ELECTRON MICROGRAPHS MADE FROM L FORMS OF PROTEUS AND TWOHUMAN STRAINS OF PLEUROPNEUMONIA-LIKE ORGANISMS 1 2

L. DIENES

Department of Bacteriology, M1assachusetts General Hospital and the Robert W. Lovett MemorialFoundation, Harvard Medical School, Boston, Massachusetts

Received for publication Februarv 27, 1953

The organisms in the cultures of the pleuro-pneumonia group and of the L forms of bacteriaare visible with the light microscope. Theirmorphology has remained controversial for a longtime because they are easily deformed in micro-scopic preparations and give riise to bizarre forms.It seems well established at present (Klienebergerand Smiles, 1942; Dienes, 1945) that the cultures,with a few exceptions, consist of round formswhich vary in size by continuous transition frombarely visible ones to those several micra indiameter. The exceptions are the organisms ofbovine pleuropneumonia and of agalactia in thecultures of which the small granules grow outinto fine branching filaments which later developswellings or break up into other granules. Accord-ing to Freundt (1952), branching filaments alsoare present occasionally in the cultures of otherpleuropneumonia-like strains. Reproduction oc-

curs either by binary fission as in bacteria or bythe production of small forms in varying numbersinside the larger ones (Dienes and Weinberger,1951). The size of the smallest viable element inone bacterial L form, an old Li isolated fromStreptobacillus moniliformis (Klieneberger-Nobel,1949), has been determined by filtration throughgradocol membranes. It was found to be at theborderline of visibility between 0.175 and 0.25 I,uabout the same as in the pleuropneumonia group.The electron micrographs of these organisms

which have been published have added little newinformation on their morphology (Freundt, 1952;Smith et al. 1948a, b). The fragility of the or-ganisms made it difficult to make preparationsappropriate for electron micrography. We had

1 This is publication no. 145 of the Robert W.Lovett Memorial Foundation for the Study ofCrippling Diseases, Harvard Medical School.

2 The expenses in connection with this investi-gation were defrayed by grants from the Common-wealth Fund and the United States Public HealthService.

similar difficulties with agar cultures of L formsof Salmonella and Proteus species and obtainedappropriate preparations only from the floatingcultures of the Proteus L described in the preced-ing paper (Dienes, 1953). On the other hand,repiesentative micrographs were obtained with-out difficulty from the cultures of an oral and of agenital strain of human pleuropneumonia-likeorganisms (Dienes and Madoff, 1953). A selectionof these micrographs is presented in this paper.To facilitate the interpretation of the electronmicrographs, photographs obtained with lightmicroscopy from stained preparations an(d withphase contrast from the same cultures also arepresented.3

METHODS

The simplest method of preparation gave themost useful screens. The Proteus L culture, liftedfrom broth, was smeared lightly on horse serumagar, and the screens were placed on the inocu-lated surface and lifted immediately. They wereexamined with the high dry lens of a light micro-scope, and those in which the distribution oforganisms seemed appropriate were used. Im-pressions were made directly from serum agarcultures of pleuropneumonia-like organisms. Ex-traction of the agar with distilled water, eitherneutral or made slightly acid with phosphoricacid before the screens were made, neither im-proved nor harmed the preparations. The screensweere surprisingly free of aitifacts, and the or-ganisms were sufficiently opaque to permit themaking of pictures without shadowing.

RESULTS

Photographs made with phase contrast give thebest general impression of the various types offorms present in the L cultures of Proteus. Photo-

' The electron micrographs were made by Mrs.Sokal in Dr. Jerome Gross' laboratory at theMassachusetts General Hospital.

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graph 1 shows the edge of a culture mass trans-ferred to a thin layer of transparent agar. Theround, apparently empty, places are vacuolizedlarge bodies. The largest ones do not result fromthe growth of a single organism but from thecoalescence of several smaller ones. They containliquid, and Brownian movement of granules isvisible in them. Germination of such large bodieshas never been observed, and they are probablydead structures. In addition to these forms thereare dense granules in all transitions of size frombarely visible ones to large bodies of 3 to 5 an orlarger. The proportion of empty and full largebodies varies in different cultures. Stained prep-arations (photograph 3) show similar elements.The dense granules and full large bodies aredarkly stained. The empty large bodies are visibleonly in wet preparations. In dry preparations,they appear as unstained spots.

The electron micrographs were made fromareas of the preparations where few organismswere present and from the edges of thicker cul-ture masses. The largest empty bodies are absentfrom these places, probably because they losetheir identity during drying. The largest forms inthe photographs are about 2 u in diameter. Someof these are dark and correspond to the full, wellstained bodies, visible with a light microscope.The faint shadows probably correspond to emptybodies. The smallest forms observed in the prep-arations made from the floating cultures wereabout 0.15 u. Continuous transition in size ispresent between the smallest and the largest. Thesmallest granules visible with light microscopeare considerably larger (0.3 to 0.5 ,u) than thosewhich can be seen in the electron micrographs.The stained preparations and phase contrastevidently do not show the smallest elements ofthe culture. The arrangement of the organismsin pairs or short chains, often consisting of gran-ules of different size, is similar in the photographsmade by light and by electron microscopy (photo-graphs 4 and 5), suggesting that the smallestgranules seen in the electron micrographs are ofthe same nature as the larger ones. In somepreparations made from the floating cultures,there are many round granules 0.15 to 0.25 u insize (photograph 5). In others, somewhat largergranules 0.25 to 0.5 p are more numerous. Thelarge forms do not always have uniform density.They present dark areas wnich in some cases areentirely similar to the small granules. Two suchlarge bodies are marked with arrows in micro-

graphs 6 and 7. Similar structures, large bodiescontaining granules, can be seen in stained prep-arations (Dienes and Weinberger, 1951) and withphase contrast (photograph 2). They indicatethe production of granules inside the large bodies.

The organisms were visible less clearly inscreens made from 3A L type cultures grown onagar. In micrograph 8, made from a slightlyshadowed preparation, the granules are somewhatlarger than in the floating cultures. In some prep-arations, granules of considerably smaller size,less than 0.1 u, were present (micrographs 9 and10). Their arrangement in a group or in a shortchain with the larger ones suggests in this casealso that all these granules are of similar nature.We were unable to obtain micrographs with suffi-cient contrast from these structures. They werevisible clearly in the original micrograph of no.9 and 10.The organisms in both oral and genital strains

of pleuropneumonia-like organisms appeared assmall granules in stained preparations and withphase contrast. The appearance of 48 to 72 hourold cultures in electron micrographs was similar.The whole culture seemed to consist of smallgranules. The size of the granules varies 0.2 to 1, in nmicrographs 12 and 13 made from the genitalstrain. No structure is visible in the granules,but there is a marked variation in density. Someappear dark; some are transparent and only theircontours are visible. The average size of thegranules varied in different cultures of the samestrain and, for example, it was markedly smallerin micrograph 13 than in 12.The organisms in the preparations made from

72 hour cultures of oral strains were similar butsomewhat smaller than in the genital strains. Theaverage size is around 0.3 u. In the preparationsmade from 24 hour cultures, the organisms areeven smaller, many being only 0.2 u. In contrastto older cultures, their shapes were not regularand their density was not uniform. The appear-ance of organisms in micrographs 16 and 17 sug-gests that transplanted organisms disintegrate tosmaller ones less than 0.1 p in size. The denseareas in the larger granules probably correspondto the formation of smaller granules within them.Disintegration of the organisms into small formscan be observed in the large bodies with the lightmicroscope. Whether the forms seen in the micro-graphs show a similar process and indicate thatgrowing elements less than 0.1 p in diameter maybe present in the cultures is only a conjecture and

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L. DIENES

needs further study. The disintegration of theorganisms into several smaller ones is not theironly method of reproduction. The young coloniesextend on the surface and into the agar in irregu-lar strands like bacterial colonies, and the or-ganisms at the edge of these colonies probablyreproduce in the same way as bacteria.

DISCUSSION

The morphology of two human strains ofpleuropneumonia-like organisms and of the L cul-tures of a Proteus strain, as demonstrated by theelectron microscope, appeared similar to thatseen with the light microscope. The pleuropneu-monia-like strains consisted of fairly uniformgranules, the smallest being about the same sizeas is indicated by filtration (0.17 to 0.25 ,u).In 72 hour old cultures, many organisms appearedto be empty. In young cultures of the oral strains,the granules presented dense areas and seemed tobe about to disintegrate into smaller forms. It wasindicated earlier that the interpretation of theseforms is uncertain.The gradual transition from small granules into

large bodies several As in size, characteristic ofmany cultures of pleuropneumonia-like organ-isms, was not apparent in the electron micro-graphs of the examined strains. This transitionwas visible in the micrographs made from ProteusL cultures. The electron micrographs confirm themorphology of these cultures observed with thelight microscope. It adds the new informationthat the size of the smallest granules, which seemanalogous to the larger ones according to densityand to their arrangement, is smaller than is ap-parent with visible light. It may be less than 0.1p. The viability of granules of this size cannot bedetermined in our Proteus L cultures by filtrationbecause growth can be started only by heavyinoculation. Filtrates even through coarse Mandelfilters remain sterile. It is apparent that the smallforms develop into large ones in growing colonies,but when the development of transplants is ob-served, new growth always starts from the largebodies. The viability of the granules of somewhatlarger size (0.3 to 0.5 ul) is supported by the ob-servation that similar granules grow out of thelarge bodies and form the young colonies. Asmentioned above, the size of the smallest viablegranules in the L1 was estimated by filtration tobe between 0.175 and 0.25 ,P. In the floating Pro-teus L cultures, most of the granules develop in-side the large bodies and become free by the

disintegration of the latter. It is possible that thesmallest granules represent the intracellular phaseof their development and they are not able togrow isolated on our media.

It is of interest that the only characteristicstructures visible in the electron micrographs werethose seen also with the light miicroscope. Noflagella, no tailed granules like the phage particles,and no filaments were seen in them. It should bementioned especially that filaments have not beenseen either by phase contrast or in stained prep-arations in the two pleuropneumonia-like strainsin any stage of their development on agar or inbroth. Oerskov's (1942) and Freundt's (1952) pro-posal that the pleuropneumonia group should bedefined by the development of a branching un-divided mycelium is certainly not applicable tothese strains. The fine filaments which Tulasne(1950) described in Proteus L cultures were notobserved either with phase contrast or with theelectron microscope, nor were the distorted formsconnected with short filaments, which some au-thors regard as characteristic of the pleuropneu-monia group (Sabin, 1952).

SUMMARY

Electron micrographs from genital and oialstrains of human pleuropneumonia-like organ-isms and from L cultures of Proteus strains arepublished in this paper. They show elements es-sentially similar to those visible with the lightmicroscope. However, in the Proteus L cultures,there are granules the size of which is below theresolving power of the light microscope; they areapparently similar in nature to the larger ones.No other characteristic forms were observed inthe micrographs.

REFERENCESDIENEs, L. 1945 Morphology and nature of the

pleuropneumonia group of organisms. J.Bact., 50, 441-458.

DIENES, L. 1953 Some new observations on Lforms of bacteria. J. Bact., 66, 274-279.

DIENEs, L., AND MADOFF, S. 1953 Differencesbetween the oral and genital strains of pleuro-pneumonia-like organisms. Proc. Soc. Exptl.Biol. Med., 82, 36-38.

DIENES, L., AND WEINBERGER, H. J. 1951 TheL forms of bacteria. Bact. Revs., 15, 245-288.

FREUNDT, E. A. 1952 Morphological studies ofthe pleuropneumonia organism (Micromycesperipneumoniae bovis). Acta Path. Micro-biol. Scand., 31, 508-529.

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KLIENEBERGER-NOBEL, E. 1949 On Streptobacil-lus moniliformis and the filterability of itsL-form. J. Hyg., 47, 393-395.

KLIENEBERGER, E., AND SMILES, J. 1942 Someobservations on the developmental cycle ofthe organisms of bovine pleuropneumonia andrelated organisms. J. Hyg., 42, 110-123.

OERSKOV, J. 1942 On the morphology of peri-pneumonia virus, agalactia virus and Seifiert'smicrobes. Acta Path. Microbiol. Scand., 19,586-590.

SABIN, A. B. 1952 The pleuropneumonia group,

pp. 621-633. Bacterial and Mycotic Infectionsof Man. Second Edition. Edited by R. J.Dubos. J. B. Lippincott Company, Phila-delphia.

SMITH, W. E., HILLIER, J., AND MUDD, S. 1948aElectron micrograph studies of two strains ofpleuropneumonia-like (L) organisms of humanderivation. J. Bact., 56, 589-590.

SMITH, W. E., MUDD, S., AND HILLIER, J. 1948bL-type variation and bacterial reproductionby large bodies as seen in electron micrographstudies of Bacteroides funduliformis. J.Bact., 56, 603-618.

TULASNE, R. 1950 Quelques donn6es nouvellessur la formation et les caracteres de culturedes formes L du Proteus vulgaris. Compt.rend. soc. biol., 144, 1200-1203.

WEISS, L. J. 1944 Electron micrographs of pleu-ropneumonia-like organisms. J. Bact., 47,523-527.

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PLATE I

The photographs in plate I were made from a floating Proteus L culture, no. 1 to 3 with the lightmicroscope and no. 4 to 7 with the electron microscope. The magnification in no. 1 and 3 is X 2,000; inno. 2, X 3,000; in no. 4, 5, and 6, X 6,000; in no. 7, X 9,000.

Figure 1. Dark phase contrast. The mass of the culture appears as vacuolized large bodies. Only afew of these large bodies appear dark and are full. The small granules are dark, and all transitionalforms between these and the large bodies are seen.

Figure 2. Dark phase contrast. The large body marked with an arrow contains 5 granules arranged atthe periphery.

Figure 8. Preparation stained with crystal violet. The granules and full large bodies are darklystained. The vacuolized empty large bodies are indicated only by the unstained round areas. The ar-rangement of the granules in pairs and short chains and the variability of their size are characteristic.

Electron micrographsFigure 4. The individual granules and their arrangement in short chains often consisting of granules

of different size are clearly shown. The granules in the short chains marked with an arrow correspondin size to the smallest ones visible with the light microscope.

Figure 6. A thickly covered area was selected for this micrograph. Granules of 0.15 to 0.3 , in sizeare present in large numbers connected to the large bodies with transitional forms.

Figure 6. The uneven density of several organisms is apparent. In one marked with an arrow, threedark granules are visible.

Figure 7. The uneven density of the organisms is more apparent with higher magnification. Severalof the small granules seem to be surrounded by a less opaque fringe.

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PLATE IIElectron micrographs nos. 8, 9, and 10 were made from an agar culture of 3A L type colonies of Pro-

teus. The magnification is about X 14,000.Figure 8. A lightly shadowed preparation showing round granules of 0.25 to 0.5 pA in size.Figures 9 and 10. Nonshadowed preparations. At the upper center of no. 9, there are two organisms

about 1 p& in size partly vacuolized and surrounded with smaller granules. At the right lower corner smallgranules varying in size from less than 0.1 to about 0.15 p& are arranged in a half circle probably indi-cating their production inside of a larger form. In no. 10, many dense granules are less than 0.1 pU insize.Photograph no. 11 and micrographs 12 and 13 were made from a human pleuropneumonia-like organism

of genital origin.Figure 11. Dark phase contrast. It shows small granules at the borderline of visibility. XC 2,000.Figures 12 and 18. Electron micrographs. Impressions of two different agar cultures of the same strain,

magnification X 6,200 and X 5,900, respectively. The culture consists of round granules, the size ofwhich is noticeably smaller in no. 13 than in no. 12.

Micrographs 14 to 17 were made from agar cultures of an oral strain of pleuropneumonia-like organ-isms with higher magnification. X 14,000 to X 14,500.

Figure 14. A 72 hour old culture. The organisms are of uniform size, about 0.3 p, and have an evencontour and density.

Figures 15, 16, and 17. A 24 hour old culture just starting to develop. The size of many organisms issomewhat smaller than in no. 14, and they often have uneven contours and density. Some of the organ-isms in nos. 16 and 17 seem to contain several much smaller granules.

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