similar difficulties with agar cultures of L forms of Salmonella and Proteus species and obtained genital strain of human pleuropneumonia-like

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1 ELECTRON MICROGRAPHS MADE FROM L FORMS OF PROTEUS AND TWO HUMAN STRAINS OF PLEUROPNEUMONIA-LIKE ORGANISMS 1 2 L. DIENES Department of Bacteriology, M1assachusetts General Hospital and the Robert W. Lovett Memorial Foundation, Harvard Medical School, Boston, Massachusetts Received for publication Februarv 27, 1953 The organisms in the cultures of the pleuropneumonia group and of the L forms of bacteria are visible with the light microscope. Their morphology has remained controversial for a long time because they are easily deformed in microscopic preparations and give riise to bizarre forms. It seems well established at present (Klieneberger and Smiles, 1942; Dienes, 1945) that the cultures, with a few exceptions, consist of round forms which vary in size by continuous transition from barely visible ones to those several micra in diameter. The exceptions are the organisms of bovine pleuropneumonia and of agalactia in the cultures of which the small granules grow out into fine branching filaments which later develop swellings or break up into other granules. According to Freundt (1952), branching filaments also are present occasionally in the cultures of other pleuropneumonia-like strains. Reproduction occurs either by binary fission as in bacteria or by the production of small forms in varying numbers inside the larger ones (Dienes and Weinberger, 1951). The size of the smallest viable element in one bacterial L form, an old Li isolated from Streptobacillus moniliformis (Klieneberger-Nobel, 1949), has been determined by filtration through gradocol membranes. It was found to be at the borderline of visibility between and 0.25 I,u about the same as in the pleuropneumonia group. The electron micrographs of these organisms which have been published have added little new information on their morphology (Freundt, 1952; Smith et al. 1948a, b). The fragility of the organisms made it difficult to make preparations appropriate for electron micrography. We had 1 This is publication no. 145 of the Robert W. Lovett Memorial Foundation for the Study of Crippling Diseases, Harvard Medical School. 2 The expenses in connection with this investigation were defrayed by grants from the Commonwealth Fund and the United States Public Health Service. similar difficulties with agar cultures of L forms of Salmonella and Proteus species and obtained appropriate preparations only from the floating cultures of the Proteus L described in the preceding paper (Dienes, 1953). On the other hand, repiesentative micrographs were obtained without difficulty from the cultures of an oral and of a genital strain of human pleuropneumonia-like organisms (Dienes and Madoff, 1953). A selection of these micrographs is presented in this paper. To facilitate the interpretation of the electron micrographs, photographs obtained with light microscopy from stained preparations an(d with phase contrast from the same cultures also are presented.3 METHODS The simplest method of preparation gave the most useful screens. The Proteus L culture, lifted from broth, was smeared lightly on horse serum agar, and the screens were placed on the inoculated surface and lifted immediately. They were examined with the high dry lens of a light microscope, and those in which the distribution of organisms seemed appropriate were used. Impressions were made directly from serum agar cultures of pleuropneumonia-like organisms. Extraction of the agar with distilled water, either neutral or made slightly acid with phosphoric acid before the screens were made, neither improved nor harmed the preparations. The screens weere surprisingly free of aitifacts, and the organisms were sufficiently opaque to permit the making of pictures without shadowing. RESULTS Photographs made with phase contrast give the best general impression of the various types of forms present in the L cultures of Proteus. Photo- ' The electron micrographs were made by Mrs. Sokal in Dr. Jerome Gross' laboratory at the Massachusetts General Hospital. 280

2 19531 ELECTRON MICROGRAPHS MADE FROM L FORMS 281t graph 1 shows the edge of a culture mass transferred to a thin layer of transparent agar. The round, apparently empty, places are vacuolized large bodies. The largest ones do not result from the growth of a single organism but from the coalescence of several smaller ones. They contain liquid, and Brownian movement of granules is visible in them. Germination of such large bodies has never been observed, and they are probably dead structures. In addition to these forms there are dense granules in all transitions of size from barely visible ones to large bodies of 3 to 5 an or larger. The proportion of empty and full large bodies varies in different cultures. Stained preparations (photograph 3) show similar elements. The dense granules and full large bodies are darkly stained. The empty large bodies are visible only in wet preparations. In dry preparations, they appear as unstained spots. The electron micrographs were made from areas of the preparations where few organisms were present and from the edges of thicker culture masses. The largest empty bodies are absent from these places, probably because they lose their identity during drying. The largest forms in the photographs are about 2 u in diameter. Some of these are dark and correspond to the full, well stained bodies, visible with a light microscope. The faint shadows probably correspond to empty bodies. The smallest forms observed in the preparations made from the floating cultures were about 0.15 u. Continuous transition in size is present between the smallest and the largest. The smallest granules visible with light microscope are considerably larger (0.3 to 0.5,u) than those which can be seen in the electron micrographs. The stained preparations and phase contrast evidently do not show the smallest elements of the culture. The arrangement of the organisms in pairs or short chains, often consisting of granules of different size, is similar in the photographs made by light and by electron microscopy (photographs 4 and 5), suggesting that the smallest granules seen in the electron micrographs are of the same nature as the larger ones. In some preparations made from the floating cultures, there are many round granules 0.15 to 0.25 u in size (photograph 5). In others, somewhat larger granules 0.25 to 0.5 p are more numerous. The large forms do not always have uniform density. They present dark areas wnich in some cases are entirely similar to the small granules. Two such large bodies are marked with arrows in micrographs 6 and 7. Similar structures, large bodies containing granules, can be seen in stained preparations (Dienes and Weinberger, 1951) and with phase contrast (photograph 2). They indicate the production of granules inside the large bodies. The organisms were visible less clearly in screens made from 3A L type cultures grown on agar. In micrograph 8, made from a slightly shadowed preparation, the granules are somewhat larger than in the floating cultures. In some preparations, granules of considerably smaller size, less than 0.1 u, were present (micrographs 9 and 10). Their arrangement in a group or in a short chain with the larger ones suggests in this case also that all these granules are of similar nature. We were unable to obtain micrographs with sufficient contrast from these structures. They were visible clearly in the original micrograph of no. 9 and 10. The organisms in both oral and genital strains of pleuropneumonia-like organisms appeared as small granules in stained preparations and with phase contrast. The appearance of 48 to 72 hour old cultures in electron micrographs was similar. The whole culture seemed to consist of small granules. The size of the granules varies 0.2 to 1, in nmicrographs 12 and 13 made from the genital strain. No structure is visible in the granules, but there is a marked variation in density. Some appear dark; some are transparent and only their contours are visible. The average size of the granules varied in different cultures of the same strain and, for example, it was markedly smaller in micrograph 13 than in 12. The organisms in the preparations made from 72 hour cultures of oral strains were similar but somewhat smaller than in the genital strains. The average size is around 0.3 u. In the preparations made from 24 hour cultures, the organisms are even smaller, many being only 0.2 u. In contrast to older cultures, their shapes were not regular and their density was not uniform. The appearance of organisms in micrographs 16 and 17 suggests that transplanted organisms disintegrate to smaller ones less than 0.1 p in size. The dense areas in the larger granules probably correspond to the formation of smaller granules within them. Disintegration of the organisms into small forms can be observed in the large bodies with the light microscope. Whether the forms seen in the micrographs show a similar process and indicate that growing elements less than 0.1 p in diameter may be present in the cultures is only a conjecture and

3 282 L. DIENES [VOL. 66 needs further study. The disintegration of the organisms into several smaller ones is not their only method of reproduction. The young colonies extend on the surface and into the agar in irregular strands like bacterial colonies, and the organisms at the edge of these colonies probably reproduce in the same way as bacteria. DISCUSSION The morphology of two human strains of pleuropneumonia-like organisms and of the L cultures of a Proteus strain, as demonstrated by the electron microscope, appeared similar to that seen with the light microscope. The pleuropneumonia-like strains consisted of fairly uniform granules, the smallest being about the same size as is indicated by filtration (0.17 to 0.25,u). In 72 hour old cultures, many organisms appeared to be empty. In young cultures of the oral strains, the granules presented dense areas and seemed to be about to disintegrate into smaller forms. It was indicated earlier that the interpretation of these forms is uncertain. The gradual transition from small granules into large bodies several As in size, characteristic of many cultures of pleuropneumonia-like organisms, was not apparent in the electron micrographs of the examined strains. This transition was visible in the micrographs made from Proteus L cultures. The electron micrographs confirm the morphology of these cultures observed with the light microscope. It adds the new information that the size of the smallest granules, which seem analogous to the larger ones according to density and to their arrangement, is smaller than is apparent with visible light. It may be less than 0.1 p. The viability of granules of this size cannot be determined in our Proteus L cultures by filtration because growth can be started only by heavy inoculation. Filtrates even through coarse Mandel filters remain sterile. It is apparent that the small forms develop into large ones in growing colonies, but when the development of transplants is observed, new growth always starts from the large bodies. The viability of the granules of somewhat larger size (0.3 to 0.5 ul) is supported by the observation that similar granules grow out of the large bodies and form the young colonies. As mentioned above, the size of the smallest viable granules in the L1 was estimated by filtration to be between and 0.25,P. In the floating Proteus L cultures, most of the granules develop inside the large bodies and become free by the disintegration of the latter. It is possible that the smallest granules represent the intracellular phase of their development and they are not able to grow isolated on our media. It is of interest that the only characteristic structures visible in the electron micrographs were those seen also with the light miicroscope. No flagella, no tailed granules like the phage particles, and no filaments were seen in them. It should be mentioned especially that filaments have not been seen either by phase contrast or in stained preparations in the two pleuropneumonia-like strains in any stage of their development on agar or in broth. Oerskov's (1942) and Freundt's (1952) proposal that the pleuropneumonia group should be defined by the development of a branching undivided mycelium is certainly not applicable to these strains. The fine filaments which Tulasne (1950) described in Proteus L cultures were not observed either with phase contrast or with the electron microscope, nor were the distorted forms connected with short filaments, which some authors regard as characteristic of the pleuropneumonia group (Sabin, 1952). SUMMARY Electron micrographs from genital and oial strains of human pleuropneumonia-like organisms and from L cultures of Proteus strains are published in this paper. They show elements essentially similar to those visible with the light microscope. However, in the Proteus L cultures, there are granules the size of which is below the resolving power of the light microscope; they are apparently similar in nature to the larger ones. No other characteristic forms were observed in the micrographs. REFERENCES DIENEs, L Morphology and nature of the pleuropneumonia group of organisms. J. Bact., 50, DIENES, L Some new observations on L forms of bacteria. J. Bact., 66, DIENEs, L., AND MADOFF, S Differences between the oral and genital strains of pleuropneumonia-like organisms. Proc. Soc. Exptl. Biol. Med., 82, DIENES, L., AND WEINBERGER, H. J The L forms of bacteria. Bact. Revs., 15, FREUNDT, E. A Morphological studies of the pleuropneumonia organism (Micromyces peripneumoniae bovis). Acta Path. Microbiol. Scand., 31,

4 19531 ELECTRON MICROGRAPHS MADE FROM L FORMS 283 KLIENEBERGER-NOBEL, E On Streptobacillus moniliformis and the filterability of its L-form. J. Hyg., 47, KLIENEBERGER, E., AND SMILES, J Some observations on the developmental cycle of the organisms of bovine pleuropneumonia and related organisms. J. Hyg., 42, OERSKOV, J On the morphology of peripneumonia virus, agalactia virus and Seifiert's microbes. Acta Path. Microbiol. Scand., 19, SABIN, A. B The pleuropneumonia group, pp. of Man. Second Edition. Edited by R. J Bacterial and Mycotic Infections Dubos. J. B. Lippincott Company, Philadelphia. SMITH, W. E., HILLIER, J., AND MUDD, S. 1948a Electron micrograph studies of two strains of pleuropneumonia-like (L) organisms of human derivation. J. Bact., 56, SMITH, W. E., MUDD, S., AND HILLIER, J. 1948b L-type variation and bacterial reproduction by large bodies as seen in electron micrograph studies of Bacteroides funduliformis. J. Bact., 56, TULASNE, R Quelques donn6es nouvelles sur la formation et les caracteres de culture des formes L du Proteus vulgaris. Compt. rend. soc. biol., 144, WEISS, L. J Electron micrographs of pleuropneumonia-like organisms. J. Bact., 47, Downloaded from on October 18, 2017 by guest

5 PLATE I The photographs in plate I were made from a floating Proteus L culture, no. 1 to 3 with the light microscope and no. 4 to 7 with the electron microscope. The magnification in no. 1 and 3 is X 2,000; in no. 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 a few of these large bodies appear dark and are full. The small granules are dark, and all transitional forms between these and the large bodies are seen. Figure 2. Dark phase contrast. The large body marked with an arrow contains 5 granules arranged at the periphery. Figure 8. Preparation stained with crystal violet. The granules and full large bodies are darkly stained. The vacuolized empty large bodies are indicated only by the unstained round areas. The arrangement of the granules in pairs and short chains and the variability of their size are characteristic. Electron micrographs Figure 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 correspond in 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 size are 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, three dark granules are visible. Figure 7. The uneven density of the organisms is more apparent with higher magnification. Several of the small granules seem to be surrounded by a less opaque fringe. 284

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7 PLATE II Al At.. 4W Downloaded from "UP PLATE II Electron micrographs nos. 8, 9, and 10 were made from an agar culture of 3A L type colonies of Proteus. 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 small granules varying in size from less than 0.1 to about 0.15 p& are arranged in a half circle probably indicating their production inside of a larger form. In no. 10, many dense granules are less than 0.1 pu in size. 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 of which 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 organisms 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 even contour and density. Figures 15, 16, and 17. A 24 hour old culture just starting to develop. The size of many organisms is somewhat smaller than in no. 14, and they often have uneven contours and density. Some of the organisms in nos. 16 and 17 seem to contain several much smaller granules. on October 18, 2017 by guest