FORMATION AND STRUCTURE OF THE

SPORE OF BACILLUS COAGULANS

D. F. OHYE and W. G. M U R R E L L , D. Phil.

From the Commonwealth Scientific and Industrial Research Organization, Division of Food
Preservation, Ryde, New South Wales, Australia

ABSTRACT
Spore formation in Bacillus coagulans has been studied by electron microscopy using an
epoxy resin (Araldite) embedding technique. The developmental stages from the origin of
the initial spore septum to the mature spore were investigated. The two forespore membranes
developed from the double layer of cytoplasmic membrane. The cortex was progressively
deposited between these two membranes. The inner membrane finally became the spore
protoplasmic membrane, and the outer membrane part of the inner spore coat or the outer
spore coat itself. In the mature spore the completed integuments around the spore proto-
plasm consisted of the cortex, a laminated inner coat, and a dense outer coat. No exosporium
was observed. The method of formation of the cortex and the spore coats is discussed.

INTRODUCTION

Following certain nuclear events (38, 39) the
early stage in spore formation involves the en-
closing of the spore nucleus by two membranes
derived from the centripetal invagination of the
sporangial cytoplasmic membrane (5). This in-
vaginatlon to form the initial spore septum is
associated with considerable mesosome develop-
ment (5). The association of certain peripheral
bodies with cell division and the early stages of
spore formation was observed by several workers
(Figs. 2 and 3, reference 2; 3; 7; Fig. 14, reference
19; 33) before the cytological structure of these
bodies (mesosomes) was clearly revealed by
Fitz-James (5) and Giesbrecht (7). Although
glimpses of certain stages in the development of
the initial spore septum and "wall" of the fore-
spore had been obtained (17, 32-34), it was
Young and Fitz-James (38, 39) who clearly
demonstrated the origin of the septum and Fitz-
J a m e s (5) who showed the association of meso-
somes with septum development in several Bacilluf
cereus strains, Bacillus megaterium, and Bacillus
medusa. Hashimoto (16) has shown a similar
development of the septum in Clostridium sporogenes.
Thus, the origin of the spore septum is now clear,
and considerable detail is available on the struc-
ture of the mature spore in several species (2, 4,
14, 16, 20, 26, 32, 34). However, until quite
recently very little was understood of the stages
between the early development and the complex
structure of the mature spore, and of the mode
of development of the structural components.
In 1962 Young and Fitz-James (40) described in
B. cereus var. alesti the formation of the cortex
between the two membranes of the forespore.
The spore coat apparently was formed external
to the outer membrane but within the exosporium.
This study of Bacillus coagulans confirms the
origin of the forespore membranes and the develop-
ment of the cortex between the two membranes.
It also reveals several differences in structure and
perhaps in method of formation.
MATERIALS AND METHODS
The observations reported in this study were made
with a very heat resistant strain of B. coagulans

111
isolated from c a n n e d cabbage. T h e o r g a n i s m was
grown in a m e d i u m of 0.5 per cent soya peptone
(Oxoid), 0.5 per cent c a s a m i n o acids (Oxold),
0.1 per cent yeast extract (Oxoid) a n d inorganic
salts (final concentrations of Na2HPO4, 5 raM;
NaC1, 5 raM; MgSO4, 0.125 raM; CaC12, 0.05 raM;
M n S O 4 , 0.005 raM; FeSO4, 0.005 raM), p H 7.4.
Synchronously sporing cultures of B. coagulanswere
obtained as follows: 14 liters of m e d i u m were inoc-
u l a t e d with a b o u t l0 s spores, s t e a m e d 1 h o u r to
heat-activate the spores, a n d aerated vigorously with
a s t r e a m of air for 15 to 16 hours at 50°C; 0.5 liter
of this culture was a d d e d to fresh 14-liter batches of
the s a m e m e d i u m a n d similarly aerated. Spore
s e p t u m formation occurred a b o u t 4 h o u r s after
inoculation in t h e final culture a n d spore formation
was complete within 24 hours. Samples (100 ml)
were r e m o v e d at 15-min intervals a n d chilled in ice.
A b o u t 20 to 30 ml were fixed i m m e d i a t e l y b y the
m e t h o d of Kellenberger, Ryter, a n d S6chaud (18).
T h e fixed material in a g a r blocks was d e h y d r a t e d by
passage t h r o u g h g r a d e d e t h a n o l to 100 per cent a n d
finally e m b e d d e d in Araldite (I0).
Sections were c u t with a P o r t e r - B l u m m i c r o t o m e
fitted with a d i a m o n d knife, floated onto 20 per cent
acetone in water, a n d picked u p on collodion-coated
grids a n d e x a m i n e d with a Siemens Elmiskop I
electron microscope.

Explanation of Figures

SCM, sporangial cytoplasmic or p l a s m a CHR, c h r o m a t i n , or nuclear material

membrane TRS, transverse cell septum
SPM, spore protoplasmic m e m b r a n e CW, cell wall
CM, cytoplasmic m e m b r a n e IC, i n n e r coat
SPC, spore c y t o p l a s m OC, o u t e r coat
SPS, spore s e p t u m M, mesosome
IM, i n n e r m e m b r a n e CX, cortex
OM, outer m e m b r a n e

FIGURES 1 TO 4
Electron m i c r o g r a p h s of early stages in the formation of forespores in cells of B. coagulans
fixed with OsO4. O n l y spore-forming ends of the rods are shown.

FIGURE l
An early stage in the d e v e l o p m e n t of the spore s e p t u m showing t h e i n v o l v e m e n t of
the cytoplasmic m e m b r a n e a n d mesosome. Note also t h e dense outer m a r g i n of the
cell wall of this organism. X 140,000.

FIGURE 2
A later stage shows completion of the spore s e p t u m , a n d t h e double unit m e m b r a n e
n a t u r e of the s e p t u m . X 110,000.

FIGURE
T h e forcspore is almost free in the c y t o p l a s m of the s p o r a n g i u m . T h e two m e m b r a n e s
s u r r o u n d i n g the forespore c y t o p l a s m are showing evidence of separation at several
points (arrows). X 90,000.

~IGURE 4
T h e forespore is free in the cytoplasm of the s p o r a n g i u m . T h e c h r o m a t i n material
of the forespore is evident. T h e m e m b r a n e s are showing separation at m o r e points in a
vesicle-like fashion. Mesosomes occur at other points of the s p o r a n g i u m where di-
vision m i g h t be expected to occur if sporulation h a d n o t c o m m e n c e d . T h e particulate
debris on the outer surface of the cell wall in Figs. 1 to 4 suggests a n outer layer in
the vegetative cell wall of this species. T h e walls in Figs. 1, 3, a n d 4 also h a v e a zoned
a p p e a r a n c e . X 110,000.

112 THE ~OURNAL OF CELL BIOLOGY • VOLUME 14, 1965

D. F. OHY~. AND W, G. MURRELL Spore Formation in B. coagulans 113
 To overcome possible permeability difficulties
during fixing and staining some mature spores were
disrupted by shaking with glass beads before fixa-
tion.
RESULTS
Development of the Forespore
The progressive development of the forcspore
is shown in Figs. l to 4. T h e spore septum formed
by the invagination of the sporangial cytoplasmic
m e m b r a n e is composed of two typical Bacillus unit
membranes. T h e typical Bacillus unit m e m b r a n e
described by Fitz-James (5) consists of a dense
backbone line bordered by two lighter zones with
an over-all thickness of 140 to 150 A. Fitz-James
suggests that the inner dense line, typical of a
variety of animal cells, is obscured by the dense
cytoplasm. T h e general appearance of the cyto-

FIGURE 5
An enlargement of the left hand portion of the spore septum of Fig. 2 which shows greater detail
of the double membrane structure of the spore septum. The connection of the spore septum with
the mesosome, in the upper right corner, is only partly resolved. The dense backbone lines of the two
membranes show evidence of cross-banding at this stage. The inner line (adjacent to the cytoplasm)
of the unit membrane is partly resolved, but is not nearly so dense as the dense backbone lines of the
spore septum. X 320,000.

FIGURES 6 To 8
Electron micrographs of thin sections of cells of B. voagulansfixed with OsO 4.
FIGURE 6
A typical dividing vegetative cell shows development of the transverse cell wall. The
large mesosome forms part of the invaginating plasma membrane. Cell wall material
has been deposited between the invaginating membranes. X 90,000.

FIGURE 7
An oblique transverse section of a sporulating cell which shows well the double mem-
brane structure of the spore septum, and the structure of a mesosome which appears
to be enclosed within the forespore. The mesosome is probably joined to the spore
septum at its centre, but the connecting membranes are poorly resolved. )< 90,000.

FmUnE 8
A well resolved spore septum at a later stage shows complete separation of the two
membranes and a small mesosome clearly contiguous with the inner membrane of
the forespore. X 120,000.

114 THE JOURNAL OF CELL BIOLOGY • VOLUME 14,, 196~

D. F. OHY• AN]) W. G. MVnR~LL Spore Formation in B. coagulan8 II~
 plasmic or plasma membrane of B. coagulans is
similar, but the over-all width of the plasma
membrane is 85 to 105 A comprised of a centre
dense line and two outer light zones of 25 to 30 A
each, with a thin line next to the cytoplasm of
10 to 15 A thickness (Figs. 2, 4, 5 and 6). The
thickness of the double membrane was 170 to 210
(Figs. 2 and 5) compared to 280 to 300 A
((5), his Fig. 16).
The description of the cytoplasmic membrane
as used by Fitz-James (5) has been followed closely
in this paper. It is appreciated, however, that the
"inner dense line" of this membrane and of the
inner and outer membrane of the forespore of B.
coagulans is resolved in many instances (Figs. 2, 4,
5, 9). In such cases the plasma membrane resem-
bles that described by Robertson (25) for animal
cells, and by Glauert et al. (9) for B. subtilis. This
resemblance is more marked if the lighter zone
between the "dense back-bone line" and the cell
wall is disregarded. The correct interpretation
probably depends upon whether the osmiophilic
layers are the "lipid lines" as discussed by Fitz-
James (5).
The initial spore septum appeared particularly
flexible in the early stages, but began to "round
up" at the stage when the forespore moved towards
the centre of the sporangium (Figs. 3 and 8).
The two unit membranes are showing evidence of
separation at several points in Figs. 3 and 4 with
the suggestion of deposition between them of
material of lower electron opacity (Figs. 8 and 9).
Mesosomes occur associated with the initial
spore septum in many instances (Figs. I, 2, and
7), and with the inner membrane of the forespore
at later stages in its development (Fig. 8). They
are also observed at several other points on the
sporangial cytoplasmic membrane, presumably
where normal cell division would occur if sporo-
genesis had not commenced (Fig. 4). The striking
difference between membrane development in
spore septum formation (Fig. 2) and ordinary
cell division (Fig. 6) is again evident in this or-
ganism (cf. Fitz-James, 5). The sporangium shows
a fairly homogeneous granular appearance unlike
that in some other organisms such as B. cereus
(2, 38, 39) and B. rnegateriurn (2) which are usually
filled with large granules believed to be of
lq~OtrRE 9
A later stage in spore development than Fig. 8
shows a definite zonation of the cortical material
between the two well defined membranes. The sporangium wall. a X 80,000. b part of the same
middle more dense zone is similar in density to the micrograph enlarged, X 160,000.

116 TH~ JOURNAL OF CELL BIOLOOY • VOLUME 14, 196~

/3-hydroxybutyrate. The cell wall is quite thick
(ca. 250 A), and shows on the outer surface a
considerable amount of dense granular material
(Figs. 1 to 6). This may consist of adherent par-
ticles of the growth medium as suggested by
Glauert et al. (9) but the outer zone of the cell
wall is sufficiently well defined to suggest an outer
layer in the wall of this species (Figs. 1 to 4).
Development of the Forespore into a Mature
Spore
As the terminology of the cytological compo-
nents of the mature spore is still being resolved
(cf. 16), it is necessary to define the terms used
FIGURE ]0
The cortical region between the two membranes
is much more developed (the membranes are 750 A
or more apart) and shows a gradual increase in
density outward until a zone of reduced density is
reached. The two membranes of the spore are not
so well resolved as that of the sporangium. The
outer membrane is vaguely resolved just beneath
a broken ring of very dense material which is
possibly the initial development of the dense outer
coat of the spore envelope. X 90,000.
D. F. OHx'~ ~ND W. G. ]V[IYRRELL SporeFormation in B. eoagulans
in this paper. The membrane enclosing the cyto-
plasm and nucleus is called the "spore proto-
plasmic m e m b r a n e " rather than "spore wall"
(17, 27, 37). The spore protoplasm (nucleus +
cytoplasm + spore protoplasmic membrane)
is surrounded first by the cortex, then the inner
coat and outer coat (or coat 1 and coat 2 respec-
tively, 27). The terms "envelopes" or "integu-
ments" are used collectively to include the layers
external to the spore protoplasmic membrane.
The mature spore develops from the spore
cytoplasm and nucleus which are initially en-
closed by the two unit membranes. The stages
in this development are shown in Figs. 8 to 1 I.
The two membranes gradually move apart as
cortical material and eventually the coat layers
are formed. The spore cytoplasm remains im-
mediately enclosed within the inner membrane
clearly visible almost to complete maturity of the
spore. Note in the membrane the typical dense
backbone line cf 25 to 30 A bordered by the 2 less
dense zones of ca. 25 to 30 A thickness (Figs. 9
and 10). Sections of completely mature spores
(Fig. 12) usually show only poorly the inner
membrane (now described as the spore proto-
plasmic membrane), but this is probably due to
difficulties in fixing and staining of the mature
spore rather than to the disappearance of the
plasma membrane. T h a t the spore protoplasmic
membrane is the same inner membrane of the
spore septum would seem certain from the pro-
gressive stages seen in Figs. 8 to 11.
The outer membrane of the septum of the
early forespore gradually moves away from the
inner membrane as the cortical material is de-
posited between them, until finally its identity is
lost in the outer regions of the complex spore
envelopes (Figs. 10 and 11). In the intermediate
stages there are 9 differentiated zones about the
spore cytoplasm (Fig. 9). First can be seen the
inner unit membrane of 3 zones with the outer
less dense zone apparently merging into the corti-
cal substance or 4th zone. Next is the cortical
region about 300 A thick. This consists of less
dense inner and outer zones with a centre zone
very similar in density and granulation to the
sporangial cell wall (Fig. 9). Eventually the thick-
ness of the cortex increased to ca. 1200 A (0.12 #)
(Fig. 11-13). Outside the cortical zone are the
three layers of the outer membrane, that nearest
the cortex again appearing thicker than usual
due possibly to its blending with the cortical
material (Fig. 9). As the gap between the two
117
m e m b r a n e s increases the cortex assumes a n ap-
p e a r a n c e of several zones of varying electron
opacity (Figs. 10 a n d 11). This m a y result from
uneven p e n e t r a t i o n of the fixative into the nearly
m a t u r e spore. Even at the stage of d e v e l o p m e n t
shown in Fig. 10 it is possible to make out density
differences h a v i n g the zonal a p p e a r a n c e of the
Structure of the Integuments of the Mature
Spore
W i t h the fully m a t u r e spore of this organism
it has not b e e n possible to o b t a i n sections in which
the details of all the components of the spore
protoplasm a n d its integuments were clearly
identifiable (Fig. 12). This m a y be due to poor

Fmt~m 11
A slightly more mature spore than in a previous figure which shows clearly the inner membrane,
the very thick cortical region, the first two or three repeating layers of the laminated inner coat,
and the dense outer coat. a X 60,000. b part of the same micrograph enlarged, X 290,000.

outer m e m b r a n e . I n Fig. 10 a broken ring of p e n e t r a t i o n of OsO4. M u c h more information

electron-opaque material appears just external o n the n a t u r e of the morphological components
to the outer m e m b r a n e . This is p r o b a b l y the of the m a t u r e spore was, however, o b t a i n e d from
initial d e v e l o p m e n t of the outer coat, w h i c h is sections of spores which h a d b e e n disrupted by
possibly laid down o n the surface of the outer
shaking with glass beads (35), w h i c h h a d been
m e m b r a n e of the spore septum. I n Fig. 11 the
h e a t activated, or in which g e r m i n a t i o n h a d been
lamellae of the inner coat are becoming evident,
initiated (23). These sections show clearly that
the cortex now being surrounded by a l a m i n a t e d
i n n e r coat a n d a very dense outer coat. A t no the envelopes of the spore protoplasm consist of a
stage in the d e v e l o p m e n t of the spore was there cortical region a n d two spore coats (Figs. 13 to
observed a m e m b r a n e h a v i n g the loose flexible 15). T h e cortex in these preparations is p r o b a b l y
characteristics of the exosporium of B. cereus largely residual insoluble m a t e r i a l a n d appears
(2, 16, 26, 27) or Clostridium sporogenes (17). to have at its i n n e r surface a m e m b r a n o u s zone

].18 THE JOURNAL'OFCELL BIOLOGY • VOLUME 14, 1962

or at least an orientated insoluble boundary,
which fixes more OsO4 (Fig. 15), and which is
distinct from the protoplasmic membrane. In the
section of the heat activated spore the proto-
plasmic membrane was again visible (Fig. 13).
The cortex shows a less dense zone about a more
dense inner one. The inner coat, with a thickness
of about 375 A, can be seen as alternate light
and dark zones having a repeating distance of
about 75 A (Fig. 14). Altogether, up to 7 zones
have been observed. The outer coat is a strongly
electron-absorbing region of ca. 200 A thickness
(Figs. 12 to 15). The outer membrane was at this
stage masked by the very dense outer coat or the
laminations of the inner coat.
DISCUSSION
These studies have confirmed in another species
the peculiar type of cell division involved in
sporogenesis, and have revealed further detail of
spore development and of the structure of the
spore envelopes. The nature of the process of
sporogenesis poses two very important questions.
First, what are the chemical substances initiating
this type of cell division and operating in the
nuclear control mechanism responsible for this
new phenotypic expression of the vegetative
cell nucleus? Secondly, what are the mechanisms
of the synthesis and deposition of the chemical
substances composing the cortex and coat layers
of the envelopes of the spore protoplasm? There
is very little information available about the
first point (22). With regard to the second, two
hypotheses have been suggested for vegetative
cell wall formation. First, mesosomes have been
suggested as the site of synthesis and extrusion of
polymerized wall material (5, 28). Secondly, cell
wall synthesis may involve the synthesis of wall
components in the cytoplasmic membrane itself
with polymerization of the wall material over its
surface or synthesis in the cytoplasm and poly-
merization at any point on its surface where the
chemical components are extruded through the
plasma membrane.
Fitz-James (5) has discussed the function and
structure of mesosomes. Although the present
work tends to support the importance of these
bodies in the formation of transverse septa and
development of membranes, M u r r a y (21) has
suggested that they are not essential for cell
division as they have not been seen in several
bacterial species. The fact that mesosomes are
associated with the formation and early develop-
D. F. OHYI~ AND W. G. MUURELL Spore ~brmation in B. coagulans
ment of the spore septum and apparently not with
later stages in the deposition of the inner and
outer coats suggests that mesosomes are involved
only in the active synthesis and unfolding of cyto-
plasmic membrane material itself, and not in spore
coat synthesis. We have looked for mesosomes, both
on the inner and outer membranes, during the
various stages in spore formation, and have ob-
served them only on the inner membrane (Figs. 7
and 8), yet certainly in the intermediate and later
stages of spore formation cortical and possibly
coat material is being deposited between the
membranes. The presence of mesosomes on the
inner membrane does not exclude the possibility
that this membrane only is involved in the syn-
thesis and extrusion of cortical material by the
first mechanism postulated for cell wall synthesis.
If this membrane is involved in the synthesis of
the cortex, one would expect the cortical material
to be somewhat similar in composition to vegeta-
tive cell walls. It has been shown recently that the
site of a, e-diaminopimelic acid (DAP)-hexosamine
peptide containing material of the spore integu-
ments, i.e. the material equivalent to the vegetative
cell wall in Bacillus and other species, is the cortex
(35). The evidence for this is the release by lyso-
zyme and spore lytic enzymes of material con-
taining DAP, hexosamine, and glutamate coinci-
dent with the disappearance of cortical material
seen in electron micrographs of thin sections of
crude spore coat preparations. This had been
suggested by Mayall and Robinow (20). Thus
the cortex, in part at least, contains material
similar in composition to that of the cell wall,
and the presence of this material just external to
the inner membrane suggests that the cortex is
synthesised and deposited by a mechanism similar
to that for vegetative cell wall formation. The
accumulation of cell wall substrates in the cortex
places these in a favourable position for assembly
into the cell wall of the germ cell (20, 27) as the
cortex is broken down by lyric enzymes during
germination (24, 31).
The cytological evidence of the origin of the
forespore membranes suggests that they are
lipoprotein in composition like the vegetative
cytoplasmic membrane (8, 36) and hence are
likely to control permeability of the forespore.
There is a distinct possibility that, in the early
stages of the forespore, before any marked changes
in permeability of the integuments of the spore
protoplasm have occurred, both the spore pro-
toplasm and the sporangium are synthesising
119
120 T H E JOURNAL OF CELL BIOLOGY • VOLUME 14, 1962
material w h i c h diffuses into the region between
the two m e m b r a n e s of the forespore. However, at
later stages changes in permeability m a y cause
the outer spore m e m b r a n e a n d the s p o r a n g i u m
to become involved more in the f o r m a t i o n of the
two outer coats. T h e m e c h a n i s m of f o r m a t i o n of
these outer coats which are largely protein (29,
35) is still obscure.
T h e r e is evidence of material occurring as
l a m i n a t e d entities b o t h within a n d outside a n
outer m e m b r a n e or exosporium, similar in com-
position, appearance, a n d staining properties,
a n d sometimes contiguous with the l a m i n a t e d
coat of the spore. This is of sufficient i m p o r t a n c e
in regard to the m e c h a n i s m of formation of the
spore coats to w a r r a n t elaboration. H a n n a y
(14) has shown t h a t the protein crystals of Fowler's
bacillus, a strain of B. cereus, are formed within a
m e m b r a n e which also encloses the spore a n d t h a t
this m e m b r a n e bears some resemblance to a u n i t
plasma m e m b r a n e (cf. his Fig. 10). T h e crystals
of Bacillus thuringien~is a n d other parasporal bodies
m a y r e m a i n a t t a c h e d to the spore after their
lytic release from the s p o r a n g i u m (1, 13, 30) a n d
are p r o b a b l y enclosed by a n unresolved m e m b r a n e
(1). H a n n a y (13) has also shown clearly t h a t the
parasporal body of Bacillus laterosporus L a u b a c h
is contiguous with the l a m i n a t e d coat of the spore,
consisting of lamellae adjacent a n d parallel to
similar lamellae of the spore coat, suggesting a
crystalline type of outgrowth of the coat itself
(cf. H a n n a y , 13, his Figs. 12 a n d 13). I m m e d i a t e l y
s u r r o u n d i n g the parasporal body of Fowler's
bacillus, a crystalline body with the lattices of the
crystals orientated at different angles, is a lami-
n a t e d layer similar in a p p e a r a n c e to the spore
coat. This l a m i n a t e d layer resembles, also, certain
m e m b r a n e systems a n d " l a t t i c e " structures oc-
curring in the sporangial cytoplasm external to
the m e m b r a n e t h a t surrounds the spore a n d the
parasporal body (cf. Figs. 8 a n d l l of H a n n a y , 14).
T o r t u o u s m e m b r a n e structures in the cytoplasm

FmuR~ 1£ TO 15
Sections of a mature spore in the sporangium, a heat-activated spore, a disrupted
spore, and a germinated spore of B. coagulans.

FIGURE 1~
Transverse section of a sporangium containing a mature spore. The inner components
of the spore are poorly preserved and of low contrast. The dense outer coat of the spore
is resolved but the adjacent laminations of the inner coat are hardly resolved. There is
no membrane present equivalent to a typical exosporium. X 90,000.

FIGURE 13
Section of a mature spore which was heat activated for 8 rain at 110°C. The inner
membrane or spore protoplasmic membrane is evident again, surrounded by 2 cortical
zones, the 1st slightly more dense than the outer zone. The 5 or 6 laminations of the
inner coat can he seen surrounded by the very dense outer coat. No membrane equiva-
lent to a loose exosporium is present. X 100,000.

FIGURE 14
An enlargement of the coat region of a section of a spore germinated in glucose (0.05
M) for 60 min at 50°C. The dense outer coat and the laminations of the inner coat
can be seen. Four repeating light and dark units of 75 A thickness are present. )<
290,000.

FIGURE 15
Section of a disrupted spore after washing, showing the dense outer coat, the inner
laminated coat, and residual fibrous cortical region. This region shows a n u m b e r of
partly resolved cross-banded layers as has also been observed by Robinow (private
communication). The inner margin of the cortex is well defined, and has a membrane-
like appearance which does not resemble the spore protoplasmic membrane. X 210,000.

D. F. OHYE AND W. G. MURRELL 8pore Formation in B. coagulans 121

a r o u n d electron-opaque particles a n d in continuity
with the spore coat have also b e e n observed in
B. subtilis (34). These l a m i n a t e d entities are ap-
parently composed largely of protein together
with some basophilic lipid or phospholipid soluble
material. T h e crystals of B. thuringiensis are largely
protein (15), the parasporal bodies of B. latero-
~porus phosphoprotein (6), a n d the staining a n d
solubility properties suggest t h a t in other cases
these inclusions m a y be protein also (11-13).
This evidence suggests t h a t the l a m i n a t e d outer
coat of the spore is a product of the metabolism
of the s p o r a n g i u m in the later stages of spore
formation, a n d t h a t this occurs as a type of ori-
entated layering or crystal d e v e l o p m e n t within
or associated with a m e m b r a n e , most probably
the outer m e m b r a n e of the forespore. T h e mech-
anisms possibly involved in the formation of the
spore coats could be some m e t h o d of deposition
of substrates (synthesised in situ or in the sporan-
glum) onto a t e m p l a t e m e m b r a n e or by the split-
ting of such a m e m b r a n e . O n the other h a n d the
outer m e m b r a n e m a y invaginate (evaginate?)
a n d form concentric lamellae a b o u t the cortex
by a m e t h o d such as occurs d u r i n g the develop-
m e n t of the myelin layers a b o u t the axon in the
nerve cell (25). No evidence of the latter type of
d e v e l o p m e n t has yet been observed. T h e a p p a r e n t
occurrence of the spore coat external to the outer
m e m b r a n e in B. cereus var. alesti (40) m a y per-
haps suggest t h a t a process of evagination is
involved in this species. Certainly, m u c h more
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123