STUDIES OF THE FINE STRUCTURE OF MICROORGANISMS

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1 STUDIES OF THE FINE STRUCTURE OF MICROORGANISMS II. ELECTRON MICROSCOPIC STUDIES ON SPORULATION OF Clostridium sporogenes TADAYO HASHIMOTOI AND H. B. NAYLOR Laboratory of Bacteriology, College of Agriculture, Cornell University, Ithaca, New York Received for publication January 24, 1958 The sporulation process and spore structure in bacteria is a subject which has attracted the interest of many bacteriologists. Conclusions reached by investigators in this field have been so diverse that the need for clarification of the mechanism of sporulation, at least from the morphological standpoint, has been expressed by many of the workers (Knaysi, 1948, 1955; Delaporte, 1950; Bisset, 1950; DeLamater and Hunter, 1951; Robinow, 1950, 1951, 1953; Fitz-James, 1953; Chapman, 1956; Van den Hooff and Aninga, 1956; Smith and Ellner, 1957; Dondero and Holbert, 1957). The impressive application of the ultrathin sectioning technique to the study of this problem by Robinow (1953) and Chapman (1956) has opened up a new approach which may help to resolve some of the controversial points. Undoubtedly much of the disagreement over the sporulation process has been due to the limited resolving power of the light microscope which has led to subsequent "customary guessing" (Knaysi, 1952a) about the morphological changes that take place within the sporulating cell. Through application of the ultrathin sectioning technique, full advantage can be taken of the superior resolving power of the electron microscope. However, the preparation of specimens for ultrathin sectioning involves rather drastic treatment of the cells which increases the danger of producing artifacts. Indeed, the persistent occurrence of the "explosion phenomenon" in bacterial cells is an "exasperating difficulty" (Chapman, 1956) which is frequently encountered with this technique. Before the work to be presented here was undertaken, a systematic study was made of the various factors that might cause distortion of the cells and thus obscure the delicate fine structure. As a result, 1 From the Department of Bacteriology, Tokushima University School of Medicine, Tokushima, Japan, on Standard Vacuum Oil International Fellowship ( ). a technique was developed which yields reproducible results and a minimum of cellular explosion with various gram-positive bacteria (Hashimoto and Naylor, 1958). Employing the improved technique, the sporulation process of Clostridium sporogenes was investigated. The results obtained may serve to settle some of the disagreements concerning the sporulation process in bacteria, particularly the number of spore membranes produced and the time and order of their appearance in relationship to the development of resistance to simple staining. MATERIALS AND METHODS C. sporogenes obtained from the Cornell culture collection was used in the present investigation. In order to assure a high degree of synchronization of spore formation, young vigorously growing cells were prepared by making three serial transfers of the culture at 4 hr intervals in thioglycolate broth (Difco). The young cells were collected from 15 ml of the final transfer by centrifuging and were used to inoculate 40 ml of fresh thioglycolate broth which had been boiled and rapidly cooled to the incubation temperature of 37 C just prior to inoculating. Preliminary experiments showed that the cells in a culture prepared in this manner started to sporulate in about 16 hr and became stainable with the malachite green spore staining technique in approximately 24 hr. In 30 to 36, hr sporulation was almost complete. Photomicroscopic examination of the culture at various intervals indicated that satisfactory synchronization of spore formation was attained. At appropriate intervals, 12 ml aliquots of the culture were removed. Cells were prepared for ultrathin sectioning and electron microscopy by the improved technique previously described (Hashimoto and Naylor, 1958). Sections were cut with an experimental ultramicrotome equipped with a glass knife. This machine was designed 647

2 ... " ::,, C:BeE.u... *: : j *., :.. ^ t1 ;,... :,,.' l..: ::.-..'. 3.. ':.>^._ i< ':.: ; X :... SS.A. ': I. e:.... ma... ffi....- l :;: '..:.i ^ : e. e:.:: e 5_ -. w:, X *,...,..a.: X...s.a, :.,. 4.- s a,.:b.r.' :. P648 HASHIMOTO AND NAYLOR [VOL. 75.7:R:..,n..-, u.:. W.""' &.x.. :. i 'ie li I,,.X IA/4 I :;:.:, r.::. :.::: o os e :.^.: t. ar>6 x g *-r *._ g g X X S - * *0.:::..3S6- f t: u. 2' :2,: '.. -: Ds.M :..:...<. :::::..::...:.::: :.:. >..'... :: :.:..;ke ;46-:.. :... ::;.X.-.:5S:. FF...,iCstsES. > ;:...:..!....:..... ': 8. :.; S..'X... :: ;',S, w...:...c.::.. :... o:< : :.: ::.::;:: ::.:..:... ::: *: _.: :-:. -;:.. : :.... :::.:,.: w.. Figures 1, 2, and 2a. Electron micrographs of ultrathin sections of Clostridium sporogenes. Figure 1 shows vegetative cells sectioned in different planes. In figure 2, the first membrane to appear is shown in the lower right portion of the section. Note the wrinkled appearance. The upper left portion shows the first outer membrane completely formed and the partially formed second outer membrane. In figure 2a arrows O1 and 02 point to two separate membranes. and constructed by the Cornell Engineering RESULTS Physics Department (Knowlton, 1955). The sections were observed with an RCA EMU-2b elec- above, the cells of C. sporogenes multiplied rapidly Under the experimental conditions described tron microscope with an objective lens aperture for 16 hr before the sporulation process was of 50,u initiated. Figure 1 shows cells taken from an The light microscope was used for making 8 hr culture. At this stage, no structural differentiation was discernible with ordinary staining parallel observations of cells stained with gentian violet, safranin, or methylene blue, and of cells methods employing gentian violet or methylene stained by the malachite green spore-stain blue. The areas of low electron density in the technique. cytoplasm of the cells may correspond to the * ' ::.. :.:. :.:.... J. :.' *.,.' *',:n *.:' *:; **''.' :" ::... S. :. :'. :.: ;.23. _' ".. :... _.,: :. 11 *.;.:: _ E.,'., :. siea;::: :. : :....:.:.. s.:.: z R.t jf /[ > tj. : X RE > 5is >t!sb's' ' w o :: 4;in'! x ^;.:. - R... s. i leig fflr@.. A..... XE.

3 19581 FINE STRUCTURE OF MICROORGANISMS. II , I,.S"0 0.4%,o.. % 1.w all--.wwar ;a Downloaded from 7 l! Figures to 7. Electron micrographs of ultrathin sections of Clostridium sporogenes in the intermediate stages of sporulation. In figure 3 the two outer membranes are complete except at the ends where openings still remain. Figure 4 is a cross section of a cell in approximately the same stage of development as the cell in figure 3. The apparent flexibility of the two outer membranes is evident in figure 5. Figures 6 and 7 show the exosporium which begins to form after the two outer membranes have been completed. nuclear material described by Chapman and Hillier (1953). The first indication of sporulation was the appearance of a very thin membrane (0, right lower corner in figure 2).2 This mem- 2 The abbreviations used in figures are as follows: S. W., sporangial wall; S. C., sporangial cytoplasm; Cr., cross wall; N. E.?, nuclear element; E., exosporium; 0, original membrane; O1, first outer membrane; 02, second outer brane enclosed a portion of the area of low electron density and the process seemed to be one of cytoplasmic condensation in situ. Almost simultaneously with the completion of the first membrane, a second membrane began to form in close proximity and parallel to the first membrane (O1, 02). Figures 2 and 3 show this process. In membrane; I, inner membrane; and S, spot of unknown nature. on March 21, 2019 by guest

4 ,650 the upper left corner of figure 2, a portion of the enclosing membrane appears as a single layer while the second membrane has already formed along the sides and appears to be merging in the area nearest the center of the sporangium. In HASHIMOTO AND NAYLOR figure 3 the formation of the second membrane is nearly complete except at the ends. These openings at the ends do not seem to be due to artifacts since most of the cells at this stage showed the same thing and complete formation of the two membranes was evident in cells observed at a slightly later stage of development. Whether the second membrane in this double arrangement was formed by a splitting of the original membrane or was formed independently is not clear since the two membranes were so close together when they first appeared. Each membrane was approximately 50 to 70 A thick and the membranes were separated by a distance of about 100 A. The close association of the membranes is evident in most of the electron micrographs presented here. At this stage of development the spore membranes were regularly found to be much folded as indicated in figures 2a and 5. The initial enclosure was always slender and elongated as shown in figures 2 and 3. Figure 2a also indicates that there are two separate membranes involved rather than a single thick membrane in which the edges have a greater electron density than the internal portion as is commonly observed in [VOL. 75 various structures in the cells of higher animals. In a later stage of development, the two membranes come very close together to form the outer spore coats (figure 8). Figure 4 shows a cross section of a cell that was presumably in the same stage of development as the cell in figure 3. It isnoteworthy thatof the material of low electron density contained within the sporangial cytoplasm, only a small portion is enclosed by the spore membranes. Also, no difference in texture between the enclosed cytoplasm and the sporangial cytoplasm could be detected at this stage. Following the completion of the two enclosing membranes, a third membrane indicated as E in figures 6 and 7 begins to form. The distance between the new membrane and the original membranes is much greater at the ends than at the sides. This membrane is the exosporium which was never observed to appear before the first two membranes were completely formed. In parallel with the samplings for electron microscopy, stained preparations were observed with the light microscope. During the formation of the outer membranes and the exosporium, the forespores stain deeply with gentian violet or methylene blue. They never take malachite green but stain only with the counterstain, safranin. Thus it is clear that the formation of these membranes or coats has nothing to do with the resistance of the spore to basic dyes. From this stage on, the cells start to take on Figures 8 and 9. Electron micrographs of ultrathin sections of Clostridium sporogenes in the late stages of sporulation. The various membranes are discernible in figures 8 and 9. Figure 9 is a cross section through the sporahgium and the spore showing the lamellar nature of the exosporium. Note the smoothness of spore texture at this stage as compared with earlier stages. The spots of low electron density in the spore cores are particularly noticeable in figure 8. Magnification of figure 9 is the same as figure 8.

5 19581 FINE STRUCTURE OF MICROORGANISMS. II 651 the characteristic drumstick shape (figure 8). This transformation is not due to shrinkage of the sporangium, but rather to the swelling of the spore to such an extent that the outer membranes and the exosporium are forced outward until they assume a regular oval form. Up to this stage, these membranes appear irregular in outline as shown in figures 5, 6, and 7. This phenomenon is readily accounted for by the apparently flexible nature of the membranes. In figure 8 the membranes are shown to be tightly stretched and the space between the exosporium and the two outer membranes is considerably narrowed. It is at this stage that the spore acquires resistance to staining. Also at this stage a fourth membrane or inner spore coat appears (I in figure 8). It is formed around the spore core leaving a narrow space between it and the outer membranes. This space is uniform all around the core and is of lower electron density than any of the other structures with the exception of small areas (S in figure 8). A change in the texture of the spore cytoplasm also occurs at this stage. It becomes more compact and smoother than the cytoplasm of the sporangium. This is evident in figure 8. Figure 9 shows a cross section of a cell of approximately the same age as the cell in figure 8. Here it can be seen that the inner coat is a very thin membrane, but the outer membranes are fairly thick, and appear to be rigid. The close proximity of the outer membranes is indicated in figures 8 and 9 by arrows (O1 and 02). The exosporium is a thin membrane which appears to be somewhat lamellar in nature and comes off in discernible flakes at various sites which are especially evident in figure 9. During the course of this study, it was observed that although the spore was always formed at a terminal position within the sporangium, when two sporulating cells were attached in a chain, the position of the spores with respect to each other could be any of the three possible combinations of proximal-proximal, distal-distal, or proximal-distal. DISCUSSION Much of the confusion and lack of clarity concerning the sporulation process and the structure of spores of various bacteria is gradually becoming eliminated due to the introduction of new techniques and the improvement of older methods. The development of the ultrathin sectioning technique and its application to the study of the sporulation process (Chapman, 1956) and to the study of the morphology of spores (Robinow 1953; Van den Hooff and Aninga, 1956; Dondero and Holbert, 1957; Hannay, 1957) has revealed that certain differences exist in the mode of sporulation and the structure of mature spores among the various Bacillus species investigated. For example, spores of B. cereus have three membranes while those of Bacillus megaterium lack the so-called exosporium and have only two membranes (Chapman, 1956) whereas Robinow (1953) reported two coats in both. Chapman (1956) also reported that B. megaterium forms the inner spore coat first and the outer coat later while B. cereus forms three coats simultaneously. The time of first appearance of a spore coat apparently differs also. In the case of B. megaterium, Chapman stated that "while the endospore cytoplasm is becoming distinct (apparently both micromorphologically and chemically, as well as physiologically) the spore coats are formed about it." This implies that the first spore coat or coats were formed after a definite forespore or primordial region was observed in the sporangial cytoplasm. In the present study, it was found that this is not the case with C. sporogenes. The first observed indication of sporulation is the enclosure of a portion of cytoplasm, including some material of low electron density which may be nuclear (Chapman and Hillier, 1953), with a single flexible membrane and later with two membranes. The enclosed cytoplasm was of no greater electron density than the unenclosed cytoplasm. The fairly sharp contours of the enclosed body when stained with basic dyes suggests that the membranes as well as the spore primordium are

6 652 HASHIMOTO AND NAYLOR [VOL. 75 highly chromophilic at least in the early stage of formation. The exosporial wall remains appreciably chromophilic even after the spore proper becomes resistant to basic dyes. The cortex of low electron density observed in the spore of B. megaterium by Robinow (1953) was not found in the spore of another strain of B. megaterium studied by Chapman (1956). Thus, differences in spore structure may exist even between strains of a single species. In C. sporogenes a narrow but definite area of low electron density always occurs between the inner membrane and the outer membranes. To our knowledge, the most recent work on the sporulation process of anaerobic bacteria was that of Smith and Ellner (1957) with Clostridium perfringens. From observations with the light microscope, they concluded as follows: "The sporangium progressively contracts until, finally, the cytoplasmic membrane of the sporangium appears to envelop the forespore wall by a process of lamination. Thus, the wall of the mature spore is composed of at least two layers." No evidence was obtained for the shrinkage of the cytoplasmic membrane to form a spore wall in the present study. Instead, the exosporium is formed around the spore leaving considerable cytoplasmic substance between it and the sporangial wall. This takes place before any noticeable change in the texture of the spore occurs. The inner membrane which encases the spore core appears last. This nearly coincides with the change in spore texture and with the loss of affinity for basic dyes. At this time the spore begins to grow in size and, as it swells, the surrounding membranes are forced outward in all directions, especially to the sides, which causes the sporangium to take on the characteristic drumstick shape. Indeed it is not the sporangium that shrinks but rather it is the spore that expands to occupy most of the sporangial space. There is very little difference in total surface area between young cells and the cells that contain mature spores. Approximate surface areas were estimated from photomicrographs of cells with intact cell walls using the formula proposed by Knaysi (1952b). The question as to what structure or property of the spore is responsible for resistance to staining has not been convincingly answered. The present study indicates that at least two possibilities exist in C. sporogenes; either the spore core becomes chromophobic, or the formation of the inner membrane and the layer of material of low electron density establishes an effective barrier to dyes. The results of this study tend to favor the second possibility since the acquisition of resistance to dyes coincides with the formation of the inner membrane and the region of low electron density. Robinow (1951) has reported that B. cereus spores become readily stainable when the inner membrane is broken which also lends credence to this view. The significance of the area of low electron density between the inner membrane and the two outer membranes is not clear. The recent work of Mayall and Robinow (1957) has cast considerable light on the nature of a similar region in spores of B. megaterium. They showed that the electron density of the material could be increased by treating the spores with lanthanum nitrate and that this region occupies approximately 50 per cent of the space within the spore coats. In spores of C. sporogenes, the region of low electron density occupies a somewhat smaller proportion of the total space. Treatment of these spores with uranium acetate, silver nitrate, or mercuric chloride produced uniform electron density throughout with no differentiation of internal structure. Similar regions also appear to be present in spores of Bacillus polymyxa (Van den Hooff and Aninga, 1956) and Bacillus laterosporus (Hannay, 1957). The constant occurrence of this region and the symmetrical positioning of the spore core within it may be significant. There is a great temptation to speculate that the material present in this space may play a role in protecting the most vital portion of the spore, which is presumed to be located in the core, from unfavorable environmental conditions. The nature of the small but sharply defined spot (S) of low electron density in the spores of C. sporogenes is unknown. It may correspond to the peripheral bodies observed in spores of B. megaterium by Chapman (1956). The rather striking difference in the mode of sporulation in C. sporogenes compared with that in various species of Bacillus with regard to the order of formation of the membranes and the number of membranes which enclose the spore was surprising. Although minor variations could be due to differences in the degree of refinement of techniques used by the various investigators, it seems certain that the spores of different species of bacteria differ in structure and mode of development.

7 1958] FINE STRUCTURE OF MICROORGANISMS. II 653 ACKNOWLEDGMENT The authors express their appreciation to Dr. Benjamin M. Siegel of the Cornell Engineering Physics Department for allowing free access to the necessary instruments in his laboratory for carrying out this investigation. SUMMARY The sporulation process and the structure of the spores produced by synchronized cells of Clostridium sporogenes were studied using stained preparations for light microscope observations and ultrathin sections prepared by an improved technique for parallel observation with the electron microscope. In the mature spore, an exosporium, two outer membranes and one inner membrane were observed. The two outer membranes appeared first and enclosed a portion of cytoplasm containing an area of low electron density. The exosporium was formed next and the inner coat appeared last. With the formation of the inner coat, a change in spore texture occurred, and it was at this point that the spore lost affinity for basic dyes. In this organism, the cytoplasmic membrane of the sporangium did not shrink down to form a spore coat. Differences in the mode of spore formation between C. sporogenes and species in the genus Bacillus have been discussed. REFERENCES BISSET, K. A The sporulation of Clostridium tetani. J. Gen. Microbiol., 4, 1-2. CHAPMAN, G. B. AND HILLIER, J Electron microscopy of ultrathin sections of bacteria. I. Cellular division in Bacillus cereus. J. Bacteriol., 66, CHAPMAN, G. B Electron microscopy of ultrathin sections of bacteria. II. Sporulation of Bacillus megaterium and Bacillus cereus. J. Bacteriol., 71, DELAMATER, E. D. AND HUNTER, M. E The nuclear cytology of sporulation in Bacillus megaterium. J. Bacteriol., 63, DELAPORTE, B Observation on the cytology of bacteria. Advances in Genetics, 56, DONDERO, N. C. AND HOLBERT, PAULINE E The endospore of Bacillus polymyxa. J. Bacteriol., 74, FITZ-JAMES, P. C The structure of spores as revealed by mechanical disruption. J. Bacteriol., 66, HANNAY, C. L The parasporal body of Bacillus laterosporus laubach. J. Biophys. Biochem. Cytol., 3, HASHIMOTO, T. AND NAYLOR, H. B Studies on fine structure of microorganisms. I. A study of factors influencing the "explosion phenomenon" in ultrathin sections of bacteria. J. Bacteriol., 75, KNAYSI, G The endospore of bacteria. Bacteriol. Revs., 12, KNAYSI, G. 1952a Cytology of sporulation. In Symposium of the biology of bacterial spores. Bacteriol Revs., 16, KNAYSI, G. 1952b In Elements of bacterial cytology, 2nd ed. Comstock Publishing Co., Ithaca, N. Y. KNAYSI, G On the structure and nature of the endospore in strain C3 of Bacillus cereus. J. Bacteriol., 69, KNOWLTON, K. C The development of a microtome for the electron microscope laboratory. Thesis, Cornell University, Ithaca, N. Y. MAYALL, B. H. AND ROBINOW, C Observation with the electron microscope on the organization of the cortex of resting and germinating spores of B. megaterium. J. Appl. Bacteriol., 20, ROBINOW, C. F The structure of Bacillus spores. Bacteriol. Proc. 1950, 33. RoBINOW, C. F Observation on the structure of Bacillus spores. J. Gen. Microbiol., 5, RoBINOW, C. F Spore structures as revealed by thin sections. J. Bacteriol., 66, SMITH, A. G. AND ELLNER, P. D Cytological observations on the sporulation process of Clostridium perfringens. J. Bacteriol., 73, 1-7. VAN DEN HOOFF, A. AND ANINGA, S An electron microscope study on the shape of the spores of Bacillus polymyxa. Antonie van Leeuwenhoek J. Microbiol. Serol., 22,