india, vrus: recent;detailed a half century previously by moore. in the century after jenner's...

THE PRESERVATION OF VACCINIA VIRUS'L. H. COLLIER

Vaccine Lymph Unit, Lister Institute of Preventive Medicine, Eletree, Hertfordshire, England"These are the chief methods in use for preserving lymph; and as all occasionally succeed, each

has its partisans; and as all occasionally fail, each has its enemies."-James Moore, The Historyand Practice of Vaccination, 1817.

INTRODUCIJON The survey which follows has been planned toThe problem of protecting the living virus in cover a rather wider field; a number of phases in

smallpox vaccine against the influence of heat the development of the subject have been dis-has exercised the ingenuity of vaccinators for a tinguished, which follow each other in roughlyconsiderable time, and the history of attempts chronological order, and emphasis has been placedat its solution provides a fascinating record of on those events which serve as landmarks in ourdevelopment in laboratory technique. knowledge. Because of its historical interest,

It is possible that during the many centuries however, the very early literature has been dealtwhich saw the practice of variolation established with in more detail than is customary in a reviewin the East, notably in China and India, primi of this nature. The reader's attention is drawntive attempts were made to preserve the virus not only to the multiplicity of techniques usedfrom the deleterious influence of the prevalent to solve this problem, but also to the variableclimatic conditions. Drying as a means of results obtained by different workers usingpreserving foodstuffs, such as fish and meat, similar methods.must have been known to the physicians of those TI FIST PHASEtimes, and the virus of smallpox presented itself Preservation on ivory "points" and glass. Fornaturally as dried vesicle crusts which could be many years after the introduction of vaccinationconveniently transferred from one patient to at the close of the 18th century, the vaccineanother in whole or powdered form. Since virus virus was either propagated directly from humanin the liquid state is susceptible to the influence to human or from animal to human, or wasof temperature, but will survive for long periods preserved in small quantities for use as required.in dried crusts (26), it is quite likely that the Writing in 1817, Moore (66) gives the followingvalue of these as vehicles for the causal agent of summary of current methods of preserving thethe disease was soon appreciated. It is believed, vrus:for example, that the ancient Chinese practisedvarolaionbynb otin o poderd sab "When pointed quills or bits of ivory are well

variolahtienonsb inhalation of powderedscabsand repeatedly moistened with lymph, they pre-through the nose (58). serve the virtues of the Vaccine for a long time.Whether or not the use of dried crusts was a They are more certain, however, the more recent;reasoned attempt to preserve variola virus, the but when wrapt in lint and secured from air,history of the preservation of vaccinia itself is heat, and moisture, they have sometimes con-

almost coeval with the general use of this agent tinued efficacious for several months."as a prophylactic against smallpox. And later,

It is not proposed to give a complete account "There are several other methods in use for pre-of such experiments, as an exhaustive survey serving vaccine lymph. A drop is sometimes in-would require a volume to itself. Several short closed between two bits of square glass: or it mayreviews of the subject have been published al- be deposited in a small cavity hollowed out of theready (21, 31, 32, 54), but these have been con- centre of a piece of ground glass, and coveredcerned mostly with the history of attempts to accurately with a flat piece of the same size.preserve the virus by various drying methods. Lymph desiccated on glass is brought to a proper

state for use, by mixing it up with a particle of1 This review is based on the introduction to a cold water by the point of a lancet. Vaccinethesis accepted in December, 1952, for the M.D. crusts also, when powdered and triturated withdegree of the University of London. cold water, are often efficacious .... Crusts have

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19541 THE PRESERVATION OF VACCINIA VIRUS 75

been transported to the tropical climates, and breathing on the glass; Warlomont rightlykept for many months without losing their proper- pointed out the danger of infection from such aties. Ingenious methods have also been devised procedurefor attracting the lymph into small capillary glass The first use of glycerol. At about the middle oftubes, and sealing them hermetically. By this the 19th century, glycerol was adopted as ameans the lymph has been found liquid, and ef- vehicle for vaccine lymph. Although the methodficacious, after being transported across the i oAtlantic." I said to have origiated m Germany, the earliest

reference I have found to its use was in 1850 byJenner himself sent lymph dried on threads to Cheyne (17), an English physician. He advocated

de Carro in Vienna, who found it still active on mixing lymph with glycerol to prevent decompo-arrival. From Vienna, in 1800, equine lymph of sition and claimed successful results with lymphItalian origin was sent "across European and treated in this way and stored for two months,Asiatic Turkey, and over the whole line of deserts presumably at room temperature. On the otherto Baghdad", on this occasion sealed in liquid hand, Seaton (83) in 1868 condemned the methodform between glass plates (83). We do not know in the following manner: "I think it scarcelyhow long the journey took, but the lymph necessary to advert, except for the sake of depre-survived the severe conditions to which it must eating it, to the proposal which has been made ofhave been exposed and on arrival was success- keeping lymph liquid for a longer time by mixingfully used to vaccinate a child. Seaton (83) states it with glycerine. What effect, if any, glycerinethat the virus was then propagated by arm to might have in retarding the decomposition ofarm inoculation, and in this manner survived the lymph I have not thought it worth while to en-sea crossing from Basra to Bombay, whence it quire: because it is with lymph, pure lymph, aswas distributed throughout India. This assertion pure, as concentrated, and as pungent as can be,has been quoted by later authors, but Moore that we should vaccinate, not with mixtures and(66), writing at a time closer to the event, compounds. But I happen to be acquainted withgives a more circumstantial account of the last some results of using the mixture, and they ex-stage of the journey; he says that after 30 or 40 hibited, as might be expected, a large relativefruitless attempts to send dried vaccine to Bom- amount of failure."bay by sea, a batch was eventually received Today, a century after Cheyne's experiments,which proved still potent after the voyage. glycerol is used extensively in the manufacture

In the early 19th century, therefore, the trans- of smallpox vaccine; as will be shown presently,mission of vaccine for long distances was at- however, these early misgivings found moretended by many difficulties and hazards. This recent expression on less empirical grounds.situation saw no improvement for many yearsto come since the methods of preservation used TH SWOND PHASEin 1868 (83) were almost identical with those Drying of lymph from the liquid state in bulk.detailed a half century previously by Moore. In the century after Jenner's discoveries, pre-Some contemporary opinions of these various served lymph was largely put up as individualmethods are available. Thus Seaton (83) stated doses, dried, as we have seen, on glass, threads orthat lymph kept in capillary tubes in liquid ivory. In 1881, however, Reissner (73) publishedform was better preserved than when dried on the first description of a method for dryingglass squares or on ivory "points". The latter vaccine in bulk, his object being to facilitatemethod, however, was advocated by Warlomont the distribution and use of vaccine derived from(89) who coated the ivory with gum arabie to animals. He dried calf lymph on glass micro-prevent it absorbing the lymph. He claimed that scope slides in a desiccator and ground thevaccine thus prepared lasted much longer than product to a fine powder which was kept inglycerinated lymph which only retained its boxes or in small tubes. To make it serve for morepotency for 5 or 6 days. The same author drew vaccinations, the dried vaccine was diluted byattention to a danger in the method whereby the addition of an inert powder; quite good re-lymph dried on glass was "reconstituted". sults were obtained with material which hadIt seems that the necessary moisture was often been stored for two weeks (74). This lymphprovided by the vaccinator or "sage femme" was used by Hager (43, 44) who thought it


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76 L. H. COLLIER [VOL. 18

possible that drying the infective agent in the dried material in vacuo. He compared theassociation with the dermal cells of the host deterioration rates of dried and glycerinatedmight be responsible for the preservative effect. lymph on storage at 37 C and found that, whereasSchulz (82) also obtained good results with this the latter was inactivated after 20 days, thematerial, but Bauer (4), although acknowledging dried lymph still produced lesions in the calfits stability on storage, thought that the neces- after 30-37 days.sity for reconstitution before use was a draw- It now began to be realized that the advan-back; he also stated that it caused severe vaccina- tages gained by drying lymph were partiallytion reactions when used undiluted. Schoen offset by the fact that it could not be sterilized(81) particularly advocated the use of dried by a period of contact with glycerol, as was thelymph rather than arm to arm propagation, common practice. Carini (16) therefore recom-drawing attention to the danger of transmitting mended the collection of pulp from the calvesother diseases by the latter method. with aseptic precautions to reduce the number ofAlthough at this time increasing emphasis bacterial contaminants, or the lymph could be

was being laid on the use of lymph derived from reconstituted with glycerin-water and left foranimals, humanized vaccine was still employed, 24-72 hours before use.and Warlomont (89), 1883, mentions several Achalme and Phisalix (1), 1909, thought thatworkers, himself included, who dried pustular the active agent was killed on storage by liberatedmaterial from human vaccinations in bulk. enzymes from the dermal cells present in glyceri-Successful results were obtained with material nated lymph and considered that drying, bystored for 90-130 days. He also gives the first preventing enzyme action, should preserve theaccount of a vacuum drying method, used by virus from attack. Whether or not this assump-Verardini, who processed 300 human pustules tion was correct, they successfully preservedat a time for 5-6 days under a vacuum of 10-15 calf lymph by drying it on porous plates ("as-mm Hg. This dried vaccine survived best at siettes poreuses") over HpS04 in vacuo, evacua-0C to 6C. Unfortunately, no reference to a tion being performed with a water pump. Driedpublication by Verardini was quoted. lymph was better preserved as large pieces than

The introduction of potency testing in animals. in powdered form, and they recommended thatUntil the beginning of the present century, the it should be kept sealed under vacuum. Thesepotency of lymph could only be assessed by ob- authors tested the potency of their lymph byservation of the success rate with human vac- inoculation on rabbits' ears or on the calf. Theycinations. Calmette and Gu6rin (14), 1901, then did not express their results quantitatively butpublished a method of potency testing by rabbit stated that after storage for one year at 37 C,inoculation; later the technique was improved the desiccated material gave a comparable re-enormously by Gu6rin (42) who introduced the action on the calf to that produced by freshinoculation of serial dilutions of virus. Vaccine lymph. This procedure was therefore advocatedlymph could now be titrated with some accuracy, as being the method of choice for preparationand many subsequent reports of tests on stored of smallpox vaccine in hot countries.vaccine acquired much greater value. The use of dried lymph in the tropic. The years

In 1902, the first investigation in England of a between the opening of the 20th century and themethod for drying lymph in quantity was re- outbreak of the first World War saw a great in-ported, and animal tests of stored material were crease in activity in this field. Both German andused. Blaxall (7) dried calf lymph in porcelain French workers were concerned to find a way ofdishes over sulfuric acid in vacuo. After 24-48 sending lymph to their overseas colonies withouthours, the product was ground in a mortar and loss of potency during the voyage or during sub-then dried for a further period to get rid of any sequent storage under adverse conditions. Theabsorbed moisture; the loss in weight due to difficulties of setting up overseas stations wheredrying was about 68%. The lymph was stored at lymph could be manufactured nearer the place ofroom temperature and was tested by inoculating use were numerous. Apart from the shortage ofcalves with the undiluted material. Positive re- skilled technicians, it was considered that animalsactions were obtained after storage for 5 months. vaccinated under tropical conditions gave poor

Carini (16), 1906, described a similar method yields of lymph, and that the dangers of second-of drying lymph with the refinement of storage of ary contaminants were great (90). Suitable


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1954] THE PRESERVATION OF VACCINIA VIRUS 77

vaccinifers were often difficult to obtain because After 14-18 days all lymph stored in the liquidof decimation by trypanosomiasis (75). state was completely or almost completely in-Even if lymph could have been produced active, while the dried lymph was still potent

locally, the problems of internal transport were after being held at this temperature for twoconsiderable. Ziemann (92) refers to this difficulty months. Even so, irregular results were oftenin the Cameroons, and Ross (79), 1913, relates obtained with the desiccated material, somehow lymph might have to endure "a month's batches proving more resistant to heat thansojourn in a hot postbag on a porter's head in the others.sun". The transport of lymph on the Ivory Leger (61) reported poor results with lymphCoast in water cooled cloth bags is described by dried at the Paris Vaccine Institute by theSorel and Arlo (86) in 1912. This method was Achalme-Phisalix technique. He observed onlytried in French Equatorial Africa as late as 1929, 12% successful vaccinations with this lymphbut fresh lymph sent in this way, or packed in after storage for two months but stated thatthermos flasks, rapidly deteriorated (11). Similar even this result was better than that obtaineddifficulties were encountered in the Phillipine with the Swiss lymph prepared at Berne andIslands (80). tested under similar conditions. By contrast,

It is not surprising, therefore, that there were Ross (78) was very favorably impressed with thenumerous trials of dried lymph under tropical stability of lymph dried by the Achalme-Phisalixconditions. Camus (15), 1909, was among the proes. At Nairobi he recorded 18/23 successfulfirst workers to send bulk-dried vaccine over- vaccinations with desiccated lymph which hadseas, and he also used the method of drying been stored at the prevailing temperature for 14lymph in thin layers over sulfuric acid; he added months. He later reported that 14/20 vaccina-10% gum arabic to the lymph before drying, not tions were successful with similar material afterto improve its keeping properties, but to facili- 3 years' storage (79).tate reconstitution which was otherwise difficult. Dried lymph, therefore, yielded variable re-Camus adopted elaborate precautions to protect sults in different hands. It was generally noticedthe virus; it was dried in the dark, and the prod- that reconstitution of this material was difficult,uct was stored under vacuum in glass tubes, and that grinding with pestle and mortar wasprotected from "all rays" by thick tinfoil. This necessary to get a good suspension. An explana-material was sent by post to the Sudan during tion of the irregularity of results was advancedthe hot season and gave a vaccination success by Voigt (88) who suggested that differentrate of 86-89%. This appears to be a creditable operators might well vary in the thoroughnessresult, but Camus considered that the lymph with which they ground the material. He recog-had lost too much potency as a result of drying, nized clearly that the necessity for grinding thethat the vaccinial eruptions were not as good lymph was a serious drawback and drew atten-as those produced by glycerinated lymph, and tion to the danger of respiratory infection as athat the dried material did not give a good re- result of this process. As protection for the oper-action on the calf. ator, he advocated the wearing of a mask.Joyeux (51) described the use of Camus' During the short time remaining before the

lymph in French Guinea; used on arrival, after First World War, further accounts, mostlya month's journey, 89.5% of 105 vaccinations favorable, of the use of dried lymph in the tropicsperformed were "successful", but few of the were rendered by Sorel and Arlo (86), Manteufelreactions were normal, poor "takes" being ob- (64), Kersten (56), and Ringenbach (75).served in most cases. A calf was also inoculated,but although only a sparse eruption occurred, The drying of lymph with the aid of refrigerartt.it was later shown to be immune to revaccina- After the war, during which little further work

tion. on this subject was reported, the problem wasCarini's method (16) was used in Berne by again tackled, but now with the aid of new

Tomarkin and Serebrenikoff (87) in 1910 for the techniques. D'Arsonval and Bordas (25),large scale production of dried vaccine. This 1919, contended that it was dangerous to exposecompared favorably on storage at 37 C with lymph to sulfuric acid vapor during the dryingglycerinated, lanolinated and vaselinated lymph process. They used a moisture condenser, cooledderiving from the same batch of crude pulp. by liquid air, or solid C0, and acetone and


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claimed that vaccine dried in this way gave good time gave poor results. Like Hager (43), Ottenresults under tropical conditions. attributed the resistance of the virus to dryingAt this time, Wurtz and Camus (90) described to its being enclosed in epidermal cells.

a new technique whereby the lymph was first Later, a paper was published in both Englishfrozen and then dried over H2104 or P205 while and German (71, 72) confirming his earlier work,thawing. By this means, they hoped to bring and laying stress on the optimum time forthe virus from "1'6tat d'activitV" to "l'Ttat de harvesting the crude pulp. This was, generallysommeil". Subsequent manipulations were done speaking, 3 days after inoculation. Lymph thusin a dry atmosphere, and the dried lymph was obtained lost more weight on drying than didstored in vacuo. Although stated to preserve its that removed on the second or fourth days, andactivity after several weeks' exposure to a tem- it tended to give better results when stored. Itperature of 37 C, the authors enjoined strict was thought that on the third day, the inflamma-precautions to be observed in its use. Thus it was tory reaction had reached its height, with moreto be kept as cool as possible in transit and water in the tissues, and a greater concentrationground in a chilled mortar with cold glycerin- of virus. The technique now adopted was towater when reconstituted. They also stipulated dry the lymph in thin layers over H2S04 for 24that the actual vaccination should be performed hours in vacuo. The dried material was thenin a place sheltered from the sun. removed from the desiccator and ground to a

OUen'8 methOd. By far the most detailed study fine powder, which could be transferred to glassof the desiccation of lymph from the liquid state tubes in measured amounts. The tubes were thenin vacuo was made by Otten. Although he used evacuated to a pressure of 0.25 mm Hg andthe early technique of drying over sulfuric sealed. Lymph dried in this way lost about 80%acid, his investigations are reviewed separately of its weight as moisture. It produced 86-92%from those of others who experimented with this successful vaccinations after storage at the pre-method, both because they were carried out at a vailing temperature for 1V-24 years, and itlater date, and in view of the remarkable results was further observed that the dried lymph waswhich he achieved. more effective after storage at 37 C for one year

His first paper on the subject was published than glycerinated lymph kept at room tempera-in both Dutch and German (69, 70) and dealt ture for 1% months.with preliminary experiments in which buffalo Samples of lymph which had been dried bylymph was either dried at room temperature Otten no less than 18 years previously wereover sulfuric acid or was frozen and dried at examined in 1950 (20). This material had been-15 C. As a result of storage experiments he stored throughout at an average temperatureconcluded that nothing was gained by freezing, of 22 C, and different ampules yielded from 30and henceforward most of the work was done to 85% successful vaccinations. The titer ofwith lymph dried by the first method. different batches varied from 1/250 to 1/8,000

It is perhaps unfortunate that Otten relied as tested on the guinea pig cornea. By this time,on the percentage success rate of vaccinations most of the lymph used in Indonesia was beingwith his dried lymph as an index of its potency, dried by Otten's method, and the Pasteur In-so that no clear quantitative estimations of stitute at Bandoeng could produce 10.8 milliondeterioration rates were given. His results are doses per annum (20).also rather difficult to assess because individual Although this method of drying lymph hassamples were stored for varying periods at differ- been highly successful in Indonesia, it may beent temperatures, which makes comparison diffi- stated here that attempts by the writer tocult. Nevertheless, it is obvious that his dried reproduce these results using sheep lymph metlymph retained its potency much better than with little success (19). In one experiment, Ot-glycerinated control suspensions. He concluded ten's strain of virus in the form of buffalo pulpthat the life of dried lymph held at 37 C could (kindly sent by Dr. R. Green from the Institutebe measured in months, while that kept at 41- for Medical Research, Kuala Lumpur) did not45 C remained efficacious for some weeks, survive better than a batch of sheep lymph driedprovided always that the glass containers re- under the same conditions. In addition there is amained vacuum-tight. Lymph which had been decided risk of airborne scatter of finely powderedexposed to the atmosphere for any length of vaccine when handling the end product, while


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reconstitution is difficult, due probably to crude pulp. Vaseline was mentioned by Harveydenaturation of the slowly dried protein. (46), 1905, who also referred to the danger of

The large scale use of dried lymph. By the late growth of secondary contaminants in this me-1920's the use of dried lymph was becoming dium. He considered that vaselinated lymph didgeneral in many tropical countries, notably in not keep better than glycerinated material. In aFrench Equatorial Africa (11, 12), French East later paper (47) he stated that the use of vaselineAfrica (59), Indochina (35) and in the Belgian was introduced by Colonel Bamber, I.M.S.,Congo (60). Boulnois (9) described the successful but gave no reference; the method, however, wasresults obtained in India with dried vaccine known in 1890 (4).although he did not state its source. He concluded King (57), 1920, was the chief advocate ofthat dried lymph afforded a high degree of pro- lanolinated lymph. He stipulated that thetection against variola, both in India (9) and in lanolin should be neutral in reaction, anhydrous,Africa (10). and free of chlorine and glycerine fats. He said

Untoward reactions to dried lymph. Since most that lymph prepared in this manner kept betterof the dried lymph from the Continental labora- in India than vaselinated material and con-tories had undergone no preliminary treatment demned dried lymph as being too variable in itsto remove contaminating bacteria, it is perhaps behavior on storage.surprising that there were few reports of untoward Carefully controlled large scale trials in Indiareactions following its use. It is possible that of glycerinated and lanolinated lymph werethe drying process itself helped to reduce second- reported by Cunningham (23) and Cunninghamary contaminants. This was noted by several and Cruickshank (24). They came to the un-authors, including Voigt (88), who made use of equivocal conclusion that glycerinated lymph wasthis phenomenon in the preparation of vaseli- superior to the lanolinated variety, both in itsnated lymph, which was notoriously liable to keeping properties and the type of vesiclegross contamination. Voigt dried the crude pulp produced.and then added the powder to the vaseline,hoping in this way to minimie the number ofextraneous organisms. Addition of "protectie" substances to liquid

Boulnois (9) mentioned that in 14,000 vaccina- suspension of vaccinia virus. The beginning oftions performed in India with dried vaccine the next decade saw a new approach to thethere were no deaths. One case each of tetanus problem. Hitherto, lanolin and vaseline had beenand post-vaccinal encephalitis was recorded, and added to liquid lymph only because they werethere were 16 cases of abscess, one being severe. considered to be suitable inert vehicles. DuringBoulnois remarks that this record is much better the 1930's, however, a more positive attitude wasthan would be expected from a consideration of adopted in that various substances with definitethe standard of care after vaccination. physical or chemical activities were added to fluidGauducheau (38) thought that dried lymph suspensions, and, it was maintained, protected

which had been stored for long periods tended the virus against high temperatures.to give a generalized eruption similar to variola, Morosow (67) contended that glycerinatedbut I can find no other references to the occur- lymph became acid in reaction on storage, andrence of this type of reaction. that this harmed the virus. As a buffering agent,Table 1 summarizes the methods used for he added egg white in concentrations of approxi-

drying lymph from the liquid state. mately 26% or 40% and claimed that glyceri-The use of vaseline and lanolin. Reference has nated lymph thus treated showed improved keep-

been made in the preceding paragraphs to the ing qualities. He suggested that the lysozymeuse of glycerinated, lanolinated and vaselinated present in egg white exerted a bactericidallymph. Glycerol was used to reduce the number influence on contaminating organisms.of contaminating bacteria; when mixed with the Some workers added sugars of various kinds toground pulp, it acts over a period, the process calf lymph. Silber and Wostrouchowa (85) as-being known as "ripening". It was generally serted that saccharose preserved virus held atagreed, however, that glycerol also damaged the 37 C. Akasawa (2) tested many sugars in thisvirus (1, 24, 57, 68, 88). Attempts were made, way, including glucose, mannose, galactose,therefore, to find other suitable vehicles for the fructose, maltose, lactose, salicin, arabinose


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TABLE 1Summary of methods used for drying vaccine lymph from the liquid state

DATE ORIGINATOR AND METHOD U1ED BY OPINION

? ? JennerDrying on threads

? UnknownDrying on ivory "points" Warlomont, 1883 Good

? UnknownDrying on glass slips

1881 Reissner Hager, 1883 GoodDrying in bulk over H2S0O4 at atmospheric Schulz, 1889 Good

pressure Bauer, 1890 Doubtful

1902 BlaxallDrying over H2S04 in vacuo

1906 CariniBlaxall's method, but dried lymph stored in Tomarkin and Serebreni- Goodvacuo koff, 1910

1909 Achalme and PhisalixCarini's method, but dried lymph stored in Leger, 1911 Poor

large pieces Ross, 1911, 1913 Good

1909 CamusCarini's method; gum arabic added Joyeux, 1909 Indifferent

1919 D'Arsonval and BordasDrying over refrigerated condenser

1919 Wurtz and CamusLymph frozen, and dried while thawing

1926; Otten Collier, W. A., 1950 Excellent1932 Essentially Carini's method Collier, L. H., 1952 Poor

and xylose. None was effective, but he reported lecithin, and egg yolk at 25-30 C was noted bygood results with a preparation termed "Ja- Hirano (48), and among other substances testedpanischer Kandizucker" in 30% concentration. Yaoi and Arakawa (91) found that 5% glueLong and Olitsky (63) tried the addition of prolonged the life of vaccinia virus, but that the

cysteine hydrochloride in 1/2,000 concentration addition of gum acacia or agar to lymph resultedto suspensions of testicle-passed vaccinia. They in rapid deterioration.hoped that this reducing agent would prevent The use of culture virus. At about this time,oxidation and inactivation of virus stored in the virus propagated by tissue culture or in em-liquid state and reported that the addition of bryonated eggs became available. Eagles andcysteine to virus suspended in broth, Ringer McClean (29), 1929, found that virus cultivatedsolution or distilled water prolonged its life at in chick embryo tissue deteriorated rapidly even37 C. Anaerobiosis was increased by storing the in the cold room. They were particularly inter-suspensions under layers of petrolatum. ested to discover a way of preserving such sus-The preservative influence exerted by milk, pensions at low temperatures, and to this end


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investigated the effects of adding cysteine influence of a number of so-called protectivehydrochloride, glycerol, agar, gelatin, borax substances on vaccinia purified by iso-electricbuffer solution, fresh minced tissue, Hartley's precipitation (6). Virus suspended in 0.1%broth and "hormone" broth (65). The influence gelatin preserved its activity unimpaired forof storage under anaerobic conditions and of 1% many months at 5-10 C. Good results also at-phenol as a bactericide was also investigated. tended the use of 1% peptone, but glucose,They concluded that anaerobic storage in the glycerol, serum and cysteine hydrochloridepresence of 0.25% agar at i1 C gave the best (1/1,000 or 1/2,000) were found to have littleresults for virus grown in chick embryo tissue or no preservative influence. No mention isand plasma. Phenol did not impair the infectivity made of storage under anaerobic conditionsof the virus unless the suspension was frozen. with cysteine, as recommended by Long andVirus grown in Maitland and Laing's kidney Olitsky (63), and no storage temperature abovemedium was also well preserved at 41 C under 10 C was used.anaerobic conditions. Eagles (27) described a number of long termGoodpasture and Buddingh (39) working with storage experiments using elementary bodies

egg cultured vaccinia found that although virus suspended in phosphate buffer (pH 7.3), 0.25%suspended in 3% mucin, 30% gum arabic, agar-saline, broth, and saline alone. Most sus-glycerol-saline or egg yolk deteriorated rapidly pensions in broth or in 0.25% agar survived wellat room temperature, it survived with un- at 4C for many months, sometimes for morediminished titer after 6 weeks' storage at 25C if than a year. Again, the influence of highersuspended in normal inactivated rabbit serum; temperatures was not investigated.living virus was still present after storage at37 C for 4 weeks. Further similar experiments THE FOURTH PHASEwere later recorded, in which both rabbit andox serum were found to be effective preservative Dhehing of vaccint a virus from the frozen state.agents (13). The history of thiismethod of preserving protein

Gallardo and Sanz (36) found that virus grown and other solutions is reviewed by Greaves (41)in chick embryo tissue culture or on the intact and Will not be fully considered here. The methodchorioallantoic membrane, and suspended in was first described by Shackell (84), 1909, andTyrode's solution or in physiological saline had his discovery not been neglected for manyrespectively, would maintain unimpaired potency years, it is probable that far more would befor 14 months at -10 C. By contrast, Gastinel known today about the preservation of biologicaland Fasquelle (37) reported that glycerinated substances. Briefly, the method consists in freez-suspensions of egg-cultured vaccinia deteriorated ing the solution, either in a cold bath or by usingseriously during storage for 6 months at -20C the rapid extraction of heat occurring duringalthough dried material survived well at this evaporation of moisture under high vacuum.temperature. At room temperature, however Drying then takes place under high vacuum fromboth glycerinated and dried suspensions showed the frozen state, the continuous evaporation ofmarked falls in titer after one month. Neither water vapor in the presence of a suitable con-the method of drying nor concentration of denser keeping the product frozen until theglycerol was stated. process is complete. By this means, untoward

Experiments with purified virus. After the effects due to concentration of potentiallypioneer studies of Eagles and am (28) deleterious agents such as salts are completelyand Craigie (22), 1932, on the purification of avoided (30, 84). The frothing which is liablevaccinia virus, Amies (3) reported that ele- to occur when protein solutions are exposed tomentary bodies suspended in nutrient broth re- a high vacuum can be eliminated by freezingtained some activity for several weeks at 37 C. the liquid before reducing the pressure (30, 84)Even so, the infectivity showed a steady decrease or by centrifuging the material during the evacu-at this temperature as judged by intracutaneous ation process (40). The method was shown totitration in rabbits. Control suspens~ions in yield a product of high solubility, and denatura-physiological saline deteriorated even more tion of proteins was minimal. From about 1935rapidly. onwards the technique was used increasingly for

Behrens and Ferguson (5) investigated the the preservation of a wide range of more or less


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labile biological agents such as complement, without quoting actual figures, that a series ofantisera, blood plasma, bacteria and viruses. cultures of bovine virus dried 15 years previously

Rivers and Ward (76), 1933, first used this had conserved "all their virulence". On the othermethod for preserving vaccinia; they dried tissue hand, a dried chorioallantoic culture lost allcultured virus from the frozen state. A prepara- activity after only 6 months, although kept attion with an original intracutaneous titer of 4 C.10-5 still produced a reaction at a dilution of Hahn (45) described the production of a10-3 after storage for 4 weeks at 37 C. Later, egg combined yellow fever-smallpox vaccine, con-albumin or 2.5% gum acacia was added before sisting of 5 parts of embryo mince infected withdrying, and material treated in this way was yellow fever virus and one part of a % suspensionsuccessfully used to immunize humans by the of sheep lymph. The mixture (which also con-intracutaneous route (77). Experiments in which tained 15% gum arabic) was shell frozen andculture virus was mixed with an equal volume of dried in 0.5 or 1.0 ml quantities, being then sealedhorse serum before freeze drying were described under dry nitrogen and stored at 0 C. The titersby Lloyd and Mahaffy (62). Their preparations of both viruses were found to be satisfactorywould retain undiminished titers for up to 14 after storage for 16 months at this temperature.days at 37 C. Unfortunately, the initial titers of the prepara-In 1937 Kaiser published the first of a series tions were not mentioned.

of papers on the freeze drying of vaccinia (52). All the work with virus dried from the frozenOriginally, approximately 1.5% glucose was state described above had been done by the pre-added to partially purified virus to protect the freezing technique using methods similar to thevirus against protein denaturation. The material so-called "lyophile" (33) or "cryochem" (34)was treated with 0.3% "zephirol" before drying processes. In 1951, however, a brief account ofto reduce bacterial contamination. This dried the application of Greaves' centrifugal freezevaccine retained a high titer after storage for 20 drying method (40) to the preservation of vac-months (53). Further investigations into meth- cinia virus was given by the author (18). Byods of preservation were later described (55). contrast with the findings of earlier workers (3, 5,Lymph was ground in a cooled ball mill and 27), peptone and broth could not be shown tocentrifuged to get rid of extraneous matter; protect purified virus in the liquid state; freezegelatin which had been partially broken down by drying, however, exerted a marked preservativeautoclaving was added in 10% concentration to influence on such preparations and on crudethe supernatant fluid containing the virus, which vaccine pulp. Better results were obtained whenwas then dried from the frozen state. "Zephirol" the dried suspensions were sealed under highwas used as before. Eagles (unpublished material) vacuum than when nitrogen packing was used.stated that dried vaccine prepared in this manner This communication was a preliminary ac-was used extensively by the German Army dur- count of experiments described in a doctoraling the 1939-1945 war. thesis (19) to be published elsewhere, in which

After reviewing different methods in current the development of a stable smallpox vaccine isuse for preserving viruses, Hoffstadt and Tripi fully recorded. Using purified virus freeze dried(49), 1946, described storage experiments with in 5% peptone, a vaccine has been producedthe freeze dried infective agents of Shope's which has yielded 100% successful primary vac-fibroma, vaccinia and infective myxomatosis of cinations after storage for at least 4 months atrabbits. Of the three, vaccinia survived best. 37 C, or for at least 12 months at 22 C. SuchA review of the preservation of microorganisms material also offers the advantage of easy re-

and of biological substances in general by freeze constitution.drying was published by Fasquelle and Barbier A summary of methods used for drying vac-(31), 1950. They described their own work on the cinia virus from the frozen state is given inpreservation of vaccinia and stated that virus table 2.propagated in different animals varied in its It is hoped that this review of attempts toresistance to drying and storage. They thought solve the important problem of preservingthat bovine virus was more readily preserved vaccinia virus has conveyed some idea of thethan that derived from the rabbit, whereas egg complexity of the task and of the variable resultscultured virus was the most labile. It was stated, obtained with different techniques. It may be


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TABLE 2 5. BEHRENS, C. A., AND FERGUSON, W. W.Summary of reports on the drying of vaccinia virus 1935 Preservation of purified suspensions

from the frozen state of the virus of vaccinia. J. Infectious Dis-eases, 56, 84-8.

DATE MATEIAL AUTHOR 6. BEHRENS, C. A., AND NIELSEN, F. A. 1935Purification of suspensions of the virus of

1933 Tissue culture virus Rivers and Ward vaccinia (iso-electric point method). J.Infectious Diseases, 56, 41-48.

1935 Egg culture virus + Rivers and Ward 7. BLAxALL, F. R. 1902 The effect of desicca-egg albumin or tion on calf lymph. In Annual report ofgum acacia the medical offer of the local government

board. (Appendix C) 1900-1901. London,1935 Tissue culture virus Lloyd and England.

+ horse serum Mahaffy 8. BOLLNGER, 0. 1879 Ueber animale Vaccina-tion. F. C. W. Vogel, Leipzig, Germany.

1937 Partially purified vi- Kaiser 9. BouLNois 1936 Epiddmiologie de la variolerus + glucose dans l'Inde et controle de 1'efficacit4 du

vaccin & sec. Rev. M6d. Hyg. trop., 28,1942 Partially purified vi- Kaiser 185-195.

rus + gelatin 10. BouLNOIs 1937 Dur6e de l'immunit4 vac-cinale laissee par le vaccin sec et ses conse-

1951 Lymphandchickem- Hahn quences pratiques pour la prophylaxie debryo yellow fever la variole au Tchad (A.E.F.). Ann. M6d.vaccine + gum Pharm. colon., 85, 1081-1093.arabic 11. Boyh 1929 Sur l'emploi du vaccin sec pour

la vaccination antivariolique dans l'Afrique1951 Lymph: purified vi- Collier, L. H. Pquatoriale Frangaise. Bull. Off. int.1952 rus + peptone, Hyg. publ., 21, 1528-1530.

broth, etc. 12. Boyt 1932 Variole et vaccine en Afrique9quatoriale Frangaise. Bull. Off. int.

wondered, with some justification, how much real Hyg. publ., 24, 1421-1426.progress has been made since Jenner was success- 13. BUDDINGO, G. J. 1938 The use of serum as

ful in sending to Vienna virus dried on threads. a protective diluent for vaccinia virus.

Itl is sathending to Vi virs dried uitetre- 1 Am. J. Hyg., 27, 530-7.It is rather chastening to reflect that quite 14. CALMETTE, A., AND GUOWN, C. 1901 Re-cently, along with a description of a modern cherches sur la vaccine expdrimentale.technique of freeze drying sapox vaccine, Ann. inst. Pasteur, 15, 161-168.Hornibrook and Gebhard (50) also advocated its 15. C wus, L. 1909 Quelques modifications Apreservation in the form of impregnated threads la preparation et A la conservation du vaccinwound on steel needles. sec. Compt. rend. soc. biol., 67, 626-629.

16. CARINI, A. 1906 Vergleichende Unter-REFE:RENCES suchungen fiber den Einfluss hoher Tem-

1. AcHmmE, P., AND PRIsAEX, MARIE 1909 peraturen auf die Virulenz trockener undContribution A l'6tude de la conservation glycerinierter Kuhpockenlymphe. Centr.du vaccin dans les pays chauds. Bull. Bakt. Parasitenk., Abt. I., Orig. 41, 32-40.soc. pathol. exotique, 2, 431-433. 17. CHEYNE, K. R. 1850 (Correspondence.)

2. AKASAWA, S. 1936 tVber Zucker-Glyzerin- Med. Times, London, 21, 227, 248 and 266.Kalblymphe (Z-G-Lymphe). Kitasato Arch. 18. COLIMER, L. H. 1951 The preservation ofExptl. Med., 13, 118-126. vaccinia virus by freeze-drying. In Freez-

3. ARIs, C. R. 1934 The influence of tem- ing and drying, pp. 133-137. Institute ofperature on the survival of pure suspensions Biology, London, England.of the elementary bodies of vaccinia. Brit. 19- COLLER, L. H. 1952 The preservation ofJ. Exptl. Pathol., 15, 180-185. vaccinia virus. M.D. Thesis, University

4. BAUER, M. 1890 Die Schutzpockenimpfung of London.und ihre Technik, mit besonderer Berlcksich- 20. COLLER, W. A. 1950 Preservability of drytigung der Impfschdden, ihrer Verhtitung vaccine lymph. Documenta Neerl. etund Behandlung. Ferdinand Enke, Stutt- Indones. Morbis Trop., 2, 41-43.gart, Germany. 21. COLLIER, W. A. 1952 Vaccins antivario-


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liques sees. Pr6paration du vaccin d'Otten other substances-the cryochem process.& l'Institut Pasteur de Bandoeng, Indon6sie. J. Tmmunol., 34, 469-490.Bull. World Health Organisation, 5, 127-147. 35. GAIDE AND BODET 1930 Trans. Congr. Far

22. CRAIGIE, J. 1932 The nature of the vaccinia East. Assoc. Trop. Med., 8th Congr. Citedflocculation reaction, and observations on by Collier, W. A. (21).the elementary bodies of vaccinia. Brit. J. 36. GALLARDO, E., AND SANZ, J. 1937 Subcu-Exptl. Pathol., 13, 259-268. taneous smallpox vaccination with bacteria

23. CUNNINGHAM, J. 1922 Report on the rela- free vaccine. Am. J. Hyg., 25, 354361.tive values of lanoline and untreated 37. GASTINEL, P., AND FASQUELLE, R. 1941glycerine lymph. Govt. of Madras, India, Remarques sur la conservation d'activitdG.O. No. 1421.P.H. 16th October, 1922. de l'allantoido-virus vaccinal. Compt.

24. CUNNINGHAM, J., AND CRUICKSHANK, J. A. rend. soc. biol., 135, 30-32.1924 An investigation into the relative 38. GAUDUCHEAU, A. 1917 Recherches sur lavalues of lanoline and glycerine as vehicles variole-vaccine. Bull. soc. pathol. exo-for vaccine lymph in the tropics. Indian tique, 10, 260-268.J. Med. Research, 11, 1173-1203. 39. GOODPASTURE, E. W., AND BUDDINGH, G. J.

25. D'ARSONVAL, AND BoRDAs, M. F. 1919 Tech- 1936 The protective action of rabbit serumnique de la conservation du vaccin. Bull. for vaccinia virus at high temperatures.acad. med., Paris, 82, 225-227. Science, 84, 66-67.

26. DOWNIE, A. W., AND DUMBELL, K. R. 1947 40. GREAVES, R. I. N. 1944 Centrifugal vacuumSurvival of variola virus in dried exudate freezing. Its application to the drying ofand crusts from smallpox patients. Lancet, biological materials from the frozen state.i, 550-553. Nature, 153, 485-487.

27. EAGLES, G. H. 1935 The preparation and 41. GREAVES, R. I. N. 1946 The preservationtesting of elementary body suspensions of proteins by drying, with special referencefrom vaccinia filtrates, and their possible to the production of dried human serum anduse in smallpox prevention. Brit. J. plasma for transfusion. Med. ResearchExptl. Pathol., 16, 181-188. Council, Spec. Rept. Ser. No. 258.

28. EAGLES, G. H., AND LEDINGHAM, J. C. G. 42. GUhmN, C. 1905 Controle de la valeur des1932 Vaccinia and the Paschen body: in- vaccine Jenneriens par la numeration desfection experiments with centrifugalised 6l6ments virulents. Ann. inst. Pasteur,virus filtrates. Lancet, i, 823-826. 19, 317-320.

29. EAGLES, G. H., AND MCCLEAN, D. 1929 43. HAGER, O. 1883 Ueber das Reissner'scheCultivation of vaccinia virus. Brit. J. Vaccine-Pulver. Berliner Klin. Wochschr.,Exptl. Pathol., 10, 35-45. 20, 744-746.

30. ELSER, W. J., THOMAS, RuTH A., AND STEF- 44. HAGER, 0. 1883 Ueber das Reissner'scheFEN, G. I. 1935 The desiccation of sera Vaccine-Pulver. Berliner Klin. Wochschr.,and other biological products (including 20, 760-762.micro-organisms) in the frozen state with 45. HAHN, R. G. 1951 A combined yellow-fever-the preservation of the original qualities of smallpox vaccine for cutaneous application.products so treated. J. Immunol., 28, Am. J. Hyg., 54, 50-70.433-473. 46. HARVEY, W. F. 1905 Vaccine lymphs in the

31. FASQUELLE, R., AND BARBIER, P. 1950 A Punjab. Indian Med. Gaz., 40, 85-90.propos de la dessication sous congelation. 47. HARVEY, W. F. 1920 Vaccine lymph pro-Sem. H6p. Paris, 26, 4762-4766. duction, preparation and preservation.

32. FASQUELLE, R., AND FASQUELLE, J. 1949 La Indian J. Med. Research, 8, 257-269.vacuna desecada y la lucha contra la viruela 48. HIRANO, N. 1937 The influence of severalen los pafses c~lidos. Bol. Ofic. sanit. substances on vaccine virus in some cul-pan-amer, 28, 1118-1125. ture media with special reference to en-

33. FLOSDORF, E. W., AND MUDD, S. 1935 Pro- hancement of vaccine virus. Kitasatocedure and apparatus for preservation in Arch. Exptl. Med., 14, 328-342."lyophile" form of serum and other bi- 49. HOFFSTADT, RACHEL E., AND TRIPi, HELEN B.ological substances. J. Immunol., 29, 389- 1946 A study of the survival of certain425. strains of viruses after lyophilization and

34. FLOSDORF, E. W., AND MUDD, S. 1938 An prolonged storage. J. Infectious Diseases,improved procedure and apparatus for 78,183-189.preservation of sera, micro-organisms and 50. HORNIBROOK, J. W., AND GEBHARD, W. H.


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1951 Dried smallpox vaccine. Public 66. MooRE, J. 1817 The history and practice ofHealth Repts. (U.S.), 66, 38-43. vaccination. J. Callow, London, England.

51. JOTEux, C. 1909 Vaccination antivario- 67. MoRosow, M. 1930 Zur Methodik der Zu-lique aux pays chauds avec de la lymphe bereitung eines thermoresistenten Pocken-dess6chee. Compt. rend. soc. biol., 67, impfstoffes. Zentr. Bakt. Parasitenk., Abt.624-626. I, Orig, 117, 247-251.

52. KAISER, M. 1937 Zur frage der Herstellung 68. NoGucm, H. 1918 Further studies on theeines bakterienfreien Trockenimpfstoffes properties of pure vaccine virus cultivatedgegen Pocken. Zentr. Bakt. Parasitenk., in vivo. J. Exptl. Med., 27, 425-442.Abt. I, Orig., 139,405-416. 69. OTTEN, L. 1926 Droge vaccine. Geneesk.

53. KASER, M. 1938 Weitere Erfahrungen Ober Tijdschr. Nederland-Indie, 66, 642-670.Trockenimpfstoffe fMr den Pockenschutg. 70. OTTEN, L. 1927 Trockenlymphe. Z. Hyg.Zentr. Bakt. Parasitenk., Abt. I, Orig., 142, Infektionskrankh., 107,677-696.359-366. 71. OTTEN, L. 1932 Dry lymph (2nd communi-

54. KAiSER, M. 1942 Die geschichtliche Ent- cation). Mededeel. Dienst Volksgezond-wicklung der Herstellung von Trockenimpf- heid Nederland-Indie, 21, 196-205.stoffe, insbesondere fr den Blatternschutz. 72. Om7N, L. 1933 Trockenlymphe. (II. Mit-Arch. ges. Virusforsch., 2, 279-35. teilung). Z. Hyg. Infektionskrankh., 114,

55. KAxISER, M. 1942 Bericht fber Versuche, 705-719.einen Trockenimpfstoff fur den Pocken- 73. REISSNER 1881 Ueber eine einfache Me-schutz herzustellen und uber den Einfluss thode zur Aufbewahrung thierischen Impf-von Kilte und Trockenheit auf das Vac- stoffs, als Grundlage einer allgemeinencinavirus. Arch. ges. Virusforsch., 2, Einffthrung der animalen Impfung. Deut.426-459. med. Wochschr., 7, 409-413.

56. KERSTEN, H. E. 1914 Versuche mit einer 74. REISSNER 1881 Weitere Erfahrungen fberneuen von Ponndorf-Weimar hergestellten getrockneten Kllberimpfstoff. Deut. med.Tropen-Trockenlymphe. Arch. Schiffs- u. Wochschr., 7, 647-648.Tropen-Hyg., 18, 564-570. 75. RINGENBACH, J. 1914 Emploi du vaccin sec

57. KING, W. G. 1920 Vaccination in the en Afrique Equatoriale frangaise (Moyentropics. Tropical Diseases Bureau, Lon- Congo). Bull. soc. pathol. exotique, 7,don, England. 17-18

58. KLEBS, A. C. 1913 The historic evolution 76. RIvERS, T. M., AND WARD, S. M. 1933of variolation. Bull. Johns Hopkins Hosp., Further observations on the cultivation of24, 69-83. vaccine virus for Jennerian prophylaxis in

59. LASNET 1929 Sur la question de la variole man. J. Exptl. Med., 58, 635-648.et des vaccinations en Afrique Occidentale 77. RIvERs, T. M., AND WARD, S. M. 1935Frangaise pendant l'ann~e 1926. Bull. Jennerian prophylaxis by means of intra-Off. int. Hyg. publ., 21, 260-263. dermal injections of culture vaccine virus.

60. LEAGUE OF NATIONS 1934 Monthly Epi- J. Exptl. Med. 62, 549-560.demiological Reports, 13, No. 7-8, p. 152. 78. Ross, P. H. 1911 Some experiments with

61. LEGER, M. 1911 Discussion of paper by vaccine prepared according to the method ofRoss, P. H. (78) (q.v.) Bull. soc. pathol. Achalme and Marie Phisalix. Bull. soc.exotique, 4, 285-286. pathol. exotique, 4, 283-285.

62. LLOYD, W., AND MAHAFFY, A. F. 1935 Cul- 79. Ross, P. H. 1913 A further experimenttivation of vaccinia virus by Rivers'method. with vaccine prepared according to theProc. Soc. Exptl. Biol. Med., 8S, 154-156. method of Achalme-Phisalix. Bull. soc.

63. LONG, P. H., AND OLITSKY, P. K. 1930 Ef- pathol. exotique, 6, 144-145.feet of cysteine on the survival of vaccine 80. SCHOBL, O. 1920 Note on the keepingfects.ofcysteine on the survival of vaccie properties of dried and pulverized vaccinevirus. Proc. Soc. Exptl. Biol. Med., 27, virus. Philippine J. Sci., 17, 55-57.

380-381. 81. SCHOEN, E. 1896 Die Blattern in Afrika64.cMANT ymhE1912 Einige VersucheOst kt. und die Schutzpockemmpfung daselbst.

Trockenlymphe in Deutsch-Ostafrika. Centr. Bakt. Parasitenk., Abt. I, 20, 641-Arch. Schiffs- u. Tropen-Hyg., 16, 370-371. 653.

65. MCCLEAN, D., AND EAGLES, G. H. 1931 82. SCHULZ, M. 1888 Impfung, Impfgeschaft undThe conservation of vaccinia virus grown Impftechnik. Berlin, Germany. Cited byin vitro. Brit. J. Exptl. Pathol., 12, 103-107. Collier, W. A. (21).


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83. SEATON, E. C. 1868 A handbook of vaccina- bewahrung der Lymphe in den Tropen.tion. Macmillan and Co., London, England. Schiffs- u. Tropen-Hyg., 14, 429-456.

84. SHACELL, L. F. 1909 An improved method 88. VOImT, L. 1911 Die Versorgung tropischerof desiccation, with some applications to Schutzgebiete mit Kuhpockenimpfstoff.biological problems. Am. J. Physiol., 24, Erfahrungen und Versuche. Arch. Schiffs-325-40. u. Tropen-Hyg., 15, 497-521.

85. SIBER, L. A., AND WOSTROUCHOWA, E. I. 89. WARLOMONT, E. 1883 Traitd de la vaccine1932 Ueber AD-Vakzine. (III. Mit- et de la vaccination humaine et animale.teilung). Thermostabile Pockenlymphe. J.-B. Bailliere et Fils. Paris, Brussels.Z. Immunititsforsch, 76, 59-69. 90. WuRTZ, R., AND CAus, L. 1919 Le vaccin

86. SOREL, F., AND ARLO 1912 Essais A la sec. Technique de sa preparation. Bull.C6te d'Ivoire du vaccin sec de l'Academie acad. med. (Paris), 82, 12-16.de M6d6cine de Paris. Rev. Med. Hyg. 91. YAOI, H., AND AAKAwA, S. 1938 On thetrop., 9, 126-129. protective substances for the preservation

87. TOMARKIN, E., AND SEREBRENIKOFF, NADINA of vaccine virus. Japan. J. Exptl. Med., 16,1910 tlber die bakterienfeindlichen und 465-477.konservierenden Eigenschaften einiger Auf- 92. ZIEMANN 1908 Schutzpockenimpfung in denbewahrungsmittel der animalen Lymphe mit Kolonien. Berliner Klin. Wochschr., 45,besonderer Bertcksichtigung der Auf- 123-126.


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india, vrus: recent;detailed a half century previously by moore. in the century after jenner's...

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