenomena of photosynthesis were studied with the Warburg

]~mchemmal ]tnvestxgations, 35

apparatus, so that a series of light intensities could be applied simultaneously. At low light intensities the rate of photosynthesis was the same as under statibnary conditions of illumination. Light saturation, however, was reached at lower light intensities and showed a lower rate of photosynthesis. The light saturation-rate in the induction period was strongly dependent on temperature. The saturation value is higher for illumination periods of l0 sec. than for periods of 5 sec. The various observations show that in dark a cyanide-insensitive-dark system is par t ly inactivated, and is re- activated during illumination. I t lays at hand to assume that in- the dark this catalyst is reduced by ~he respiratcry activity, and that it is only active in an oxidized state, as was concluded also for the system of energy transfer.

j. Dx T~MI'~-, Alkaloidvorming door Claviceps purpurea (Fr.) TuL in saprophytische cultuur. (,Formatmn of alcaloids by Ctaviceps purpuvea (Fr.) Tul. in saprophytic culture). Thesis, Amsterdam 1945.

Many hundreds of isolations from Spanish, Dutch, Canadian, Polish and Hungarian ergot were tested. Alcaloid formation .in saprophytic culture is in principle possible. The absence of s~le~o- tium formation in cultures does not prohibit alcaloid formation. The percentage of alcaloid forming cultures from Spanish commercial ergot was twice that from the ergot of other origin. The ability to form alcaloids decreased rapidly as the isolations grew older. Attempts to avoid the degeneration by using a number of different media in stock cultures were in vain. In peptone-maltose Claviceps demonstrated a scantier growth but more often alcaloid formation than in asparagine-saccharose. This was connected with a stronger deterioration in the latter solution. In asparagine- saccharose with tertiary calcium-phosphate alcaloid formation appeared more often than in the same solution with secllndary phosphate. As an acid solution is better for the preservation of the in water soluble alcaloids this was probably due to a stronger alcaloid formation in the more alkaline solution.

The interference of the many factors that influence the formation of alcaloids in saprophytical culture (and which not all could be regulated) caused a great irregularity in the results.

The cultures exhibited also great differences in mode of growth,, colour, spore formation, formation of pseudosclerotia and other' qualities.

When newly isolated strains from ergot of good. quality (Spanish) were used, .andthe chemical assay f~]lowed directly on the filtering off of the mould deck, theh it was possible to demonstrate a measu- rable quantity (at least 0.01 rag) of alcaloids in more than half of the cultures. The highest outputs o rS0 ml cut~kreswere 1.'50rag alcaloid in ~ the mould, 0,21 nag in water soluble aicalbid Jn th~

36 Biochemical Investigatlon~.

mould; and 0.90 mg alcaloid in the filtrate. "The conditions of the experiment did not give an explanation for those isolated cases of high output, found in different cultures. These quantities must be compared with those of MARTIN, who regularly obtained 2.45 mg alcaloid in the mycelium from 50 ml nutrition agar, and those of B~I~SY, who obtained 7,4 mg in the myce]ium from 50 ml agar.

In some experiments a strong influence of different kinds of water, used as solvents for the food substances, on the alcaloid formation was observed. A connection between these results and the composition of t h e different kinds of water could not be ascertained. In extensive experiments, in which traces of r~any elements were used, n o indication was found to attr ibute these differences to the presence of micro-elements.

In experiments with crude vegetable extracts some influence was found on the growth of Claviceps, but more on the rate of growth than on the final output. No influence on the alcatoid formation was found, not even if extracts or products from rye and other cereals were used. Indole-containing substances - - such as tryp- tophane which is closely related to lysergic acid - - h a s no favourable influence on the formation of alcaloids.

The mould crop was greater in diffuse light than in darkness, but greater in darkness at the bptimal temperature of 25 ° C. than in diffuse light at room temperature.

Cultures from single ascospores or conidiospores, or combined cultures, did not form alcaloids with more ease than the cultures of isolations from fragments of sclerotia, that were commonly used. They also showed no other'differences. This, and the results of infection experiments on rye makes it probable, that Cla}~iceps is homothallic. Polyploidisati0n with camphor or acenaphthene is perhaps possible. The clones with considerably increased length of the conidia demonstrated usually a decrease until normal d imen- sions were reached. They were not better sui ted for alcaloid formation.

Experiments were made to disinfect chemically, according to SCI~WEIZER, culture media made of rye. By a thorough disinfection t rea tment substrates, prepared according to SCHWEIZER, were in some cases free from infections. In extensive experiments with those media prepared from germinating or from ripening rye and disin- fected with chemicals, a good development was never obtained.

No factor was found, which forced the moulds to form alcaloids though they were in principle capable of it.

J. MOLDER, The development of sulfanilamidopyridine-resistant strains of pncumo¢occi in vivo. 'Antonie van Leeuwenhoek 6, 221, 1939~I940.

In mice the pneumococcus strain America type I may become resistant against s~lfanilarnidcpyridir.e within a few days, in spite of the fact that this strain originally is highly ~ensitive to the drug.

Biochemical Investigations, $•7

The young resistaiit strain is at first less ~ru:lent than the sensitive strain, but after several mouse-passages it tan easily regain its full virulence. I t is possible .tO make the strain wholly-resistant against .sulfanilamidopyrjdine. The young resistant strain may cause a protracted bacteraemia in mice when tile latter are treated with the drug. Later on this phenomenon for the greater par t disappears.

In clinical prac t ice i t is of great importance to count with the development 'of drug-resistance, and i t will be wise to treat serious pneumococcal infections immedia te ly with substantial doses of sulfanilamidopyridine, probably bet ter still in combination with an anti-serum.

P. LoPEz CARDOZO, De gevoeligheid van dysenteriebaciHen voor sulfanilamideverbindingen in vitro en in de cliniek. (The sensitiveness for sulfamilamide compounds of dysentery bacteria in vitro and clinically). Ned. T. voor Geneeskunde 85, 417, 1941; Cf. also: Chemotherapie bij bacillaire dysenSerie. (Chemotherapy for b~cillary dysentery ). Thesis, Groningen 1940.

The chemotherapy of Shigdla infections has been seldom studied either clinically°0r experimentally. For this reason a few in vitro experiments are-communicated so far as they are of clinical importance. Bactericidal effect has been demonstrated f o r sulphapyridine and sulphamethylthiazol. Sulphanilamide has less-;bactericidal action and acts mainly as a bacteriostatic. The significance of traces .of fresh •blood is considered. The value of chemotherapy is pointed out, in which respect the importance of maintaining other therapeutic measures is stressed.

H, W. JULIUS and K. C. W~NKLER, On the action of sulfanilamide. II. The action of sulfanilamide on catalase. Antonie van Leeuwe.n- hoek 7, 25, 1941.

According to MELLON, LOCKE and SHINN, the baeteriostatic action of sulfanilamide is d u e to the inactivation of (bacterial) catalase and the resulting ac'cumulation of hydrogen peroxide. The probabili ty of this theory i s discussed.

Catalase activity: w a s s t u d i e d by means of Photobacterium Fischeri, as an oxygen indicator. By adding peroxide to the tested' cultures of bacteria it has been demonstrated that:

I. Bacterium .coli, Photobacteri~m Fischeri and Streptococcus haemolyticus contain catalase. II. Sulfanilamide does not inactivate the catalase in blood-. III, Sulfanilamide does not inactivate bacterial catalase nor does it affect the production of catalase in the growing Culture containing the drug. So the conclusion is drawn that the assumption of catalase inactivation t.o b e t h e essential factor in sulfanilamide action on bacteria will not lead to the solution of the problem,

38 Biochemical Investigations.

H. W. Jtn.zus and A. SALOMON, On the action of sulfanilamide. I I I . The difference between sulfanilamide and sulfapyridine. Antonie van Leeuwenhoek 7, 77, 1941.

I t has been tried to establish whether the difference in therapeutic effect between sulfanilamide and sulfapyridine .is due to the action of a pyridine derivative. Various substances were administered to animals infected with pneumococci along with sulfanilamide. Pyridine itself improved only slightly the action of sulfanilamide. Step b y step pyridine derivatives were t r ied. Toluene-sulfonamino-pyridine, inactive as a therapeutic, greatly enhanced the effect of sulfanilamide. So the action proper to sulfapyridine must be attributed to the fact that another activity, next to sulfanilamide resides in the molecule.

Other drugs may have a similar effect~ This was noted for chinine hydrochloride and ace.tanilide. The original sulfanihmide drug has unmistakable advantages. The possibility of finding drugs that might supplement its action in the cases of infections were it h a s proved unsatisfactory is of great importance.

H. W. JULIUS and K. C. WINKLER, On the action of sulfanilamide. IV. I s the sulfanilamide molecule al{ered before action? Antonie van Leeuwenhoek 7~ 153, 1941.

The problem of alteration of the sulfanilamide molecule as the co~¢ditio sine qua non for ifs action as a germicide is dealt with experimentally.

1) Some drops of a culture solution containing sulfanilamide and inoculated With cocci are brought in a fresh medium when the decrease in bacterial numbers may be expected. The cocci develop in the same way as if they had remained in their Original environment.

2) When an inoculated culture solution is re-inoculated after a lapse of three hours, the freshly introduced cocci pass a same lag phase as the cocci of the initial inoculation.

The sulfanilamide as such is the active molecule.

K. C. WINKLER and H. W. JULIUS, On the action of sulfanilamide. V. The action on anaerobic growth. Antonie van Leeuwenhoek 7~ 163, 1941.

T h e action of sulfanilamide on B. ,cull is not influenced by different oxygen pressures. Sulfanilamide was shown to act on B. cull and Str. haemolyticus (strain ARONSON) cultured anaerobic, ally: a). by addition of thioglycolate, b). in evacuated Thunberg tubes. The obtained growth curves with anaerobic cultures show the same characteristics (lag time of action, influence of inoculum, counteraction by para-amino,benzoic acid) of sulfanilamide action

Biochemical Investigations. 89

as the aerobic controls. The mechanism of sulfanilamide action must be the same in both cases.

The presented facts are incompatible with the claim of MAIN, SHINN and MELLON as to the an~icatalase activity of sulfanilamide; no hydrogen peroxide being formed in" anaerobic cultures.

K. C. WINKLER, On the action of sulfanilamide. VI. The action on bacterial respiration. Antonie van Leeuwenhoek 8, 10, 1942.

The oxygen consumption of resting B. colt or Streptococcus haemolyticus (strain ARO~SON) was not influenced by addition of sulfanilamide. When both organisms were cultivated in media containing sulfanilamide and suspended in sulfanilamide containing buffers the oxygen consumption was the same as in control susisensions of normal cultures. The same conclusions held true with regar d to dehydrogenation velocity for various hydrogen donators. The inference is that sulfanilamide does not interfere with bacterial respiraticn: The cause of sulfanilamide action must be looked for in some other process of bacterial metabolism.

K. C. W!NKLER and H. W..JuLIus, On the action of sulfanilamide. VII. A sulfanilamide activating substance in hbrse blood~ Antonio van Leeuwenhoek 8, 86, 1942.

A sulfanilamide activating principle was found to be present in red cells of the horse. This activator substance is active in the rather high dilution of 0,5 % haemolysed red ceils. The substance or substances are present in the red cells, not in their cell membranes. They seem to be of a protein nature or ads.orbed on the protein (haemoglobin). In some media no sulfanilamide action is obtained without the activator. In other media sulfanilamide action, though clearly present, is markedly enhanced. So it must be emphasized, that the substance under discussion is an activator and not a ,,conditio sine qua non" for the sulfanilamide action an0 ~{s characteristics. The substance is activating sulfanilamide against streptococci, staphylococci and B. colt. The .substance is not present in human blood or in the red cells of sheep; rabbits or mice.

H. W. JULIUS and K. C. WINKLEi~, Off the action of sulfanilamide. VIII . The mechanism of its action. Antonie van Lee'uwenhoek:8~ 139, 1942.

The theory of KUHN and'0thers, who explain the action of-sulfanilamide and its antagonist p. aminobenzoic acid, by supposing the latter substance to be the prosthetic grouping of a bacterial enzyme,, is discussed. Varidus facts Which are not in accordance with this hypothesis are considered, It is emphasized that the

4O Biochemical Investigations.

growth promoting property and the sulfanilamide antagonizing property of p. aminobenzoic acid need not necessarily be correlated. If p. aminobenzoic acid counteracts sulfanilamide this does not necessarily mean that the sulfanilamide acts by counteracting p. aminobenzoic acid, as for many kinds of bacteria it is by no means proven that p. aminobenzoic acid is a growth factor.

Experiments show that: 1). B..coli grows in synthetic media without p. aminobenzoic acid. Still sulfanilamide inhibits growth in this medium and p. aminobenzoic acid is antagonistic. 2). Cysteine is antagonistic to sulfanilamide with streptococci. 3). Peptone is strongly antagonistic for sulfanilamide. This action is not due to p. aminobenzoic acid. 4). p. Aminobenzoic acid is strongly adsorbed by B. coil. This adsorption cannot be due to the apoferment. Salfanilamide is either not adsorbed or if it-is, adsorption is within the experimental error.

I t is still unknown which part of the bacterial metabolism sulfanilamide interferes with, by supplanting p. aminobenzoic acid or not. Various considerations lead to the assumption that sulfanilamide interferes with protein metabolism. Within this scope some alternative hypotheses about sulfanilamide action are offered.

I{. C. ~rlNKLER, On the action of sulfanilamide. IX. The action on the synthesis of amino acids by bacteria. Antonie van Leeuwen- hoek 9, 115, 1943.

Studying the action of sulfanilamide on bacterial nitrogen meta, bolism, it was shown that: a). Sulfanilamide does not alter the rate of gelatin-hydrolysis by papain or by the proteinase of B.pyocya- neum and B. prodigiosum, b). Sulfanilamide does not influence the synthesis of aspartic acid from fumaric acid and ammonium chloride by resting B. coli. c). Addition of single amino acids does not counteract sulfanilamide, d). Addition of single amino acids merely accelerates growth slightly; a marked acceleration was obtained only by adding var ious amino acids simultaneously. e). The addition o$ such an ,,optimal" mixture of amino acids did not exert any influence on the action of sulfanilamide on growth. As the growth acceleration shows that the bacteria are saved an important output of en%rgy in synthesis as a result of the supply of the amino acids, the author concludes that sulfanilamide action cannot be due to interference with the synthesis of amino acids from inorganic nitrogen.

Considering these facts, t he -au tho r expects sulfanilamide to perform its action on bacteriM growth by interfering Withprotein anabolism, anywhere in the synthesis of protein from ammo acids.

Biochemical Investigations. 41

H. W. JuLIus arid K. C. WINKLER, Oil Lhe action of sulfanilamide; X. The mechanism of act ion of sulfanilamide derivatives in vitro. Antonie van Leeuwenhoek 10,~ 1, 1944--1945.

The better activity (in vitro) of various sulfanilamide compounds as compared with sulfanilamide itself is only quantitative, i.e., an equal activity is obtained with lower concentrations. I t is shown that the activity of the drugs studied is so narrowly related to their absorption in ,the bacteria (B. coli), that probably the varying activity of the compounds studied is due to differences in ad- sorbability. For different drugs the absorbed amount was equal for concentrations ~ i th equal activity.

The concentration of p. aminobenzoic acid which re-establishes growth - - in cultures containing the compounds studied in concentrations of equal activity - - was equal in all cases, This fact corroborates the hypothesis, that activity and absorption are correlated and shows that the mechanism of action (in vitro) is the same in all cases.

J, D. VERLINDE en J. ZELDENRUST, Over her voorkomen van necrose en de o6rzaak van de dood bij pneumonieen, welke met sulfapyridine zijn behandeld. (On the occurrence of necro~s and the cause Of death in pneumonia which has been t rea ted :wi th sulfapyridine). Ned: T. voor Geneeskunde 86, 2520, 1942.

The occurrence of necrasis of inflamed lung tissue in 4 cases of fatal pneumonia, which" has been treated wffh sulfapyridine is described. The possibility is considered that this. necrosis might be a result of local anaphylaxia and t h a t the fatal results might be entirely or partly an outcome of general anaphylaxia. Based on experiments taken with rabbitS, the assumption is defended that the cause of death by pneumonia of human beings who have been treated with sulfapyridine might be a general, and the.necrosis in the lungs a local anaphylactic reaction. In fact rabbits can be sensibilized with filtrates pre]~ared from the infected" lungs of rabbits, The reinjection of a large amount of such an extract (5cc) can cause death-by shock. Extracts of iungs from rabbits which have been t reated ~ with sulfapyridine cause death by shock with ever~ a ' tenth of this amount. Rabbits which have recovered from pneumonia are also sensibilized. With. these animals shock occurs after injecting a large quanti ty of lung-extract from an uninfected rabbit just as well as after injecting a small amolmt of lung-extract from one that has b e e n treated with sulfapyridine. A chemo. specific anaphylaxis against sulfapyridine could not be shown.

42 Biochemical Invostigations.

J. ][). VERLINDE and .J. ZELDENRUST, A phenomenon resembling anaphylac~cie shock s, fter treatme~lt with. 'sulphapyi'icline. Antonie van Leeuwenhoek 10, 17, 1944--1945.

Two patients on which a successful'operation of the stomach had been performed developed fever Some days after the operation, notwithstanding a.prophylactic treatment with sutphapyridine and both of them died rather suddenly respectively 9 and l 0 days after the operation. On obduction in both cases hemorrhagic serous- cellular bronchopneumonia were found in the caudal parts of the lungs, all the organs were very hype~aemious and the heart did not show any alteration. To account for the fatal course the possibility of chemospecific anaphy]axis to sulphapyridme .has been considered. We succeeded in inducing in guinea-pigs by means of su!phapyridine a shock, which, however, did not result in death. Such a shock could be induced as early as five days after semi- bilization. The adding of the filtrate of inflammated lung tissue resulted in a deadly shock. I n this connection the surmisa] was made that also in the patients a sensibilization by sulphapyridine had occurred a n d that :the pneumonia, which as such could not sufficiently account for the death, has furthered the arising of the shock. The various facts are pointed to which disagree with the identification of the phenomenon observed ~vith an anaphalactc shock. It is mentioned, however; that there is a certain agreement with the. phenomenon of SANARELLI=SHARTZMAN and that of GLAUBACH.

A. B..:GREVENSTUK, Synthese en chemotherapeutisch onderzoek van sulfanilamido-pyrimidinen. (Synthesis and chemotherapeutical investigation of sulfanilamido-pyrimidines). Thesis, Gr0ningen 1942.

In order to investigate the influence of substitution in the pyrimidine nuclefis on theact ivi ty of the three isomeric sulfanilamido pyrimidines (2, 5 and 6), a number of substituted ~ulfanilamido- pyrimidines were synthetised and ~ tested o n chemotherapeutic activity. I n order to obtain, the substituted amin6pyrimidines the following three amino-chloro-methylpyrimidines were:prepared: 2-amino-4-methyl-6'chloropyrimidine~ ~5-arrdno-2-chloro-4-methyl, pyrimidine; 6-amino-2-chloro-4-methylpydmidine, :Further some amin0chloropyrimidines and one amino-chtoro-dimethylpyrimidine were made-.

An investigation of the therapeutic activity of.the sulfanflamido- py~imi~ne was made i n white :mi~e: that h a d been infectedwith pneumoc0cci. The mice'were.injected intraperit, oneallyin a manner t ha t t he "m0ctflum ~ontained: about 20000 organisn~s, Once a day 0.4 mlof a 10% suspenslon of the:drug in Water was a ~ e r e d by a stomaCh tube. The treat-ment was continued for five days.The effe6t of ~ e ¢0mpound in i~ec ted mice was :traced by suspen~g_ a l o o p ~ :of blood from-the t ~ t ~ c e daffy ~ ascites agar ami

Biochemical Investigations.

cotmtlng ~he .~es~t'mg:-pfieumococcus eolor~es, By ¢ o m p ~ g the course of the septicaemia with that in a mouse treated with dagdnan and in one only infected with an equal dosis of pfieumococci the activity :of ~ the-compound :cOuld be deduced.

The three isomeric sulfanilamido-4,methy!p~idines-have an equal: or, larger activity as sulfanilamidopyridine; .consequently the position of the sulfanilamide "group has no influence in ~ i s case. The presencd of a methyl group in position 4 :-of the: p ~ d i n e nucleus proved to: incre.ase, the adtivity of the compound'; this was especiallythe case with the 6-sutfani lamid9p~dines. The~ritro- duction of a methyl group in 'position. 5 decreases the.actiVity. The presence of: se~veral other ~ . . . . . . methylthio, phe'nyt:thio, anilino) has the .same eft ,s the toxicity in ~ome cases. The last pr0pe~ty positionof the sUlfanfi- amido group.

J. DS J o ~ , He~erocyclische derivaten van sulfanilamide en hun che~notherapeut~sche act~v~te~t. (Heterocychc derivatives of sulfanilamide' an.~l: ~he~ ~ cheni0thempeut~cal' activity}: Thes~s, G'ro~

ningen i9"42

A number of compounds, related to. s~anilamide, were ~y~:the~ tised a:nd, tested onchemotherapeutic acU'vity. Various s ~ - amid0thiazqles,, with sabstituents in, the positions 4, or 5 in:the thiazole nucleus~ land 2~sulfanilamidot.hiazo~e,, were prepar.ed. These N'-der[vati~es, of sulfanilamide were obtained by reaction of N-acetylsulfanflyl~ ch!oride ,width t he appropriate ~ e s .~.,:the presence ~f- a ~ y ~ o u s pyridine. N',derivatiwes of Sulf ~ a m f d e : of some substitutediselen~oles, oxazoles,, isoxazoles a~d triaz01es:were prepared by 'the same method.

The new products were i investigated~ on their-chemQthe~apeu~c activity against mouse septicaemia caused: bypneumococci

As is.well-known, the Substitution of.a methyl group in position 4 in 2-su~anilamidot~'azoie, has no appreciabl~ i effect~ bn ~ e ac, ti_vity:: Substltution. ~y ~the ~ehyl~)tert. ' ~h~yl:-or ~ h : ~ ! . ~oups espe~alIy~increases the t0~¢ity~ widowS-: .~n~i)at.ing ~e~activi~y~ Subst~tu:tion o f a :ea~bo~i: go 'p. , either. ~: ~ior,5 :pJ~sf~ioa, :res~t~d

44 Bioche$ical !nyes'in\ga~'ions.

meric with 2-su]/anilamido-4-methylthiazole, proved to be very toxic.

H. VELDSTRA, Nieuwe voOruitzichten voor de chemotherapie van :b£cteri~te infectieziekten. (New prospects for ttie chemo- the rapy of bacter ia l infectious diseases). Chem. Weekblad 39, 506, 1942.

The action of sulfanilamide is inhibited by p. aminobenzoic acid, thus .by a structurally related substance. The antagonistic factor in yeast is p l o b a b l y a s well p . aminobenzoic acid. Mc. INTOSH a n d WHITBY were the first to point to the possibility that the action of sulfanilamide might consist in the elimination cf some mechanism or enzyme essential for the bacterial metabolism. I t had in fact been evidenced that p. aminobenzoic acid is an essential growth factor for many microbes. Substances related to p. amino' benzoic acid may also inhibit the function of Sulfanilamide, although in a slighter measure. Sulphopantothenic acid and pantothenic acid act antogonistically as well. Assuming t h a t displacement reactions play a part, ' it is needed in order to combat a definite microbe to detect its growth factors and to synthesise structurally related substances', which, however, cannot act as such. Probably a special pat tern ot :active points is more important than the structure, of the Whole of the molecule.

In order to detect any occurrence Of antagonistic actions, growth factors for green plants were synthetised by the author, e.g. a- naphthalene sulphonic acid in comparison with a-naphthalene acetic acid.-No inhibitbry action could be traced. Further investigations were carried out with other compounds of the naphthalene series and with the sulphonic acid derivatives of hetexo-auxine ~[nd related compounds.

j. j. DuYVEN~ DE WIT, A. JAARSVELD, B. C. P. JANSEN, A. VAN LUIJK, R. L. UYKEN, H. K. OOSTERHUIS en J. IR. WYBRANS, De isoleering van een bactericide en fungicide stof uit een 1~enseel- schimmcl. (The isolation Of a bactericide and fungicide substance from a Penicillin,.m). Ned. T. voor Oeneeskunde 88, 718, 1944:

The authors isolated from Penicil~iu~n expansum Wesfl. a bactericide and fungicide substance termed by them expansine. Empirical formula: CTHe04. The growth of Pythium rna~nillatur~ is inhibited in a dilution of 1.000.000; staphylococcus aureus in a dilution of 1 : 100:0G0;IE. coli a n d V. cholerae in a dilution of 1 : 20~000. In contradist inct ion to penicilline expansine is rather toxic:" 1 mg dayly is t h e tolerated dose for an adult rat.

Serological Invest igationg

S E R O L O O I C A L I N V E S T I G A T I O N ~

4:5 ̧

J. REURINK, Natuuflijke Agglutininen. (Natural aggiuf~inins). Thesis, Amsterdam 1941.

Two theories exist as to the nature of the natural antibodies. The theory of the physiological ripening maintains that the origin lays inl an acquired property which develops gradually postnatally. According, to the other theory the natural antibodies develop postnatally as a response to the presence of bacterial antigens produced by saprophytes or commensals. I t has been established that the normal hen serum can agglutinate a great number of bacteria of various kinds, whilst in serum of newly hatched chickens no agglutinins whatever occur. The latter develop shortly after the hatching. The mode of flocculation and the behaviour in heating indicate that the natural agglutinins in hens belong to the O-type. By means of absorption experiments it could be established that these agglutinins are very specific. I t was possible to cultivate out of the intestins of sound hens a bacteriurn?which agglutinated strongly with normal hen serum. I t appeared that this bacterium (Escherichia anindologenes)had properties in common with Shigdla sonnei, which bacterium as well is agglutinated strongly by normal hen serum Although the results of these investigations do not permit the author to reject the ripening theory, he points to the fact that the latter in connection wi th the bacterial character of the natural antigens (character of. the fiocculation, thermolabili ty and specificity) does not contradict the theory of the active formation of the natural antigens.

I. J. LE COSQUINO DE BUSSY en J.-J. VAN LOGHE~a, Natuurli'jke agglutinatie van staphylococcus aureus. (Natural agglutination of St~zpkylococcus aureus), l~ed: T. voor Geneeskunde 87, 1164, 1943.

The occurrence of naturM agglutinins in m a n and in rabbits of various ages w~s st~idied, bo th by direct me thod and by that: of CASTEI~LANI. The hypothesis is advanced that the antigens which are considered as the physiological growth stimuli responsible for the development of the cellular defence apparatus (GLIMSTEDT), also induce the production of humoral antibodfes.

W. A. L. D]~I~Iq.~R, C. VAN DER M ~ R and R. TH. SCHOL~r~Ns, The electrophoretic behaviour of. Bacterium :typhosum in relatioa to the clianges of the antigenic struCtttre obse~ed in ~e s~00f&- rough variation. Antonle van Lee'uwenhoek 8, 53, 1942.

Reasons are given f~r the classification of the Vi antigen a.mong the O-antigens, A description is given o f a micro electrophdresis cell with which measurements of the migration velocity of bacteria

46 Serological Investigations.

can be easily performed. The pH-electrophoresis Curves of different strains o{ B. typhosum, determined by the method described, leads to a classification in four different groups corresponding closely to four different antigenic types. !Most likely the ~ .antigens are amphoteric substances with iso-electric points between pH 2.0 and 2.5 (nucleo-proteins?). After heating up to 100 ° C. the Vi antigen rest, even at p H < 2 , possesses a negative charge. In all probability it contains groups strongly acid in character (phosphoric acid, sulphuric acid?). I t is supposed tha t the somatic antigen IX is an amphoteric substance with an iso-electric point above pH 4.5. A provisional diagram of the localisation o f the antigenic components on the bacterial surface is given.

J. H. BEKKER, The antigenic properties of bacterial spores. Antonie van Leet~wenhoek 10p 67, 1944--1945.

Bacterial spores are antigenic. Spore antigens differ from the antigens of their bacillary forms. Antigens of the spores of various kinds of spore-forming bacilli also differ mutually.

S. J. c. DUNLOP, Is haemoagglutinatie en haemolyse bet resultant van de werking van 66n enkel antilichaam? (Is hemagglutination and hemolysis the result of the'action of one single antibody?) Thesis,. Leiden 1941.

The author puts the question, whether hemolysis and hemagglutination are symptoms of the action of one and the same antibody. Rabbit serum immunized against sheep erythrocytes was separated into fractions of proteins by means of various methods (according to DOLADILHE and MAZILLE, THOMSEN, RONDONI, PIETTI~E), a n d in these fractions the content of hemolysines and hemagglutinins:was estimated.

The results Of his exeriments lead the author to the conclusion, tha t hemagglutination and hemolysis do not depend on the same antibody.

S. J. C. DUNLOP, On a bacterium that, while retaining its full biochemical and serological properties, develops on.the agar plate in two colony forms. Antonie van Leeuwenhoek 7, 33, 1941; Cf. also: Acta Leidensia IS---l{}, 167, i940--1941.

A bacterium has been described which had the property of continually developing on t h e agar plate in t w o colony forms, tha t had the same biochemical and serological properties and did not show any morphological difference either. The cause of this var iety i n development is left unexpla-ined.

Serological Investigation~. ~7

J. H. BEKKER and H. H. V, INK, Vi-antigen in B. coli and Vi- agglutinin. Antonie van L¢euwenhoek 19, 12, 1944--1945.

The Vi-agglutinin in normal serum occurs in 4--8 %, the Vi- antigen in B. coil out of ieces of heal thy persons in 7--8 % of the cases. The authors could not, however, prove a connection between these two phenomena. The authors present some reasons why such a connection need not to b e considered as absolutely impossible after all.

J. D. VERLINDE and A. J, VAN DEN HOVEN VAN GENDEREN, The biological identification of native and of cooked proteins. An,tonie van Leeuwenhock 9, 32, 1943.

In rabbits specific precipitating sera of high titer (up to l -: 30.000) can be induced with native as well as with cooked proteins by means of single subcutaneous injections with the protein concerned emulgated in vaseline-lanolin. In the experiments muscle protein of cattle and dog serum and hen albumen have been used. The proteins, either cooked or native, are dried, pulverized and emulgated as such in vaseline-lanolin on a waterbath o f 40--50 ° C., some saline or distilled water added if being needed. The injection follows immediately. The precipitating sera against cooked proteins give a precipitating reaction with the homologous native protein as well as with the cooked protein which is dissolved in"-NaOH.

ONG SIAN GWAN, Sur la production d'anficorps au moyen d'un afit]g~ne enrob$ ~ dans la larmline-vaseline. (On tile production of antibodies by means of an antigen emulgated in lanolin-vaseline). Antonio van Leeuwenhoek 9, l, 1943.

Subcutaneous injection of rabbits with proteins emulgated in a mixture of vaseline and lanolin leads t o the formation of a much larger quant i ty of precipitines than obtained with t~ae usual methods of preparing precipitating serum. By night the formation of pro-" cipitines is much stronger than by day.

ONG SIAN GWAN, Differentiation des prot~ines de poumon normal et de poumon intoxiqu~ par le phosgene au moyen de la r~action anaphylactique. (Differentiation 'of the proteins of .normal lung and the lung poisoned by phosgene by means of the anaphylactic reaction). Proc. Kon. Ned. Akad. van Wet. 45, 774, 907, 1942

Proteins of the normal lung and those of the lung intoxicated with phosgene of the same kind of animal can be differentiated by meanso f the anaphylactic reaction. The proteins of the intoxicated lung of different k inds of animals', contain an identical grouping, formed under influence of the phosgene,

48 Serological Investigations.

ONG SIAN GWAN, Serologische verschillen tusschen eiwit ten of l ipoiden van cte normale en door phosgeen vergiftigde long. (Sero- logical differences between the proteins or l ipoids of the n6rmal lung and the lung poisoned b y phosgene). Versl. Nect. .Akad. van Wet. 52, 40, 1943.

The following results show the observed serological differences between the proteins or the lipoids of the normal lung and the lung poisoned by phosgene:

1. An ant iserum against an extract of normal hmg gives a positive precipitation reaction with the corresponding extract, while it does not precipitate an extract of a poisoned lung.

2. The complement fixation with the ~ame serum performed with a corresponding extract is much stronger than that with an extract of a poisoned lang.

3. An antiserum against an extract of poisoned Iang gives stronger complement fixation with a corresponding extract than that with an extract of normal lung.

4. An antiserum against lipoids of normal lung gives a stronger complement fixation with the corresponding lipoids than that with the tipoids of poisoned tung.

This result is much clearer when a syphilitic human serum is used.

ONG SIAN GWAN, Over de vorming en de eigenschappen van amboceptoren tegen een longextract . (On the production and propert ies of amboceptors aga ins t a lung extract) . Versl. Ned. Akad. v. Wet. 52, 270, 1943.

1. The organs of a rabbit, inoculated with a normal lung extract contain antibodies. The acquired immunity is general.

2. The complement fixation shows the presence of identical molecular groupings in the lung proteins of different animal species, after the action of phosgene.

3. An antiserum against a lung extract gives a positive BORDET- WASSERMANN reaction and a positive complement fixation with the lipoids of lung extracted by means of acetone, ether, petroleum- ether and alcohol.

4. The simultaneous presence of two antibodies or of two antibodies and two antigens may either intensify or decrease the phenomena due to complement fixation, as shown in the case of a mixture of two antigens.

ONG SIAN GWAN, Over een positieve reactie van BORDET- WASSERMANN, verkregen met een serum tegen longweefseJ en een vaccine-immuunserum. (On a posit ive reaction of BOI~DET-~TASS~R - MANN, obtained with a serum against lung tissue and a vaccine- immune serum). Onderzoekingen en Mededeelingen ni t her l n s t - t a u t voor Praevent ieve Geneeskunde 2, 1943.

Sera of rabbits which have been immunised with lung tissue of the pig give a positive complement fixation test with a watery

Serological :Investlga~on~. 49

extract of the lung, with pure albumens isolated ~rom t h e l u n g and with lipoids extracted from the lung. Moreoveri. these sera give a positive complement fixation test with the ,antigen of BORDET-RUELENS, used in the reaction of BORDET-~ASSE~LMAN._N. The serum of a rabbit, immunised with dermovaccine or neurovaccine g ives a positive reaction of BORDET-WASSERMANN tOO, as well as a positive complement fixation test with albcanens and lipoids f rom the lungs of the pig. I t is true that normal sera of rabbits also give a positive reaction with the antigens mentioned, but the values (expressed in /complement units, according to BESREDKA) for the sera of the immunised rabbits are considerably higher. The results cannot be explained by the FoRSSMAN-antigen as cattle, pigs and rabbits do not possess it. I t is assumed that .the lipoids of these three species of animals have the same molecular grouping.

W. AEG. TIMMERMAN, Are-the toxic properties o.f a staphylococcal filtrate manifestations of one or more constituents ? Antonie van Leeuwenhoek 8, 41, 1942.

The experiments show, tha t under certain well-defined conditions both the lethal and the necrotic potencies of staphylococcal a-toxin can be destroyed, without much damage to the haemotytiC power, After 3 days incubation at 37 ° C. with 0.25 % of ~formalin, t h e lethal activity disappears, whereas the haemolytic potency remains high. The-same applies for an incubation period of 4 daysa t 22 ° C. with 0.4 % of formalin. Under these conditions it is possible to obtain a modified toxin showing solely haemolytic power..This would apparently not be possible if both the haemolytic and the lethal.properties were caused by the same toxic principle. These findings, therefore, strongly support the view, that haemolytic and lethal powers are caused by two different constituents of the toxic filtrate. The same findings were obtained when necrotic and haemolytic activities are compared. The experiments do not permit to answer the question whether lethal and necrotic activities are manifestations of one or of two' constituents.

A. PONDMAN, Bleedgroepbepalingen in de praktijk.i (Blood group estimation in medical practice). Geneesk. Bladen uit Kliniek en Laborator ium 37, 291, 1940.

A blood g roup estimation is usually a very:simple procedure, sometimes, however, it is difficult or next to impossible to attain a decision. I t is always needed to t e s t the red :corpuscles as well as the serum they are derived from. A descriptior, is given of the blood group estimation such as it has been indicated by the Rijl.cs, Inst i tuut voor de Volksgezondheid, Utrecht. Difficulties may ~lse owingto infection, lysis, cold-agglu~ation, difficult demonstration

~0 Serological :InvestigationS.

~f agglut'mins,:'the presence o f subgroups. As to ~ e latter case a or fl agglutinins may be present, having-a limited activity with respect :to the red blood corpuscles of group A o r B a n d which can also agglutinate the red corpuscles of the O ~oup. To account for this the assumption is made for each red,corpuscle .to possess the O antigen, on the .surface of w~ich ~he A, B,or A and B antigens occur. So the O ant.igen is more or less separated from the serum. When the covering is Very dense an O a g g l u t ~ may be expected, ~his is the so-cMled~special aorB agglutinin. The special agglutinin ,is harmless when present inthe-btood of a donor, in the blood of the receiver, however,, it may be harmful. A thorough theoretical knowledge and a large expelJence should be r e q ~ e d in the per- .former of blood group estimaticn.

S. J. c. DUNLOP, On b~cteriophage anti-Flexner. Ant~onie van Lecuwenhoet~ 9~ 41, 1943.

The Flexner strain KB of FLu from 1921 has givenrise to two strains the Flexner strain 38 which has grown SR and the Flexner stra.in 39 which has grown R. The Flexner strain 39 is at ~this ~omen t a spontane0usly agglutinating, 13~s0-sen~ifive str.ain, while the Flex_u. er strain 38 apparently has kept up i ts original properties and is. inagglutinable and lyso-resistant. Both strains belong to the type X o.f AND REwEs and II~MAN. The Flexner strain 38~ h0wever, appears 'to agglutinate 'very,specifically with a X serum. This strain can also be lysed by a s~ecial group: of Flexner phage and not by others. The Flexner strain 38, however, is probably no variation o f the strain KB Of FLu. The results of BuRN~T and McKIE as to the correlation between antigenic Structuxe; Of the Fiexner strains and their sensitiveness to some groups 0fbacterio- phage anti-Flexner could be confirmed in a high measure.

j . D~ VERI~INDE, The complement fixation test in vaccinia with ant igens/ f rom the brain, , he testis and the skin. Anton~ie van Leeuwenhoek 7~ 111, 19.41.

Eight antigens prepared from the bra~, the testis and the skin of rabbits after the tissues had been inoculated with vaccinia virus, have been compared in the complemen.t~ ~a~tion:testwith vaccinia- immune-serum. It was rare that a suitable antigen could be pre- .pared from ~the brain, Antigens from:Lhe:,testis~ and thee:skin were always active, butut he activity of the former surpassed £h~t :of~the latter. The best results were ~btained with a , s ~ e extract 1 ,:100 of fres.hly removed testis; when frozen: thisextract may.:be~ kept for some weeks. In .the sera of vaccinatec~ rabbits~ ~ d ef:a:monkey complement:fixing antibodies ,could.be :deCe~ted regaflafl~,~e~en th,r6e~;months gf,t6r vaccination., In.sera of~men.,:who h~cl been

~ro!og/e~t~ ~nVes~g~ions,

revaccin~ted more. ~an , :three months earlier this was only r l are y the case,

J. D. V*ZRgX~DS, Ma~Hegta~on. ~o~ax~ques ~-~Stopathol0g4, quesi .obtenues chez des.. l~pins.ino.culds, par, vole s0uscutande-avec l~ neurovaccine'et le virus de l'herp~s. (Cerebrospinal:.o~d>=hi~sto, ~,~ho~ogica!: manifestatiOnS.: obtaine~ in rabbits ~inocul~t~d:. s~b. cutaneously wi~h ~he:. nem:ova¢cine andl the ~ S lot. he rl~eS),. Ondetzbekinge~n en M~declceilngen uit her inSti~udtv66r l~a~;ce~i- fibre Geneeskunde~l; ] ~43o

Neurovac.cine _and t h e virus. 0£ iherpes.cannot :be: sho~w~',i.n~ ~ e brain of th.~ r.~bbi~ a, fter. stkbcutane0us moculatign,: iIf . t h ~ v~us~es have been incorporated .~ .yase~ne-!ano~,-s.esa:.m. ~-ofl or glyce.~ol, they can always been shown in ttie blood and Often in the brain after subcutaneous inoculatio~r:Tlze~a'bbits often die alterS--9 days, sometimes w~th cerebral symptoms..I-Ilstologmally an acute menm- girtsor men/~goencephal~tis of the same t_~e ds the "v~ccinal or herpetic mehingoericephalitis wRh n/ononuclear and ~ pbiynuciear peri~ascular ~infil~rations -are ~oun&

J, V~N ~s.R-He,DaN, De.toepassi,ng van-her v~schijnsel d~ p.hagocytos.e der Brucellos¢ n, (The practiced use ofthe 10heno .~.e~on o£. phagocy~osis i.,n bl-uceHoses)~ Tijdscbrift ~roor ~ iergenee~te 67, 910, 968, 1940.

By. a.dding .~q~oi~ tp. blood the co~p!ement becomes .inactiye, As, the complement being absent , the norm~,o.ps.onin~ a~e:i:nact~v,e and the specific tropins axe not yet it/activated, it mi'ght be expected that the phagocytosis reaction, carried out with" Iiquoid blood, would: ~ i s h specific ~esults..Suc:h~w~s.actuaUy .the case .in man. By addi!ng liqu'oid, to the blood of. cow.~ .horse anclgoat, howeyer, the tropine reaction appeared to-~be eomplbtely inhibited.~ In blood of rabbits, its action varied ~in, ~trength~ Theinadtivatioi~ of! ~h:e tropins W~s not cansed b y a changg, of the white, blood :corpuscles, but by ~he aCtion of the blo~:d :liqufd, atthdug~i~t.~ ~ : : ~ < ~ n e d t e ~ witll: inactivation -of the compiem~nt. :The l !~q~l ~ :still !~'tS ' i ~ Very g," eat dflii~ioff (end ~COncen~'tati~fl :ii: !400~ ~ to.~8000 i.

specific, p h ~ C~ytosis stlmtilating 'agents, 1he :t~6p,~s' WhiCii~7~e:- ther/n0stabfle in.: :cbfltra~t : @i{h. the: ~0n:specifie:8~ghnifl~.~ :I£~:~he ~ *era" to"' ~ '"':"be tes~di~ '..the"":i :trop :> <iins: are" k~ ~t:O'h... p . . . . . . . d~fii~:~'"~:i'Sff~;~e~:i:~:~:g.. ,ry.~,~ g The: re~cfi'6~i: ',can' thuS' be ~carried. but ~/t : .any,'m0m~t'

Yo~ the phag0cytoS!!~S:blb6d .of;:inafi i S (cow, ~orse, sheep and gO)it), ~ a ~ "be=:dse~.

In eXP~H~i'ts ~ t h goats W~ch =had'been'injected, ~ t h ! ~ &

soon asS or6 days after injection) the ~gglutinins and amboceptors still I t o 3 days earlier. 'The three ~n~'bofl~es:'remai~ned"present during. sever~ .~noi~thS,

52 Serologlcal Investigatlons.

The investigation of the reaction of the blood of 62 men, 112 cows and 66 horses learned tha t the tropin reaction possesses next to a great sensitiveness a high specificity. In some cases of brucellosis the agglutination and complement fixation tests were negative or merely slightly positive, whilst these Sera gave a strong tropin reaction.

An important diagnostic :value to the positive tropin reaction is a t t ached in man and cow~ In the horse it is 0f lower value. The positive results in a number 'of horses that do not clinically suffer from brucellosis, are probably connected with the frequent occurrence of latent in{ectionsAn horses. The tropi n reaction in the diagnostics of brucellosis is an easily practicable quick methcd, that can be recommended for practical use.

j . VAN DER HOEDEN, A tropin-reaction for the diagnostic of brucellosis. Antonie van Leeuwenhoek 7j 211~, 1941.

Description of a serum test called ,,*ropin-reaction"' for ~the routine diagnosis of brucellosis in man and animlds. I t is based upon the heat-stability of specific tropins, in contraclistinction to the heat-sensibility of the unspecific opsonins~ With due regard to latent infections, especially in- the case of highly exposed individuals (occupational infections) and in horses, the tropin-reaction is a simple, specific and extremely sensitive diagnostic method, which may prove a valuable addition to the agglutination- and ¢omlSle- ment-fixation reactions..

J. H. BI~I<KER and H. H. VmK,. Salmonella antigens in Baaeriu~ colli and paragglutination. Anthonie:van Leeuwenhoek 8,1~4, 1942, Cf. also: Salmonella-an~igenen in colibacilMn en paragglutinatie. Ned. T. voor Geneeskunde 86~ 2629, 1942.

The phenomenon, of paragglutination with typhoid serum, of B. colifroln]the intestins of healthypersons and of patients suffe~ng from typtloid or paratyphoid fever was observed and s tu~ed. Evidence could be Obtained that the paragglutination was t o be at tr ibuted to the presence Of a common Salmonella antigen (XXVII I ) , so tha t it is wrong, to make a distinction between paragglUfination and co-agglutination.Salmonella antigens Were found in variou~ combinations with B. colt and different combi- nat.ions colild aiso be ascertained f o r t h e game person. The attention is drawn to the presence of the Vi-antigen with B. coli, also from healthy persons and the possibility, of its connection with the Vi-agglutinin in the blood of healthy persons is pointed at.

Serological Investigations.

J, MtrLVBR,: L.~BIJ LMER en L., VAN TuI~ss, De i~ntigene stmctuur van .den in Feb~u~tri 1939 geisoleerden stam van influenzavirus. (The antigenic S~UCture of the, strain of influenza .virus isolated in February 1939 at Groningen). Ned. T. voor Geneeskunde 85~ 1743, i 941.

The influenza strain isolated in the Groningen epidemic of February ! 939 was studied in detail for its antigeni c structure, Use was made of ferret immune sera and of the specific strains WS, Talmey, Gatenby and ChriStie. The strain was found to be closely related with the Christie s~rain, isolated in England in 1937, less closely with the Talmey and remote from ~he WS and Gatenby strain. The~ epidemic from which the strain was isolated has been accurately described as to its morbidity, course of illness and nature and number of secondary bacterial infections.

W. J. BRUINS SLOT,. Infectieuse mononucleose. (Infectious mononucleosis). Geneesk. Blad'en uit Kliniek en Laborat0rium 38, 79, 1"940.

A survey is given of the history of. the development of infectious mononucleosis and its relation to the gland~ar fever of PFEIFFER. The identi ty of both diseases is doubted. A diagnosis can only be established with centainty by means of morphological ~nd serological blood investigation. There exists a relative and a~ iabsolu~e mononucleosis, t h e latter amount ing to 60--90 % of the white blood corpuscles. Serologically it is impo~ant tha t aggiut~nins against sheep erythrocytes are formed (reaction of PAUL and BUNNELL ) which cannot be abs0rbed by cells from the kidney of guinea-pigs, but can be absorbed by cells f rom cow blood and can be distinguished in this. manner from FORRSMAN. antibodies.

The investigations of the au thor Comprehend twenty cases of infectious mononucleosis, observed i n the last seven years. They were scattered c~ses, slight epidemics not having been observed. The aetiology has not been ascertained as yet. Accordizlg to NYFELDT and SCHMIDT' and NYFELDT B.~ (List~rdtla) mo~o~toge~e,s homi~is is isolated from blood, respectively spinal fluid of patients with infectious mononucleosis. T h e serum from some of these patients, hoWe~ver, did not show any increase of aggiut.,inion against Lis~rdla mono.cytoge~es. F o r clinical reasons the author claims infectious mononucleosis t o b e probably a virus disease.

J. H; B:EKKER, De beteekenis van absorptieproeven voor de reactie van PAUL ~en BUN{CELL. /~he significance 0f .absorl~tion tests for the reaction of PAUL ~nd BUNNELL). Ned. T. voor Genees- kUnde~85, 2197, 1~4I.

The au thor describes a method by:means of which the. agglutinins against~ sheep red b l o o d cells f o u n d in/normal serum, i n serum of

54 Sexologi¢~l Investigations.

putie~ts who have received .a therapeutic serum "rejection, as well a s i n s e r u m of patients suffering fromM~onudeosis infeaiosa, can b e :di~fgrdiifiated:. The method i~ based on absorptio n with.kidney cells of the guinea-pig and wi th ox red blood cells. Kidney cells of the guinea-pig absorb the agglutinins from normal serum and from serum of patients who received a serum injection, a n d not those of the . s e ~ m of patients ~Suffermg f rom Mononucleosis i~- /~tiosa. Ox .red bk)od cells abs&b t h e agglutinins after a serum in]ec.tion.and in Mo~,onucleosis in/ectiosa, but a s a r u l e n o t the ag61utinins against sheep red bl6od cells found in nora/a1' serum.

A. PONDMAN, On the preparation o~ vaccine against typhus fever and the experiences g.athered therewith, Antonie van Leeuwenhoek 10, 57, 1944---1945.

Experiences have been gathered_in, the culturing of Rickettsiae in order to arrive at the preparation o f a vaccine against typhus fever. The strains are kept active by iiassage through guinea-pigs. The susceptibility of these animals may strongly Vary an d depends on their batch of origin. The fertile hen's egg is used as a growth medium for the- Rickettsiae, After initial growth in the-egg the organism is further grown 0n a suspension of the yolk;:sack "in. saline. When growth, was profus e the production of a toxine might cause difficulty, :whicl~ however, .could mostly be overcomeeither by dilution of :the growth medium or by incubation at low tempera, t~re: The vaccine is purified by centrifugating of ~he yolk sac suspension; Before application it has t o pass a cont ro l on sterility, on harmlessness a n d o n its immunizing value.

S, L. BRUG, Op Rickettsia gelijken:de vormsels in de menschelijke long. (O~I corpuscles resembling Rickettsia in ¢~e human lung). Ned. T. voor Geneeskunde 85, 4636, 1941.

Description of intracellular granules found i n sections as well in smears of human" lungs. The granules greatly resemble Rickettsia. In 21 lungs collected at random they could be found in every case either,in the smears or in the sections or in both. Probably they are normal elements of the lungs. ,When one is searching for virus in human material they may be mistaken for Rickettsia. Also ~in spleen and kfdney these corpuscles were found.

S. D. LIEM and F. H. VA~ THIEL, The complement-fixation test for'Chagas' disease employii~g a dried culture antigen. Acta Lei- dehsia 15--1:6, 259," 1940--1-941.

A complement-fixation test for Chagas' disease, in which a dried culture antigen of Trypanosoma cruzi il used, is described. The antigen is checked with- good r e s e t by means 'of s e r av f rabbits immurdzed with this parasite and of dogs infected with it.

Serological Investigations. ~5

Sera of 3fi7 ,persons not suffering fromChagas' disease are tested, 2,7 pgrcent cross reactions :occur with Wassermann-positive:-sera. S t rumapat ien is don't lead to a positive: reaction.

A positive reaction was shown in 4 out of fi: (tiff per cent) ulcus mone patient sera, I t has been proved by means of the immuni. zation of a rabbit with Dmelcos vaccine that the positive reaction was not a fortuity.

The antigen is very probably tenable unlimitedly. In practice its use yields advantages over the antigens used up to now,

I t deserves recommandation to check the value of the ar~igen in sera of patients claimed to be suffering from Chagas' disease on account of the establishment of the presence o f Trypanosoma cruzi and not silffering from an ulcus molle infection. This research must be performed in countries where Chagas" disease is endemic .

MEDICAL BACTERIOLO6Y AND SEROLO6Y

J. H. BEKKER, De beteekenis van de fluorescentie-microscopie bij her onderzoek op tuberkelbacillen in sputum. ~The significance of the fluorescence microscopy in the, examination of sputum for tubercle bacteria). Ned. T. voor Geneeskunde 85, 3399, 1941.

The author describes the results of a comparativeAnvestigation of 1200 sputa, examined according to the ZmHL-N~LSEN method and by means of the fluorescence microscope. 183 Sptrta appeared to be positive according to the ZIEHL-NEELSEN stain, as well aS on application of the fluorescence microscope. 4 sputawere found t o be positive only by means of the ZIEHL-N~-ELSEN method; in three, cases this result was confirmed by means of animal and culture tests, but as to the 4th sputum, the presence o f tubercle bacteria could not beproved , neither culturally, n o r by means of animaltests, nor clinicaUy, 13 sputa were ascertained to be poskive only on applying the fluorescence microscope and in 7 cases this result could be. confirmed b~¢ means of cu l tu reand animal tests. Out of - the remaining fi sputa 3 came from patients w h o were certainly not suffering frOm tuberculosis'and as to the' o ther 3, the pa t ien ts suffered from pulmonary tubemutbsis; closed or not. With a view to these "details, 4t may be considered as probable that t h e fluorescence microscopyis a more sensitive method to prove the presence of tubercle bacteria in sputa, but that on the other hand the reliability of ,the re'suits decrease.

J. H. BEKKER en A: TASM'AN, H e r ,verband tusschen, de zuur- vastheid en her fluor~scentievermdgen-van d e n tulS~r.kelbkcfl. (,The relation between the acld-fastness,, and the fluoresc n g action of the tubercle bacteria). Geneeslmndlge Gids 1i9, ~23; 19¢I.

The conclusion of some authors that the acid-resistance .~nd the

56 Medical Bacteriology and Serology.

fluorescen~ing action of. the tubercle bacteria are due to different priii~ples seemsno t to be adequate. After ungreasing with alcohol, e ther and chloroform t h e t u b e r c ! e bac ter ia loose their acid- resistance b u t also their fluorescenting action. Therefore these two phenomena seem to depend on one and the same principle e.g. the lipoid cover of the bacillus.

G. P. F. MUNrCIK, Hat aantoonen van tuberkeibacillen m~t her fluorescentiemicroscoop. (The detection of tubercle bacteria with the fluorescence microscope). Tijdschrift voor I)iergeneeskunde 69~ 287, 1942.

The paper contains a short description of the various apparatus used in fluorescence microscopy, a list of the s t a in ing methods in use and the work carried out in-this field. Most of the workers have compared this method with t h a t o f ZIEHL-~ELS~r¢, and studied in how far a greater number of positive results could be attained, and a shortening of the time needed for the examination.

The advantages of the fluorescence method are: 1. great gain of time. 2. possible gain in positives. 3. s imple staining method. 4. the use of a dry system. The disadvantages are: 1. the need of working in a darkened room. 2. t h e great sensitiveness of the method, so tha t the greater gain in positives may not always be ascribed to the tubercle bacteria. 3. the preparations stained with auramine lose their fluorescent activi ty ra ther quickly.

j. H. BEI~KER en H. H, VINK, Her onderzoek op tuberkelbacillen in weefselsneden met den fluorescentie-microscoop. (The examination of-tubercle bacteria in tissue sections with the fluorescence microscope). Ned, T. voor Geneeskund~ 87, 10, 1943.

Description and discussion of t he t e s t for tubercle bacilli in tissue sections by means of the fluorescence microscope. The fluorescence microscopy is to be preferred to examination with the ZmHL- NgELSEN stain, because of the simplerstaining technic, thelsfronger contrast, the detect ing of more bacteria, and the t ime gained.

D. MULDER, Her kweeken van tuberkelbacillen bij de bestrijding der rundertuberculose. (The culturing of tubercle bacteria in the eradication of tuberculosis among cattle). Thesis, Utrecht 1943.

The purpose of this investigation was to introduce a method for the detection m as complete as possible m of ~nimals with open tuberculosis. For that, reason the cultivation of tubercle bacteria on an artificial medium from Sputum and from other exudates was studied.

Medical Bacteriology ~nd S~olog3r.

Smears: of the sputum were s tained after ZmRL.NE~LSE~ ~ d examined for acid fast bacteria. If these were:not found, the~sputmn was treated with s~furic acid and cultivated on the LO~WENSrEIN medium. Every precaution was takeh to prev~ent • acid fas t Sapro- phytic bacteria to be mistaken for tubercle bacte~a.

To prove the efficiency of the cultivation method the detailed results of the examination of 43 cows, one horse and one goat, were recorded. All these animals showed a positive tuberculin test, but the repeated bacterioscopical examination of the smears had no results. I t was possible to cultivate tubercle bacter ia in ever~Tone of them within 20 to 30 days.

The results of the examination of smears has been compared by: I. the staining method of JOTTEN-HAARMANN. 2, the fluorescence microscopy, 3. the cultivation method.

With the cultivation method many more Cases of open tuberculosis were detected than with either of the bactefioscopical methods. In a few cases the results of the inoculation of sputum in guinea-pigs were compared with those of cult ivation The lat ter method appeared to be not inferior to t he first.

AmOng 235 cows, all giving a positive tuberculin test and r e siding on farms, where notwithstanding systematic measures against tuberculosis had been applied, a higher nnmberof positive reactors had been found than the year before, 23 cases of open tuberculosis had been detected, by the cultivation m e t h o d fM 150 cows, indicated b y clinical examination as highly suspected of severe tuberculosis, only 6 open cases were found by bacterioscopical examination. Among the remaining 144 another 22 cases of open tuberculosis were detected, by the cul t ivat ion ,method.

The conclusion of the author is t h a t t h e cultivation, method should b e employed obligatorily in the systematic-eradicatio~ of tuberculosis among cattle.

C. F. vA~ OX'EN, Een kweekmethode voor tuberkelbaci]len toe- gepast bij de bestrijding van de rundertuberculose. (A cultural method for the tubercle b~llh,used m t~e fight agmnst the b0vme tuberculosis). Verslagen Tubercmose Stu~lie-CommiSsie 20, 19, 1944.

An effective separation on the ca~tle-farms of animals i~lfected with tuberculosis, according to the tuberculin reaction, from ~0n- infected animals is very difficult, Notwithstanding the measures taken~ new infections continually arise among the full-grow~ and especially among the young cattle.

Among the m e a n s available for th~ detection of spreaders the cul tur ing of "tubercle bacilli on the medium-of L6VCE~STE~ iS ~ S cussed]n detail. Apparently the culture me.thod furnishes themeans to detect the remaining spreaderS of baciUi. ~Fhe results :obtained by means of this: culture method are compar~ed w i t h ~ those of :the bacterioscopic examinatio~ of sput~um under the f~uorescenee

Medical Bacteriology and Serology.

microscope. It appears that with the culture method many more open sufferers are detected.

The results of the culture method and of the animal test are compared. Here the number of open sufferers detected are of the same order. The animal test may be slightly more sensitive. I t is pointed out that ~nitially one may have mastered the technique of the culture insufficiently which fact enhances these differences.

This method may be applied successfully on cattle farms where a larger group of :reacting animals can be found and where re- peatedly new infection is detected among the young cattle.

J. H. BEKKER, De beteekenis van de verschillende laboratorium~ methoden voor her onderzoek op tuberkelbaciUen bij den mensch. (The significance of various l abora to ry methods for the detectio~t of tUbercle bacter ia in men.) Verslagen TubercuIose Studie-Com- missie 20~ 3, 1944.

The quickest way to detect tubercle bacteria in matter taken from tuberculous patients or from such suspected of being tuberculous, is the direct microscopic preparation, the results of which canbeincreased by concentrating the matter in some way or other. In a considerable part of the cases, however., this method will leave us in the lurch. A much more sensitive method is to try to cultivate the bacteria out of the matter and better still to demonstrate it with the aid of the guinea-pig. While offering a great measure of sensitivenessand reliability, both methods, however, have the disadvantage of being of long duration to which is added, for the gminea-pig experiment, the intercurrent mortality of the test animals. It s tandsto reason that the best results are obtained by applying the available methods side by side; the results can thereby supplement each other; this is therefore the method followed in the State Institute for Public Health.

J. P. BIJL en J. D. yERLINDE, De beteekenis van de virulentie der tuberkeibaciUen. (The significance of the virulence of tubercle bacilli). Verslagen Tuberculose Studie-Commissie 17, 17, 1942.

In order to ascertain whether the virulence of the tubercle bacilli circulating among the population has decreased in the last 50 years, it has been investigated whether the length of life of the guinea- pigs infected with tubercle, bacilli has undergone any change. Reliable data have been collected in literature and treated sta- tistically. As the duration of life of the guinea-pigs which had been injected with a same amount of a same culture varied wideI, y, its average has been determined for every group of animals injected with a same material Out of these data the average length of life of the group of animals which had been infected in a same year

Medical Bacteriology and Serology. b~

has been determined. The entire material concerns 382 ~ n e a - pigs ~ v i d e d over 37 grot~ps. S t a t i s t i c ~ y no change i~ the virulence could be ascertained.

A. CH. Ruvs, Kritische beschouwingen over de bestaand'e op- vattingen over de pathogeniteit van de vogettuberkelbacil veer den mensch. (A critic~I treatment of the .existing ~ew$ sbon~ the pathogenicity of the ~avian tubercle bacillus for men), Verslagen Tuberculose Studie-Commissie 15, 43, 1941.

This review of the literature shows that Mycobaaerium avium can be recognised as a separate variety and tha t even its strains of weak virulence may be determined by various methods. Not all reports on avian tubercle bacilli in disease of man are complete enough to judge a b o u t their pathogenicity. Clinical features characteristic for this infection are nowhere reported. Neither the reaction on tuberculin, nor the serological examination of t hepagen t can furnish any indication as to the existence of avian infection in men. The hypothesis of the activation of a latent infection with this otherwise non-pathogenic micro-organism under definite corn ditions might explain some of the rare findings ~ avian tubercle bacilli in men.

J. VAN DER HOEDEN, Tuberculose bij zoogdieren, veroorzaakt door her vogeltype van den tuberkelbacil. (TuberculOsis in mammals, caused by the avian type of the tubercle bacillus). Tijdschrift veer Diergeneeskunde 68, 335, 1941.

A review of literature learns tha t cases of tuberculosis caused by the avian type have been sporadically observed in various species of mammals. In swine it has been regularly observed, The author 's investigations offer the following results:

[~an Cow Sheep Goat Swine Horse Dog Cat Wild animals in zoological gardens

i i i

strains ~xamined human bovine avian

i,,,,,,,,,,,,,,

914 137

8 17

169 14

8 6

12

842 72 0 133 0 3 0 15 0 118 0 12 3 5 0 6

0 12

0 4

2 56

2 0 0

Nearly all cows and horses with avian infections had severe forms of tuber¢ulos!s (mi'liary dissemination, disuse catarrhal ][~ng

60 Medical Baoterielogy and Serology.

tuberculosis, large extension), Contrarily to most of similar cases stated in literature the avian infection in ~swine had mostly caused no striding processes, although a few cases with more severe extension were met with. Both goats had grave cavernous lung processes, characteristic for tuberculosis in these animals. The results of the investigations substantiate the presumption that the tuberculosis of birds may be a source of danger for our domestic animals and that in the fight against tuberculosis in cattle we have to pay attention not me~ely to the bacteria of the bovine type b u t to the avian type as well,

j . VAN DER HOEDEN, De typen van tuberkelbaci l len bij zoogdieren in Nederland. (The types of tubercle bacter ia in mammals in the Netherlands), Verslagen Tuberculose Studie-Commissie 15, 59, 1941.

The natural store for the so-called , ,human type" of the tubercle bacteria exists in human beings, for the bovine type in cattle and for the av ian type in poultry. If conditions are favourable, these types may also cause tuberculosis in other kinds of animals. The bovine type proves to be the least specific. Outside their-natural medium the tubercle bacteria usually retain their characteristics for a long period (ex.: bovine bacteria from sputum from a woman, who had been suffering from tuberclflosis for thir ty years). There are however exceptions (ex.: from a veterinary surgeon with bovine infection, after 27 years a strain was c~t iva ted from a fistula which immediately grew eugenically as a human one, but had wholly retained the bovine virulence).

ONG SIAN GWAN, De remmende werking van meta len op den groei van tuberkelbacfllen. I. Arseen, ant imoon en bismuth. (The inhib i tory act ion of meta ls on t h e growth of tubercle bacter ia . I) Versl. Ned. Akad, v. Wet. 53, 345, 1944.

Arsenic, ant imony and bismuth inhibit the growth of tubercle bacteria. The inhibitory action of ant imony and bismuth exceeds tha t of arsenic. Bismuth preparations of different puri ty give identical results. The difference in the inhibitory action of arsenic, ant imony and bismuth in a determined concentration is in accordance with the relative position of the elements in the system of elements.

ONG S:AN GWAN, De remmende werking van metalen op den groei van tuberkelbacillen. II. Bismuth. (The inhibitory action of metals on the growth of tubercle bacteria. If. Bismuth). Versl. Ned. Akad. v. Wet. 53, 353, 1944.

Tubercle bacteria tolerate bismuth; a~ter a protracted period they are not killed, growth continues. They can be t ransmit ted

Medical Bacteriology ~nd 8~r.o|og~; 61

on fresh, m ed i a a n d t h e y m a y 'infect guinea-pigs.~ It. i s possible to slow "down the g t 0 w t h r a t e . o f t t i b e r c l e b a c t e r i a or even t o inhibi t g rowth altoget.her b y . . . . * . . . . adding b i smuth dur ing g r o w t h : T h e mh.lbitory act ion of b i sm u th on tuberc le b a c t e r i a resul ts f rom the absorpt ion of t h e m e t a l b y the bacter ia ,

J. D. ~rERLINDE, De complementbindingsreactie van antistof tegen tuberculeus weefsel ~ met een antigeenmengset bestaande uit tuberculeus weefselex'tract en tubercuhne. (The complement fixing reaction of .antibodies against tuberculous tissue with a mixture of antigens, consisting out of tuberculous tissue .extract and tuberculin). Verslagen Tuberculose Studie-Commissie I?, 98, 1942.

Th e serum of rabb i t s in jec ted once subcu taneous ly wi th e i ther a boi led sa l ine ex t r ac t of tuberculous bov ine t issue or . the same e x t r a c t unboiled, bu t f i l tered t h rough a Seitz filter, o r tubercuhn , or a m ix tu r e of bo th emuiga ted in vaselin-lanolin, .already af ter 7 days contains complement f ixing ant ibodies against tub,erculln.With a mix tu re of bo th ' ant igens in 72 %. of the cases more, of ten even cons iderably mote complement uni ts are f ixed t h a n would have been the case a f t e r separa te appl iance of the. ant igens (Oi~G's phenomenon) .

Serum

,,,,,,, ,,,,

Rabbit 1092

. 1093 ,, 1212

,, 1213 . 1172 ,, 1173 ,, 1216 ,, 1217 , , 1151

,, 1165 , , 1214

1.215 Normal

o J

Imraunisation. with

,,,, ,,

Boiled tuberculous bovine tissue extract

idem Seitz-filtrate tuberculous bovine tissue

idem tuberculin idem idem idem Boiled tuberculous bovine tissue extract Jr- tuberculin

idem Seitz-filtrate tuberculous bovine tissue ~- tuberculin

idem-

Bound complement units

Antigen

K T KT ~KT (K.+,T)

125 lO 260 + 1 1 5 15 0 250 + 235

100 0 125 + 25 250 0 500 -b 250

0 10 30 -i- 20 0 o~ 0 0

75 o loo + 25 75 o 75 0 75 lO 125 + 40

0 lO 2O -t- 10 75 0 75 0 7~ 0 75 0

10 0 125 + 115 15 0 150 + 135

l~edlca]r ]~c~eriology and Serology.

J. D."V~-RLJ~D~-~ Over de pathogene werking van!mycoba~te~b~. (On thepathogenic action of. mycobacteria).. Verslagen Tuberctflose Studie-Commi~sie 18, .3, 1943.

The mycobacteria as far as th'ey are: reckormd ampng the acid proof saprophytes and the bacillus 'of JOH~E are harflIyor not at all pathogenic for the usual-t-es-t animals. As far as they possess Some pathogenic properties they can evoke, in rabbits and guinea- pigs histological changes reminding of tuberculosis which are lir~ited to the injection spot;: somegeneralisat~on is'rare. Dead tubercle bacilli h a v e similar prop.erties. When, _however, the former saprophytes living or dead and the dead ..tubercle bacilli are injected with fat .or oils, generalisation with a proliferating character does ensue. As all mycobacteria are able to evd~e es- sentially the same change~ it is probable that they po.ssess p_atho- genic constituents in common. The virulence ,might be dependent on their na tura l covering with .lipoids.which evidently not in all mycobacteria are equivalent in activity .against the body's natural 'means of resistance.

J. D, V]~RLINDE; Over de a ntigene eigenschappen van tuber- culeuze weefsels en hun verband met tuberculine-allergie. (on the antigenic, properties of tuberculous tissues and their connection with tuberchlin allergy).'~Verslagen-T~berchl6~e--Sth~i~C0m~nissie 19, 17, 1944.

1. ]?he antigenous .action 'of the tuberculous caseous.material, ,,cheese", is for the greater part not specific for the species, for a minor part it is.

2. Tubercfil0hs ,,cheese~~ of man, cattle, goat, pig, and rabbit are serologically identical, or they at-least contain serologically identical groups. Tuberculous tissue of horse and dog is divergent in this respect•

3• In the serum of normal rabbits and horses, sometimes in that of c'atfle anti-bodies against tuberculous horse ,issuej. not against tuberculous ,,cheese" of cattle can b.e detected•

4, Neither ,,cheese"-antigen nor ,,cheese"-antibodies can as a rule be traced in the-blood of the tuberculous animal. Yet, the tuberculous animal is able to form,,cheese"-antibodies after having been given ,,cheese"-antiboaies parentererally. Antigen and antibodies are either included in' the tuberculous process or they are present in the blood in such a small quantity that they cannot be detected by the usual sgrological'reac.tions. The fact that the entire individual is sensibilized speaks for the:latter ~ew.

5, Guinea-pigs may give a positive reaction of MANTOUX ~dter • " ° " " J ~ " ° " r having been senmbilized with ,,cheese -antigen, wlth a n~xtu e

of- ~¢he~se'~-p, ti6e~---a~l-~tab~rv~-i.n:-~n~ ~ s o m e t ~ ~ h tuberculin alone by injecting these substances emulgated in vaselin- lanolin subcutaneously. Thel'tr'~Ii6~ on the skin is more distinct

Medlcal~ ]3acteriology and ~erology. 68

if ins tead of tuberculin a mixtu.re consisting of equal p a r t s Of tuberculin and ,,cheese"-antigen is .injected subcutameously, After. the sensibflizifig injection has beer~ repeated about six weeks later or perhaps also after a .very large, dose of ,,cheese"-antigen,. the sensibilization is sufficiently strong to cause a distinct reaction of MANTOUX with tuberculin only.

6. The tuberculin reaction is supposed to be brought about by .the formation of a complex tomcally composed, of an agglutination of antibodies against the tuberculous cell (,,cheese~'~antibodies) to the antigen of the tuberculous cell (,,cheese"-antigen) and tuberculin. The fact that the tuberculous process sensibilises the entire individual explains that through the formation of such a complex the skin reaction takes place in the skin, the focus reaction in the tuberculous process and the general reaction throughout the entire body.

J, ~). VERLI'NDE, Over her voorkomen van tuberkelbacfllen, en anaerobe micro-organismen in tandenborstels, (O~othe occurrence of tubercle bacteria and anaerobic micro-organisms in tootli- brushes). T. voor Tandheelkunde 48, 779, 1941.

In the long run a mass, consisting 0ffoodrests, tissue:~etritus afld t00th-paste accumulates at the base o f the hairs of a ~o~h-brush$ aerobic andanaerobic mouth bacteria will thrive here. Ir~ t~e to0ih- brush of a sufferer f rom lung tuberculosis living tubercle ba/ct~ria were found; in three of four other tooth-brushes anaer0bic micro- organisms (Fusi/ormis, Actinomyces, B. melaninogenic'~m, Le"ptO, thrix lanceolata) which are possibly.of importance for the affection of mouth and teeth, were detected. Therefore it is important for a good hygiene of the mouth to pay special attention to the hair base when cleaning tooth-brushes.

J. VAN DER HOEDXN, Tuberkelbacfllen in zu.ivelproducten (Tubercle bacilli in dairy 9roducts 1. -V, erslag~n Tuberc~ose Studie- Commissie_14; 77, 1940.

The examir~.ation of dairy products, prepared f~om non-heated milk in the country produced the.following results; a. Two out of 15 samples of butter milk contained liying tubercle bacilli of the bovine type: They originated from stables where ,the cattle were suffering from tuberculosis. b. In none of 72 sa~aples of whey-butter tubercle ,bacil~ w e r e f 0 n ~ . c. In 2 out of 24 samples of farni-butter tubercle l~aciili o f / h e bovine type were found. These-2 samples had been prepared from: I. milk from cows from various farms whichha& been milked in the ca t t l e market;: 2. milk from. a stock of cattle where serious t.ubercu.losis was prevalent, d, Bovine tubercle bacLUi were found in one ¢mt of ~ 56 i~¢ cheeses

Medical ~acteriology a.nd ~erolog~.

originating from 42 farms. The infected Cheese had been~ made 4 weeks earlier at a farm where the cattle had been infected in a high degree with tuberculosis for a considerable time. Three months later living tubercle bacilli could no longer be ascertained in :that cheese.

In order to test the resistance of tubercle bacilli in dairy producfs,. fat cheese had been made from milk to which had been added 1 : I00 milk f rom a cow with tuberculosis o f the -udder which contained m a n y tubercle bacilli, though it did not show any maoroscopic alterations. Then but ter was prepared from a mixture of the same milk in the ratio 1 : 20. In the thus obt'ained but ter milk, which was kept at room temperature, the t~bercle bacilli pr~ved virulen t after 32 days (pH = 3.6). In the salted but te r -kept at d 6 ° C. t h e y had still retained their virulence after 151 days; after 180 days living, though less virulent.baci~i were still found;~after one year no more could be detected. In the fat cheese kept at room temperature during 82 days and subsequently at 4--6 ° C., the tubercle bacilli after one year were still present in large numbers and at high virulence. After 17 months they had died off.

So dairy products prepared from non-heated milk m a y constitute a source of danger to human health. This danger is most imminent in the country, where these products are consumed by a.small group of individuals and So:may cause ever recurring infections with all the evil consequences of this. The problem of the danger inherent in the consumption of dairy products containing tuhercle bacilli deserves full coi~sideration. For concerns which manufacture dairy products from non-heated milk it would be worth considering to ask for a guarantee tha t the cattle is free from tuberculous infection or to h/tve pasteurisation of the milk made obh'gat6ry.

j. H. BEKKER, A new acid-fast bacillus isolated from a patient, suspected to be suffering/rom tuberculosis. Antonio van Leeuwen- hoek 9, 81,. 1943. Cf. also: Een zuurvaste bacterie ge~soleerd uit een van tuberculose verdachten patient. Verslagen .der Tuberculose Studie-Commissie,. 18~ 18, 1942.

From the catheter urine of a patient, the right kidney of whom had been removed at an earlier date in connection wi th a clinically diagnosed tuberculosis, an acid-fast bacillus, was isolated. On a closer:investigation this bacillus appeared to belong to the' group of the So-called saprophytic acid-fast bacilli, bLit its properties are different from. all acid-fast bacteria described h i ther to . The new- fOund bacillus was called Mycobacterium Behkerii.

J. H. BEKX~R, De intercurrenCe sterfte van ca~ae :bij hot onderzoek op ~mber~elbaciUen. (The intercfirrent'morf~lity- o~ guinea*, pigs during the tracing of tubercle bacteria). ~ G.enee~k~ndige Gi.ds SJ, ~!7L 49~4,

lI~ ~Aia:"ma.v, eS:iigati0n, ,oft-he i~tex:~l~eat: :m0tta~,'.ty ~of, ,g~ne~-~pi'gs d.~:ring th~-t~aciiig of tubercle" bac~,:it. ~,ppeared~that,the' md~ali'vy w~s .cA~s~d ;':m',a.merely: :sli'ght d~egree-~by '.the injecte~ mater.i~. Climatic and diethetic conditions play: a..~r~greater .pal,.: So'me: measures, which might ihduee-a eh~nge herein are discussed.

J, ,D. VX.RI,INDX,. The sensiti.v.eness of ,the guinea~iffand the rabbit for .Mycobaaerium Bek.ke.rii. 'Antonie van .Leeuwenhoek 9~ 129, 1943.

The guinea=pig ~anct t he r~bbit: may be i~e~ed b y ,~ .acid ..fast micro-orgam'sm(:jsotated by BEKm~R. Mter intmpe~it0nea~ and subcutaneous inoeula.~i0n, i a .gran~la.ti0rI . ~tiss~ue; ,especialIy in: the liver is e:x6ite6, which can hardly be distinguished from pr0]iferr ative tube rmfl0sis as found in bovine tuberculosis in horses and avian tuberculosis in swine.

j, D, VERLIND~, Experimenteet onderzoek ~aar de pathogene werking van den Bacil van "B~.ItXZR. (Experimental investigation of the pathogenic action of My¢obaaer~urn. Bek#erii.), Verslagen Tnberctilose S~udie-C0mmi~si'~ ~" 1~8~'"3~, i 9~3.

Mycobacterium Bekkerii is b y its nature pathog~entc for the guirieapig. After subcutaneous and intraperifoiaealinjection generalisation occurs. A definite .influence.-o,f paraffinon, the virulence can be no.ted./Dead B.C.G~is but sl.ightly pathogenic and causes at most a'l'6calprocess, speadily ~healed, Para~in may'exoke a generalised_ disease a.t least after p.eritohe~l, injection:

The bacillus Of JoHms, can* be pathogenic for the~ guinea-pig ax~d evencause "slight general~sati0n (small ~aclei ,in ,liver~" and spieen t. when 'paraffin: is used {he generd]isafion bec0mes~ idisfinc, i. .Tl~e increase in~ the. density ~of the~dissiminati0n shows best ir/~fl~6 ~llve~ ~, which tee~ns with ~ miliary-:and:subrhiliary sma~ :nuciei, ,~fii ~¢h often are confluent. Aftei injeeii0n of ~tead. B.C2G~ ini,paraifin, however ~, nuclei are met with only,-sp-oradi-cally. This might indicate, that living bacteria (Mycobacteriur~ Bekk~rii and the bacillus of JOHNE) Wm ''~ rnul~tip!y~,iu . the g~ia~a:~ig. ; The,'~t~ffi~n 'w6~d ~'ct then: in t~v6-wky~: ' i1 the '~ t~sfer 5~ the bkct6ria :thr~'g~6fft "the body; 2:7: fhe proi~e'tion 0f':the :l~adte~a aga~s~,"~hd'0~A~aS? !~f' defence of the ~ host, so that they migl~t '~de~el~p.'bet~er ' :~n: ]t~;presence., P.res~, mab!y :the:aqi&:p~o,of. sa2r, ophy~gs ,v~hi'gh ,s~i~,_,~pg,s~.o~:s~e p~thogenic p, r0per, ty: ,aide : a l so :a~d . ,upon~-,ia:.J~t~-ways':, ~o~ ttle

need the paraffin. Suchbacteriain~y 'l~sse~s id their capsuies ~ e necessary lip0ids which are-~as:str0ng~ protective as paraffin, In its-histologiCal aspect..the investing:ion sul:/stantiates the 'co=-

66 Medical Bacteriology and Serology.

¢eption ~ha t .many mycobacteria possess a definite pathogenic property in common, evoking a s a m e type o f proliferative inflammation, characterised by the occurrence of epitheloid cells, giant cells and lymphocytes.

W. A. P. SCHOl~NER en H. BOHLANDER, Bacteriologisch en epidemiologisch onderzoek van modderkoorts (Sehlammfieber),in Nederland. (Bacteriological arid epidemiological investigation .of mire-fever ~Schlammtieber) in the Netherlands). Ned. T. voor Geneeskunde 85, 4390, 1941.

In two boys , who had gone out in pursuit of voles, mare-fever was found, a leptospirosis ,up till then not observed in. the Netherlands. T h e underlying leptospira, L. grippotypkosa, was demonstra ted in the kidneys of the common vole, Microtus arvalis (Pallas). I t Still remains to be proved, whether this disease only occurs in,the Rhine-Valley near Huissen o r whe ther in other regions of th i s country too certain morbid phenomena should suggest the possibility of mire-fever.

W. A. P. SCHOFFNER en H. BOHLANDER, Voor~geZette waarnemingen over modderkoorts in Nederland, een laboratorium- infectie. (Continued observations of mire-fever in the Net~herlands, a laboratory infection). Ned. T. v6or Geneeskunde 86, 1341, 1942.

A laboratory infection with mire-fever i s described, occurring af ter a bite of a wild vole whose urine contained innumerable leptospirae. The illness ran a highfever , ending by crisis and folloW- ed by a brief recurrence. Leptospira gri#potyphosa could be demonstra ted in the patient 's blood on the first day of illness, but no more in the blood 'withdrawn during the last (5th) fever attack. In con t r ad i c t i onwi th the known facts, in this case again the leptospira appeared in the urine, in which after two months it could still be demonstra ted intermittingly.

W. A. P. SCHOFIrNER und H. BOHLANDER, Die ersten Ergebnisse der ScMammfieberforschuiag in den N.iederlanden. (The first results of the ~investigation of the mire fever in the Netherlands). Antodie van Leeuwe/ihoek 9, 19, 1943.

For more than a year the occurring of mire fever:in the Nether- lands has been investigated. In t h a t per iod 21 cases have been reported. From 479 examined mice 146 (31%) :were carrier of Leptospira gri~potyl~twsa. The" spreading is n o t uiiiversal; it fluc- tuates from 1--I00 %' According to place And time.

P. Mr3NT~NDAM, ~V~.-A.P. ,ScH~'ITI~.NER ell H'~ ZEISLING, Modder- kbort~, in Friesl~nd.. (Mi~e ~fever ,i,n. Friesl~nd). ,Ned. ,T, voor Genees- kunde 87,.397, I948,

In central Friesland, where a strong epizoStia with L. grippo- typhosa was fo~, d~ aanong voles, 9 eases of-mire fever ha~;e been diagnosed ~. The patients are exclusively larra Iabourers; who were occupied with the raking a n d c a t t i n g of gTass.

W. A. P. SCH~F~ZR und H. BOrm~NDER, L~ber eine Epizootie ua~er.zahmen RaVen und die dageger~ gerichtete Sdllu~zimpfuilg de~ Personals. (On an epizootic among t, ame rats and on the active immuniza~on against it of, the .staff). Anton, ie van ,Leeuwenhoek 7, 1,. 1941.

Among the staff o f a big commercial laboratory a case of WEzz's disease occurred. The infection was supposed .to be caused by the bite o f a tame rat . ~ This aninaa] forme, d part ,of a group of 50 rats, Among these 15 leptospira carriers Were found.From the remaining 3000 rats a random test was carried out with 42- °animals; 18 leptospira carriers were .found. ,Consequently 0n an average 36 % of tlie adult animals were infected. The stMf of t h e laboratory was immunized actively by administering the home-made'vaccin two times intravenously with an interval of a week; the fi~t~fime I mi, the second.time 3 ml.

P. H. v&~r THmL, Her gebruik van liquoid (Roche): i~ de prac~jk' ~/ande diagnostiek tier ziek~e van WEIL. (The 'use o~ liqut~id (Roche) in :.the diagnosticat practice of .1flue W~IL'S disease). Ned..Tijdsch~,, voor Geneeskunde 85, 3018, 1941;.Cf. also: Aeta Leidensia 15--16,. 328, i940--1941.

I t is possible to demonstrate Ieptospirae i n blood collected in 1 % tiquoid: (Roche).in normal saline, by doub!,e centrifuging; The ad~iti0n of :acid buffer flUid, a s done in RuYS ;double-centrifuge method, i s impossible, When carried out wi.thin 3 d a y s after collecting the blood~ the sodium oxalate method of RuYs is preferable to the liquoid method. When fhe centrifuging process cannot bedone before 3 days, '~ter taking the-blood, it is better collectedin ~qndid. if it iS:cbilected.t9 o longag6, I one mus~: put up with 0no-stage centrifuging., Undo1: equ.al circumstances, the second centrifuging !can be:~earried~out dnr ing a longer peffod~ with blood stored up with liquoid than with oxalate blood. Both in temperate zones and'ia~'~the-tr6pies.it is possil~te:~o, isol~te leptospirae;by a~imal expenmerrt -from liquo~d or oxala~e "blood ~ up, ,to: kt Ieast:,6~days a ~ e r ~orward'mg t,o~lab0ratories,

68 Medical Bacteriology and'Sezulogy.

P. ~ VAN THI~-L, Ist es m6glich Leptospira ~cterohaemovvhag~ae dutch. Kulturversuehcn. aus Oberflt~¢henwasser zu isolieren? (Is it possible to isolate Leptospim iaevohaemo.'haglt~e out. of surface water?) Antonie van Leeuwenhoek 7, 137, 1941.

Because the antagonism described by APPELMAN and VAN THEEL between the pathogenic Eeptos~ira icterohaemorrhagiae, and Lepti- spora bi/lexa under definite cor~ditions does n o t occur -in the egg agar medium of ZUELZER, it has been tried to isolate by means of this nutr ient medium Leptospira icterohaemorrhagi, ae out of surface water, in which merely a small number of Leptospirae occur. ,Mthough the water under examination had been supplied.with numerous Leptospirae, it has not been possible to d e t e c t their presence by me~ns of injection of this heavily contaminated water in guinea-pigs. Neither has it been possible by means of a combination of the filtration method of VINZE~T and the egg agar method of ZImLZm~. As Leptos~ira icterohaemorrhagiae as yet cannot be cult ivated from surface water, the methods described by the author have to b e applied.

P. H. VAN THIEL und W. L. C. VEER, Biologische Methoden zum Isolieren yon Leptospira ictevohaemort, ha, glae aus Wasser. (Biological methods for the isolation of Leptospira icterohaemorrhagiae from Water). Antonio van Leeuwenhoek 7, 221, 1941.

I t has been tried to improve the bathing method,of APPELMAN which aims at t h e isolation of pathogenic Leptospirae from surface water. Three new biological, methods have been described: I. a centrifagatlon method, 2. a flooding of the guinea~pig, 3. a subcutaneous transflooding of the guinea-pig. The 2nd and 3rd method are recommended. By these means it is possible to detect a single Leptospira in 50 ml of water. W h e n compared with the bathing method, the lat ter methods offer the following advantages: a). a 9--19 times greater possibility to detect pathogenic Leptospi-' rae in surface water; b). a smaller number of guinea-pigs needed for the experiment.

L. DE BLrECK and JAC, JANSEN, Listerellose bei Tieren. A. Listerellose bei Ferkeln. "(Listerellodis i.n animals. A~ LiStereli0sis in pigs), Autonle van Leeuwenhbek 9~ 93; 1943. Cf. als0: L. DE BLIECK en JAc. JANSEN, Listerrellose bij biggen. (Listerellosis in pigs). Tijdschrift voor Diergeneeskunde 69, "67.3i 1~42.

Out of 5 days old pigs a bacterium was isolated ~that proved ,to be a Listerella. In the livers 0f the pigs multiple necrosis was found. The bacterium was pathogenic for pigs, ..rabbits, mice,, rats and guinea-pigs and ext remely pathogenic for canaries. The same changes in the liver were observed in experimentally infec ted rabbit~, rats and mice. In the pig and the rabbit encepha~tis could

Medic~l Bacteriology ,and~ Serology. 89

be ~provoked.~ The Stra~:proved to:be identical~w/th~e List, rdla. str~i~ isolated, b3 ~ 'KAPSENBERG from a child .that had died of encel~hali'tis.

, 1 T JAc.. JANSEN und C. F. G. x~. VAN DER HURX, Listerellose be~ . L .

Tieren. B . Llsterellose beJ der Ziege. (Listerellosis in animals. B. Listerell0sis in the goat). Antonie van Leeuwenhoek 9, 104, 1943.

From the mesenteric iymph glands of a goat, having died as the r.esult Of a hemorrhagical necrotising inflammation of the large mtestme, a Listerdla was isolated, identical wi~h a Lis~erelta strain isolated from pigs.

A. CH. Rw'x'S, De waarde van typebepaling voor he* epidemiologisch ,onderzoek bij diphtheriC. (The value of the determination of type for the epidemiologic~aI investigation of diphtheria). Ned. T. voor Geneeskunde 84, 3969, 1"940.

In the autumn of .1939 and the spring of 1940 ,diphtheria in Amsterdam was nearly exclusively caused by the mitis ty.pe. In a few cases connected epidemiologically an atypical gravls strain was g o w n , corresponding with a strain isolated 15y SIEM~-NS in 1936. In the same period m a rigidly secluded group of Jewish children from Germany an outbreak prevailed caused by the normal gravis type. Two groups of carriers, later shown t,o carry different types, for some mon~ths lived together in a institution. In 16 out of 54 children a change of type was observed. It is un- desirable that diphtheria carriers should be nursed together, as the carrier period ca.n be lengthened by reinfection.

A. L. NOORDAM, .ECn diphtheric-epidemic, verodrzaakt door her gravis type . van Coryneba~te~ium di'phth~iae in een kindertehuis te Amsterdam~ (An epidemic of diphtheria caused by ~he gravis t~rpe of Cor)mebacteri~m ~dSphtheriae in ~ children's home at ~ Amster- dam). Ned. T. ~'0or Geneeskunde 84, 3962, 1,9~t0.

Report of an outbreak of diphtheria caused by the gmvis type o f C.. dip, h~heriae.in a children's home, from which ,the,children could not be evacuated any more. Of' 96 children, 26 coI~tracted the' disease, 1.9 o f whom had received complete immunisation 4 months before. Of 8 .patients the SCHICK reaction had been found negative just before the onsef" of the illness. 30 children were isoiatect as carriers. The Value 6f immunisati0n is discussed. The p0ssible :usefulness of the ,,injection de rappeJ~' is pointed out,

7O

CH.:G. J: DO~N~CXX en J..~. ~n~K~,B~acte~o!o~sehe.~dlphtherie di.agnos~)e k door middeL van d~ C~UBSR ~ !I~', E laat, iee n te~ur- indicatoren-plaat welke reeds bij macroscopische ,aflezing aaJa* vnjzmgen geeft. (Bacteriological dmgnostics of diphtheria by mean.~ of the CL~UB~R6 III plate, ~ tellurite-indicator-plate~ which furnishesmacroscopic indic~tions)~ Geneeskundige Gids 19, 719, 1941.

The CLAUBERG indicator plate already by macroscopical readings furnishes important indications~ for the bacteriolo~cal diag~ os,tics of diphtheria. The propertie s o f the in~cat0r ~plate, are ~scus~:d~ and compared w i th those of the L0~F~LER serum med!um.~ ~T~..e results are reported of a comparative test covering ~I !6 samples, wherein the LOEYYL~R and the indicator plate have been used side by side. The indicator plate appeared to indicate a number of positive cases only slightly higher tt~an the LOEYFI~R plate. The use of both plates together, however , ga~e a large increase of positives.

A. P. vA~ DER WEY, De prognose van diphtheric, in verband met her bacteri~type. (The prognosis of 'diptitheria •in Connection with the type 0f bacteria~. Ned. T. voor Geneeskunde 87-~ 203, 19~3 ~.

The malignant, forms of diphtheria are caused, more often b y the gravis :type than by the miffs type. The latter'is more laryngotrope than the . former. The ~intermedius tsrpe seems to resemble the gravis type more ,closely than the mitis~type.

A. CHA~LOTT~,~RpYS, On the behaviour of /~. ~yphi' in surface water. Antonie van Leeuwenhoek 7, 93, 1941.

Several t~phoid infections were ~raced to direct or indirect contact with contaminated Water from which fypho~d bacteria c6uld be isolated. Especially~ during the winter monfhs-typhoid bacteria could be isolated from surface,.water. In experiments the disappearance of typhoid bacteria •from contaminated ~water is accelerated much more by the influence of fight than by the action of protozoa. The definite phototaxis :of t ~ h o i d bacteria m~y :c~use the rapid decrease of the microbes from the depth of smfface water as weli.

K. c. WIN I~L~R: ~en W, J.. QUA~S VAN U~ORD, Typhusg~vaar door 'her eten. v~ffaardbeie~n en Sla. (Danger of typhoid ~hr6iigli the eating of s~awbernes and salad), l~ed. T. voor C~n~eskunde 8§, 1200,-1941.

The importance of strawberries and salad as vehicles of typhoid for the Spreading Was studied by examining their infestation with

M~dic~l ,~ Bact~flol~gw,sn~'~ol0gy. 71

coli:bae-~eria,as ari,indicator ~o~faecal ' ¢o~taimk~ati'ofl2,.It~was fotmd f h ~ :>.~-iO~. stmWbdrfies iiea~!y alWay~ ~ ie011'iba~e~ -:ate found~ 0i~" salad ' i~:gdfidi-a] ~ ' they ate: ab~dfit;"b~ :On" Botli: "p~0dflcts cola bacteria r~em~in alive for at .least 24 hours; c. T~fie juice of straw- bett'i*~'- has a ~0wth '~ibi~in~g adtio~.,: thai of saI~d has:fione; d. By washin~ wi th w~ter ~ coLi' bacteria' ar~°n6t "tem~o~ed~ 'but 'thei~ number is'diminished; e. Stmwberrie~ and salad :can be efficiently desinfected with .1 %..hydrochloric a.cid (salad~ also with vinegar) without floss of fia~/0'ur. The- c0mm6n ~ a d sauces cannot be relied upon ~or,~ thispurpose.

G.D. I-I~-MME$, Overbrenging.van typhus dp.or kaas (The tra~ls~ mit~ug .of typhoid bij cheese). Ned. T. voor Geheeskund,e 8~," 203,, 19~3.

Description of the coming down wffli typhoid' of 8 persons, from eating cheese. 40 days after the making of this cheese, typhoid bacteria could be grown from it.

B, ~N: HAv~ en A.. P. VAI~ DXR W~Y: Over bet kwceken van gyphusbacteriSn ui{ bloed en b~enmerg. (On. the culturing of .typhoid, bactezia ou t o~ blood and bone m a ~ o ~ Ned. T. voor Ge~eeskunde 86r Noodnnmmer *vf, 87, ]944.

Next to the culturing of typhoid bacteria out of the blood the authors isolated these-bacteria als0 from bone m~row by means of puuctlon of the sternum (0.5 cc marrow punctate added to 1Oc ox bile). The bacteriological test of the bone marrow offered positive results, during a period.'of more ,than a.week When, the blood was already negative; it may tfius .be of value in the,,phase when the blood iS alre'~dya~egative and- the culturing out Of feces and urine "not"yet, positive.

~f. CLARENBURd ~- S@lmoffelIa a~sTdee~ gekw~klr uit eon'voedi~ga- preparaat. (Salmonella ~ aberdeen cultured ~Ezom:~.nutrient preparation). Gehe.'esku'ndige Gads 18,' 10.40, '1940.

The investigation.,.of a s t r ~ of .paratyphoid c,~,tured from a nuirient, preparation .is. desoribed.. The straiii appeared t0 belong to'~he'~.t~pe,Sal.mo~ella aberdeen. The 3.~.chiidr..ex~ 0f:the family from" which the nutrient preparation had. been derived,, suffered from paratyphoid. In the ,feces o~ these patients Salmoneltapara~ @#hasum .-]3 hkS 'been ~ defected. ~'A' 6ausal" 'Cohnect'i0li b~twee"n the oee~rrenee' 6 f m ~ disease '~ Symi~oms"alad-~he' .pa~ta~b~: i~i6 pr'Oparatio~n'"'co~ilff ' ~Of- be ~'e~tablisjaed. The""'pathogemi:eit3~:~/f Sal~Onetl~.~berdeen for men is not'est'gMish'ed~,~s'yOt,

72 Medical: Bac~er~elogy,-an@ ~Serolol~.

~'!-~: H..VINK, R, ,~H. SCHOLT~'N~S en ;C., ,&, v ~ H,~s, .~,e~.ge:val van !nfecti e met Salmonella m~ntev~deo. (A Cas,e-of ~,fection ,~,t~a Salmonella monUvideo). Ned., T. voor Geneeskunde ,87,: 1433,: 1943.

Description of a case of paratyph0id,fever c.aused bv Salmonella montevideo (ant igen formula VI, V I L ' g , m, s).

A. Cm Rux, s, E~,aringen met den voedingsbodem van LEIFso~ voor her isoleeren van dyserfteriebacillefi. (Experiences ~¢i~h the nutrient medium of LEIFSON-for the isolation of'dys'entery b~hil~.j: Ned. T. voor Geneeskunde "84, 3562, 1940.

For the detection of bacillary dysentery b y examination of the feces the LEIYSON cul ture medium is indispensable. This medium must always be used freshly prepared, and~ subcultures must be taken after 24 and 48 hours' growing~at 37 ° C.

E. A. TILLEMA, Een orienteerend onderzoek over den voedingsbodem van LF.IFSON 'voor.bacteri~ele dysenterie. (A preliminary investigation of the nutrient medium of LEIFSON for bacterial dysentery). Ned. T voor Geneesk~nde 84~ 3564, 1940.

Some results are recorded concerning the cMtivation of pathogenic bacteria of t he intestines on t h e sodium~desoxycholate citrate plate of L~I~SO~. This plate proved to be pre-eminently suited for dysentery bacteria, n o t so however for t h e group Of the paratyphoid and typhoid bacteria.

R. TH. SCHOLT~NS,In Nederland .Waaxgenomen gevallen vail dysenterie, veroorzaakt door B. dysenter, iae NewCastle. (Cases of dysentery caused by B. d~senteriae type "New Cable-noted in the Netherlands). Ned. T. voor Geneeskuade 88, .242, 1944.

Bacteriological and seroiogical description of cases of dysentery observed in the Netherlands, caused by .B. dysenteriae type New Castle (agreeing with: the type 88 of BoYI))~.

R. TH. SCHOnT~NS, Een" 0hbekend t 3 ~ dysentetiebacterie a ls oorzaak eener epidemic, (Aft unknown type of dysentery baCteri~im as the cause of an epidemic). Ned. T. ~oor Genceskunde 84, 1,613, 1940:

I t w a s pr.oved that an outbreak o f 20 cases of dysentery was c~used by a type.of dysentery bacterium unkalpwn_ :t~,tKe pr~esenL Th i s t y p e . i n view ~of its biochemical properties beiongg:t0,,the group of lactoSe-f.ermenting dysentery bacteria. 'Ser0io~caiiy if takes a separate position.

Medlc~l/Btmteriol0g T,.an& ~zolosy. ~

On, omg~gc(ihv.estlg.~.~gn ~. 0f;.b~9~la~. dyseht ery in.i~eide~ and i~' ndiglibburh60d). 'Th"6sls,. Leide~ .1940

A survey:'isgiven of the ~ t o r y , tlie clinfc.,:~he.~p~t~emiology; and~ the b~a~t~ri6tdg~ :of :B: ;dy~i~#er~e: The ~tiiho~ a~i~es: a~ :the~:dori~ dffsio}i ~h~t ~B: dy~ent~ria'e SOnne '6¢e~ii'g endemic a'lly ~,~ei46n~u~i i~s"n~ighb6ytho6tt ~and prbb~ibfy ' i n all of: fh~ ~ l~:eih6i~Iafi~. :~'iit~ e~ia-¢l.hsibri is ~tibstantiated by the oecurrelfce"of tilgh gggitit~tti0fi fliers a,galnSt B . "dj~sent'erif~ sonne in S~ra "of i~efs6n~ :i'n "fi6,nn~il healthiTl~esekgglutiiiins are'deemed' by the autilbr as true rest i~ggliitmi'ns.

C. D. WESTERMANN, Over het voorkoraen van Shigell~ d.ysenteria, type New Castle en her kweeken van dysenterie baeteriea uit. de faeces. {On the occurrence Of Shigella dysenteria~ tyl~.. New Castle, ancl th'e culturing ~ Of 'dysefiteq} bacteria ~r0m feces). Ned. T. voor Geneeskunde :88, 2~t2, 'I94~.

The auflaor isolated:at Amsterdam from ieceGof patients in-3 case sSMgella ambigua and m !08 eases SMgelld dysenteriae type New( Castle.: The suit~bfli:ty :of the :medium of .,~I~sO~ ~for the diagnosis, of bacillary dysentery is emphasized again.

J. D. Vxm, mDx en H. D. Bo~-~, De verwekker van hgl~atitis epicl~mic s, (Tim cause of hepatitls'epidemica).Nehl. T. ~;obrGenee~: kun'de 87, ]3"04; 19~3.

In four patients suffering from hepatitis epidemica t t i e ' a u ~ r s succeeded in isolating ~ filterable virus from-the blood during the period of fever and from the urine during the ie~t~.ric stage. The virhs.'is p athogemc f0r the guinea-pig after in-traPeri.t6~ae~, "mtracar- dial,.subcutaaeous and. intracerebra!.moculation.. The.onl, y morbid symptom in the guinea-pig after an incubation time. of:~= 7. days, is a fever period o.f some days:: :In the ,anima!s~,the V i~...s can.::be showk du~ing the/fever period' ~i~ :the blo,od/.aa6'in~he .Iige;F. - ~ flie-l%dbd the Virus is e~'cr&ted, wit]6' the Ufilie apparently i0r':~ sho~ time. !~'the hvers 6t .the g~u~ea.-pig~a.reas6f fa,t~yi,~g~de~4

60n~al&cent men. arid guhiea~pig~ neutra~izii~g: ~ t i~b3d i~a /~ be strewn.

A fiiter~ble v..ix~S ~as' a cau~atix;e-ageiit ~ 6~_,epidemih - li6#~iti~S-~. Ant~nie ~/a~L l~efiV~enh6ek I0, gK 19~-19~5~

ln-Ao .ur patle~ts, sufferings,from, epidemic -hepatitis :we: su¢ceede@_ in isolating from ..the b!ood:~d~g dck~ ffc~e~:,p~:~od~nd::~em~:~ brine d~ring the, jauncli'ce, a filteraJ~l~., virus., :whieda is ~,at.hoge~i¢

74 Medical Bacteriology and ~Serology,

to t h e guinea,pig and which can be inoculated into this animal in different ways, b u t by preference intraperitoneaUy. Fever which sometimes lasts only one day is the only morbid symptom observed in guinea-pigs..In these animalsthe virus .can be shown in theorgans ~nd ha the bloodduring the fever period; af ter that it is excreted with ~the u.r. ine during apparently a short period. The virus has been grown on the ch0ripallantois of the .chick embryo so far duririg 20 passages. The virus is resistent to glycerol, drying and low t.em.peratures, not to formaline and heating. I n the livers of the guinea-pigs focal chafiges (degeneration, dissociation, necro- biosis, yellow liver-atrophy) may be found.:After pulling through the disease immunity occurs, whilst in the serum of recovered patients and guinea-pigs neutralizing antibodies can be detected.

J. VAN LOOKEREN CAMP,~GNE, Phgumo.coccus-onderzoekingen bij kinderen. (Investigations of pneumococci in childreri). Ned. T. voor Geneeskunde 85, 622, 1941.

The results are communicated of a type investigation in 157 cases of pneumococcal infection in the Groningen Children's Hospital observed during the years 1938--Ig41. After type 1, p~eponderating mainly as a causative.organism Of empyema, type 6 follows in freq~uency; the types 19, 23, 12, 7, 18, 3 and 14 were also comparatively freqLient. The practical importance of the type-examination, though having diminished since the in- troduction Of sulfapyrieline, remains considerable for the combined chemo- and serotherapy of meningitis, septic pneumonia and peritonitis.

J. MULDER,-Pneumococci-types bij pneumococcie8n van vol- wassenen in Nederland. (Types of pneumococci in pneumococcises o:f adults-in the. Netherlands). Ned. T. voor Geneeskunde. 85, 692, 1941.

From March 1935 to September 1940 in Holland 560 pneumococcus strains Were typed (NE~F~-I,D method) from various pneumococcal infections in adults (over 12 years). T3)pe 33 of Etinger Tulczynska (related with type 19) occurs rather frequent!y in the bronchitis group and in otitis media. In lobar pneumoma and empyema type 1 takes the first'place with 58.3 %' a s a causal orgamsm. Then follows the types 2 (12 %), 7 (9 %), 3 (9 %), 5 (6.5 %), and 4 (2.5 %). The h igher types of CooPEm are practically of no significance in lobar pneumonia.In meningitis the types 1 arid 3 prevail, but higher types are relatively frequent. In bronchopneumonia ar/d bronchitis type 1 is absent; types 2, 3 and 6 are rather frequen~ here, andso are the higher types of COOPER. 7.3 -% of the strains could not be typed. The types.. 25, 27 ~and .32 d id not ,occur among the 560 strains.

Medicel Bacteriotogyand: Serology. 75

J,: H: BExxtZR, ,,BotUlisme, iiz .Hees.,, (B0~m atHees)~, 'Ned2 ~.T. voor 'Genosslmnde.88/-N~0odsummer VII., 69,, ~19,~4.

Report of the bacteriological investigation of an: epidemic of boiulis~ at H~es, caused by, the eat}ng of Sm0kedp0rk.58persons partodk of:thiS f0od, 24 feli~ill out of Wl~ich 9 clied. The isolated *oxiii: was not ne~ir~ized, by:.it/e an~ti'-bbtUlhiUs Sera~ A o~ B; other antiLbb~ulinussera vCef~ riot av'aflable. The i~ossibility.that

here one Of. the' 6ther~.-~ypes or a new type-may have c~useff the epidemic .is left open.

J. H. BEKm~R,-Botulisme imNederland. (Botulism in the Nether- lands): Goneesk.undige, Gids 22, 303, 1944.

After a description of the bacteriology, the epidemiology, the clinic, the therapy and the prophylaxis of botulism all epidemics which ,have become known in the Netherlands are reviewed. Since 1899 17 epidemics of botulism have become known, in.8 the ¢linical diagnosis .has been confirmed~ bacteriologically, in the' Others the diagnosis has been merely clinical. All together, the epidemics bear on 76 patients, out of which 21 died, thus an average letality of 2,7 %, whilst the letality of the various epidemics varied :between 0~ to, t00 %.

j. j., VAN LOGH~M, The classification of the plague-bacitlus. Antonie van Leeuwenhoek 10p 15, 1944--19~6.

The author ,proposes .to-classify the plague-bacillus, together with the nearly related rodentium bacillus, in a new genus' Yersinia (Y. pest's, Y . rodenti~) and to place this~ genus.with .other un- classifiable genera into the family of the Baaeriaceae.

R. ABDO]~LRACHMAN, Vibrio research .in the Heinz in connection with t.im E1 Tor problem. Ant0nie van Lceuwenhoel~"ltJ,, 93, 194~--1945.

Water Vibrios occurring i,n many water sources can easily be differentiated from V. choierae. Sex~eral times V: vbolerae-lilce Vibrios were isolated fr0m the stools of people sufffer'mg f~bin gastroenteritis.

In 1905 GO'TSCHLICH and afterwards other investigators as WeB isolated at E1T0r from:the' feces of pilgrims who were not k~feriiag from cholera, vibrios'Which aec~)rding to some of them:were identicM ~itli :file a'ctUal" V.-ch'oZerae, but which in ithe opmi0n of o :thersmus~ no't be ,donsidered" ~ ,s~ich. .I-a 1937~! 938 at Macassax3and its surroundings from. thes.tools o/.people s~ier'mg from dhole~aqik~ gastroehteritiS, ..as well as f rom waters, whiqh ,we~e.:..pmbabl~ ~lluted: ,with. flie:~eces, of ,'~hdse :people, D~:,Moo~ .isoiatdd::ge~s

76 Medical BacteriolosN. and:'SerolosN~.

which irt~.very respect were like,the E1 To~ vibrio.. Yinally the Author in 1937---1938, in a water sample derived from the Zam-Zam well at Mecca (Hejaz), the w a t e r of which is drui, k by thousa:r, ds; of p'flgrims without any harmful effect,, detected germs whicl~ ~ere abso!ut41y, identic-With V. El T o t of GOTsCHLiCH,~The E l T0r Vibrio is never foundin, the Stools of people bound for the Hejaz, but several t imes in the fec4s Of home-bound pilgximS.

These ~acts seem tb warrant the foll6wing conclusions: a. V . E! Tot forms a group of vibrios of its own, neither identical with V. cholerae nor with water viblios, b. This group of vibri6s may be subdivided into 2 types: I. The apa'thogeuic type which is found in the Hejaz and surroundings; this vibrio may be called: Vibrio El Tor, type Hejaz. 2. The pathogenic type, which is found at Macassar and surroundings; this may be called: Vibrio El Tot, type Macassar,

]3. TE~ HAVE, Onderzoekingen over de groeP der ka,pselbacteri~n, in her bijzonder in v~rband met de ozaena. (Investigations of the group of capsulated Bacteria, more especially in connection with ozena). Thesis, Amsterdam 194~.

Recent literature dealing with the system of the Klebsiellae suggests that difficulties may arise, when it comes to separating tl~e genus Klebsiella from other genera, particularly from Aerobaaer and Citrobacter; the grouping of the species is likewise fraught with difficulties. All the same the opinion is gaining ground, that there is room for the species t~l. pneumoniae and Kl. ozaenae besides the well-defined species Klebsiella rhinoscleromatis.

383 capsulated strains of Klebsiella have been examined, 24 belonging to Kl. rhinoscleromatis, 23 to Kl. pneumoniae, 330 to Kl. ozaenae and 6 to Kl..paulum [ermentans. Provisiohally, the author has introduced Klebsiella paulum #rmentans as a new species. With the 330 strains of K1. ozaenae (of which 3 belonged to the capsulated type C,' 294" to D, 3 to E , . t9 to A, 5 to E, 3 to F and 3 to the remaining group Y) it was possible, without exception, to distinguish them from the Other species; the same also applied to {he 25 strains which were certainly not of ozenaI origin. For the identification of the Klebsiella the author considers the agglutination of the decapsulated ¢ ..variant of paramount importance. However, ~he finding of an efficient method of decapsulation has failed. It is taken that the age of'the strains plays an important part in the appearafice of ¢ -variants. In most cases the s-variants (ectoplasma po[ysacch'a- ride free) immediately 0riginated'from the I(-form; sometimes, however, an ¢ form (decapsulated but with ectoplasma still containing some potysaccharide) was intercalated as an intermediate stage. The characters of the-54 ozena $.variants collected led to the ~,es,tablishment of a .preliminary division into 4 types on the basis of. agglutination.

In 300 ozena :patients capsulated bacteria have been found 248

~edleal B~cterlology ~nd-' ~erologg:

Canes. By ,far. the majority ha.rboured Kl. ozaenae,, t;O-sho,wed,KL [meumoni'ae and 4-.KL :paul ur~ termenta~, I~l. rCWn~sdeeomatis m ~ never reef with.. In cases .of. ozena:the ~ most~ predominant bacteria are the Klebsiellae. The notion tha t these ~re-.closely,. con,netted with ozena is supported by the results o~ .t~e agglutination tests carried outv~ith sera from patients.. In ozeria thg;rea ! coccobacil|us foetidus of PEREZ has been met with orfly sporadically; agglutination tests with sera from patients did not tell in favour of a close connection between this ba~ter.ium and ozena, B. proteus and related organisms are ,only of importance as . the , originators o f fetor. Corynebacteria'found in ozena shou,ld-'be considered as saprophytes. The view is he!dthat there,is, no sufficient gr0und.to:consider any of the microbes found, as the fundamental ,cau-se Of ozena,

I, J. :idE Cos~uiNo DE B~dssY, J,.. J, VAI~ LOGHI~M en4k. K. VIS~ER, Staphylococcus aureus in den gezor~den, neus, (Staplvflocoa$us aureus in .the heal~hy nose). Ned. T. voo'r Geneeskunde. 86, .9.629,, 10~2

The authors examined 1559 children and adults for the.presence of Staphylococcus aureus in the healthy nasal cavity, and obta~ed in the various groups 36--63 % p0sffive lesults.oThe commensal strains of Staphylococcus aureus which they could' g~:gw were un- distinguishaSle bacteriologically (bj~ examinati0h( for pigment, haem01ysins, coagu]ase, toxin and antigen~ic StrUcture) from.strains of'StaIbhylococcus aureus from'patho~0~:c prodlidts.

J. C. ~/~RHAGE en W. H:~KMAN', Een epidemie van s~reptoeocPus infec~ies o p een..chLru,rgische afdeeling,:-veroorzaak~.doo~ ee~i baciitendrager. (An epidemic o~ infections by. Streptococci .cau~d by a carrier), l~Ied. T v6or Genees'kunde -87, 53@, d~°48.

Description of an epidemic 0t wound infecdon~ in ~ a : ~ c M department, caused b y haemolytic streptocbdci, Which 's~a~tedin the throat of a germ carrier. These haemolytic streptococci have caused various clinical pictures, v/z., purulent wound infections with an exanthema wi thno characteriStiCs o~ scarlet fev~r, erysipe- ]as; wound scarlet fever and ordin~ryS~arlet .feVer

~IU~Nz.PIcK, Be0bachtungen an, eider: 'Kfil~:uv' v o n : ! . ~ 0 e ~ Maolyaca ($Chaudin~):. IOl~serv~fiotmf-~of ' a; ~ t i~o *~of~:En~u*~oeb~ hisloly~i~a (S~haudinn). ,~k~tonie v~m~ee~iwe~0el~-,~ laL.!.9~L

An. adequate=, enr/c.hment me:t.hod~, for~..~nt.~y~oeb,~.:..~iCt~lfft@~.: li,ver: infusion ~ u:.nt0 which starch ~ s u s p e ~ t . ~ : i . .nS~,has. , .~e~ added. The vegetative :foi-m-of Entamoeba '.h~s$~y~ia~:$sjp~ed> The cysts, of Entatcweba his_to!yt, ic~ :shove:as a: biol0~c~" rek~it~fi after supply of defribinatedsheep, bl0od ring formatieia on dist =anCe;

78 Medlcai ~acterlology ,and geroiog~:

Thus tl~e diagnosis of ,,cysts" is made easier. Pyocyanin is a vital stain for Entamoeba histolytica; it makes structure and nucleas more clearly visible a n d "increases the moti l i ty of the amoebae. Some observations of a twisting of the stem o f a .psgudopodium give" rise to the assumption that an ectoplastic excystation spot exists a n d that the amoebae do .not slip ont nakedly.

F: J. A. PAESI, Observations on ,,healthy" human carriers of Plo, smodium vivax. Antonie ~¢an Leeuwenhoek 10, 77, 1944--1945. Cf. also: Ned. T. voor Geneeskunde 88, 670, 1944.

The number of plasmodia in the blood of sufferers of chronic malaria tertiana varies greatly. An examination of the blood of 15 childern carried out from November till June (Sundays excep~ted) brought to light that these variations occur more or less reguikrly and that even negative phases may alternate ~ i th parasitic re-. cidives. Based on thesefindings it is advised that for the detection of plasmodium carriers examination has to be follov:ed by a second and third examination after l0 and 12 days,

PIK GING Hoo, Aangeboren malaria. (Congenital malaria). Ned. T. voor Geneeskunde 85, 1542, 1941.

The problem of congenital malariais discussed in connection with a number of observations recorded in literature~ A case is'described in which the possibility of congenital malaria .must be seriously reckoned with.

The child's mother got her first at tack one d a y after delivery. On its 20th day of- life malaria parasites were found, in the child's blood. I n the period during which t h e child's blood contained malaria parasites, there was at first no fever, afterwards some slight pyrexia; the spleen was not enlarged. Tile child also had a congenital heart lesion.

S. L. BRU~, Exo~erythrocytaire malariaparasieten. (Exo~erythrq- cytar malaria parasites). Ned. T. voor Geneeskunde 85, 2745, 1941.

Description of a ureparation containing three structures probably exo-erythrocytic piasmodia, obtained f ro~ smears of human lung tissue. The patient, suffering from paralysis, had, 10 days before, been inoculated intravenously with blood containing Plasmodium viva~..)it the, time of death, as some days before, benign tertian parasites were found in his blood and he died during the acute stage of malaria. The E-E-forms are reproduced. In Smears of the spleen, the liver~ the kidney, the bone marrow and t h e brain rio parasites were found.

Med~l ~cterlolo~ ~nd "~rolog~.,

G.. VAN DER MW.ER, en S. lw. : BRtJ~, ~ , u ~ . 0~st~%.als ~ara~. iet bij den mensch ~ (Pn~umocys~is,. a parasite of man}, Ned. T. voor Geneesku~de 86, 2666, 1942.

The search for plasmod~a in a three months old chfl'd, Who died from malaria, rev~ealed in the smears o:f the' l u n ~ the ~presei~ce 'of a considerable nu/flber of Pneumo'cys~t"iS: In the:=~:meai:S Col0t/red wffh KIEWIT DE JONGE'S stain Several cysts Of :5'":7/~! diameter with 8 elements within could be demonStrated::Als0 uhl:ipe:cysf~ with 4, 2 and ' l nuclei were ~ound: All these . fb~s d idno td i f f e r from Pneumocystis carinil a's found in m~ny animal Speci~ s. Besides these classical cysts the writers describe other ~ st.tuctures which theyconsider to belong to the lifec-ycle of the paresiS.e: i:' in. the smears there were groups ot .nuclei, each with. a mSi-et~r less ill defined protoplasm, the whole often together With ~anumbet 0f cysts embedded in a pale pink matrix; 2. C'OlTespondifig iwith these groups honeycomb=Hke stnlCtures were f0~nd in ~ sections Of the longs, each combcell containing a small mass Of :pto~0pla'sm With a .minute nucleus..Smears of the lungs of 78 postmortems were thoroughly examined. Only once a similar cyst was found, but this was an unmistakable one.: In the lungs of,rats and mice, both wild and tame, and in one guinea-pig out of I4 the same cysts were found and also the honeycomb-like structures.

As to the systematic position oi the parasite the authors consider it to be uncertain. They are convinced, however, that it is not a Coccidium.

J.-~D. VERLINDE~ De actieve medewerking ~van! bac.teri~n b lj tandcaries. (The active c0-operation of bacteria "in tooth caries),, T. voor Tandheelkunde 49, 506. 1942.

A number of aerobic and anaerobic bacteria: have been iso/a~ed in pure culture from Carious elements; with these:it wasatte~npted to cause Caries in vitro on sterilized elements. Thfs ~ucceedet!: fbt eight kinds ,of bacteria, among which2aerobic (S'[r~p~beO~¢us acid~ lactici and Dit~lococcus crassus) and 6 anaerobic strains (Fusi/ormis, Vibriothrix tonsillaris, 3 kinds of Lel~tothrix, Diplococcus tonsillaris) in. sugar containing 'substrate but only ,if the enamal::had been pierced before. Th~ enamel was not, affected ~by-the bacteria.

j . MULDER) R, VAN DEN BERG en ~. VAN K0,LL.~I~i~, De influen~- epidemie "va~ Febru'axi-Maa~ 193ff i~' he~ g ~ t z 6 e ~ it% !G~griiugen. II. Bac~enologle (The iix~luenza'epldemy of Fe~SruarytM~[4c~ 1@~9 m the garrison of Gronmgen..tI Bac~ermtogy): ~ed~..~:~ o o r

Geneeskui~de: 8,4, ~2141, i940'.'.

T h e 1929 o~,tbreak of ,influen,za' mi the ga~is~m:~gf G.toB~g~.n' lasted from aboat February ,l~st to abau.t March~6~h~,.OLfl~s.ol~$ 36 % go~ actu~l!y:ill, Tile. ilhiess.had a yew,mild:co ~2s¢,a~d wa~

90 ~[edical Bacteriolog3~ and" Serology.

characterized clinically by a pyrexia lasting 1 to 6 days, a dry cough being the main clinical symptom. The number of patients with complications was only 4 %, mainly tonsillitis, ofitis media and two cases of bronchiolitis and bronchopneumonia respectively. In 3 of these cases a haemolytical streptococcus type 5 was cultured. In the pa t i en tw i th purulent bronchiolitis H. influenzae was found besides. In 4 patients the diagnosis was confirmed bacteriologically by transference on a ferret. Of one .patient the ferret strain was transferred on mice. The direct transference of the disease from man to mouse did not succeed, neither tha t ' f rom the.first ferret on a mouse. Transference of the first and Subsequent passages upon mice succeeded easily. -The virulence of this virus strain is at present moderate, about a thousand times weaker than that of the strain WS isolated in Londen in 1933. The antigenic structure differs distinctly f rom that of strain WS. T h e sera of 3 patients showed a distinct increase of their con ten t in immune bodies against the strain WS and the isolated strain.

j. MULDER, Haemophilus influenzae and influenza-ultravirus met betrekking tot de etterige bronchitis. (Haemophilus influen~.ae and influenza ultravirus in connection with purulent bronchitis). Ned. T. voor Geneeskunde 84, 2806, 1940.

Haemophilus influenzae of the PFEIFI~ER type is fotmd .in the greater par t of cases of ,,ordinary" and (non-fcefid) chronic punflent bronchitis (bronchiectasis), often as the numerically prevailing organism. The pathogenic properties of the PI,'EIFFER type in the bronchi are not yet strictly proved, but it can be considered as very probable that this group'causes acute and chronic purulent bronchitis. I t is improbable that ordinary purulent bronchitis and bronchi°litis are primarily caused by a still unknown brochiotropic ultravirus, and the infection with the Haemophilus group shotlld be of a secondary nature only.

L. BIJLMER, Aetiologie der influenza. De isoleering van her influenza-virus tijdenS de epidemie van t941 te'Groningen. '(Etiology of Influenza. The isolation of the influenza virus during the epidemic of 1941 at Groningen). Diss. Groningen 1943.

T h e experimental studies on influenza, during the epidemic of J~nuary-February 1941 at ~ Gr0ningen (HOlland), were carried out according to the technique evolved by WlLS0~ SmTH, ANDREWES andLAIDLAW, Of the National Inst i tute for Medical Research at Hampstead (London). The investigatigns were carried out in a_ laboratory specially built, in 1940 ior the study of e:~perimel~tal influenza in fe r re t s a n d since !941. incorporated in ~ the' Insti tute for Hygiene and :Bacteriology ;of the Sta te University,

MediCal Ba~terlology ~nd Serology. 81

DUring the period of January 9th to February 7th, material from 13 patients, partly throat~washings, partly suspensions of sputa, obtained during the first days of illness, was.inoculated' in ferretsj which were kept under rigid measures of isolation. The temperature was read twice 'daily and the :clinical symptUms were recorded

In 5 ca~es the ferrets developed a fypicalinfluenza infection, with the characteristic . fever peak on the second or third day and catarrhal symptoms of the nose, sometimes of the conjunctiva, In all these cases influenza antibodies against the .virus strain WS could be dem0nstrn.ted ' in the ferret convalescent serum by means of the mousd protectiQn test.

In two other cases the signs in the ferrets were doubtful, but the ferrets' convalescent serum contained antibodies against the influenza virus~ so that these animals must be assumed to have gone through a subclinical infection.

In 4 of the 6 remaining cases in which the ferrets did not show any reaction, the patients' convalescent serum neither showed any rise in antibodies. In the remaining 2 both the mouse protection and complement fixation test of the patients" sera gave positive resultsi so that missed ferret inoculation must be assumed

Three virus strains were isolated, from three of the enumerated cases. After some ferret passages (3, 4 and 5)~the virus was adapted to. mice. The strains produced lethal pulmonary lesions in the mice after 9, 3 arid7 passages respectively.

In the course of the ferret and mouse passages the virus suspen~ sion was filtered through a collodium membrane (average pore diameter 0,6 #). The throat-washings and sputa from the patients were not filtered before being inoculated in the ferrets, in order not to reduce the virulence of the pathogenic agent.

-The nature of the pulmonary lesions of the mice was checked microscopically by sectioning the lung. Hereby the Criteria described by STm~UB were accepted.

The analysis of the three isolated influenza virus strains did not showany mutual antigenic differences. The new strains were not

• • . - - • . • " * f . ~ , found to be identical wlth any of the Enghsh ,,specl m .stratus, The immunological study of the serafromfifty influenza patients

demonstra~ted, that in 43 cases the influenza virus had been the etiological agent; among these were 13 cases of pneumonia, following an attack of influenza.

Prophylaxy and therapy by means of vaccine and immune serum are discussed. A multivalent anti-influenza serum was preparedby hyperimmunising rabbits with various virus strains. Some prophylactic effect of rabbit immun.~ serum, intranasally a ,dministered to mice infected with influenza virus,-co~d~ be noted.

The aim of this study m a y not:be .consideredre.~ched by the ascertainmeflf of the etiology in a single influenza epidemic..This work mhst be :seen .as a preparation for the Stud}; of an inflt/enia pandemic,

82 Medical Bacteriolo6qr and Serolog~

]~. E. KULSDOM, Over de epidemiologie van meningo~occOSis en poliomyelitis anteri.0r acuta. (On the epidemio!0gy ofmening 0- coccosis and poliomyelitis anterior acuta). Ned. T. voor Genees- kunde 84, 398, 1940.

The s tudy of the occurrence of meningococcat infection makes it probable that~'~linical-cases of this infection-are always independent casualties, in Which the germs havesucceeded in Overpowering an organism lacking in its normal means of defence. This fact and our' knowledge of t h e distribution of. meningococei among human individuals also gives rise to the assumption that biologically ihe symbiosis of man and meningococcus may be characterized as commensalism. A study of Statistics and literature of acute anterior poliomyelitis furnishes data in support of t h e view that fo r the virus of this disease similar considerations are valid 'as for the meningococcus.

CH. G. J. DORNICKX en H. PE~TERS, Nekkramp bij militairen. (Epidemic meningitis among military men). Ned. T. voor Genees- kuude 84, 4155, 1940.

During the mobilisation of 1939 32 cases of epidemicmeningitis have come to knowledge in the mobilised Dutch armies, The opinion displayed in Holland by BUL, PEET-ERS and SI~STROP ~, that epidemic meningitis is .an illness preferably attaining soldiers and more especially recruits, is sustained by the collected data. For the control of the d~sease the testing Of smears from contact- persons for the presence of meningococci is of no use. The great importance of sulfapyridine for the t r ea tmen t of the disease i~ pointed out .

J. J. TH. Vos, Encephalitis toxoplasmotica. Ned. T. voor Genees- kunde 85, 2401. 1941.

The section of a 5 weeks old child, which had suffered from in- distinct cerebral symptoms showed a chronic necrotising inflammation in several parts of the brain. Some of t he lesions showed signs .of calcification, Which made it probable that the disease had set in before birth. At first the cause of the efice~halitis was unknown, ~but after the publication of cases of tox0piasmotic encephalitis by WOLF and CowEN the author re-examined the sections and Was able to demonstrate th e toxoplasma in several foCi~ Some were lying in so-called cysts, others free. A/similar case has been published by C. C. DE LANCE. Here a child 3.5 montlfs old was concerned, the disease of which in~'tially Was described :as a case of hyclrocephalus. Later the disease Was identified b y W0T.ir a n d CowsN as a toxoplasma meningo-encephalitis.

~edidal ~a~teriolog~r and Ser&logy. ~3

], D.. V~..mD~ en'F. :WEMm.NC~, Herpesvinm in den liquor cerebrospinMis-.~an ~een lijder aan sclerosis multiplex. (Her~es virus in the.liquor .cerebrospinalia of a sufferer from sclerosis muL tlplex). Ned T. -voor Geneeskunde 86, 3209, 1942

A vires taken from the cerebrospinal fluid of a patient suffering from dis~seminated sclerosis has been isolated. This was ii~oculated in rabbits and from cross immunisation experiments i t proved to be the virus of herpes. The patient suffered at the same t ime from repeated herpes and must be regarded as a virus carrier. The blood serum~ contained a large amount of neutralizing antibodies: In animal experiments the strain pr._oved to be strongly anligenic.

E. F. J. H. FALGER, iVieningo-er~cephalitis na rubeolae. (Me.ningo- encephalitis after rubeolae). Ned. T. voor GeneeskUnde, 87, 109, 1943.

Descrip.tion of a .fatal case of meningo-encephalitls after rubed!ae in awoman of 26. i n general the neurological complica~i0ns after ruI~eolae begin very .acutely. Practically without excep.tion lymphocytes occur in the cerebrospinal fluid, which remains sterile. The anatomical changes in the central nel'vous system are probably caused by an anaphylacficreaction on the rt~be~lae;toxallergene.

A. C. DROGENDIJK, Bijdrage tot de casuis~iek van her zoster- varicellen vraagstuk. (Contribution to the casmstics of the zoster- varicellae problem). Ned. T. voor Geneeskunde 84'/2931, 1940.

Description of two cases of herpes zoster f011owed/by vadce]~ae in persons who had been in contact with fhe patient.

A. C. DROGENDIJK, Over hot aetiolggisch, verband tusschen zos-ter en Varicellael IOn the~ae'tiological ~elatidh betWeen zoster and. ~aricellaei. Geneesk. B1a~len uit Kl~ni~k en-Lab0~a~o'rium 37j 325, 1940.

The author discusses the possibility of ,a ,close~ relation :be~wee~ the zoster and varicella vims-wifkreference tO literatme:study and his own observations

J. D. VX'RLINDE, De in, flood van.vimlentie en herkomst var~ hot" vac~inevizus op l iet .0ntstaa~:'~ean • experiment~ele ~hcephalitis. (The'influence of Virulene~e aa4i~~origin~Qfthe v~¢.cineviTus on the causing, of exi~rimentM enc~p~Mit~s~: ~led~.. ~T: .V0o~ Geneeskunde 84~ 214, 1940.

En'cephali~. ~can~ be ~csused ~ by:inocelating degs" cutaneQ~s!y wf~h :vacoit~ia. ~ s d ~ g ~s, l~,terit ,g~aa~iid:m~O-xication., ~Th~

MedI~A1 ~adter~ology -and- ~eiolog~

vitu~.~nce of three different StrainS of dermovaccine appeared tO have no influende on fhe~causing of eX~perimen~alleneephalitis. Neurovaccinewas st.ronger ence:pha]itogenie ~tha'n. d~r/n6'vaccine, Ih herbivorous animalg encephalitiScould hot be induced in this w/zy, ~hot even in calves after vaccinatitm with the .original Cowpox vxrus. _ . . . . . . . . . . .

J. D. -~.ERLINDE, Over de pathogenese van de encep]~al-itis postvaccinalis. (On tlle p~thogenesJs o~ the ~encephalitis postvaccinalis). Maandschr. voor:'Kindergeneeskunde 9, ~68, 1940,

It is possible to induce a post-infectious encephalitis in the course of dog-clistemper by-inoculating dogs with the distemper virus during a guanidine-intoxication. The same result~is obtainec~4n dogs vHth the d~stemper virus by vac~nating ~hem against small- pox during a guanidine:intoxication. W i t h rabbits, calves and monkeys experiments did n o t succeed. Probably' in these animals and perhaps also in men, another form of alit6-intoxicatiOn from the intestinal tract plays a role in' the etiolbgy 'of po~±vadcihal encephafitiS, sevemi ~ strains o f dermovaeCinel. bi~ especi~,"y rieur.ovaccine"were found Go have enEepha~toge,nic pr@effles. Preventive. and Curative measures should be-directed kgain~t predisposing metaboiic factors.

.B.H. WoRM6OOR, Koepokken. (Variola',bo~ne):..(Cow pox. Variola hovina). Ned. T. voor Genee~kunde.86, 2185, 1942.

The:development of yaccme pustules is described in, tw.o dakry- men who pr0b~ab!y had transmi,tted the virus themsel'ves from a vaccinated infant Upon the cattle.

G~ F.. Bo~m~,. De ~esreactie-van, ,CH~DI~_t~. :,(~T~he syphilis, reaction 05 CH.~DI~X). ~le& T. vOOr Geneeslmnde :86,-1279~ ./.94~,.

The technic of the micro-syphilis reaction of CHEDIAK.is described. After: mentlo,mng thegoo~ r~sults~ofe~xtensl~e.ex~emmen s ¢l~where, :theresutts :of. ~ person~ mvestlga~ons' are g~ven:.: ,I~J'¢on, eluding, attention is calle d ±o t h e Vafious~: '~,V~tages ~ef-the.t~st~ a,o. its special Suffability fo~ gt.oup~.~esCmgs for syphilis.

ea~ed.~o~. ,~. ~ ~ ~ p c . ~ flrie-d!~:~loe~i ~Oh e ~ a i a i m i ~ o ~ ~ ~F~

Medical Ba¢~d~logy,~sd Serology.

bloQdd, tops, against the W~ss~AN~,re~ctien~ .a :gai~, 0t2: %:and a,,lbss: .of: l. % ~wer:e: obtairied;~hereas i n I',5 % of .the cages a nora specific reaction. :was seen..The reaction ,b,f Cmz~i,Ai~, therefore, is q~ite praetieabie ~for.. the detectior~ .of syp~g , i~-ca:ses~ wliere diff ic~ies a r e en.countered m.c~rying:0ut the Other reactions, if only one keeps in mind that in case of a p0si~ve readin, g it remahis necessary to submit the blood.to a complete serologic examination,

ANIMAL PATHOL06Y

j. D. V~ZI~mD~, Aetiologische, epidemiologische en se~,ologische on.derzoekingen over adenitis infecti0S~ equo~um. (Aetiological, apidemiologieal and serological investigationsof adenitisinfectiosa eq~lorum). Tijdschrift voor Diergeneeskunde {}7. 6~6, 1940.

Studying strangles of army horses two forms were met with, v/z~, ,one in:=which an unc0mpliCated .cata~.h o f the larynx-, and trachea and one i n which abscesses of the ~trophm3mg.eal and submaxillar glands dominated. In either form streptoc.occi c0uld:be isolated from the purulant discharge of the nose or the glands, Two. out of eight, strains of the "strePtococci ~verged somewhat in; their fermentation of lactose. The cultures were .pathogenic for nnce and rabb~ts. Culturally xdentmal, stratus AlJi~eared to dff er widely in agglutination tests. T h e atypical strains were strongly aggla~inated by: all e~amined sera of convalescent horses, :the t,ypica~ strains .o~y i n Sma41 measure" and then. hardly' by other homologous sertim

J. D. V:~RLINDE, En'cephalbmyelit/s equi: Mtmndschr., voor Kindergeneeskunde .8,. 1B1, 1940:

S':mce :!938 many~ :c~s~s o f encepha!i/tisj~ man ha~e bee~ observed,: caused ..by .:the.:v',m~S ef h0rse encephalitis which., in this continent occurs epizeeti¢alty in horses: Atthe~$hpeople of all ages are sehsitive for the virus, most of:the c~ses, are' found i~.'chitdren. This d!sease, unknown in our country, is discussed iv. a gen.eral s~tV~y-~in'~hich ~t~.importa~cd'for men is poin~t:ed but:.

~#ct Jx~s~, Goe~aardlgb droes, ..aden,~tiS equor~,m., ~1~] st~gles,.:a~den'itis e qhoru~)~.i ~ij~iSchr~ 'vobr I)i'bxgenee~lmn~d6:67,

46 Animal Pathology.

mild case, resembling strangles, five times from the discharge Of t h e nose and once.from'abscesses Caused by Serum injections: Two identical streptoc.oc¢ii which colored bloodagar slightly green, were cultivated, one from the k idney-and the spleen and one f r o m a bursted gland. The m a j o r differences between the cultures of Str. equi, Str. pyogenes and Str. viridans were:

i i

strains

Str. equi ~tr. pyogenes Str. viridans

h e m o - l y s i s

+ +

p r o - ducing green colour

+

sorbitol lactose

+ +

trehalose litmus milk

i

pale acid,- clot

+ pale, some acid

Some strains cultivated from the throats of persons, suspected of being infected with strangles, did not agree with the above strains.

JAc. JAI~SXN, Shigella equuli (B. pyosepticus equi)-infecties. (Iiifection by Shigella equuli (B. pyoseptious equi)." Tijdschrift veer Diergeneeskunde 68, 687, 1941.

Shigella equuli does not only occur in foals and pigs, but also in full-grown animals. The anther diagnosed the disease in an 1 { year old horse which had died from acute septicaemia. From the pus of a case of chronic fnniculitis in a 9 years old horse, Shigella equuli was isolated. I t has also been found in the organs of a pig, which had died from acute septicaemia.

L. W. JANSSEN, Her mend-"en ldauwzeervraagstuk.bekeken van de chemische zijde. (The problem of the foot a n d mouth disease looked a t .frbm the chemical side). Tijdsch.rixct veer Diez- geneeskunde 67, 10, 1940.

The author gives a summary of his conception of the nature of the virus of foot and mouth disease and of.the disease itself. Starting from the size of the virus, the resistance against chemicals differing from bacteria, the inactivation by pH between 4,0 and 6,5, the insensibility for bacterial substrates, the lacking of metabolism, the predilection spots and the connection between the virus and the cells in which i t is formed, the author had reached the conclusion, that this virus must be a dead protein.

In his own experiments, the influence of .chemical. and physical factors was established. Tke virus was resistant to the action of .alcohol and ether at a low temperature. It behaved as a dead

Animal Pathology. $7

protein/a~6mot as a living protoplasm, t h a t is disturbed b y the Iipoid extraction. Toluol, chloroform and ether are rather good b~ct:eri~, poisons, but they i n t e ~ r e bu t ,little wf th enzymatic reactions. They are good conservatives for the vires. The virus be- haves aS a protein dr enzyme also when shaken.with these solvents. Th'einactivity of ferment poisons (KCN) which con6pletely stop the respiration of living organisms in a much smaller concentration, is p u t forward as a-sufficient proof of the dead nature of the virus.

In di,fferent ways, recorded 'before, i t was. possible to obtain a protei n, fraction containing the virus in a .very pure form. One gram oI .this is sufficient to infect a milliard guinea-pigs. Exami- nat ion .of the chemical structure of the protein, considered as virus made it likely that it i s a nucleoproteid containing ribose. The observed increase of the' virus is not due to division of, an inde- pendant micro-organism, but the virus is a pathological prodnct of the plasm of the epffhelial., cells of the Skin. These cells are not hostcel ls , but they have a~disturbed'metaboli.sm. The normal :epithelial cells-produce keratin fibriUs, the sick cells produce virus instead of keratin. This conception agrees with the histological findings.

L. DX BLIECX en JAc. JANSEN, Enting tegen mond~ en ]dauwzeer met crygtai~iolet-vaccin alsmede met bij '37°;C. gedood virus bij de cavia. (Inoculation against ffoot- and mout]~ di~ase with crystal ~iolet vaccine and with virus ldHed at 37°C '. in the guinea-pig). Tijctschriff voor Diergeneeskunde 69, ~7, 194- °.

Immuni ty against foot- and mouth disease could be induced in the guinea-pig by in6culation with virulent bl0od, ~ kept for l 0 days. at 37 ° C. The same ~esult was attaified by adding crystal violet. For both methods the results Were Better with a filtrate of epithelion than with blood. A single subcutaneous injectionsufficed for the immunization of the guinea-pigs. After vaccination a humoral imrhunity is formed, setting in a f te r 3 days and lasting for at least 52 days. A fur ther injection iff the humoral immune guinea-pig causes a local reaction and .complete immunity.

A. F. VAN DER SCHE~R, Eenige waarnemingen,ovef in Nederland voorkomende mas~titisstreptococcen bij her ru~nd. (Some observations on in the Netherlands occurring mastitis streptococci in ca~cle). Tiidschrfft voor Diergeneeskunde 67, 76, 1940.

A. F VAN DER SCH~.ER, Over mastitis veroorzakende strepto- cocce~. (On Strept ococcicausing mastitis). Thesis~W~agening~n, 1941.

The biochemical and serological char ac÷eristics of the strepto- ¢~occi ¢~using mastitis .in ca~tle are described, The n~w .aame.of


Streptococcus pyosel~ticu* is suggested for the haemoly~ic group of C-streptococci.

The detection of the haemolytic property was greatly ,simplified by the use of stab-ctfltures in blood-agar-slants. For the serological differentiation the .precipitin test of LA~CEYlELD was used. Sera were prepared "in the usual way. For the preparation of extracts a method was worked out.

The examination of 312 s{rains of~ Str. agalactiae showed' that the slimy consistence of the colonies of 80 t o 90% of the strains on horse-serum-agar is of great importance for 'a quick recognition of the species. Moreover about 60 % Of the strains with non-slimy colonies can be recognised by the colonies ,,in shape of ~wound thread". Nearly 50 % of the strains induce haemolysis. The area of haemolysis is usually small, but about l0 % of the haemolytic strains produce very large areas. 77 % of the streptococcal mastiffs in cows a~e caused by Str. agalactiae.

The examination of lq5 strains of Str. dysgalactiae showed that the colonies are never slimy or in ,,shape of wound thread". On horse-serum-agar, about 95 % of the strains cause a turbidity in the agar under each colony. After removal of the colonies from the surface of the agar this phenomenon is very conspicuous. I t was shown that some 30 % of the, strains show a slow production of acid from salicin and some 20 % cause a weak hydrolysis of sodium hippurate. B y means of the precipitin-test and reciprocal ab~ sorption-tests it was proved that Str. dysgalactiae belongs to the serological group C and that the deviating Strains just mentioned should in fact be regarded as Str. dysgalactiae. 14 % of the number of cases of streptococcal,mastitis was caused by Str.dysgalactiae.

83 strains of Str. uberis were examined. The surface colonies on horse-serum-agar often show a close resemblence to those of Str. agalactiae. Even slimy colonies may occur. A few strains may cause a turbidity in the agar like Str. dysgalactiae. It was demonstrated that some 30 % of the strains do not form acid from inulin. Moreover the hydrolysis of sodium 'hippurate is extremely weak in several cases, so that the result may easily be recorded as negative. B y means of the precipitin-test and the absorption-tests it is proved that these deviating strains should indeed be regarded as Str. uberis. More than fi0 % of t h e strains contained group specific precipitinogens. Group specific serA are only obtained from strains containing no type-specific precipitinogens. The serological group is not indicated by a letter, becalise it is considered likely that Str. uberis will prove to belong to group E. This supposition however cannot be substantiated, because a comparison with American sera was impossible. The number of cases caused by the species was 7%.

68 strains of Str. lbyosepticus offered data in complete agreement with those presented by other authors. Acid is formed from sorbitol but-not from trehalOse. T h e precipitin-test w i th .~oup C serum is Mways positive. In cows this species caused 2 ~ of~ttre

A~aimal., ~atholog¥. ~9

cases 0f mastit-is. In o, ther animals~especiMly,in. :horses $~r.:#2osep, ticus occurs more or less frequently ,in various, in.fec¢iom,

Str. t~yosepticus var. l, umanus was isolated .from cow-milk only once, but not in connection with mastitis. Acid is formed from trehalose, but riot from sorbitol. Group C serum is precipitated.

Str. l~yogenes could not be isolated frOm:covcmilk'. Biochemical tests Cannot serve as m~ans o!'a definite differenfidta'Onof these strains from S#r. t~yosepticus vat. humanus. A group specific serum prepared from Str. pyogenes is considered as a group A getum.

H~rse-sertim-agar is' an excellent, medium for growth and ~ola, t ion of mastiffs strept0cocci. By the ,addition of 0.05: to' 0.1% aescuiin this agar becomes excellently suited: :for diagnostical purposes. The hydrolysis of aesculin is easily detected und~r:•a quartz lamp. When only non-aesculin splitting strains are Con, sidered, the characteristics fgr th e colonies of, Str. aga!actiae and Str. dysgalactiae become specific. Sir. uberis and other streptococci, with colonies showing the same characteristics, all decompose aesculine.

L. F. D. ]~. LOURENS eI1 A. F. VAN DER SCHXER, Over her verloop en de genezingskans van de mastitis door Str~ptococ~s dysgalaaiae en Streptococcus,pyogeues. (On the course and, the chance of recovery of mastitis by Streptococcus dysgalactiae and "Streptococcus pyogenes). Tijdschrift v0or Diergeneeskunde-68, 283, 1941.

A mastitis caused by Str. dysgalacgae is acute. Alatent infection however of the udder may Occur. The thus inducedmastitiS can nearly ~ways: be cured by repeated(milking~ preferab!y combined with an auto-va¢cin treatment. It rather often occurs in connectiofi with Other dis:turbances. The danger of spreading the infection after t,he mastitis has appeared is rather small.

The mastiffs caused by Str. pyogenes was severely acute m .more than half of the number of 'cases'examined. in the other c~s-es: the mastiiis was dhr~,onic, with0ut acute initial stage, Tlle. quarters affected by Str: t~yogenes hardlywith0ut exception, must be. given up f0rl0st. ,The after-effects of ~he maqtitis (emaciati0n):\m~y possibly, b~ combated by opening of the ~eats: The infe, eff0n by Str. ~ pyogenes spreacls easit3~ to other quarterg of thesa~ne ' c0w. Hygienic measures to prevent, a possible spread of the. infeetiorr to other cows are advisable. In exceptional cases the mastitiS by Sir. t~yogenes may occur during the period of refreshing.

W. v~,N DE~ BERG, ::Een. nieuWe mathode, tear onclerk~malng !van streptoc0ccen e_n :staphylococcen. "m .de melk .van :kl'.mi%cb.no . ~ uiers. (A new meih0d for the ::dis:tLuction.',of Strept, OrCOC~¢$:,ma~ s%aph~lococci in the milk of clinically n0rmal udders): '_Tijd~c'~ voor Diergeneeskund~e -67," 8, 1940.

Under aseptic precautions | - -2 ml milk is brou6htstra~gh~ from


t h e teats in to a sloped tube with serum agar. After incubating for 18 hours, the result can: a l ready be noted.

~." VAN DE R HOEDEN, Komt Brucellosis suis in Nederland voor? (Does Bruc, ellos~s suis occur in the Netherlands?). Tijdschrift voor Diergeneeskunde {}7, 226, 1940.

Among 87 Brucella strai.ns isolated by the author from men and animals not one of the suis type could be detected. Serological tests of 486 slaughter swine in merely l fi cases gave an agglutination against Bruedla but the tffers Were of no conclusive value. I t is possible tha t those feeble reactions have been caused by a svmtomless infection with Brucella Bang, originating from cattle. IJp tfli-now Bruedlosis suis is not met with in the Netherlands.

C. J. D~- GIER, Is de ,,Teschener Krankheit" ($chweinel~hme. Encephalomyelitis) een afzonderlijke ziekte of wordt zij veroor-. zaakt door her virus van de varkenspest? (Is the ,,Teschener disease" (Schweiuelfthme, Encephalomyelitis) a separate disease or is it caused by the virus .of the swine-plague ?). Tijdschrift voor Diergeneeskflnde 68y 695, .723, 1941.

The author poses this question in view of the occurrence of swine-plague with obvious paralytic symptoms, , whilst no ma- croscopicaUs/ visible pathological changes are to be found, but merely perivascular infiltration and morbid growth of glia in the central nervous system. As t h e diagnosis of the Teschener disease is based on these symptoms it is not out of the question tha t both diseases are identical. Paralytic symptoms also accompany other diseases of pigs (Aujeszky disease, salt proteid intoxication, vita- mine A def ic iency)and so do perivascular infiltrations and morbid growth Of gha (bronchopneumonia, swine influenza, swine pox, paratyphoid, Aujeszky disease, worms aud excessive feeding with proteins). This is of significance, .as. such pigs might be used as test animals for the Teschener disease.

H. H. VINK, M, ucormycose bij een varken. (Mucor mycosis in a pig). Tijdschrift voor Diergeneeskunde 68, 312, 1941.

The au thor mentions a case of mucor mycosis, caused by Absidia Lichthei~i in a pig. The progress was localized in both submaxiUar gl~mds and the left inguinal gland. Histologically a fairly strong eosinophily is-~ecorded

An/msl Psthology. 91

A. ~ BAUV~RY-ASMA'N, De beteekenis der seroreaetie bij~ honc~en met hetrekJdng tot leptospiren infecties. ('~he sigaHication of the seroreaction in dogs :in connectign with ,the hffectlon by Lepto- spira). Tijdschrift voor Diergeneeskunde 67, 799, 1940.

In 280 dogs, taken at random, the agglutination-lysis test against leptospira has been carried out. About 40 ~/o were ascertained as positive. These dogs were divided in those with a low.and those wi th a high titer. In the latter the rate of Let~tospira icterohae~dor- rhagiae to Leptos~ira canicola, was. 1 : 2, a same rate as has been found in clinically sick animals. The low titer might perhaps be caused by a slow repeated immunization.

In about 50 cats the seroreaction was negative. This does not exclude the possibility of an infecti6n, although not making ff probable.

The progress of the titer of dogs which had suffered from leptospi.rosis has been followed. The increase is rather quick, after 4 ,5~7 weeks t h e maximum is reached, t h e n a decrease follows, but much slower. A titer may remain for years. This is of value for the serological test.-Only a repeated examinatl~on of the blood Can tell us something about the patient.

JAC. JANS.EN, Over Cl6stridium Welchii-infecties. (On infections by Clostridium Welchii). Tijdschrif/~ voor Diergeneeskutlde 68, 562, 1941

From a phlegmon in the wall of the stomach of a .dog, Cl. Wdddi was isolated i.n pure culture and also from the urine of, a living dog. and from the bladder of the same dog after its death. The surroundings of the bladder and the prostate were phlegmono.us. The latter strain digests glycerol but not inulin.

JAc. JANSEN, Konijnenpest. (.Rabbit's~ plague). Tijdschrift voor Diergeneeskunde 68, 967, 194].

In rabbits a filterable virus was detected which was very pathogenic when inoculated subcutaneously or intranasally. Contact infection was easily proved..Tt~e virus ha§ been cultivated on , the egg membrane.

JAc. JANSXN, Experimer~teel onderzoek van konijnefisterfte door een filtreerbaar virus. (Experimental investigation of mortality in rabbits caused by a tflterable virus). Tijdschrfft valor Dier- geneesk-unde 69, 504, 194~.

A spontaneous acute disease with hi'.gh mortality (rabbit plague) was observed in rabbits. A filterable vlrus proved to be the'caase. The virus w~s ascertained, in blood,,-urine~ gall and-muc~s o f :th'e

,92 Animal Pathology.

aaose. I t is .possible to t~ansmi,t the d i sease to rabbits by. subcutaneous, intradermal, intranasal, conjuncti~val, intravenous inoculation and by infection per os and by contact.

M. F. POLAK, ]~pid6mie survenue' parmi des souris blanches k la suite d'une infection par le CorynebacZerium pseudot~berculosis murium. (Epidemic set in among white mice as the result of an infection with Coryneba~terium pseudotuberculosis murium). Antonie van Leeuwenhoek 1O, '23, 1944---1945.

An epidemic attaining white laboratory mice has presented the clinical picture of pseutuberculosis of the liver. The cultures of the isolated organism have revealed Corynebacterium l~seudotuber- culosis murium which for various reasons is considered as the causal agent. This micro-organism ~has merely provoked the pneumonia with foci regularly described in li terature when it has been administered per os with the food.

H. VERVOORT and A. CHARLOXTE RUYS, The recognition of Psit- tacosis. Antonie van Leeuwenhoek 6, 11, 1939--1940.

The psittacosis virus seems to be endemic amongst cage birds in Amsterdam, In animal experiments it is of low vinflence. I t gives only occasionally r~se to isolated human cases or small out- breaks, The results of animal experiments and complement- fixation tests with material from human cases and infected birds a re recorded. A description is given of a reliable staining method for the virus. The value of the complement-fixation test is discussed.

L. DE BLIECK, Immunisatie tegen Coryza infectiosa gallinarura. (Immunization against Coryza in.fectiosa gallinarum). Tijdschrift voor Diergeneeskunde 69, 204, 1942.

By intravenous injection of living or formalized cultures of Haeonophilus coryzae de Blfeck fowls can be immunized against- c olyza caflsed by t h a t organism. Against coryza caused by the so-called Nelson bodies complete immuni ty can ,be induced by intravenous, intramuscular and intrabursal (bursa Fabricii) injection of exudate from the cella infraorbitalis. Intravenous immunization gives the best results.

A Bos, Weer nieuwe gevalten van eendenpest. (New cases of duck" plague). Tijdschrift voor Diergeneeskunde 69, 372, 1942.

New cases of duck plague were diagnosed. I t caused/an important mortali,ty in some large duckfarms nei6hbourin 6 each other. 0 n

ALrilmal Pathelo~]. ~$

5 farms in the course of some months-2600 o~ 57~}(~ ~rl~mals died. Other animals fell ill but recovered and appeare~t immune afterwards. The symptoms were lameness, soon followed by paralysis (especially of the legs, creeping locomotion); further V i01en~ thirst, sometimes Stinkfng discharge from the bill, diarrhoea With ~eenish and yellow coloured sloppy stools. Incubation period together with period or progress of the disease takes about a week. Dissection: petichia of the heart andsometimes also of the other internal organs and in the trachea. Fibrinous or fibrin~6purulent peritonitis copho- ritis with large haemorrhages in the egg follicles,

The virus could be kept on in a series Of 18 passages through ducks in the course of more than a year, Intramuscular injection of heart's blood conserved in 50 ~o of glycerol preserved at 5 ° C. suffises for the keeping on of the virus.

Ducks wich had rema'med negative after inoculation app~ear~ed to be immune to superinfections, so did a recovered duck from one, of the i~fected farms. No infection~ could be. provoked in fowls, pigeons, rabbits, guinea-pigs, rats and mice.

The causative agent of the ducks plague ~s assumed ~o be 'a separate kind of virus and not a variety of. fowl plague virus.

C. J. DE GI~R, Her cultiveeren van Tricho~nonas foetus. (The culturing of Trid~omonas foetus). Tijdschrift voor Diergeneeskdnde 67, 902, 1940.

The medium for the culturing of Trichomonas foetus, such as it has been recommended by SCHOOP and OEHL~RS is preferable to those which contain more protein. The liquid from the, allantois also is very .suitable as ~i culture medium and quickly leads to success. Scrapings of cotyledones mixed with 0.9 % saline, have. no value as a culture medium.

A, Bos, Die Trichomoniasis der Tauben und ihre Bel~$~npfung: (The trichamoniasis of'pigeons and its combating). Thesis, ~treGh.t. 1941.

Trichomoniasis in Holland i~ one of the most Widespread infectious diseases of pigeons. To, obtain a pure ~ culture TARozzI- broth is most suitable. In :this medium a l~ure ~ulfi/re ~ of Tricho- ~nonas hepatica can be cult~ired ~con~ineously for, 1, year .~and a half in 162 generations...The egg-medi.am Of L b c ~ . i s very ~:e~.:sw!ed to obtain apure ctflture from material c, 0ntamin£tdd w i ~ bacteria° In blood i~roth and bloodm'flk the par:agate imul.tiplids as, we~, Culturing on a solid medium had no.,success.

94 Animal Pat/koiog~¢:

JAc. JA~s'xl% P~kken bij de kauw. '(Pox i~ ~a tame jackdaw). Tijdsct~ift voor l)iergeneeskunde 69, 128, 1942.

Spontaneous pox was observed in a tame jackdaw, (Colaeus monedula). The virus was ascertained as t h e canary-pox virus.

TECHNICAL ,MICROBIOLOGY

T. FOLPMERS, New enrichment methods for the cultivation of Bacterium coli and_ faecal Streptococci in water samples~ Antonio van Leeuwenhoek 6, 22, 1939--1940.

The results of a series of tests extending over a period of three. years in. the Rotterdam water works ar, e reported. For the detection of Bacterium colt very satisfactory results were obtained with:

1) a medium ~ontaining 1 % glucose, 0.5 % glut~m~.c, acid neutralised by NaOH, 0.5 % ammonit~mtactate, 0.5 % NaC1, 0.3 ~o K2HPO, and tapwater ( p H = 7.0). Cultivation at 45°C. in completely filled stoppered bottles.

2) a medium contaimng 0.3 % bacto-tryptone, 0 ,1% sodium formate, 0.04 % K,HPO 4 and 0.0001% crystal violet, dissolved i n destilled water (pH = 6). C~tivation is performed at 45 ° C. in completely filled stoppered bottles. After 24 and 48 hours gas production is observed and a test for indole formation .is made. As a rule they coincide.

Fo.r the selective enrichment of S.treptococcus/aecalis best results were .obtained with:

1) a,medium containing 1 % pgptone.(Poulenc-, Difco of Bacto), 1% lactose, 0.5 %, NaC1 (pH = 7.0). Litmus added up to a deep

purple colour. Cultivation in test-tubes filled to a depth of 10 cm or in Erlenmeyer flasks at 45 ° C.

2) a medium containing 1 % peptone (Poulenc~ Difco or Bacto), 1 % caffein, 0.! % glucose, 0.3 % Liebi'~s beef extract, 0.5 % NaC1 (pH ---- 7.2). Incubation at 37 ° C. in completelyfilled stoppered bottles.

T. •OLPMERS, AI1. improvement in the bacto-tryptone, sodium formate medium for the detection of B. col~. Antonie van Leeuwen- hock 10, 28, !944---1945.

An improvement of the bacto-tryptone, sodium formate medium ~(cf. Antonie van Leeuwenhoek 6, 22, 1939--1940) which has beeI~ put On trial for nearly a year in the Laboratory of the Rotterdam, Waterworks, cons"ists in lowering the contex~t Of crystal Vi61et to half of the original amount and raising.the pH {o 6.8.

Techni~l,, M~robiology 9~

T.. I~OLPMERS, On the d.isa, p.pearence, of*,B,, e, oli, and ~fae'cal Stre.p. tococci (Enterococci) as the redult of,.slow sand:filtration,, Antonie van Leeuwenhoek '7, !04, 1941.

DUring three successiveyears countsweze mt~de ofStre~tococcus /aecati's and B. colt in-th~ prefiltered Water (prirdatyfiltrate)and: the sloWsand filtered w~ter (~econdar 3 filtrate) of the-Rotterdam Wate~orks. The faecalstreptdcoc~i merely appeared 'in the st~0xl- dary ffl'tmte in the winter inonths-and in'h~ghest, :nimibersvSh~n ~ the tempe'rature Of .the water Under the ice de~k" sinks beldw l°C; In summer not' any streptococci' car/ ,be detdcted ~ "ill ~ Sdmpl~s,iSf 100 ml of the primary filtrate,. The number O f , B . ~ l i " i n ~ e a s e d , as' well at low temperaturefi. In secondary filtrate'sttept6cocciap'pear later 'in the year, than B.' .colt aiad ~ as soon as the~ tee in. the fitters is melted they disappear at a much earlier ,date 'than B . ~oIt. Protozoa which are the r~atural enemies of s~feptococci encyst as a result of low temperature and this: entai]s "the 'increase of stPepto- coccl in Winfer.

The bacthrlological situation especially in w~tit~e. ~ may be dange- rous and properly applied and bacteriologically controlled chlori- nation will be essential.

P. C..FLU, Are bacteriophages significant for the .self~put'i~ iea fion of surface:water? Antonie van Leeuwenhoek 7, 39; ~941~; Cf. als~ Acta Leidensia 15~16, 63, 19~t~1941.

As early as 1926 the author has Claimed that in, the self-purifi- catlon ,of~water (under experimental Jcoadifi0ns,~e~iz~d,,m-~e laboratory) bacterioph£ges ~take no part, at 'le~/t n~oi an-dasily. discernable one. The investigation: of SCHU.UR~tA~ on"the :si~fi~ dance of typhoid phages in t-l~e' self-purification of the ~ a { ~ i ~ ~t~e Tjfliwoeng river (Netherl:ands East-Indies) induced. ,tiie, author to attack the problem once more from the experimental, side.. The results of these recent investigations aHow~no"dther :conclusion tl~an that under'circutnsiances,and colidifi0ns~:as:h~e ~ b~en fully described, bacteriophages do not act on the ~self-p~ication of water. I t might be conceived:that the high t~pe~at~re 6f ,~he Tjfliwoeng ri~er, and~ iisi~igh Conten~ of c611oidi~:~la F :,~oW the phage~t0 play~ a certain part, al~oug~a-the r:~s~ts.:o!, t:he" d~j:~p.~'~ experi~eilts, ma'ke this hardly' p~o,l~a:b}:e~ :Ai,: .~d~:'e~e,nts.,~u~~-n,.' in.fluence is. heR, her ,pr.6v.ed nor mad.e probable ~Ahe~ ~si.d.ts::.o~ SCH'U.UaM~N'S, expeiiments ~ t h ' ~he, u nt~ea~ed ~Ti~'.'~bepg' , Ware!:;, because of his l a ~ to exciude 'th~ ~cfion of t]~ ,i~ro,tozda

~.. AB~O~C~mA~N~ Can,~we t use, the,~!Sbiga~bac~eri'cpI~ge as indicator for, a faecal-pollution of water ?: Antonie ~n~L~euwe ~z~hc~ ~ 9~ 148, 1948;, Cf. a ~ : W~t~ontlerzoek met: een -:bacteriQphaag-,

~6 Technical -Microbiology

mef~aode. (Water control by means of a bacterophage method t. Thesis, Utrecht 1942.

In an important percentage of stools of heal thy persons Shiga bacteriophage occurs. The Shiga bacteriophage occurs during some lapse of .time in water which has been polluted 'by human faeces. The Shiga bacteriophage could be ascertained in water by means of the enrichment method of NYBERG. The Shiga bacteriophage method for indicating the pollution of strongly polluted water is as sensitive or sometimes less so than the methods of EIJI~MAN and of CLEMESHA. For the exmniliation of water in several stages of purification in Water works the Shiga bacteliophage method is ' as sensitive or even more so than the other methods. The Shiga bacteriophage does not act strongly in the self-purification of polluted water. I t may be .used as indicator for a faecal pollution of water and this method may be applied along with the other bacteriological methods in water control. I t m a y be advanta- geous to s tudy more closely its limits and its benefits in the practice of wa te r control.

H. DE GRAAF, A revision of the procedure for the Voges-Proskauer test. Antonie van Leeuwenhoek 7, 92, 1941.

The recommended method is the following: The medium consists of o 0 . 1 % 2 % glucose, 0.I ~. K2HPO 4, 0.05 % MgSO~, _ NHtC1 (or (NH,)~SO,) and 1 % CaCO3. The culture'xs incubated for 24 hours (or if necessary for 48 hours) at 37 ° C. To three d~ops of the culture solution in a test tube three drops of a 4 n NaOH solution and one drop of a 1 % creatine solution are added. The mixture is placed in a waterbath at 45--50 ° C. and is shaken n o w and then. In case of a positive result the red c01our will show in one or two minute~.

JAN SMIT, B. M. KROL and A. J. VAN WIJK, The B. coli test in the routine analysis of raw milk. Antonie van Leeuwenhoek 6, 1, 1939--1940.

I t is emphasized, tha t the estimation of coliform bacteria is i i~portant in the routine analysis of r a w milk. Various tests recommended in the l i terature are described and stress is laid on the fact, tha t only those which m a y be completed within 24 hours are of actual value. A prescriptibn is given of a satisfactory test using a broth of nearly the same composition as the well known Endo agar, and a comparison is made of the results of this test wi th 3 o ther tests in investigating 30 samples of raw milk. I t is :proved tha t a considerable number of the coliforms found are true B. coti which have los t the power of indole formation.

Technical Microbiology. g7

N. SAV~J,. Eenige vergel/jkende.onderzoekimgen over t/et aantoonen van coli-bacteri~n in gepast~uriseerde melk. (Some com- paritiv.e investigations of the means o1 detecting B. coli "m pas. teurised milk). Chem. Weekblad 38) 114, 1941. Officieel orgFan van den Alge'meenen Nederlandschen Zuivelbond 36, 55, 194].

Compared are: The method of the milk decree 19I 6 (enrichment in acid broth), the method of an official control instance (enrichment in meat ~" broth with lactose and neutral red and cultures on Endo plates), and the method of the laboratory of t he Cooperative Plant of Milk products at Bedum (enrichment i n a peptone-lactose- brillant green-gall medium and culture on eosine-methy|ene blue plates.

The resalts vary, especially when the fermentation test is nega= ~ive, I t ,became evident that on the Endo plate red colonies even those with a metallic lustre are often not due to B. coli. On the other hand on the eosine-methylene blue plates colonies with a metallic lustre are always formed by bacteria of the coli group.

• Therefore it seems desirable to base the research for B. coli in pasteurised milk on: 1. the occurrence of gas in a lactose containing medium. 2. the metallic lustre of colonies on cosine-methylene blue plates.

C. F. ~VAN OVEN, Kleine plaat methode. (LittTe-plate method). Handelingen "van her Genootschap tot bevordering van melkkunde 1940, I, .p. 3.

VAI~ OYEN'S modification of FROST'S little plate method "is recommended for the bacterial count in ~ailk of good bacteriological quality. 0.1 ml milk is mixed with 0.1 ml broth-peptone agar and spread over a surface of 20-× 50 mm. Incubation at 28--30 ° C. for 20 .hours. After drying the slides are stained with carbol-thionin and t heaga r decolorised in running water. The colonies are counted by meatls of the microscope (50 × ), under a cover glass" of 20 × 50 mm divided in spaces of 1 mm ~, resting on t h e agar slide. This method is not suitable for pasteurised milk as the bacteria develop too slowly.

A. PASV~EI~, De toepassing van de rolcultuur b~j her bacterioio- gisc~i onderzoek van melk en~melkproducten. (The use o1 JULIUS' ceUn~ method in the bacteriol6gical' con~Q1 of milk and milk products). Officieel orgaan van den Algemeenen Nedeflandschen Zuivelbond 35, 473, 1940.

Fo r the estimation ot the. bacterial~ount of milk JULIUS' method was appli.'ed~'(Ant.oni.e van' Leeuwenh~ek 5,28, 1938--39).~In small cylindrical glass j a r s w i t h a cov6~ Of a luminium 3 ml liquified gelatine or agar is inoculated with a small quanfit~y of m ~ a f t e r which ,the ja r , . a re placed .m .~it app.aratus: in which, they are-r~- volving rap idly.it~pund their, cy.¥~ d~ical ¢,xis .to.s~lidify, the medium

3~ Teehni~al Mic~biology.

in a thin layer on tl~e cylindrical wMl. Af~er ~bcubati0n~he~olonie~ • are counted. Non~]~ctic acid bacteria in Soi~r milk pr0ducts are estimated on a mectium cont'aining no sugar, unto whi~h0.05 % Ca~Os has been added to preve~f.,a d'ecrease :~ pHcaused b y t h e presence of lactose in the inoculum. The sour m~k lJrodudts have to be solved in four ~imes their volume of a solution-of ;~0 gsodium- hex~6taph0spha te and 22 ml n N a O H i n 1,coo ml of.,wa~er~ The q,6un~ingl o~L bacteria in butter, was performed' by means ,of this lnethod~ Using. ~the butter serum as inoculum. The methofl ,is:also suitable for the counting of yeasts and molds, when an appropriate medium is used.

Jaarmerslag van deix Gezondheidsdienst voor vee in Friesland. (Annual' Rep.ort' of ±he sani ta ry service" for 'c~ttle in~ 'l~,r~e'slar/d). Officieel orgaan ~ van, den Algemeenen Nederland~¢hen Zui~elbo~id 36, 535, 1941.

The value o/ . the reductase tes.t is discussed, A~ter .the. tubes had been .read for the reductase test tl~e same tubes were j~dged after 12 hours incubat-ion. I t appeared that peptonisation and acidification are of no importance for tl~e classing of the milk:for making cheese, as th.e milk samples of the best bacterial quality m,ay induce these changes.. The gas formation did not correlate Width the results Of the coli .test in freshmilk. Although the fermentation may be improved by addition of retreat, as. the coagulum retains the gas bubbles, the test gives no reliable in- dilations.

A. K. VAN BEVER en J, S T ~ u B, De beteekenis van ' STOl~C~I- reactle, oprOmi~g en phosphatasebepaling voor he~ toezicht o p d e ~ melkpasteurisatie. (The significance of the, reagtion:o~ S~O~CH, raising of the cr~eam and estimation of phosphatase for the. control of tl~e pasteurisation of milk). Chemisch Vqeekblad ~8~ 210, 194L

The importance of the phosphatase test fort,he, hygienic co~tro.l of milk pasteurised d'uf~g a few seconcls at higtl temperature (Voltana arid Stassano milk) is ,emphasised. The phos~fhatase tegt should be negative. As the temperature and time interval between the killing of the tubercle bacteria and a negative phospha~ase test is but 'small, a reliable cofltrol is" necessary and in the ~e~ting ot a new appa'ratus the cav.ia test m u s t n o t be eliminated.

Jaarverslag v~n den Gezondheid~di,'enst ,root, ~ee ,.in, l~r~eslan~10 (Annual l~epor~-~, of the s ani~ary,'service for, ca~fle-in! ,F,~esl~nd)~ O-~ficieel or gaan. van den/~lgemeenen~ Nederlandschen Zuivelbond 35,"549, 1"9~0~

The possibility ,~,f estimating the bacteriologicM'qu'~:ty ~Sf was,s~,udied. Qf. milk sample~ c0~it~'zizing :l~sY ~hafl~'~0:~~0~¢t~fl~

T, ecJ/.al¢~l l~li~-~biology 99

pet-~ml accordi~g,'fo ~h~e plate method (I~ffco,~trgar-.,22 ~ C.) 28%. shd, wed" a bacterial: ~ot~tl,t~of :more~ t~am ~480.00ff'~a the Breed test. Out of m ~ ' s~mp]e~ ~itt,'whi~h '5i'.000 '-"i 00.000 w~te"fb~d Vcithl the plate' m~eth0.d 42 %-,had adco~g'-t~,"~.he 'B~e.~d;'/test to ~e ~las~ed-~ '~0r/tai'nin'g mor'~,.than 480.000 ~ a . c t e ~ i ~ n ' t h e milk'. samples %vere divided according-to the Bree~dteSf: ~h class I with a, ~bacted'al~ c~unt be/ew. 600.000 and class H. with ~ c~aat %hove 600.0~0:: .12 % 'of ~,%he s.amp!es' coniaining-less t,h~p 50.000 ba.ct~,n'a a~9ordi~g ,to:the _plate method :~ad-to be,)place d in. c~as~ I~J~h. e Breed count is:therefore.unrelial~]~e. Best zesults a~e tb,' .be.e~,pec~ed with ~'3 ~reect'classes, v~'z., ~ ;I ' b e l ~ '2,~0:000, i~I 240~:~,~,:--48~:.0{J0 and III::dbo~e ;4~0.000, When claSsed~ according to..~h,is sche~u~ 72 % of:the samples with aplate count of less'than 50.000belong~ to:class i :a:nd ~ ,~y 4:%, tO class ill.. W,ifl~milk s~mples o~ 5h.OOo'-~-' ~ 1.00:000 ~i~ese numbers Were:SSP/o :a~ad 5 %: Th:e =-iltde p,l~_t¢ test of 'F~OST-VAN OY~-N ga~e merely half .the an~otmt.0flbact:eria found with the plate test.

With :the reductase test nearly all samples with ~' plate count of, less ..:tl~"an. 200.,.~00 reduced, in less .than 4 h o~rs, but so did a l~g.e~nmber o~ sample~, with a high bactt/,ial, oount~..TqCrLSON'S method, .(~tur,~ing~e~ery hour), ,gave.better results a~d 3, :clasps are recommended (less than 4 hours, '4~-6 hours and¢ more than 6 hours).

~. VAN.BEYNUM en: J: .W. PE~Ei Ee~l mefllede ~v0~l ~ l~e~ a~nt~tle~ v,a~'boterzuurbac~eriSn; speciaal gesohikt voor hot onde~e~-vau m~lk..(A method for the detection of butyric ar~id~ bacteria in m iL~:,), ~e~lag.en van.L~ndbouwkundige;onderzbekingew 46, 379,. 1940 an~d Jaarverslag Proefzuiv.elhaerderlj over 19~0~ p, 9;

A method for the det'.eet~bn of'butyric acid bacte/ia in milk i~ described. The'ferment:~i6ti test is" 6arri~cI ont I~i Vtghap~d: ~,~bes, one end of whlch I/as a rubber .stopper and the other Is cot.t0rl pluggedTb 50 ~hloI ~he ~~~ .to JJ~'teSfedane, a~ded i mlof ~ tL.25~/~

and ~ I ml Of 0 1 ~ HC1 to inliivlt the g~owm 6r pumracu?~ Clt~sz~- altL ~ or 5~-£ubes are hlled~ w~.~ ! ~0,; ~r~ of ~h~.s m-m~..u.re a~d. o ..~ th"o

Ii 'q~l 'baseline After ~astedHsa~ ~on :~J_~u+/ng ~0 t/ffh~te~' ~ifS0 C, the tubes~ate mdub'at:ed at 35.~--~.0 ~C.~ Th,e test is , p o s ~ t i ~ = ~

me~hod 1, also smtabl~ for t~t .d, ~ect~6.fi 0f, l~d t~c~c~d~ ,o : than~ fla~ '~;si~BSta:aces.-Ste~sed ~ ~ ~o, ~ used _ ,

100 Teclinlcal Microbiology.

J, ,VAN B~Yx4u~t en .J.W.' I~XTT.~, De invloed:-can de ~roeding van her vee op de besmetting der melk met boterzuu~bacteri6n. (The influence' of the feed 9f the cows on the. cont~mlnation o~[ milk with butyric acid bacteria). Verslagen van landbouwkundige onderzoekingen 46, 397, 1940 and Jaarverslag Proefzuivelboerderij over 1940, p. 27.

The milk of the .experimental farm has been tested with the above method. When the. cows were in the stable the 'milk was pmcticaJly free from butyric acid tiacteria, if they werefed on hay and artificially d.ried grass as a rotighage. 'When a silage containing butyric acid is fed, the milk is strongly colitaminated. There 'is also some contamination of the milk of cows in the same stable; not receiving silage. When the feeding Of silage is brought ~to a close, it may last some 3 weeks before the milk is free from butyric acid bacteria. The milk of cows in the pasture generally does not contain butyric acid bacteria.

C. I. KRUIS.HEER, P. C. DEN HERDER, W. C. SMIT en A. DE HAAN, Her bacl/eriologisch-ch'emisch kwaliteitsonderzoek der Nederland- sche keuringsboter. (Bacteriological and chemical gra~ting tests of controlled butter in the Netherlands). 's-Gravenhage, Algemeene Landsdrukkerij 1940.

About 1000 butter samples derived from the Laboratory for control of dairy products have been studied as to the correspondence of organoleptic tests performed by experienced butter testers with the results arrived at by means of laboratory tests. All butter had been prepared from pasteurised cream and'had been stored before ripening during 7.. days at 13 ° C. EStimated were: the amount of catalase, total counts of microbes on casein agar, counts of moulds, yeasts, fat-splitting and protein-splitting micro-organisms; coliform bacteria, lactic acid bacteria on china blue-lact0se-agar; and non- acidifying microbes. The chemical estimations bore oh: visible water drops, NaCI, iron and copper compolmds. The correlation between the store and estimations according, to tti~ laboratory tests has been calculated and although in the fresh butter samples no strong development of fault~"in odor or'tas.te C0ffl d occur, a correlation could be established between the score arrived at organoleptically and the amolmt of catalase, total count of.micrQ- organisms on casein agar, count of molds, visible drops Of Water and the occurrence of the metals mentioned. The correlation was less" close with the number of yeasts, Of fat-spli'tting-, of protein-.

~splitting- and of coliform bacteria. N o c0i-relafion existed with the count of lactic acid bacteria. T h e m u t u a l correlations between'fiie figures thus obtained have been calcttlated. The flgu~es ~for summer and for .winter Were Compared. Upper lhfiifS for good' buffer have been established.

Teahnic~L i~c~ol~iola~], 10

J.~vA~ B ~ en $,. Pv':.,Ps.~lz~ Omierz6bk~genl.over zuring en a~omavorming-blj pfactij.l~u~dls,.,in gebruik'-'bij:cfle borer. bereiding, .en:methoden van ond~zoek, van zu~csel~ (Ibvestigations on souring trod ar.oma formation in" butter starters), Verslagen van landb0uwk~ndige onderzoekingen 47, 1 ~, 1'941 en Jaarverslag l~,oefzuiv~Iboerderij over 1940, p: 59.

The rate of-acid formation is estimated in sterilised milk which has been inoculated with 0.0067 %, of the s ta~er and., incubated at 21 ° C. in a Water-bath. The interval nee.de,:for, the a~idity to increase from 40 tot 60 ml o. 1 n per 100 ml is measured In this intervkl the curve representing the increase of acidity is .nearly a stra:'.~ht line and extra- and, intrapolati0n of the titration data is permitted: With good starters the increase in .acidity will take 2.0 to 2.3 hours.

The best aroma production occurs in ~tarters with a not too rapid citric acid decompositi0n and a weak reduction of a-ceto~in and diacetyl to butylene glycol.. In this case the prodizc.tion of C4-compounds is high. The reducti0n.-.to glycol begins when the citric-acid is still present and continues after all citric.acid has been fermented. The authors introduce a new characteristic number R, the reduction percentage = the percel~tage of C4-compounds present in the form of butylene glycol. R irmreases with time as HAMMER'S creat]~-test may. indicate also.

In the starters- tested there was a great v~iance in the rate of increase of R. At the onset of the total decomposition of citric acid R was 9~100 %, a Week l a t e r i t varied from 13~100 %. For a. good starter R must be low. When the-decomposition of the citric ac id is .too rapid, the quanti ty of C~-compounds is=low and reduction is, S t rong. With starters composed of pure cultures of aroma betacocci and lactic acid streptococci, the reduction is always low. The formation of the C~-compounds and most of the acetic acid comes to a close when the citric acidis wholly fermented.

K. HOLWERDA, Over den invloed van her diacetylgehalte ~¢an de. borer op de beoord:eeling "van de ~euk en smaak hiervan bij de boterkeuring van der~ Bond van Co6peratieve Zuivelfabrieken in Fr_ iesland. (On the ,influence of the diacetyl ' content of but ter on the score}. Officieel orgaan yan den Algemeenen. Nederlandscher~ Zt~ivelbond. 36~ 608, 619, 1941.

On the average unsalted but ter kept. 7 days. at114°C, wi th .a high diacetyl content scored hi~hest. When the diacetyl content was low the but ter scored tow and more faults were observed. I t seems that 0.8,. mg diacetyl pe rkg is the under lim~it for, good butter. Af~ter keeping the butter for a fortnight' at 14 ° C. thetendeney is still, the same,~but ,diacetyt and Score c0~relate less closely,because in many samples .faUlts begin' to-ddvelop. Iri salted' 'ba t te r the diacetyl con~ent is lower.

t,0~. Technical ~ Microblology.

J. ~VAN B~VNu~ en J..W.~t~TrE, De.waarde~ van. de kreatine proe~ voor de prak~ijk. (The value :6f the creatm:test for practical ~use). V~eekblad voor zuivelbereiding, en handel 49~ 4.1, 1943.

The use of the creatin-test for the periodical control of starters in the dairy is advised. The 'test should be carried out ~n three consecutive days in the same sample, kept ate20 or 21 ° C. Wi th a s tar ter of good flavor ~he tes t must 'be negative after three 'days; otherwise the reduction of the aroma substance is too ~ stro~ig..The ffhportance of Using weakiy reducing s tar ters was proved. "In f ac t but te r of soured c r e a m made w i t h an aromatic starter had a very good flavor and contained 1.4--2.6 mg diace ty l per k g butter . Wi th a strongly reducing starter, however, bu t te r w i thou t flavor was obtained, which contained merely 0.3 mg diacetyl. However, when the but te r is contaminated the effect of a good starter may b e nullified as yeasts may reduce the aromatic compounds.

j. VAN BEYNUM en J. W. l ~ r E , De verstreF2~ing van zuursels voorde boterbereiding. (The supply of starters for the preparation of butter). Weekblad voor zuiv~lbereiding en handel 49, 181, I943.

Aromatic starters need a careful treatment. Contamination with bacteriophages may affect the. acid formation and together With the presence of other lactic acid bacteria this m a y hamper the aroma formation.,

S. G. WIECHERS eII H. BLUMENDAL, Her verband ~usschen de kwaliteit van de boter, hare bereidingswijze en hare bacteriol0gi- sche kwaliteit. (The relation between the quality of butter, its mode of preparation and its bacteriological quality). Handelinge~ van het Genootschap tot bevordering van melkkunde 1940, II, p. !2.

I t has been studied in what measure the quali ty of but ter ~ cor- relates with the number of lactic acid bacteria in the buffer, the starter, the pasteurised cream and t he soured cream. No correlation could be found, in so far as the but ter "was no t heavily contaminated. The starters were tes ted and the rate of acid formation was determined 'by titzation after 20 hours, incubation at 15 ° C. A presence of noxious yeasts, molds and lactic acid bacteria may be detected micrdscopieally in the ripe starters, kep t at 35 ° C, for 2 days. The judging of the-s tar ters is of great importance for the control of the butter.

J. VAN BEY~UM en J. W. Ps~E, Her aantoonen van ~asvormend~ bacteri~n in kaas. (The'detection of gas f0rming ~ad~ria in cl~eese).. Verslagen van landbouwkundige onderzoekingen'.48, 765, 19~2 and Jaarverslag l~oefzuivelbQerderij. ~o~er 1942, "p. 35.

Ear ly gas formation in. cheese is caused b y coliform ba~teria~

Tee, hni'c~t M~tc~obiolo~. 105

wMeh ferment t~e; 1Rctose~sti;~ presen~in-- this-stage~ Their mimber is esti, ii~ated ~y, means~of ~¢-',.C0~.,I~Y's technique, St~bseti~ent.:gas formation may be ;caused by, CI: tyrobutyri~u'm,, Pr, O~ionib~cterium Shermanii or:Lacto~acillus, bi/errnentans; all, Capable of, ferme~iting lactates

The=number of spores, o f C/. tyrobutyricura is estimated with fermentation test :by ~inoc~ating .the.- cheese in dextrosd~peptone- broth-and cultivating under,anaerobic cox/diti~)ns at 30 ° C. ,Use is made, of glass tubes Which are evacuated, When the tubes are opene~ ..the gas. shoulcl be explosive ,and:the liq,uid shemld smell of bhtOTic acid. The propionic acid bacteria are to be estimated according to the above abstfadt-gf/d Lactobacillus bi/ermentans (see below) is detected hy inoculation, of the.cheese in peptone- KsHPO/-2 % C~cium i.actate medium in tubes,Which are:co, acuated aftei~var.ds.: Incubation at 30 ° C. A slow ~e~mentation occurs;, the iermentation'ggs consists of .nearly equal ~)arts'of Hs andCO~ and the ,c~tu, re medium obtfins a sweet, odour. The .fresh medium, has a pH of 5.6. I t is.more selective ,if the pH is 4.5, bu t the deve[op, men~ of the bi~cteria is then. very slow.

For inocu.latio~ of the tubes dilutions.- of the cheese are made (0.1 g--0:000~000.1 g) in sterile water after solving the cheese" in a warm sol:ution of 2 % sodium,citrate. From each.dilution 2 or 3 tubes of each medium are "inoculated..The ~ paper is illustrated with many. photographs o~ cheeses.

j , ,VAN BEYNUM en J. W. PETTy, Propionzuurbactdrien in GSud~ sche 'en Edamuier l~aas. (Propionic acid bacteria .in GoUda and Edam 'cheese). Verslagen van landbo~wkuntlfge onderzoekingen 47, 1101 ~, 194-1 and ~aarverstag Proefzui, velboerderijover 19~1, p. 2~.

Countings of these bacteria ha-cheese were made by inoculating varioUs dilutioris of .thee cheese :'myeast autolysate-peptone-sodium lac~.ate-agar ,or silica gel. T~he.bacter~a may be easily -recogmsed by .the :size ,and shape of their~cbloni'es (see&- ,a~d ,!ens-sh~pe): Generally: Edum:cheese contains a merely smal! number, of,propionic acid-bacteria, whereas in Gouda fkrm, cheese larg~e n, umbers could be found, although i t /a few cases inxhe~e of ,this,type:.they als0 occurred in a low number. This great variety appeared to be due to the pH of the,cheese.

In a series of experiments wi.th cheeses _of. varying pH it appeared that the ~umber'~f,prop'ionic ,£¢id)bacteria was ~gh~r W~aen- the pH was~ h,{ghex: This ¢e~rela~ion wa's ra,t_ke~close and,,t£e~C~ta about this correlation and abou( the natur.al 'riseof pH in the cheese during keep~g .otfer, ed the, me.am ~o ,foreten',~the Rr~zle, of, ~ e r o p - ment ;of the.,pro,~.'ome aci&ba ,c.te~ia. I~ the p H ~ ~-eheese ~ ~ox~: ~tay is, ,~nde~. 5~O ~o: ~,o,wbh,wi~tak,~pl, ace, ~.0ver 5=:~ ..then,ala,~bii~' a~i~, g, row:th will~.ensue. ~I~, the pI-~, :ef,[a -:cheese ',0f"~:w~ks' 4s~-~o~, :5 .~:,~ ao gi, owth ,wiU: oecm'/~ ~,i't .~s, 5,2~5. 5,3: ~thei~ ,n.umb~.~,i~ -~ ~ . t O some

104 Te~nlc~l Microbiology.

hundreds:'of thousands; if it-is above 5.3. milliofiS ~v/lI.h~ fou~td afterwards. N6 growth is observed until the ISth day and i~may last .till..the 9th week at a temperature of 18 ° C.

The p ro , ion ic acid bacteria seem to be of no importance for the cheese-ripening process. If present in a large number they.wili give rise to a subsequent blowing of the ,cheese. For the format/on of a small number .of gas holes .in the chee.se their number has to be ~erely moderate. As the fermentation gas (CO~) is wate~sol~ble the holes can only be formed when small preformed, holes occur in the curd, due to included air..oe to fermentation gas from E: coll.

J. w. PETvE en J. VAN BEYNUM, Boekelscheurbacte.rien. (~,,Boe- kelscheur" bacteria). Versta~n landbouwkundige onderzoekin~en 49, 315, 1943 and Jaarverslag Proefzuivelboerderij over 1949-, p. 181.

The properties of a bacterium, ~ often found in. Netherland cheese causing gas formation have been. studied. Generally its .action in cheese is not very -pronounced, but i n some cases it causes serious subsequent blowing. This organ.ism, LactobaciUus bilerment~s, originally discovered and described but not named by BOEKHOUT and OTT DE VRZES is a duplo-rod form. The colonies in yeast autolysate-sodium lactate agar are shaped like those of the' pro- , ionic acid bacteria but are much smaller. The agar is torn-by the gas development. I t shows all properties of a lactic acid bacterium. In sugar media the pH decreases to 3.4--3.9. Inactive lactic acid if formed from hexoses; maltose, rhamuose, sorbitol and mannitol. Lactose, sucrose, raffinose, pentoses, glycerol, starch, dextrine, salicin and dulcitol are not fermented. Lactates are fermented and decomposed to acetic acid, alcohol, C02 and H2; during this process the pH increases. Thus in a sugar medium two dissimilation processes may take place simultaneously. When, however, the formation of lactic acid is so rapid tha t the pH sink s below 4, t h e lactate is not decomposed. Only when the pH is kept at a higher leve lby addition of a buffer mixture, the decomposition of lactate will set in and then the bacterium pedorms two d~fferent dissimi -~ lation processes one after the other. Hence the name Lactobacillus bi/ermentans has been chosen.

J. vAN BEYNUM en ]E. A. Vos, K~n men ,,laat-lo~" met bromaten bestrijden? (Can subsequent blowing be prevented by bromates?~ Weekblad voor zuivelbereiding en-handel 49,' 229', 1'944.

The possibility of preventing butyric acid ,fermentation in. Gouda and Edam cheese by means of oxidising agents h as~been studied. Danish investigators had .drawn the a t:tention~ to t ,b~orhates And iodates. In accordance with the Danish results .the. authors found that bromates and iodates in. very low concentrations, "m,hibited

Teclinical~ MiCrobiology, 10~

~e , gm~cth of pure eultufe~ ~of b~a~yric acid :b~¢teri~ in ai, tifieial media.~Chlora~es:did n6t everr ,in a-co.aceatration of 0,1' %. The three salts, however, were ~,th0ut any',df.ect, i n cheese. Even 150 g bromate per 100 l'of cheese milk did not prevent the butyric aci d fermentation in the cheese experiments.

It appeared that this had to be ascribed to an earlyd:ecomposition of the bromates and iodates in the cheese bytactic acid 'i~acteHa. Furt.her it was shown that both. salts have a retarding effect on .the,lactic acid fermentation, which i.s a disadvantage in cheese making. Antibut D (trade.name of a substance to be used "m preventing the,butyric acid fermentation), however, was effective. Ahaiyses showed that it Contained 34:% KBrO~ and 64 % ]KN0s. It 'is t h e nitrate, that prevents tlae butyric acid fermentatioia in cl/eese, not f-he br0mate. Experiments were carried~ out in wtiich even 2.5g KNOs: per 100 1 was effective in preventing the but3~ri~ acid fermentation. This is ,in accordance with earlier .expe~JTnehts 6f BOE'KHOUT and OTT DE VRIES.

B. VAN DAM et'J., G. WAR~MIUS, ~Rapport entre la teneffr en humidit6 du lair s ec'maigre, (provenant de lair ~cr&m~):et le d~ve- loppement d~une flore microbienne dans ce lair. (Relation bet-~een the moisture content of milk powder (derj~ed from sklmmed milk) anxt-the development of micro-organism%'m,'this milk). Antonie van. Leeuwenhoek 10, 123, 1944--19~5.

Experiments, have been carried" our With the aim of following the number of germs and the moist~e content of milk powder when-~.kept, at various degrees of relative moisture. Initially the milk ~owder has .been. stored in layers of varying thickness. Later. the smnples of'different .fabrication have been stored in thin layers. When the milk-powder is stored in surroundings the moisture of which it"can, absorb;-than initially a strong decre~e ,in,the total number of germs will: occur.,, Subsequently during a long, period the ,number of germs remains fairly stable and increases a~ew, when~he moisture, content, has~ atta'med ,~ definite level.. Below this criticalmoisture~-cofitent the, development ,of a ,corisiderable microbial flora, is not possible. The critical moi~tare content does not depend on. the vapour pressure ,oL the air, . out o~'i which the powder,has absorbed water.-The moisture content, wh,ich; allows, the ,occurrence, of visible growth, of moulds, is, all- ~he :lower, the lower the relative moisture is ~hereat the powder is ,kepf stored. &t ,a re~tive moist.ure .of. .1£, 0,%~ ,90 %, and 85 % mou!d'n~" ~p¢curs be fo, re,~the moisture~ con~e~L of: the. m~lk ~powder is, i~: equil~,'~bn~-um with, ,~he':moist ~lWe Qf ..~e,surrGund~,x!g air. At- a~reldtive~m0is.t~e of 75 % the equilibrium has already set in -before ~e~d~dopmen~t -of m ou!d,s..B~re attain,'mg ~ the'e;q~,b~,inm at a,,rel~ ,fiv¢-m0i~ure of ~5,% s0me;milk p o n d , s o~y:,~ereas96.'m m0.i~re~af, te,~,,a ~ much longer 15eriod ai~_d then up to a corltent of about ~0 %, and

106 Technical Microbiology,

then tha t coiltent decreased and at tained a normal value, corresponding with tha t of other powders, which absorbed the water directly, up to tha t normal content.

J. J. GHIJSEN, Beschrijving van proefnemiugen op laboratorium, semi-technische en technische schaal met een versneld vlasr0ot- proc~d~. (Description of expariments on laboratory, semi-technical and technical scale with a procedure for accelerated flax retting). Mededeeling Nr. 64 van het Vezelinstituut T.N.O. Delft, 1942.

A flax-rett ing process has been worlced out, by which a considerable shortening of the retting-duration can be obtained with respect to the ordinary hot water method. In this process the flax is inoculated with a certain volume of retting-water, obtained from a former retting, which has been aerated previously.

A. D. J. ME1zusE, Ervaringen met een versneld vlasrootpro¢~d6 in de praktijk. (Experiences with the method for accelerated flax- retting when applied practically). Mededeeling Nr. 71 van her Vezelinstituut T.N.O. Delft, ]942.

The advantages effected by the shortening of the retting- duration in using the quickened flax-retting process according to GHUSEN consist chiefly in a more regnlar distribution of the work. The tending of the installation necessary for the GHIJSEN method is very simple. The quality of the ribbon obtained by the GHIJSEN method (starting from various kinds of flax) is practically equal to that of the classical fl~x-retting method.

A. J. KLUYVER, De microbiologische grondslagen der voedsel- conserveering. (The microbiological ,basis of the conservation of food). Chem. Weekblad 38, 383, 194i; Pharmaceutisch Weekblad 77, 1370, 1941.

The paper contains chiefly a discussion of the action of high a n d low temperatures and more especially the harmful action of high temperatures on bacteria. The ioss of the reproductional power is taken as a criterium for the death of the bacteria. By means of graphs the letality of the bacteria and the spores as a function of t ime at Various temperatures is x4sualised. Moreover of importance are: the original number of microbes, the state of matur i ty of the spores present, the chemical composition and especially the pH of the surrounding medium. Some restflts arrived at under practical conditions are discussed.

Besides this the advantages or disadvantages of temperatures beneath and just above the freezing point are discussed. Here as well the initial number of microbes is of importance and also the degree of moistness.

The influence of carbon dioxide, ozonising, dry ing and conservation ,by means of salt, sugar and acids is shortly discussed,

. . . . . ~ J

Mycology and Plant Pathology 107

MYCOLOGY AND' PLANT PATHOLOGY

H. A. DIDDENS und J. I, ODDI~.R, Die Hefesammlung des ,,Cen- traalbureau voor Schimmelcultures", B6itr~tge zu einer Mono- graphie der Hefeartcn, II Teil; Die anaskosporogen Hefen, zw'eite H~lfte. (The collection of yeasts of the ,,Centraalbureau voor Schi~-~melcultures", II part, The anascosporogenous yeasts, second half). N.V. Noord-Hollandsche Uitgevers Maa~cschappij, Amster- dam, 1942.

,,Die anaskosporogenen Hefen, zweite H~f te" gives the classification of the Mycotoruloideae, the second subfamily of the Toru- lopsidaceae. The Mycotoruloideae are characterized by the ability to form pseudomycelium (in some cases also true septate mycelium) on which the blastospores generally are produced in. a typical ~ n r / e r . Chlamydospores and arthrospores may be present. In the first chapters the authors give a general review of the subdivisipn of the Mycotoruloideae accepted by other workers in this field, a discussion of the systemati~ position of the Mycotorutoideae within the Fungi, irnper[ecti, the definition of the genera of the Mycotor~ loideae accepted by themselves, and a discussion Of the dissociation phenomena in t h e Mycotoruloideae in relatio~ to ttieir significance for taxonomy..

The. investigation was extended over 384 strains, isolated from I~uman and animal pathogenic material as well as fro m s~/pro- phytic material.

Based on the evidence obtained theauthors divide the Mycoto- ruloideae in 3 genera: Candida, Br~t~anomyces and Trichosporon.

The genus Candida is characterized by round, oval or elongated cells and by the lack of ability to produce arthrospores. 25 species and 8 varieties were accepted, of which fi are .new species (C. cate- nulata, C. ~aponica, C. Melinii, C. ,robusta, C: Scottii, C. tenuis.) and 4 new varieties (C. ~uilliermondii var. nitropMhi, C. heveanens~s var. cur.rata, C. pellicosa var. c~lindrica, C. tropicalis vat. Rhagii).

The genus Brettanomyces possesses ogivaLly pointed, besides round to oval or elongated cells. The pronounced acid production causes a rapid die-off of the cultures. This genus contains only 4 species and 2 varieties. The treatment of this genus is based on data obtained from the publicati6n of CUSTERS.

The genus Trichosporon is characterized by the production of blastospores and arthrospores. True mycelium as well as pseudomycelium is present. I t forms an intermediate genus, between Candid, and Geotrichu~n. The relation l~et~een the number ~ of arthrospores arid blastosporcs may, even in a same ~train, be very v~riable. The authors divide the genus Trichosl~oron. into :~ specms and I variety. DescHpti0ns are given of the new Species Tricho~oron capitatu~ and Trichosporon fer~ntans..

The work is especially of great importance ~n that clarity and unity has been established in-t-he -ta-xonomy of a group in which chaotic and scattered literature has brought confusibn.

. . . . . , , , , , .

Mycology and' Plant Pafllology.

T. HO~, On t~ae iflen~-ity 0~ T - ~ J a ereeao~ ,I-Ia, mmer, et Cordea w i th . Candida pseudotropicalis (A. Cast.) Basgal. Antonie van Lcefiwenhoek.9~ 77, 1943.

Torula cremoris Hammer et Cordes obtained from the ,,National Collection of Type Cultures" in London ha.~ been studied. I t s characteris~cs were in good.agreement with those described by Hm-nmer and Cordes. A test for pseudomycelium was unmistakably positive. The strain is claimed to be identical with Ca~dida pseudotropicalis (A. Cast.) Basgal.

7F. -H. VAN BEYMA THO~. KING,A, Ueber einige Formen. v on Vert~cilli**m dahlia2 Klebahn. (On some forms of Verticillium dahliae Klebahn). Ant0nie van Leeuwenhoek 6, 33, 1939---1'940.

Verticillium atboatrum and Verticillium daMiae can be sharply distinguished. Verticillium album forms only dark coloured mycelium which may swell into black mycelial knotted masses; moreover the older conidiophores are coloured broWn at the base, whilst chlamydospores and pseudosclerotia are lacking. Verticillium dahliae develops many pseudosclerotia. Generally the mycelium mats will take on a bl~ck colour much Tnore rapidly than in V. aL boatrum which in pure culture often during a long period merely forms white mats. V. dahliae frequently occurs on many plant species.

New forms of V. dahliae are described: V. dahliae Klebahn forma zonalum, V. dahliae Klebahn forma cerebri/orme and V. dahliae Klebahn forma restrictum. Verticilli**m dahliae is thus a group form such as it is known in Penicitlium.

F. 'H. VAN BEYMA THO~ K~NGMA, -Beschreibung einiger neuer Pflzarten aus .dem Centraalbureau voor Schi:mmelculLaxres, Baarn (Nederland)..V. Niitte'flung. (Description of some new species of fungi .from th~:Centraalbureau v.oor ScJMmmelcultures, Baarn, Netherlands. VI. Communication). Antonie van Leeuwenhoek 6, 263, 1939--1940.

The following fungi were described anew: Emericdlop'sis terri- coZa, Emericellopsis terricola vat. glizbra, Peniciliiura e~glaucitm, Penicillium baarnense, Penidlliu, n ~Ovae-zeelandiae, Bisporbmyces chlamydOs.poris, Phialophora aum~t'ia~a, Pkialo~hora lutea~ol~vacea, Graphium Cartwrigh~ti~ Oos#ord "cb~orans; Spirotrichum "~usae ,and Pestatotia natalensis.

Mycology And Plant -Pathology; t@o

F, -H. v ~ B~YM.& ,XHOX KINcMA; Beschreibung elniger ne~er Pilzarten aus dem: Cg~traalb~rea.u. root ; Sch4~me~eul~ures~ Baam, {Nederland). VI I . I~i,~.eflung., (Desc~ptiom, of,~some.new species of fungi from the, C e n ~ l b u r e a u voor Schimmel*ultures, Baarn, Netherlands. VII . .C6mmun.ic~on): ,Autonie ,van Leeuwenhoek 8, 105, 1942.

The following,fungi are described anew: Ar, ackniot~s dan-kaKense, P enicillium ingelheimense ; P homa, suecica, M argarinomy~es~ deOum- bens,, ~hialophora atra. , .Ph~alophora cyda~nieds, Tritiva~hium~ ¢inn~, momeum, Tritirachium mseum and Cephalosporium la?a.oso-ni.veum.

F, H. VA~ BEYM& THO~;-KINGM~, Beschreibung.der i.m Cen.traal., bureau Voor Schimrn~elcultures v0rhandenen Artemder Ga .ts~,ngen P.hialophora Thaxter und Margar.inomycqs. Laxa, nebs.t. Schliissel zu ihrer Bestimmung. (Description of the species of the g~nera Phialophora Thaxter and Mar~arinomy~es Laxa present in ~ e Centraalbureau voor S~himmelcultures next to a l~ey 'for their identification). Antonie v~n Leet/wenhbek: 9, 51~ 194-3.

Phialopbora and Marg~rino~yces are hard to distinguish.,. There- fore both ,genera have been treated mono~raphicalty. 17, species of Phialophora and 6 species of Margarino~yce.s have been described., A key for their identification has.been composed.

Y VAN KOOT, Viruszuivqring en war iij Ons leert omtzent den aard van bet virus. (Virus-purification and what at tells us abou~ the nature of the virus) Tijdschrfft vobg" iOIantin~iel~t~n 46, 97, 1940.

A comparative study was made of the purification of the:ordinary tobacco mosaic virus and the single streak viruk o~ the :tomato. The following methods' were used: the method',of STANLEY; ffl~ tration through ~ceht~ and seP~ation o f the V/n~'S by, '~ffer¢it' saturations Wlth (NH~)2S0~ ~ that 'ofBAWDEN and PiRiE;;he~ting to 70 ° C. and seoaration Of the virus by adding I~;a~d"hhat of RISCHK0v and GR0,~Yk0; remov~'~0f~the).p.i~en~y~f6at~t~ wi~h coal. As result 'a quick" methiJd"W~S, ot~'ta~ed ~ , ~s~.hg pl/..~t,~ grown tinder moist"condffions in the' 'shade, so ~hat'the: jm'~¢.'k~ic] not contain too much. pigmeni."Th~s jhlce wag tiRered "t~irbiig!i celite, the virus precipitated With 40 % (NH4)~SO 4 followed b y a s'mgle treatment .with .I % or 2 ?/o c o~, :all:at.pI~-~--.7:~ Ykms nee~es are ,only obtained,when :the. vires is~uite ~ree" 0f pigment-.~aad when (NH;~)~SO~. is added, The.'~needles-~of ~hetom~o,si~gle~r:eak .virus ~re:some;what'-'sho~tef (l5'B).than,,'th'e"ineedles'~o~ ~h~e"0rdin~ .aL'Y tobacco mosai~ v~rus~:(20-L25 t-0[ ~ e ~ ; a 6 ~ i ~ y 'oe ~l~e ~o~ato

ifltaet, during ~long. chemical, ,puriLEh~. g ~pro~e~s,: ,~v~9 ~ae ~ac~vi~C- of the lobaec0:vimade¢~ea:s~d~,to:.a ,¢.onsidemble~ extent.~

110 Mycology and -Plant Pathology.

J. G. OORTWIJN BOTJES, De ,toepassing van een beschu~cende enting als middel ter bestrljding van vimsziekten bij de aardappel- plant. (The application of a protective inoculation as a means of control of virus diseases of the potato plant). T, ijdschrift voor- Pl~ntenziekten 46~ 181, 1940:

The presence of mild mosaic in the. variety Industrie protects this variety for infection with strong mosaic. Inoculation with both were made oh the variety Eigenheimer with same result. The damage on Eigenheimer of light mosaic is, however, still such that it is not advisable ~to use its protective power for practical purposes. The danger of. the use of protective virus inoculations is the occurrence of complex diseases. The X-virus of Irish Cobbler gra~fted on healthy Eigenheimer resulted in healthy plants, after grafting on mild or strong mosaic Eigenheimer a complex disease developed, the .plants remained small, and the leaves were very Crinkled. The reverse inoculation had no result. The X-virus present in Eersteling, Franschen,, and Magdeburger Blaue inoculated by grafting on mild or strong mosaic Eigenheimer gave the same complex disease. As the X-virus is not transmitted by Aphidae, the complex disease does not occur in practice. A weak form of the X-virus also gave the same complex disease. A second complex disease is dwarf mosaic, which developed after grafting of strong mosaic on light mosaic Eigenheimer.

J. G. 00RTWIJN BOT.JES, De invloed van bladrolziekte op de opbrengst van ve'rschillende aardappelrassen. (The influence of leafroll disease on the yield of different potato varieties). Tijd- schrift voor Plantenziekte 47~ 25, 1941.

The yield of 16 potato plants of different varieties inoculated by grafting with leafro!l was compared with that of 16 hea thy ones. The loss in yield was very different with regard to the different varieties used, and greatest withthe variety Paul Kruger (President) being 84,6 % and none with the variety Up to Date. The tubers are smaller and especially their specific gravity is less. Of a small number of varieties the yield 6f n.~turally infected potatoes was compar~ed with that of heal thy ones giving not quite the same results as that of the grafted ones.

C, M.AST~.NBROEK, Enkele. veldwaarnemlngen Over viruszidkten van lupine en een onderzoek' over haar mozaikziekte.. (Some obser~.ations in the field on virus disea.~es of ~upin and ah- in- vestigation, on lupin mosaic disease~). Tijdschrift voor-l~lante~ - ziekten 48~ ~97, 19~2.

The symptoms of mosaic disease on ,L~I~i~s l~t~us occurring the field are described,, those of the secundary form .of- the

disease on. plants grown from virus: centaining, seed~ next :~o.those of th~ pn.'mary form of. the ~ewl.y-iafected crop. The disease could

Mycology and ~la~t ~thblog~. 1tl

laot b e t ransmit te~ on the test sort iment of 8 vavietleS of american beans, but the inoculat ionon 2 dutch varieties of Phaseolus vulgaris was successful. Good results were also obtained' on 3 varieties of Pisum sativum, on Vicia ]aba, on Lat, hurus odorata, on Tri/olium pratense and on Lupinus albus, but no results were observed on Tri/olium relbens'and Tri/olium hybridum, on Lupinus mutabi'lus, on So~a hispida and on Nicotiana spp. The c o l o r e d flowers of diseased Lathyrus were ,,broken". Seed transmission occurred on lupin, for 5 %, but no s ~ d transnfi'ssion could be proved, on a variety of Phaseolus. Between,65° C. ~md 7b ° C. the virus was killed: A dilution of 1/tiC0 was stii~ active. A'sthe virus could not be trans- mit ted "to Nicotiana,or to the test sortiment of P.haseolus, it is not identical with an already known virus. Therefore it should be called Lupinus virus I. A mosaic virus occurring onLupinus albus was not identical with it.

H. M. QUANJER, Bijdrage tot de kennJs van de in NedeHand voorkomende .ziekten van tabak en van de tabaksteelt op klei- grond. (Contribution to the knowledge of.diseases of tobacco occurring in the Netherlands and of the culture of tobacdo on clay soB)..Tijdschrift voor Plan{¢nziekten*49, 3'7, 1948.

Mosaic is generally present: I t was proved again that. t he disease is not t ransmit ted with seed. After handl~ig the seedliflgs with hands, which had been irl ~ 'clos~ contact w i t k 6 l d dried, diseased tobacco, 17 % of the plants became diseased. Control measures are: destruction ~ of all rests of old plants in the fieldS, disinfection of the hands during work. Solanum nigrum m a y be a Source' Of infection. Next 'to mosaic a second virus disease.occurred genia l ly . I t resembled ,,ringspot" but differed from it throug.h the formation

• " t t '~ of typical stripes along the stem.. The name ,,ring and s ripe, ~ disease is suggested• The-symptoms are recorded in detail. The' disease could be t ransmit ted by sap inoculation. T h e ~ " i ~ : destructed at 80 ° C. during. 10 minutes. The disease'can be. trttns- mit ted from the Soil of seed-beds, seeds sown ~ in di'seased Soil "~fld handled verycare fu l ly ga~e 10 % o'f diseased plaints. A]i ~¢ari~ties of tpbacco used in theNetheriands: as Wellas' N. macr~hytla" and N. rustica were susceptible.

D. NOORDA~, Over her voorkomen v~a ,,spo~e d wilt',; in;l~ed~ere land. (The occurrence of ,,spq.tted-wilt '~ ir~ the :Ne~herlal~ds). Tijdschrfft voor Plantenziekten: 49, £17, 1943~

Spotted wilt was 'found on R@hacd~a afi4~'ana I~un :~.h and :ceuld be t ransmit ted by sap i~ocula t i~s ~ti, Sotanum '!~co'~ersicur~, Solanum 6apsicastmam, " Hi1~eastru~: f iy.~ '~~j 'N~dbEana g~ti~osat Gtoxmm.hybridum," P~sum satwum, .Tl~e syhaffcOms o a ~i!l- ~Imse' pl~i ts ~re described~

11~ Mycology and Plant l~athoiogF.

J. TXMME, N a t r o t bij t a b a k (Nicotiana tabacum). ,(S0~t, ro&,In tobacco, Nicotiana tabacum). Voorlopigo med.edeling ,ov..er een:taog nie t eerder beschreven bacterieziekte van t abak . . . (Preliminary communication about an as yet undescribed bacterial disease of tobacco). T. voor Plantenziekten 49,. 113, 1943.

The symptoms are described of a disease in the leaves of tbbacco obshrved for:~he first time in the Netherlands. Its investigatioll led to the detection of the causal 'bacterium up till then unknown. Theorganism appears to be related to Erwi~da aroideae which causes a soft rot in bulb, stem and pdtioles of Zantedesclda aethiopica a.o." Sufficient remus are claimed to be present to bring this bacterium in a separate species. The name Erwinia nicotianeae is suggested• The disease caused by this bacterium is termed .,,soft rot of tobacco".

A. JAARSVELD, De invloed van verschillende bodemschimmels op de virulentie van Rhiaoaonia soIani Kiihn. (The influence of various soil fungi on the virulence of Rhizoaonia solani Kiihn). Thesis, Am- s te rdam 1940.

The investigations have been carried out chiefly by means of cotton-plugged culture tubes, thus under completely sterile conditions. Some experiments have been carried out in flower-pots. The influence on the parasitism of Rhizoctonia solan.i on seedlings of Chinese cabbage has been studied for a number of soil fungi, :viz•, A bsidia spinosa, Cladosporium herbarum, Cvlindrocarpon didymum, Pemc,lhum ex#ansum, Pyronema confluens and two stratus of Trichoderma lignorum. All these fungi appeared to act more or.- less antagonistically on. the virulence of Rhizoctonia-solani, as well in respect of the germination as of the seedlings. In the preseflce of 2 to 4 antagonistic soil fungi the total antagonistic actioh was stronger than the action of a single one. The addition of Cylindro- carpon to two" or three-of the others macle the antagonism decrease somewhat, although Cylindrocarpon by itself acted antagonistically.. In the pot experiments rice cultures of. the fungi have ~itially beei1 used I t , appeared, however, that rice cultures of saprophytes, as well acted injuriously, probably on account of the toxic substaa.ces which had developed in them. When agar cultures had been used as inoculum the results nearly completely corresponded with the experiments under sterile conditions.

The influence of temperature on the linear growth and, :d~.. weight of the yarious fungi wasstudied; minimum, optimum and maximum temperatures were ~certained. The antagonistic .in~uen.ce ~of A*- sidia, Cylindrocarpon, pyronema and. Trichoderma appeared strongest at the optimum temperatures .of these fungi. Inversely ~..at temperatures which fa/roured the ~deyelopment, the an tag0ni~m was not always very strol~g For Trichoderma strain B .the ~a~rea of optimal temperature for the growth of the fungi was wicter t h a n for the maximal antagonistic action,

My0ology and Plant Pathology.

The antagonistically acting agens appeared not to be confined to the living f ~ g i . The filtrates as well "aCted ant~gonistically. When Rhi~oe~#:ia was inoculated in various dilutions of the filtrates, growttr ~vas most strongly inhibited-in undiluted filtrates. As a rtlle no pseudosclerotia were formed in undiluted filtrates, few in those that were s]ightly diluted and many in the strongly diluted filtrates. The filirates as well appeared to act antagonistically on the virulence of Rhizocto~ia against the seedlings.

In combinations of Rhizoctonia with the other fungi in pet-ri d ishes~nd drop-cultures various forms of inhibition occurred. No epipar~sitism, however, has been observed.

K. HARTSUIJKER, Het wetenschappelijk ondetzoek van fungiciden. (Thescientific research of fungicides). Thesis, Amsterdam 1940.

The_~mgiused in this investigation were: Ve~turia inaequalis, Ph~top~thora in/estalzs, Clados~orium fulvum, Septoria apii graveo- ten~s, Ascochyta pisi, Helminthosporium sativum, Botrytis cinema. The p~.otective fungicidal action has been ~letermined by placing spore suspensions on the dried chemical preparations, the dit. ect action by incorporating the spores in the different dilutions of the preparations and the lethal effect by bringing the spores into contact with the preparation for some time, removing them again by centrifugating and sowing them out on cherry extract.

The experimental work falls into three groups: 1) The toxicity of five metal salts, riz., CuS04, HgCI,, CdCI,~ NiSQ and ZnSO 4. In this case the direct-fungicidal and the lethal effect were determined. The salts examined were found to differ a great deal in these respects. I t was also found that the order of the fungicidal

• action of these salts is not the same in the fungi investigated. ZnSO4 proved to be least toxic and hardly lethal. The strongest effect ,was observed in the case of CuS04 and HgCI,: there being veryli t t le difference between the two. Only HgC1, was considerably mare lethal than CuS04. CdCl,Wvas found to occupy an intermediate position, since its toxicity was generally less, but never more than that of CuSO4. In the case of NiSO, the results obtained with the vain'o us fungi were widely divergent. To Barytis cinema it was the most toxic of all salts, while it was also toxic to the indirect germination of Phytophthora iniestans. The results obtained with NiSO4 migh t explain the widely divergent data supplied in the literature on the effect of nickel salts. Durir~ the determination of thelethal effect of NiSO 4, it was observed thaLhigher concentrations w e e less lethal than lower concentrations. This phenomenon, which was also observed for CuS0, in the case of Botrytis ci~erea, coilld not be explained.

2) The toxicity of a number of sdlphur compounds, viz., three different polysulphides (Ca-, NH,- and Ba-polysulphide),~a mono'- st~ide ~a-monosulphide)' and ~ colloida~ sulphu~ (coLloidal

11~ Mycology and Plant ~hology.

bentonite sulphur), A .marked. difference; between the~ dir~ ec~- and the pr,otective-famgicidal action was only observed m :the c~e,~ef the ,tlar~ ee polysulphides. The Strong direct-.fungicidal action of ~tiie polysulphide is of great importance. Ln practice (.extermin: ~tion of existing infections), The protective-fungicidal action,'of the, polysulphides was also better than ~hat'ofthe two ottier types of ',sulphur preparations, At an 'equal percentage of -total:substance :tl~e pro: tective-ftmgicidal action of the .conoidal sulphair was better tha,n that of the monosulphide.

3) The-toxicity of mixtures of polysulp~des and tend arsen~fe, The rSatefials used were polysulphides of Ca .and .NU~'i'n_com'bi, nation with powdered lead arsenate arid additional substances such as lime and ferrous sulphate.. Lead arsenate was found t o possess only a very slight fungicidal action. The fungicidal action-0f combi- natioias of 1% of polysulphide an.d :respectively 0.1%, 0.3. ~ and 0.5 % of lead arsenate was .~Omewhat better than that of polysulphide alone, although-the d~fference was only "very'slight, :T.~e quantity of lead arsenate added-did'not influence the"fhngic~d~ action to any appreciable extent..The destroyihg effect of'the lead arsenate on the polysulphides is" either -very sfig.ht, or en~tirdy corr/pensated for by the fungicidal action of Vhe ,,soluble arsenic:' formed. This fact is supported by practical data obt~ined f~om :tile literature,

:D. MULDER, Biologisch onderzoek van grondontsmet-tingsmi'dde- len. (Biological investigation of soil disinfectant.~). Thesis, Amster- dam 1943.

The aims of this investigatiQn were: a) to drav~ -up a. c.her~Qt.l~e~;,, pentic~al index for soil disinfecta.nts, b)::To~:dev.elop~.e~perimental methods to obtain this index, c) To collect data .eoiacerning-;the decrease in effectiveness of a disinfectant brought about by,,contact with the soil, d) Todetermine a dosis curativa for disinfectants:in soil, e) To test the usefulness of some new methods of soil" disin: fection,

The materials used were the ordinary seed- and soil-disinfeetants; Besides these products specialorganic mercury-compoun~ could be desposed of for the study of the,infltience of certain admixtt~r; es; the study of the difference between ethyl-and;phenylmereur, y~ compounds and the influence of different acid groupS. The d0~is toxica, in relation to fungi and the dosis to!erata,.f~ .soedl~m, gs.wdt~ determined experimentally in order to calculate the-chemoth¢~ff~ peutical index. The growth of Pythium de Baryanu~ in.0,5"2,/o saccharose .solution, without the addition of nutrition~,isa!ts; satisfied as criterion for the toxicity. Proportional,i.ty :appeared~:to exist between the rate of growth and the concent~a.ti0n. 0f~ the. poison. In the case o.f ads0rption.-expeL'=maen~ts the'gr, owth,~0f :~y- thi,im mycelium ~sed for. determinatior/of, toxi¢i.~3~,, proyJ:d.ed-~t.li.e possibility of ~n accuracy, .which surpassed, by far ~tliose of ,ehomiea~

Mycology and Plant Pathology. n ~

~ethods of~ defining the concentration. A low and consequen~I,y favorable index figure obta'med in the laboratory, however, .ofte~ coincides ,with insuf, ficient disinfectant qualities'in soil.

Therefore other qualities: than -those, which can be studied in vitro ~ . contact With fungi and plants, also influence the value of

mercUry-compound as soil-disinfectant, O~e of ~these qualities is the adsoi~tion, of the ~compour~d in soil by which its working capacity decreases. -The determination of its adsorptionwas carried' out both in garden-soil and with adsorbent coat, From the dosis. toxica l~efore and after the adsorption-an adsorption-factor can be calculated. The historical development of the seed- and soil-disinfectants~shows a parallel with a decrease of adsorption. The phenyl- mercury-c,ompounds are adsorbed ten times as stro.ugly as the ethylm~rcury-compounds. The r~sults with the various compounds is closely reIated with the intensity of attack in the experhnent in question. Therefore only restricted mutual comparison is permitted. The al~lication of formaldehyde as a dry powder, made by rnix~, g it witli~infusorial earth or ~ saw-dust, was a complete success: the disinfect-'~n was nearly as good as with formalin 4 °,/o. Complete disinfection was obtained only with formaldehyde. On the other- hand the Mghest increase in' number of seedlings ' is reached with. 0he of.the mercury compounds.

SOIL BACTERIOLOGY

G. V¢. HARMSSN, The infli~ence of the method of sampling on the accuracy of the determination of bacterial numbers- in the soil Antonie van Leeuwenhoek 6, 178, 1.939-~-1940.

Tests were made to determine the influence of the sampling and the preparatory t reatment of the samples on the counting of-bact¢~a in soil These pro*ved that the present methods are very inaccurate and give rise to considerable variance in parallel de- terminations, to . the extent even of rendering the effect of a per- fected counting procedure quite negligible. For a better prep~ra~tory t reatment of the samples a homogenisation method by rheans~f a porcelain ball mill was worked ottt, by which:means a S~spe~isior~ of l~he, sample is m~de in Water, which ~method has giVen.'satisfactory resul ts .

F. C. G~RRITS~N, Enrkele~w~a~i-gen betreffende den invloed van de temperatuur op de nitrificatie en vas~egg/ng van de stikstof. (Some Observations concerni~g the influence b~ ~mperalure on the nitrification~ and assimit~ti~ of nitrogen). Land'boziwkundig Tijdschr/ft 54, 5.78, 1942.

Laboratory tests were carried out, one series: wit~, ~a yariety of cons te~ temperatures, the other with~yarying ~tem~erat/~res in:fhe open.~ 50 rag: (~H.)iSO~,had • :beei~ sdded:~per ~'l~'O0~'g of~dry:'so~.~

116 Soil Bacteriology'.

The balance and transformation of nitrogen were checked by periodical estimations of NOa-, NH4- and total nitrogen. Nitri.- ~fication occurred even between 0 ° and 5 ° C. and,at ,5 ° C, a normal supply of nitrogen may be entirely nitrified within a fortnight. The 'experiment in the open showed that the nitrification process Was fairly ,strongly influenced by temperature. W h e n this process had arrived at its close the whole balance of nitrogen was dominated by strong perio~cal fluctuations in the ~ssimflation of N by microbes. A reason for these fluctuations could not be presented.

Y. VAN KOOT, Grondontsmetting.door stoomen en de be'/nvloe- ding van het bacterie leven en de samenstelling van. de grond. (Disinfection of soil by steaming and its influence on the bacterial life and composition of the soil). Landbouwkundig Tijdschrift 54, 532, 1942.

Experiments were carried out in a cold winter under various moisture conditions. Directly after stekming the bacterial.number was very low. After some weeks bacterial numbers had increased strongly and surpassed the original ~mounts by far..The numbers of nitrifyers increased much later when the tempera ture of the soil had risen considerably. The numbers of the protein decomposing bacteria did not ,increase so distinctly. The amount of water soluble nitrogen generally increased directly after steaming and this lasted up till the moment of increase of bacterial numbers. Then a distinct decrease in bacterial nu~pbers occurred. When the soil had been steamed under ~oo moist conditions or had been leached out after steaming, no increase of nitrogen occurred and the development of the bacteria was much slighter.

K. T. WIElZINGA, Determinatio~ of the fertility of the soil by microbiological methods. Antonie van Leeuwenhoek 6, 56, 1939--- 1940,

A review is given of t h e determination of soil fertility by microbiological methods. The quantitative and the qualitat4ve method for the de, termination of the microflorar,were discussed as well as those methods in which micro-organisms are used for ascertaining the particular condition of a limiting factor in the soil. More especiatly the use of Azotobacter cultures for this purpose was mentioned,

E. G. MuI~zR, On the use of micro-organlsms in meast~rtng ~t deficiency of copper, magnesium and molybdenum in soils. Antonio van Leeuwenhoek 6, 99, 1939---1940.

A description is given of some microbiolo .gical tests for ,~he ,determination of plant-available copper, magnemum and mulybden:,u~: in,soils. ~n th~se~investigations Aspergillus ~ige~and in a few cas~,

,Soft ~ 1 o 8 y , 117

AzotobacWr chmococcum and Bact,~,iu~, prodiglosur~ were used, From the figures obtained it. is revealed tha t soils on which the plant.:s..are.suffering from the so-called ,,reclamation disease", have .a much lower ~available copper content than those producing healthy crops. Soils on which the plants show the so-called ,,Hoog- halen disease", have a very low content of available magnesium, From these results and from experiments with cereals it is concluded that the ,,reclamation disdase" is caused by a deficiency of plant-available copper and that a deficiency of available magnesium is the chief cause of the ,,HooghaJen disease".

F, C. GERRITSEN en NORA BLUMENDAL, Een onderzoek naar de bruikbaarheid van de A spergillus-methode voor de bepaling van phosphorzuur en kali in den grond. (An investigation of the suita. bility of fhe~ A spergillus method for the estimation of phosphoric acid and potassium in soil). Verslagen van landbouwkundige onderzoekingen. 46, 219, 1940.

F. C. GERRITSEN und NORA BLUMENDAL, Phosphat-Bestimmun- gen mittels .AspergiZIus n'iger. (Estimation of phosphate by means. of A~psrgillus niger). Antonie vanLee'uwenhoek 6, 71, 1939--1940.

The estimation of phosphate in softs accordipg to the As~ergitlus method of NIKLAS entails some difficulties. Th~l~0duction of acid by the fungus varying along with the content of phosphate of the medium, the pH of the medium varies foo. The varying.lime content of the softs influences the development of the fungus In two ways: a. by altering the buffering of the medium and b. by the action of calcinm 'as a nutrit ive for the fungus. Certain humus substances mayinf luence the amount of mycelium formed. By substituting 0.6'% ammofiiumsulfate by 0.4 % urea the changing of pH in the medium is brought down considerably. By adding Ca nitrate a bet ter buffering and independence of the Ca content of the soil is arrived at. The supply of a minute dose of humic acid (Na humate) removed a source of irregularity a n d c a u s e d i n some cases an increase in mycelium of 9 to 40 %. By these means the maximum change in pH could be bxought down from 1.8 to 0.37: in tests Of' softs of vari6us origin the change in pH was on the average 0.14.

In .the presence of potassium sodium salts (5 m. tool. pro titre) appear to favour the development of mycelium (an increase 'up to 30 %).

I t is claimed that the Aspergillus method belongs to the cheapest and mos t reliable methods for the estimation of the need 0 f 'P and K in softs.

JAN SMIT and E. G. MULDRRs Magnesium deficiency as the cause of-injury in cereals. Meded. van de Landbouwhoogeschool 46, Verhandeling 3, 1942.

In order to ~scertain ~he ~ause of. the d_iseas¢ of o~ts and other

~I8 Soil "BaCtei-lology.

.cereals occurring'frequently on aci~l softs A's~ergill, us" niger wasmsed as a test organism for the estimation Of .plant available r~gnesium in soil. Its development in a magnesium, free so~tion, vchere.tl~e soil investigated was the only source -of magnesium, was recorded. A series ot flasks with known amountsOf the element served a s a standard of comparison. It proved asatisfactory method to estimate the part of the total magnesinm:,which may be assimila~d "bY Aspergillus niger. Healthy soils appeared to contain 100 ~ dr more per 3 g of soil. A striking parallelism was found to exist between the occurrence of the disease and the amount Of Mg available to the mould.

MARIE P. L6HNIS, The actio~ of manganese on the development of Aspergillus niger. Antonie van Leeuwenhoek'-10, 101, 1944 1945

The response of Aspergillus niger to the presence of manganese in the nutrient solution has been" ascertained. In an initially acidified medium which kept up its acidity or in an unbuffered nezrtral

• medium which grew strongly, acid, manganese appeared essential .for sporangial development: In suitably acidified solutions KNOs as source of nitrogen induced slightsporangial developmentin the presence of 0~05 ~ Mn, NaNOs of 0.025 ~, Mn and NH4NO ~ of 0.05 Mn in 40 ml of medium. In the acidified NH4NOsmedium the largest number of visually to be distinguished amount of sporangia could be ascertained. In acidified solutions with KNOB the ~eld of mycelium was higher inn-"the absence of manganese ~ than in c~tures whichin its presence had developed sporangia. Presumably, the development of sporangia inhibits further development of mycelinm. The weight in yield of mycelium in an acidified NaNO 3 solution in the absence of manganese was very significantly lower, than in the presence of 0.005 ~ Mn. In ,non-acidified nitrate solutions sporangia developed in the absence of mangan~e. No definite tren d,in the yield of mycelium could be ascertained. In unbuffered solutions with ammonium, salts no sporaugia developed in the absence of manganese. The response on various amounts of manganese was erratic. ,No definite trend in mycelial .developmpnt could be ascertained. Solutions with ~ ammonium salt sltitably buffered by CaCO8 produced sporangia "m the a bsenc& of manganese. They gave the highest yields in weight..The intake ,of manganese by the fungus on various" media offers slight differences. The intake is slightly hi~her~ in weary ~cid than in s~ong!y~, .acid solutions. The fact that m~nganese, is:0,~y essential in.. a hig~y acid medium "may not. be aser~bed" to. an action ~o.f t~e mediu~t such on,-the avaflibftity o L m a ~ e s e ,and must be mused by differences in the metabolism of A~gb~gi]l.~s ~ig~'.

As -~s~g~i~s ~i'g~,is sens~i,Ve, :~oi-,t~ces O:~:.~ang.anese only trader 1~g1~ly. ~ i ~hdi~ons an :A.sp~g}~[us:sf~d~ff,[~ 'not~ be s~t~ble for soft ~sts.

Soil Bacteriology. 1 !9

K..T. WIBRXN~A. Landbouwkundige en ~dbouwn~crobiolbgi- sche problemen betreffende de spoxenelementen (Oh'gopleronten). (Ag~cultural and soil microbiological problems, concerning .the micro-elements. (ollgoplcronts)). Landbouwkundig Tijdschrift '56~ 303, 1944.

Problems concerning micro-elements (oligopleronts) in a~,ri- culture a n d soil microbiology are discussed.-The name oligo- plerontic elements or oligopleronts is forwarded as apt for inter~ national use.

The paper is a review of the literature on some deficienciesi in plant growth, their occurrence and causes in connection to soil conditions such as pH, Eh and structure. The grey speck disease (Mn-deficiency), magnesium deficiency, and reclamation disease (Cu-deficiency) are discussed,

The Aslbergillus method as a means of testing the soft for these deficiencies is paid attention to.

A U T H O R I N D E X

P a g e A b d o e l r a c h m a n , R . . . . . 75, 95 B a u v 6 r y - A s m a n , A . . . . . . 91 Beeuwkes , H. , . . . . . . . . ~9 Bekker , J. H. 21, 46, 47, 52, 53, 55,

56, 58,;64, 75, 84 v a n den Berg, R . . . . . . . 79. v a n den Berg, W . . . . . . . 89 Beute , A. E . . . . . . . . . . 16 v a n Bever , A. K . . . . . . . 98 v a n B e y m a thoe K i n g m a , . F . H .

108, 109 v a n B e y n u m , J. 99, 100, 101, 102,

103, 104 de Blieck, L . . . . . . . 68, 87, 92 B l u m e n d a l , H . . . . . . . . 102 B l u m e n d a l , N . . . . . . . . 117 Boer , H . D . . . . . . . . . 73 de Boer, S . . . . . . . . . . 19 Boh lande r , H . . . . . . . . 66, 67 Bohr6, G. F . . . . . . . . . 84 B o o r s m a , H: J: . . . . . . . 22 Bos, A . . . . . . . . . . 16, 92, 93 Bouwens , H . . . . . . . . . 27 Brandwi jk , A. C . . . . . . . . 22 Brug, S. L . . . . . . 54, 78, 79 Bru ins Slot , W. J . . . . . . 53 de B r u y n , P. H. H . . . . . . 16 B u r e m a , S. J . . . . . . . . . 25 v a n der Burg, A . . . . . . . 28 Bijl, j . P . . . . . . . . . . . 5a Bijlmer, L . . . . . . . . . 53, 80 Clarenburg , A . . . . . . . . 71 le Cosquino de Bussy, I . J . . 45, 77 Custers , i~. Th. J . . . . . ". 17, 18 v a n D a m , B . . . . . . . . . 105 Dekker , W. A. L . . . . . . . 45 Diddens , H. A . . . . . . . . 107 d e n D o o r e n d e J o n g , L . E : . . 13 Dorn ickx , Ch. G. J . . . . . 70, 82 Dor res te in , R . . . . . . . 31, 32, 33 Drogendi jk , A. C . . . . . . . 83 Dunlop., S. ~. C . . . . 15,46, 50 Duyven6 de Wi t , J. J . . . . 44 Fa lger , E. F. J. H . . . . . . 83 Fl th P . C . . . . . . . 14, 16, 95 Fo lpmers , T . . . . . . . . 94, 95 Gerr i t sen , F . C . . . . . . 115, 117 Ghysen , 7. $ . . . . . . . . . 106

Page de Gier , C. J . . . . . . . . 90, 93 Gor te r , A . . . . . . . . . . . 20 de Graaf , H . . . . . . . . . 96 Grevens tuk , A. B . . . . . . . 42 de H a a n , A . . . . . . . . . 100 H~ rmsen , G. W . . . . . . . . . 116 Har t suycke r , K.- . . . . . . . 113 t e n Have , B . . . . . . . . 71, 76 van Hees, C. A . . . . . . . . . 72 H e k m a n , W . . . . . . . . . . 77 Heroines, G. D . . . . . . . . 71 den Herder , P. C . . . . . . . 100 van der Hoe d e n , ] . 51, 52, 5~, 60,

63, 90 H0f , T . . . . . . . . . . . . 108 H61werda, K . . . . . . . . . 101 v a n den H o v e n van Genderen, A. J.

47, .73 Hulk , J: F . . . . . . . . . . 70 Huls t , D. L . . . . . . . . . . 73 v a n d e r H u r k , C . F . G . W . . . 69 v a n I t e r son , G . . . . . . . . 13 Jaarsve ld , A . . . . . . . . 44, 112 Jansen , B. C. P . . . . . . . 44 Jansen , Jac. 68, 69, 85, 86, 87, 9!, 94 Janssen , L. W . . . . . . . . 86 de ] o n g e , J. . . . . . . . . . . 43 Julius, H. W . . . . . 37, 38, 39, 4! K a t z , E . . . . . . . . 31, 32, 33 v a n der Kerk, : G, J . M . . . . 28 Ke r s t en , ~. A. H . . . . . . . 34 K i n g m a Bolt jes, T. Y . . . . . 15 Kluyver , A. J. . 13, }7, 24, 28,106 van Kol lem, R . . . . . . . . . 79 v a n K o o t , Y . . . . . . . 109, 116 Kr01, B. M . . . . . . . . . . 96 Kruisheer , C. I . . . . . . . . 100 Ku l sdom, M. 1 ~ . . . . . . . . . 82 L i e m , S. D . . . . . . . . . . 54 Lodde r , J . . . . . . . . . . 107 v a n Loghem, J. J . . . . 46, 76, 77 L6hnis , M . P . . . . . . 23,27,11-8 v a n Looke ren Campagne , J . . . . . 74 Lopez Cardozo, P . . . . . . . 37 Lourens , L. F. D. E . . . . . 89 van Luyk , A . . . . . . . . . . 44 L. uyken , R . . . . . . . . . . 44 Mantenl A . . . . . . . . . 28, 24

122 A u t h o r I n d e ~ .

P a g e M a s t e n b r o e k , C . . . . . . . . 110 v a n d e r Meer , C . . . . . . . 46 v a n d e r Meer0 G . . . . . . . 79 Meeuse, A. D. J . . . . . . . 106 Mulder , D . . . . . . . . . . 56 Muider , D . . . . . . . . . . ! 14 Mulder , E . G . . . . . . . 116, 117 l~u lder ; J . . . . .36, 53, 74, 79, 80 M u n n i k , G. P. F . . . . . . . 56 N 0 o r d a m , A. L . . . . . . . . "69 N 0 o r d a m , D . . . . . . . . . 111 v a n Olden , E . . . . . . . . 18 O n g S i a n G w a n . . . . 47, 48, 60 Oortavi jn Bo~jes, J. G . . . . . 110 Oos te rhu i s , H. K . . . . . . . 44 v a n Oyen , C. F. . . 16, 57, 97 Paes i , F. J, A . . . . . . . . 78 Pasvee r , A . . . . . . . . . . 97 P~eters , H . . . . . . . . . . 82 P e r q u i n , L. H. C . . . . . . . 20 P e t t e , J . vq. 99, 100, 101 ,102 , 103,

104 P i ck , F . . . . . . . . . . . 77 P ~ G i n g H o o . . . . . . . . 78 P01ak, M. F . . . . . . . . . 92 P o n d m a n , A . . . . . . 22, 23, 49, 54 P r 6 v o t , A. 1~ . . . . . . . . . 22 Q u a n j e r , H. M . . . . . . . . . 111 Quar les v a n UHord , V~. J . . 70 ~u i spe l , A . . . . . . . . . 25, 26 R e u r i n k , J . . . . . . . . . . 45 R o t t i e r , P. B . . . . . . . . . 27 R u y s , A. C h . . . 59, 69, 70, 72, 92 S a l o m o n , A . . . . . ~ . . . 38 Savr i j , l ~ I . . . . . . . . . . . 97 v a n d e r Seheer , A. F . . . . 87, 89 Seho l t ens , R. T h . . . . . . . 45, 72 Schi i f fner , W. A. P . . . . . . 66, 67

P a g e Schui l ing , A. L . . . . . . . . 30 Sta i r , J . . . . . . . . . 96, 117 Sta i r , W. Ci . . . . . . . . . 100 s m i t s v a n W a e s b e r g h e , F. A. M. J . 20 Spru i t , C." J. P . . . . . . . . 30 S t r a u b , J . . . . . . . . . . . 98 TaR, J. D . . . . . . . . . . 24 Tas~nan, A . . . . . . 22, 23, 65 Temme~ J . . . . . . . . . . . . 112 de T e m p e , J . . . . . . . . . 35 v a n Thie l , P. H . . . . 5~, 67, 68 T i l l ema, E . A . " 72 T i m m e r i n a n , W. A e g . . . : . 49 v a n T u i n e n , L . . . . . . . . 53 Veer , W. L. C . . . . . . . . 68 Ve lds t r~ , H . . . . . . . . . 44 V e r h a g e , J . C . . . . . . . . . 77 Ver l inde , J. D. 41, 42, 47, 50; 51,/~8,

61, 62, 63 , 65, 73, 79, 83, 84, 85 V e r v o o r t , H . . . . . . . : . 92 V i n k , H. H . . . 47, 52, 56, 72, 90 Visser, A. K . . . . . . . . . 77 Vos, E. A . . " . . . . . . . 1'04 Vos, J . J . Th . . : . . . . . " 8 2 W a r I f e m i u s , J . G . . . . . . . 105 W a s s i n k , E . C . . 23, 31, 32, 33, 34 Wemsinck~ F . . . . . . . . . 83 W e s t e r m a n n , C . D . . . . . . 73 v a n d e r W e y , A . P . . . 70; 71 Wiechers , S . G . 102 W i e r i n g a , K. T . . . . 21, 25, 1"-16, 119 W i n k l e r , K. C. 24, 37, 38, 39, 40,

41, 70 W o r m g o o r , B . H . 84 W y b r a n s , J. R . . . . . . . . . 44 v ~ n Wi jk , A. ' J . . " . .: . . . . . 96 Zeis l ing , H . . . . . . . . . . 67 Ze lden rus t , J . . . . . . . 41, 42

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