Interference Contrast and Phase Contrast Microscopy of Sporulation and Germination of

Transcription

1 JOURNAL OF BACTERIOLOGY, Nov. 1968, p Copyright 1968 American Society for Microbiology Vol. 96, No. 5 Printed in U.S.A. Interference Contrast and Phase Contrast Microscopy of Sporulation and Germination of Bacillus megaterium ANTHONY D. HITCHINS, ARNOLD J. KAHN, AND RALPH A. SLEPECKY Biological Research Laboratories, Syracuse University, Syracuse, New York Received for publication 30 August 1968 The techniques of Nomarski interference contrast microscopy and phase-contrast microscopy were compared for their utility in monitoring sporulation and germination in Bacillus megaterium. The Nomarski technique permitted rapid and easy delineation of septation and engulfment during sporulation, whereas with phase contrast microscopy these stages were not detected at all. The later stages of sporulation were easily seen by either technique. Thus, of the seven stages of sporulation as recognized by the electron microscopy of thin sections, five can now be routinely detected quantitatively by optical microscopy: septation (stage II), engulfment (stage III), phase-dark forespore (corresponding to cortex formation, stage IV), phase-bright spore in a sporangium (corresponding to coat formation, stage V), and the free spore (stage VII). This means that now only stage I (axial filament) and stage VI (maturation of the refractile spore) require electron microscopy for routine detection. There was no advantage in using Nomarski optics for germination studies. Bacterial sporulation has been divided into seven distinct stages based on the examination of stained preparations in the bright field microscope, of living cells with the phase-contrast microscope, and of shadowed whole cells or thin sections of cells with the electron microscope. These stages are: I, preseptation or axial chromatin; II, septation; III, protoplast envelopment; IV, cortex formation; V, coat formation; VI, maturation; and VII, the free spore (see review by Murrell, reference 9). All seven stages can be readily distinguished in thin sections with the electron microscope; however, this technique does not allow for easy quantitative monitoring of the course of sporulation or the determination of the degree of synchrony of the sporulation process. The usual practice for the assessment of the degree of development is to follow the process by phase-contrast microscopy, but stages I to III cannot be seen by this method. By phasecontrast microscopy, stage IV appears as a phase-dark cell containing a still darker body, usually referred to as the forespore or prespore, whereas stage V appears as a phase-dark cell containing the partly or fully refractile spore. Stage VI, maturation, cannot be distinguished from stage V with phase contrast microscopy. Stage VII, the free spore, is of course easily detected with phase contrast microscopy. Thus, phase microscopy can only be used to assess relatively late periods of sporogenesis. In this investigation, we examined Bacillus megaterium cells during sporulation and germination by Nomarski interference contrast optics (1, 7, 10). The same cells were then examined with dark phase contrast. Unlike phase contrast, the Nomarski technique permitted rapid and easy delineation of stages II and III cells. MATERIALS AND METHODS Organism. The organism used was B. megaterium ATCC Culture techniiques. For studies on the growth and sporulation of B. megaterium, a portion of a standard spore suspension (6) was heated at 70 C for 0.5 hr, inoculated into a defined sucrose salts medium (SS) (14) containing the germinants L-alanine and inosine (each 100 pg/ml; NBC), incubated at 30 C with shaking to a turbidity of 70 Klett units (about 2 X 108 cells/ml), and measured in a Klett-Summerson photoelectric colorimeter with a no. 54 filter. The cells were then harvested by centrifugation in a Sorvall Superspeed refrigerated centrifuge at 2,000 X g for 15 min at 6 C. This cell suspension was placed into 50 ml of SS medium in a Klett sidearm flask and was incubated at 30 C on a New Brunswick rotary shaker (160 rev/ min). For the germination studies, stock spores were heated at 70 C for 0.5 hr, inoculated into Nutrient 1811

2 1812 HITCHINS, KAHN, AND SLEPECKY J. BAcTERioL. Broth (Difco), and incubated as described for the sporulation studies. We adopted the convention (9, 16) of arbitrarily designating the commencement of sporulation as the point at which exponential growth ends (To), corresponding to the axial chromatin or preseptation stage (stage I) as determined by exaniination of thin sections in the electron microscope. Stages I through VI are usually completed in 6 to 8 hr (T6 to T8) and represent several generation times in terms of exponential growth division. Microscopy and photography. Samples (2 ml) of cultures were removed from the Klett flasks at intervals of 0.5 or 1 hr and were stored in a freezer until examined. Cells could be held at 0 C for 1 week without damage. In preparing wet mounts for microscopic examination, the suspension was spread as thin as possible on a glass slide in order to insure optimal contrast and the glass coverslip was paraffin-sealed to minimize movement of the cells. Observations were made with a Zeiss photomicroscope equipped with phase contrast and Nomarski interference contrast optics. All photographs were taken through oil immersion objectives (phase contrast, neofluor, NA, 1.3; interference contrast, Planachromat, NA, 1.25) on Plus X 35-mm film. Contrast and resolution were enhanced by placing immersion oil between the condenser and the slide and by using a green filter. Final microscope magnification was X 1,250. Enumeration of cell types. For quantitative measurement of cell types in the population at a given time during sporulation, 200 cells were counted per sample by use of either Nomarski or phase contrast optics. RESULTS General comparison of phase contrast with interference contrast. Figures 1 to 4 show the appearance of B. megaterium during its vegetative cell-spore-vegetative cell developmental cycle, as viewed with phase contrast and Nomarski interference contrast optics. In general, the cells seen in the Nomarski system (i) lacked the light halo characteristic of the phase-contrast system; (ii) appeared larger; (iii) presented an optically flat appearance; (iv) showed a "shadow effect" reminiscent of shadowed preparations seen in the electron microscope; and, most importantly, (v) showed more detail, particularly in the early stages of sporulation. With regard to the last point, the division septa and the sporulation septa were especially well defined. In addition to some granular objects visible in phase contrast, other objects, which are not seen under phase optics were visible. Sporulation viewed under phase contrast. In general, the phase-contrast pictures of the course of sporulation in B. megaterium (Fig. 1, Al, BI, Cl, and Dl; Fig. 2, Al, Bi, Cl, and Dl) presented essentially the same details as described for this method with other sporulating bacteria (4, 18). In the early stages (Fig. 1), except for the appearance of some phase-bright granules, particularly at T-1 and To (Fig. lal, B1), the cells had the typical featureless phase-dark appearance of vegetative cells. The granules, which were relatively scarce, were probably poly-j3-hydroxybutyric acid (PHB) granules; in a previous study with the same organism and cultural conditions, the granules were shown to be PHB and were also rare at this stage (15). In the later stages of sporulation (Fig. 2), the phase-contrast pictures revealed the features which have been routinely observed and have been extremely useful in assessing the later stages. Phase-dark bodies at the poles were seen starting at T4.5 (Fig. 2, Al). These bodies exhibited a slight refractility at T6 (Fig. 2, Bl), and comparable studies have defined them as forespores (6). At T0.5 (Fig. 2, Cl), refractile spores were present in the partially lysed sporangia. Prolonged incubation to T21 resulted in complete lysis with the release of refractile mature spores (Fig. 2, Dl). Sporulation viewed under interference contrast and compared with phase contrast. Interference contrast provided much greater detail than phase contrast, particularly in the early stages of sporulation. Whereas the cytoplasm did not appear very granular under phase contrast in the early stages (Fig. 1, Al, B1, Cl), cells examined by interference contrast appeared much more granulated (Fig. 1, A2, B2, C2); this additional granulation did not appear to correspond with the phase-bright granules. The division septa were more clearly seen by interference contrast, and, unlike with phase contrast, it appeared that different stages of division septum formation could be discerned. For example, the early stages of division septation can clearly be seen by interference contrast (the lower cell of Fig. 1, A2), whereas only an indentation of the cell outline is visible by phase contrast (Fig. 1, Al). The most important and striking difference between the two methods of microscopy was revealed at about T3. Interference contrast microscopy clearly showed the presence of septa at the poles of the cells (Fig. 1, D2), whereas this could not be easily discerned in most cells by phase contrast (Fig. 1, D1). These septa were judged to be spore septa on the basis of the following criteria: the time of their appearance in the culture, subsequent events, the fact that they could also be detected by staining with crystal violet (3), and their presence, as revealed by examination of thin sections in the electron microscope, in samples taken at equivalent times with the same organism under similar cultural conditions (2). However, the prime reason for considering them as spore septa was the asymmetric positioning of these septa in the cell. By T4.5, further stages of development were

3 VOL. 96, 1968 SPORULATION AND GERMINATION BY NOMARSKI OPTICS i.l...i.. ^ ~B1 _ -~~~~~~~~~~~~~~~~... B2 C2 Downloaded from "......~~~ ~~~~~~~~~~~~~~.7si. r,ni L t'":l 4.~-.l s4 AL~ ~ ~ - on November 25, 2018 by guest FIG. 1. Comparisonz of phase-contrast with interference-contrast photomicrographs of B. megaterium cells at fouir times of development, T-1 through T3 hr. A, T-1 hr (vegetative cells); B, To hr (vegetative cells); C, Ti.5 hr (vegetative cells, probably stage I cells); D, T3 hr (stage II cells, septation). The cells at each time are the same group as seen by phase contrast (series 1) and interference contrast (series 2). Scale bar represents 5 pam.

4 *t HITCHINS, KAHN, AND SLEPECKY *: :..: *:.. Al A2..Ṫ *--x i;00 : *hs;w Msz. '"...,0d''.'.: ',:..:.: ';.:f.. 00: 1814 J. BACTERIOL. _ $' Ri :% %..,' : ::. *Gi..: '' '.:.' 7 : :: : ::.... : :!-. Alf. V. t : :.:......I... :.I".... BI C1 I.f. B2 i.~~~~~~~~~.: DlD It D2 si i..,?-x..:" 'iu FIG. 2. Comparison of phase-contrast with interference-contrast photomicrographs of B. megaterium cells at four times of development, T4.5 through T21. A, T4.5 hr (stage Ill cells, engulfment); B, T6 hr (probably stage IV cells, cortex formation); C, T9.s hr (refractile spores, probably stage V or VI); D, T2n hr (free spores, stage Vil). The cells at each time are the same group as seen by phase contrast (series 1) and interference contrast (series 2). Scale bar represents 5 pm.

5 VOL. 96, 1968 SPORULATION AND GERMINATION BY NOMARSKI OPTICS 1815 becoming visible in phase contrast (Fig. 2, Al); again, however, these stages were more clearly seen with Nomarski optics (Fig. 2, A2). The shape of the cells and the time of their appearance suggested that these cells were at stage III, the protoplast envelopment stage. By T6 and later, the stages of sporulation could be as readily distinguished in phase contrast (Fig. 2, Bi, Cl, DI) as in interference contrast (Fig. 2, B2, C2, D2). The development of refractility under phase contrast was paralleled by an increase in the characteristic strong "shadowing effect" of spores with Nomarski optics. Quantitation of early sporulation cell types (T2 to T5 hr). Characterization of early sporulation types with interference contrast microscopy allowed for quantitation of stages II and III. Cells were scored as stage II when they contained septa at one end of the cell, as illustrated in Fig. 1, D2. Cells were scored as stage III when there was a well-defined engulfment at one end of the cell, as illustrated in Fig. 2, A2. Sometimes the later steps of septation, when the septa are curving, are difficult to discern from the early steps of engulfment. Thus, a certain amount of subjectivity is involved in counting; nevertheless, reasonable quantitation was obtained (Fig. 3). Stage II cells began to appear at T2.5, and there was a period of about 0.5 hr before the appearance of stage III cells. Thus, engulfment appears to be a relatively rapid process. No sporulation types were visible with phase contrast until T5. Subsequent quantitation by phase contrast was as previously reported for batch cultures (6). Germination viewed under phase contrast and interference contrast. Germination of B. megaterium spores was examined with Nomarski and phase-contrast optics (Fig. 4). The stages during the course of germination as observed in phase contrast (Fig. 4, Al, Bi, Cl, Dl, El, Fl) were similar in appearance to those described by other investigators (5, 11, 12). These same stages could be readily detected with interference contrast (Fig. 4, A2, B2, C2, D2, E2, F2); however, interference contrast did not offer any particular advantage as a method for studying germination. DISCUSSION The results indicate that Nomarski interference contrast optics can be a useful and convenient way of detecting and quantitating the early stages (II, septation, and III, engulfment) of sporulation in B. megaterium. These stages cannot be detected by phase contrast, and even the first signs of sporulation can not be confi- n (I) w a- -J -J w o1 rt I I I T2 T3 T4 T5 T6 HOURS FIG. 3. Enumeration of septation2 (stage 11) aiid enguilfmenit (stage III) of B. megaterium by Nomarski optics. Symbols: A, cells without forespore septa; 0, cells with straight or curved septa (stage II); 0, cells with enzgulfed forespores (stage III). Units oni the abscissa refer to the time after the enid of exponential growvth (To). dently detected in phase contrast until about 2 hr after septation commences. After stage III, the use of interference contrast appears to offer no great advantage. In fact, although both methods show the same details, phase contrast shows the development of refractility between stages IV and V better. However, the use of the two methods together offers a suitable and effective tool for routine examination of sporulating cultures and for following stages II, III, IV, and V. In the phase-contrast microscope, stage IV appears as a nonrefractile forespore (for example, Fig. 2, Bl), whereas stage V appears as sporangia with fully refractile spores (for example, Fig. 2, Cl). However, for detecting the exact time interval between stage IV and V or for differentiating between stage V and VI, examination of thin sections in the electron microscope would be required. Since microscopes equipped with phase contrast can readily be adapted to add Nomarski optics, the dual examination of cells, as shown in this paper, would not present a serious problem. Just as the phase contrast-detectable stages (IV and V) can be seen by simple staining techniques (8), the Nomarski-detectable stages (II and III) can be seen by staining with very dilute

6 1816 HITCHINS, KAHN, AND SLEPECKY J. BACTERIIOL. SM 2 2 SM.P... S X Al C A2...B.,C D.B2 Downloaded from FiG,. 4. El E2 FlI F2. Comparison ofphase-contrast with interferenice-contrast photomicrographs of the stages of germiniation of B. megaterium spores. A and B, loss of refractility; C, swelling; D, elongation; E, emergence and outgrowth (note attached spore coat); F, first division. The cells at each time are the same group as seen by phase contrast (series 1) and initerferenice contrast (series 2). Scale bar represenits 5 pam. on November 25, 2018 by guest crystal violet (3). In our experiments, the adjustment of the cell to dye concentration ratio with the crystal violet technique was inconvenient and our yield of detectable forms was lower with this method than with Nomarski optics. The ease of quantitating the forespore septum formation by interference contrast optics may offer an opportunity to test more thoroughly the perennial hypothesis that sporulation results from a modification of normal cell division (8, 9, 13, 17; R. A. Slepecky, In G. M. Padilla, G. L. Whitson, and I. L. Cameron (ed.), The cell cycle: gene-enzyme interactions, Academic Press, Inc., New York, in press). Towards that end, we have been testing the sensitivity of the preseptation cells (T2) to various antibiotics which inhibit cell wall synthesis; we have found, by use of Nomarski optics, that forespore septa-

7 VOL. 96, 1968 SPORULATION AND GERMINATION BY NOMARSKI OPTICS 1817 tion is inhibited (Hitchins and Slepecky, unpublished data). To the best of our knowledge, this is the first investigation in which Nomarski interference contrast optics have been used to study bacteria. This technique may be applicable to the study of other differentiating bacterial systems. ACKNOWLEDGMENTS This investigation was supported by National Science Foundation grants GB 7275 (to Arnold Kahn) and GB 6433 (to Ralph Slepecky). LITERATURE CITED 1. Bajer, A., and R. D. Allen Structure and organization of the living mitotic spindle of Haematithus endosperm. Science 151: Ellar, D. J., D. G. Lundgren, and R. A. Slepecky Fine structure of Bacillus megateriurn during synchronous growth. J. Bacteriol. 94: Gordon, R. A., and W. G. Murrell Simple method of detecting spore septum formation and synchrony of sporulation. J. Bacteriol. 93: Hashimoto, T., S. H. Black, and P. Gerhardt Development of fine structure, thermostability, and dipicolinate during sporogenesis in a bacillus. Can. J. Microbiol. 6: Hitchins, A. D., G. W. Gould, and A. Hurst The swelling of bacterial spores during germination and outgrowth. J. Gen. Microbiol. 30: Imanaka, H., J. R. Gillis, and R. A. Slepecky Synchronous growth and sporulation of Bacillus megaterium. J. Bacteriol. 93: Maguire, M. P Nomarski interference contrast resolution of subchromatid structure. Proc. Natl. Acad. Sci. U.S. 60: Murrell, W. G Spore formation and germination as a microbial reaction to environment. Symp. Soc. Gen. Microbiol. 11: Murrell, W. G The biochemistry of the bacterial endospore. Advan. Microbiol. Physiol. 1: Nomarski, G Microinterferonmetre differentiel a ondes polarisees. J. Phys. Radium 16:9s-1 3s. 11. Powell, E The appearance of bacterial spores under phase-contrast illumination. J. Appl. Bacteriol. 3: Pulvertaft, R. J. V., and J. A. Haynes Adenosine and spore germination; phasecontrast studies. J. Gen. Microbiol. 5: Robinow, C. F Morphology of bacterial spores, their development and germination, p In I. C. Gunsalus and R. Y. Stanier (ed.), The Bacteria, vol. 1. Academic Press, Inc., New York. 14. Slepecky, R., and J. W. Foster Alterations in metal content of spores of Bacillus megarerium and the effect of some spore properties. J. Bacteriol. 78: Slepecky, R. A., and J. H. Law Synthesis and degradation of poly-,-hydroxybutyric acid in connection with sporulation of Bacillus n7egaterium. J. Bacteriol. 82: Szulmajster, J., and P. Schaeffer Augmentation de l'activite DPNH-oxydasique au cours de la sporulation de Bacillus subtilis. Compt. Rend. 252: Vinter, V Spores of microorganisms. Chloramphenicol-sensitive and penicillin-resistant incorporation of '4C-diaminopimelic acid into sporulating cells of Bacillus cereus. Experientia 19: Young, E. I., and P. C. Fitz-James Chemical and morphological studies of bacterial spore formation. I. The formation of spores in Bacillus cereus. J. Biophys. Biochem. Cytol. 6: