study of these organisms have been applied in the present work to Streptobacillus

Transcription

1 THE MORPHOLOGY OF THE L1 OF KLIENEBERGER AND ITS RELATIONSHIP TO STREPTO- BACILLUS MONILIFORMIS' L. DIENES Department of Pathology and Bacteriology of the Massachusetts General Hospital and the Robert W. Lovett Memorial of Harvard Medical School, Boston, Massachusetts Received for publication May 20, 1947 In 1945 the author published observations concerning the morphology of the pleuropneumonia group of organisms (Dienes, 1945). The methods used for the study of these organisms have been applied in the present work to Streptobacillus moniliformis and its L, variant. These organisms have been studied previously (Dienes, 1942), but improvements of technique in the meantime have made it possible to observe more clearly the form of the individual organisms and their derivation from each other. The properties of the L, and its connection with the bacillus challenge several accepted concepts in bacteriology. Most authors, taking into consideration that the L, originates from the bacillus, that it is serologically similar, and that under appropriate conditions it reproduces the bacillus, accept the conclusion that these two organisms, so different in appearance, are growth forms of the same organism. Klieneberger (1942) recently reaffirmed her objections to this conclusion; her chief objection is that the morphology of the L, is different from that of bacteria. Hence more accurate information should bring into agreement the different views on its nature. Such information is needed also as a basis to establish the biological significance of these peculiar bacterial forms. The difficulties which prevented for a long time an adequate concept of the morphology of the pleuropneumonia group of organisms are present in L, to an even greater extent. The organisms are exceedingly fragile and soft; they adhere firmly to each other; and the colonies grow into the agar. In broth, soft dense clumps are formed. The best way to overcome these difficulties, as in the case of the pleuropneumonia group, has been in the staining of the colonies on the agar. By studying several strains in various stages of development it was possible to observe the forms which comprise the colonies. The agar fixation method (Klienebegrer and Smiles, 1942), which gives excellent preparations with many strains of the pleuropneumonia group, is not applicable to small L, colonies, because they either do not adhere to the glass or, if they adhere, they are in dense clumps. Only the surface of wvell-developed colonies adheres to the glass, and some of the most successful photographs, both of the small and large forms of the organisms, were made from such preparations. This method alone is not, however, sufficient for the study of the cultures because it does not allow one to 1 The expenses of this investigation have been defrayed in part by a grant from the Commonwealth Fund. This is publication (-) 94 of the Robert W. Lovett Memorial. 231

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3 1947] MORPHOLOGY OF Li OF KLIENEBERGER 233 observe the whole course of their development. The following studies are based on three strains of Streptobacillus moniliformia and the L1 variants derived from them. The organisms in L1 colonies appear in two main forms with intermediate transitional forms. The young colonies and the central mass of well-developed colonies consist of small forms. These are transformed at the surface and the periphery of the colonies by gradual swelling into large round forms similar in every respect to the large forms present in colonies of the pleuropneumonia group of organisms and of various bacteria. The shape of the small organisms appeared most. clearly in preparations made from cultures on coagulated egg. The colonies grew on the surface of this medium without penetrating it. Impression preparations were made from the cultures following agar fixation. As mentioned above, only the surface of the colonies adheres to the glass, but in some preparations a few small forms were mixed with the large bodies usually present on the surface. The smallest organisms appeared to be distinctly bacillary. Photograph no. 2 of figure 1 made from such a preparation shows a tiny bipolar-tained bacillus. In photograph no. 1 of figure 1 three small organisms adhere to each other forming a small filament. Although they are darker stained than the organism in the following photograph, their shape is distinctly bacillary. A slightly swollen organism in the same photograph shows polar staining, which is more or less apparent in some of the larger forms. In wet stained agar preparations the appearance of the small organisms was similar. The youngest colonies consist of small bacillary forms sometime showing bipolar staining. Vigorously growing young colonies were obtained with the following procedure. The fresh medium was covered with an agar square cut from a 24-hour culture and incubated for 8 to 13 hours. The old agar square was discarded and the agar under it containing a fresh growth was studied. The organisms in photographs no. 5 and 11 of figure 1 showed the bacillary form clearly and, in some cases, bipolar staining as well. It is hoped that their shape FIGURE 1 No. 1. Impression preparation from a young L1 colony grown on coagulated egg. Fixation through the medium with Bouin's solution. Staimng with methylene blue and azur. X 3,000. Individual organisms are clearly visible, a short bacillary fiament consisting of 3 bacilli, a bipolar-stained short bacillus and consecutive stages in the development of large round forms. In one round form the polar staining remains visible. No. 2. A bipolar-stained bacillus from the same preparation as no. 1. X 3,000. Nos. 3 and 4. The surface of small L1 colonies photographed from wet stained agar preparation. X 3,000. In the lower part of no. 3, a few bacilary forms are discernible besides moderately swollen forms. In no. 4, the colony consists of small forms whose exact shape is not clearly visible. Nos. 5,6, and 11. Stained wet agar preparations. In no. 6 (X 3,000), small bacillary forms and one round body are visible. In no. 5 (X 3,000), many bacillary forms are visible, usually arranged in small clumps. One small bacillus marked with an arrow shows bipolar staining. No. 11 is the same as no. 5 enlarged to X 4,500. The shape of the organisms is only occasionally apparent, because they are parts of small clumps most of which, of necessity, are out of focus. No. 7. Well-developed colonies with moderate magnification. Stained dry agar preparation. X 500. The dense center consists of small forms embedded in the agar; the periphery consists of large round bodies situated on the surface of the agar. Nos. 8, 9, and 10. Small L1 colonies photographed from dried stained agar preparations. X 3,000. The shape of individual organisms is not clearly visible but it is apparent that their arrangement is similar to that of bacteria in bacterial colonies.

4 234 L. DIENE and structure willremain visible in the reproductions. Although the fact that the organisms are not in one plane and that they adhere in clumps makes it difficult to obtain sufficiently clear photographs. Photographs no. 8, 9, and 10 were made from preparations similar to the preceding ones with the difference that the thin agar slices were dried on the cover slips. Drying compreses the cultures vertically, and for this reason a large part of the colony is seen in shap focus. On the otherhand, the individual organism s are not so distinct as in wet preparations. The most important informations obtained from the study of dry agar preparations is that the arrangement of the organisms in the youngest colonies isimilar to the arrangement of bacteria in bacterial colonies, an observationindicating a similarity of growth and reproduction. The small bacillary forms in the photograph made from both wet and dry preparations are abouti of a micron long andi wide. The small forms in thel1 colonies are transformed by gradual swelling into large bodies. In photographs no. 1 and 6 of figure 1 the consecutive stages of this process are apparent. When the large bodies are fully developed, they sometimes appear to be filled with small bacillary formssimilar to those growing in young colonies. These small forms are tightly packed in several layers, and their actual forms can be seen only occasionally. They must be soft and fragile, because even slight mechanicalinjury to the large body destroys them. The structure of the large bodies was most clearly seen in preparations stained with safranine after fixation with Bouin's solution through the agar. Photographs 1 and 4 of figure 2 were made from such preparations. The development of the large bodies into L1 colonies in transplants has been previously described. They increase in size, their contours may become uneven, and the small L organisms grow out of them at one or at several points. The large body does not germinate as a single organism, like a yeast cell, for example, but it apparently contains numerous small organisms capable of growth. The author agrees in this point with Klieneberger (1942), but he has seen no indication of the process, postulated byklieneberger, by which previously separate organisms develop a common membrane and form a large body. The gradual swelling of the small forms into large bodies is apparent in the cultures. The development of L1 has been followed on thin agar slices under a cover slip and in stained agar preparations made from the cultures at short intervals. No other viable organisms were discernible in the cultures except the small bacillary forms, the large bodies, and the intermediary forms. The viable orgsnisms are stained intensely blue by methylene blue, and the cultures stop developing in transplants when the blue staining disappears from the colonies. Autolysis produced granules of various size which are stained pink both by methylene blue and Giemsa solution. These granules never show multiplication in transplants. Similar granules are produced in autolyzed cultures of Streptobacillus moniliformis and are regarded by Klieneberger as forms of L1 (1942). According to the experience of the author these granules do not multiply and there is [VOL. 54 no reason to believe that they represent the L1. When a Streptobacillus culture which is not grossly mixed with L1 colonies is transplanted, the L1 develops exclusively from large bodies produced by swelling of the bacilli.

5 19471 MORPHOLOGY OF L1 OF KLIENE13ERGER 235 Downloaded from FIGURE 2 No. 1. Safranine staining after agar fixation. X 3,000. Darkly stained small bacillary forms surrounded by halos are apparent in several large bodies. No. 2. Large bodies filled with small bacillary forms in a culture of Streptobacillus moniliformis. Safranine after agar fixation. X 3,000. No. 3. A large body in a culture of Streptobacillus moniliformis filled with round granules. Methylene blue and azur after agar fixation. X 3,000. No. 4 and 5. Enlargement of nos. 1 and 2, respectively, to X 4,500. No. 6. A large body in a Streptobacillus culture filled with bacilli of the usual shape. Giemsa stainidg after agar fixation X 3,000. No. 7. Large body in a Streptobacillus culture filled with bacilli of the usual shape. Giemsa staining after agar fixation. X 3,000. No. 8. Same as no. 7, enlarged to X 4,500. No. 9. A large body similar to those in nos. 6 and 7 stained with victoria blue. X 4,500. A..A.83 :.... on March 17, 2019 by guest

6 236 L. DIENES [VOL.. 54 The development of LI in the cultures of Streptobacillus moniliformis from the the bacteria has been previously described (Dienes, 1942). The bacilli swell first into large fusiform bodies and under appropriate conditions these develop into L1 colonies. These large bodies are in appearance and physical properties similar to the large bodies of L1. When they are fully developed, they are filled with similar soft bacillary forms and they develop into L1 colonies in a similar way. Transformation of the usual bacilli into L1 occurs during the development of the large body. Photographs no. 2 and 4 of figure 2 show large bodies developing in cultures of Streptobacillus moniliformis filled with small bacillary forms. In photograph no. 3 the small bodies developed into round forms, and it is clearly apparent that the large body contains many individual organisms. Although most of the large bodies develop as indicated above, in certain cultures some large bodies develop in a different way. These large bodies, a few hours after transplantation, appear to be filled with bacilli of the usual shape and develop into regular bacillary colonies. Their growth produces first a tiny dense round colony very different from the usual growth of Streptobacillus; after a few hours they lose this character and become similar to the other bacterial colonies. The development of the large bodies into bacteria was described in a former paper (Dienes, 1943). The illustrations in this paper were not successfully reproduced and are replaced here by better ones. Attention is again called to the fact that every strain of Streptobacillus and the L1 isolated from it present marked individual properties. It is often impossible to observe in a given strain phenomena easily seen in others. DISCUSSION The observations described give further support to the view that the morphology of the L1 is bacterial. The small organisms in L1 colonies are tiny, often bipolar-stained bacilli. Similar forms are visible inside the large bodies developing either in bacterial or L1 cultures. These small bacillary forms share with the parent organism the tendency to swell to round forms. The L1 is more pleomorphic and has a more pronounced tendency to autolysis than the parent organism, but it is essentially similar to the parent organism both in regard to form and to reproduction. The morphological differences between the L1 and the parent organism were exaggerated by the use of inappropriate methods of observation. The differences between them are actually not more pronounced than those between a smooth and a very rough pneumococcus colony. It was mentioned above that large bodies found in the cultures of Streptobacillus moniliformis can develop either into L1 or into usual bacillary forms. This together with the observation that the L1 for a certain period after isolation returns easily into the Streptobacilltus indicates that the L1 is apparently an intermediary link in the reproduction of the usual bacilli from the large bodies. All these observations are in agreement with the supposition that the usual bacillary forms and the L1 are growth forms of the same organism. The only characteristic in the development of L1 which was previously not noticed in bacterial variation is that the change into L1 is preceded by a morphological change

7 1947] MORPHOLOGY OF Li OF KLIENEBERGER 237 of the parent organism, by swelling into large round forms. These processes are not exceptional in Streptobacillus nmniliformis, but they are widely distributed in gram-negative bacilli (Dienes, 1942,1946). To all appearances these processes represent a complex reproductive process different from binary fission. The author has pointed out that the L1 shows many similarities to the so-called "haploform" yeast of Winge (Dienes, 1946). The morphology of the L1 is similar in all esential characteristics to the pleuropneumonia group of organisms. Without knowing the origin of a culture after it has lost its ability to return into the Streptobacillus, it would be impossible to recognize its identity on the basis of morphology. SUMMARY It is apparent in appropriate preparations that the small forms of the L1 colonies are small, often bipolar-stained bacilli. They enlarge by gradual swelling into large forms in which the small bacillary forms are again reproduced. The large bodies produced by swelling of bacteria in cultures of Streptobacillus momiliformis contain similar small bacillary forms, and when they germinate they produce an L1 colony. Sometimes the large bodies in the Streptobacilus culture are filled with bacilli of the usual shape and reproduce the usual bacillary colonies. The morphology of the L1, like that of the whole pleuropneumonia group, is bacillary, and the swelling into large round forms and reproduction by these large forms is similar in nature to the analogous processes observed in other bacteria. REFERENCES DIENES, L The significance of the large bodies and the development of L type of colonies in bacterial cultures. J. Bact., 44, DIENES, L Reproduction of bacteria in the large bodies of StreptobaciUw moniiformis. Proc. Soc. Exptl. Biol. Med., 58, Dimns, L Morphology and nature of the pleuropneumonia group of organisms. J. Bact., 50, DiBNzs, L Complex reproductive processes in bacteria. Cold Spring Harbor Symposia Quant. Biol., 11, KIJimBzEGza, E Some new observations bearing on the nature of the pleuropneumonia-like organisms known as L1 associated with StreptobaciUu moniliformis. J. Hyg., 42, KLIENEBzRGUB, E., AND SMILS, J Some new observations on the developmental cycle of the organisms of bovine pleuropneumonia and related microbe. J. Hyg., 42,