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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
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VOL. 96, 1968 SPORULATION AND GERMINATION BY NOMARSKI OPTICS 1813
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t'":L
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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.
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1814 KAHN, AND SLEPECKY
HITCHINS, J. BACTERIOL.
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... .. ... ,: .: .
T :
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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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