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journal of bacteriology nov 1968 p 1811 1817 vol 96 no 5 copyright 1968 american society for microbiology printed in u s a interference contrast and phase contrast microscopy of ...

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                    JOURNAL OF BACTERIOLOGY, Nov. 1968, p. 1811-1817                                                             Vol. 96, No. 5
                     Copyright © 1968  American Society for Microbiology                                                      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 13210
                                                           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 germina-
                                tion 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 sporu-
                                lation 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 rou-
                                tinely detected quantitatively by optical microscopy: septation (stage II), engulf-
                                ment (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 germina-
                                tion studies.
                        Bacterial sporulation has been divided into                detected with phase contrast microscopy. Thus,
                     seven distinct stages based on the examination of             phase microscopy can only be used to assess
                     stained preparations in the bright field micro-               relatively late periods of sporogenesis.
                     scope, of living cells with the phase-contrast                   In this investigation, we examined Bacillus
                     microscope, and of shadowed whole cells or thin               megaterium cells during sporulation and germina-
                     sections of cells with the electron microscope.               tion by Nomarski interference contrast optics (1,
                     These stages are: I, preseptation or axial chroma-            7, 10). The same cells were then examined with
                     tin; II, septation; III, protoplast envelopment;              dark phase contrast. Unlike phase contrast, the
                     IV, cortex formation; V, coat formation; VI,                  Nomarski technique permitted rapid and easy
                     maturation; and VII, the free spore (see review               delineation of stages II and III cells.
                     by Murrell, reference 9). All seven stages can be                            MATERIALS AND METHODS
                     readily distinguished in thin sections with the                  Organism. The organism used was B. megaterium
                     electron microscope; however, this technique                  ATCC19213.
                     does not allow for easy quantitative monitoring                  Culture techniiques. For studies on the growth and
                     ofthe course of sporulation or the determination              sporulation of B. megaterium, a portion of a standard
                     of the degree of synchrony of the sporulation                 spore suspension (6) was heated at 70 C for 0.5 hr,
                     process. The usual practice for the assessment of             inoculated into a defined sucrose salts medium (SS)
                     the degree ofdevelopment is to follow the process             (14) containing the germinants L-alanine and inosine
                     by phase-contrast microscopy, but stages I to                 (each 100 pg/ml; NBC), incubated at 30 C with shak-
                     III cannot be seen by this method. By phase-                  ing to a turbidity of 70 Klett units (about 2 X 108
                     contrast microscopy, stage IV appears as a                    cells/ml), and measured in a Klett-Summerson photo-
                     phase-dark cell containing a still darker body,               electric colorimeter with a no. 54 filter. The cells were
                     usually referred to as the forespore or prespore,             then harvested by centrifugation in a Sorvall Super-
                     whereas stage V appears as a phase-dark cell                  speed refrigerated centrifuge at 2,000 X g for 15 min
                     containing the partly or fully refractile spore.              at 6 C. This cell suspension was placed into 50 ml of
                     Stage VI, maturation, cannot be distinguished                 SS medium in a Klett sidearmflask and was incubated
                     from stage V with phase contrast microscopy.                  at 30 C on a New Brunswick rotary shaker (160 rev/
                                    the free            is of course               min). For the germination studies, stock spores were
                     Stage VII,                spore,                    easily    heated at 70 C for 0.5 hr, inoculated into Nutrient
                                                                               1811
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                      1812                               HITCHINS, KAHN, AND SLEPECKY                                    J. BAcTERioL.
                      Broth (Difco), and incubated as described for the          ticularly at T-1 and To (Fig. lAl, B1), the cells
                      sporulation studies.                                      hadthe typical featureless phase-dark appearance
                        We adopted the convention (9, 16) of arbitrarily        of vegetative cells. The granules, which were
                      designating the commencement of sporulation as the        relatively scarce, were probably poly-j3-hydroxy-
                      point at which exponential growth ends (To), corre-       butyric acid (PHB) granules; in a previous study
                      sponding to the axial chromatin or preseptation stage     with the same organism and cultural conditions,
                      (stage I) as determined by exaniination ofthin sections   the granules were shown to be PHB and were
                      in the electron microscope. Stages I through VI are       also rare at this stage (15).
                      usually completed in 6 to 8 hr (T6 to T8) and represent      In the later
                      several generation times in terms of exponential                           stages of sporulation (Fig. 2), the
                      growth division.                                          phase-contrast pictures revealed the features
                        Microscopy and photography. Samples (2 ml) of           which have been routinely observed and have
                     cultures were removed from the Klett flasks at inter-      been extremely useful in assessing the later
                      vals of 0.5 or 1 hr and were stored in a freezer until    stages. Phase-dark bodies at the poles were seen
                     examined. Cells could be held at 0 C for 1 week with-      starting at T4.5 (Fig. 2, Al). These bodies ex-
                     out damage. In preparing wet mounts for microscopic        hibited a slight refractility at T6 (Fig. 2, Bl), and
                     examination, the suspension was spread as thin as          comparable studies have defined them as fore-
                      possible on a glass slide in order to insure optimal      spores (6). At T0.5 (Fig. 2, Cl), refractile spores
                     contrast and the glass coverslip was paraffin-sealed to
                     minimize movement of the cells. Observations were          were present in the partially lysed sporangia.
                     made with a Zeiss photomicroscope equipped with            Prolonged incubation to T21 resulted in complete
                     phase contrast and Nomarski interference contrast          lysis with the release of refractile mature spores
                     optics. All photographs were taken through oil im-         (Fig. 2, Dl).
                     mersion objectives (phase contrast, neofluor, NA, 1.3;        Sporulation viewed under interference contrast
                     interference contrast, Planachromat, NA, 1.25) on          and compared with phase contrast. Interference
                     Plus X 35-mm film. Contrast and resolution were            contrast provided much greater detail than
                     enhanced by placing immersion oil between the con-         phase contrast, particularly in the early stages of
                     denser and the slide and by using a green filter. Final    sporulation. Whereas the cytoplasm did not
                     microscope magnification was X 1,250.
                        Enumeration ofcell types. For quantitative meas-        appear very granular under phase contrast in the
                     urement ofcell types in the population at a given time     early stages (Fig. 1, Al, B1, Cl), cells examined
                     during sporulation, 200 cells were counted per sample      by interference contrast appeared much more
                     by use ofeither Nomarski or phase contrast optics.         granulated (Fig. 1, A2, B2, C2); this additional
                                             RESULTS                            granulation did not appear to correspond with
                                                                                the phase-bright granules. The division septa
                        General comparison of phase contrast with in-           were more clearly seen by interference contrast,
                     terference contrast. Figures 1 to 4 show the               and, unlike with phase contrast, it appeared that
                     appearance ofB. megaterium during its vegetative           different stages of division septum formation
                     cell-spore-vegetative cell developmental cycle, as         could be discerned. For example, the early stages
                     viewed with phase contrast and Nomarski inter-             of division septation can clearly be seen by inter-
                     ference contrast optics. In general, the cells seen        ference contrast (the lower cell of Fig. 1, A2),
                     in the Nomarski system (i) lacked the light halo           whereas only an indentation of the cell outline is
                     characteristic of the phase-contrast system; (ii)          visible by phase contrast (Fig. 1, Al).
                     appeared larger; (iii) presented an optically flat            The most important and striking difference
                     appearance;     (iv)  showed a "shadow effect"             between the two methods of microscopy was
                     reminiscent of shadowed preparations seen in the           revealed    at  about T3. Interference contrast
                     electron microscope; and, most importantly, (v)            microscopy clearly showed the presence of septa
                     showed more detail, particularly in the early              at the poles of the cells (Fig. 1, D2), whereas
                     stages of sporulation. With regard to the last             this could not be easily discerned in most cells
                     point, the division septa and the sporulation septa        by phase contrast (Fig. 1, D1). These septa were
                     were especially well defined. In addition to some          judged to be spore septa on the basis of the
                     granular objects visible in phase contrast, other          following criteria: the time of their appearance
                     objects, which are not seen under phase optics             in the culture, subsequent events, the fact that
                     were visible.                                              they could also be detected by staining with
                        Sporulation viewed under phase contrast. In             crystal violet (3), and their presence, as revealed
                     general, the phase-contrast pictures of the course         by examination of thin sections in the electron
                     of sporulation in B. megaterium (Fig. 1, Al, BI,           microscope,     in  samples taken at equivalent
                     Cl, and Dl; Fig. 2, Al, Bi, Cl, and Dl) pre-               times with the same organism under similar cul-
                     sented essentially the same details as described           tural conditions (2). However, the prime reason
                     for this method with other sporulating bacteria            for considering them as spore septa was the
                     (4, 18). In the early stages (Fig. 1), except for the      asymmetric positioning of these septa in the cell.
                     appearance of some phase-bright granules, par-               By T4.5, further stages of development were
                                                                                                                                                Downloaded from https://journals.asm.org/journal/jb on 13 September 2022 by 2001:448a:7060:2392:c07:520:b407:27e3.
                                                                                                                     VOL. 96, 1968                                                                                                                SPORULATION AND GERMINATION BY NOMARSKI OPTICS                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              1813
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                                                                                                                                  FIG. 1. Comparisonz ofphase-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.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Downloaded from https://journals.asm.org/journal/jb on 13 September 2022 by 2001:448a:7060:2392:c07:520:b407:27e3.
                                                                                                                          1814                                                                                                                                                                                                                                                                       KAHN, AND SLEPECKY
                                                                                                                                                                                                                                                                                                                        HITCHINS,                                                                                                                                                                                                                                                                                                                                                    J. BACTERIOL.
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                                                                                                                                         FIG. 2. Comparison ofphase-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.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Downloaded from https://journals.asm.org/journal/jb on 13 September 2022 by 2001:448a:7060:2392:c07:520:b407:27e3.
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...Journal of bacteriology nov p vol no copyright american society for microbiology printed in u s a interference contrast and phase microscopy sporulation germination bacillus megaterium anthony d hitchins arnold j kahn ralph slepecky biological research laboratories syracuse university new york received publication august the techniques nomarski were compared their utility monitoring germina tion technique permitted rapid easy delineation septation engulfment during whereas with these stages not detected at all later sporu lation easily seen by either thus seven as recognized electron thin sections five can now be rou tinely quantitatively optical stage ii engulf ment iii dark forespore corresponding to cortex formation iv bright spore sporangium coat v free vii this means that only i axial filament vi maturation refractile require routine detection there was advantage using optics studies bacterial has been divided into distinct based on examination used assess stained preparations fie...

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