JOURNAL OF BACTERIOLOGY
Vol. 87, No. 4, p. 876-886
Copyright © 1964
Anierican Society for Mlicrobiology
Printed in U.S.A.
EFFECT OF SPORULATION MEDIUMI ON HEAT RESISTANCE,
CHEMIICAL COMPOSITION, AND GERMINATION OF
BACILLUS MEGATERIUM SPORES'
HILLEL S. LEVINSON AND MIILDRED T. HYATT
Pioneering Research/i Division, U.S. Army Natick Laboratories, Autick, Massachusetts
Received for publication 29 November 1963
LEVINSON, HILLEL S. (U.S. Army Natick Labo-
ratories, Natick, Mass.), AND MILDRED T. HYATT.
Effect of sporulation medium on heat resistance,
chemical composition, and germination of Bacillus
megateriouii spores. J. Bacteriol. 87:876-S86. 1964.-
spores, groN-n on variously,
supplemented media, had varying concentrations
of P, Ca, MIn, or dipicolinic acid. Supplementation
with CaC12 yielded spores with increased heat
resistance; addition of i-glutamate, L-proline, or
increase of the phosplhate concentration yielded
spores wvit,h leduced heat resistance. Germination
characteristics depended on both the sporulation
medium and the germinant (glucose, i-alanine,
L-leuicine. or K-NO3); pronounced differences were
demonstrable with glucose and L-alanine, which
trigger germination via different metabolic path-
ways. An increase in CaCl2 during sporulation
yielded spores witlh increased germination in glu-
cose but I-not in L-alaninie. Germination in L-alanine
was optimal with spores produced on media con-
MnCl2, but germination of such
spores wa-is minimal in glucose. An increase in the
sporulationi medium phosplhate
initial germination rate in gluicose, but not in
L-alanine. Spores produced in CaCl2-supplemented
media had increased heat-activation requirements
(increased dormancy) for germination induced by
L-alanine. and decreased heat-shock requirements
for gluicose-induced germination. An increase of
sporuilation phosphate yielded spores with reduced
dormancy for germination induced by L-alanine,
w-ith uinchanged dormancy
germinants. Spores prodtuced with added L-glu-
tamate had reduced dormancy for glucose-induced
germina.tion, a.nd increased dormancy for germina-
tion in(luced by i-alanine. Addition of CaCI2 or
L-glutamate to the sportulation medium yielded
spores with increased sensitivity to "ionic ger-
mination" (with KI). Spores from synthetic me-
dium were incapacitated for full postgerminative
1Some of the data in this paper were presented
at the 63rd Annual Meeting, American Society
for MLicrobiology, Cleveland, Ohio, 5-9 MIay 1963.
changes in oxygen-uptakie rate N-wich accompany
normal cell division.
as shown by repression
comnp)osition of the s)orulation medium affects
not only the spore's heat resistance (Sugiyama,
1951; Levinson, Hyatt, and Moore, 1961) and
its chemical composition (Slepecky and Foster,
1959), but also its germination characteristics
(Grelet, 1957; Keynan, AMurrell, and Halvorson,
1961a, 1962; Levinson, 1961; McCormick and
Halvorson, 1963). Keynan et al. (1961a) pointed
out that the germinlation agent must be con-
sidered in defining such spore characteristics as
dormancy. In this paper Nwe report that the early
rate, total extent, and requirement for heat ac-
tivation of germination of Bacillus megaterium
sl)ores are determined both by the composition
of the sporulation medium and by the germina-
tion agent used. The influence of the sporulation
medium is most marked when the germination
agents act via different metabolic pathways. The
sporulation medium also influences the ability
of the germinated spores to undergo postgermina-
has been accumiulating that the
MIATERIALS AND AIETHODS
B. megateriimn QMI 131551, the strain which we
have used for over 10 years, and with which
Rode and Foster (1962a) have demonstrated
some interesting ionic effects on germination, was
used throughout this investigation. A medium
containing 0.5%-7 of a liver fraction, Wilson's
Liver "B" (Foster and Heiligman, 1949), at an
initial pH of 6.5, was prepared. Without supple-
mentation, this medium contained approximately
0.25 mm calcium; less than 0.02 mM manganese;
and approximately 1.0 mAi inorganic orthophos-
phate. It was used supplemented with 10 mm
KH2PO4 ("basal" medium) or with other con-
SPORULATION MEDIUM AND SPORE GERMINATION
centrations of phosphate. The "basal" medium
was used as such or was supplemented with
CaCl2, AMnCl2, or with glucose or various amino
acids at 12.3 mm. The medium was dispensed
into 1-liter Erlenmeyer flasks (125 ml per flask),
inoculated with 0.5 ml of a spore suspension from
an agar slant (nutrient agar with 0.2%/, liver
culture, and incubated on a reciprocal
shaker (93 3-in. strokes per min) at 30 C for 4
days. Spores were harvested, washed (by cen-
trifugation at 4 C) twice with KH2PO4 (10-2 M;
p)Hunadjusted) and six times with water, and
lyophilized. The "basal" medium lproduced ca.
350 mg of lyophilized B. megaterimn spores per
A limited number of experiments was also
performed with spores produced on a synthetic
medium developed in this laboratory for produc-
tion of B. suibtilis spores (Donnellan, Nags, and
Levinson, 1964). The synthetic medium had the
following composition (final mm concentrations):
KH2PO4, 10; glucose, 10; L-glutamic acid, 10;
and CaCl2 in various concentrations. Glucose and
L-glutamic acid were sterilized by Seitz filtra-
tion and mixed with sterile CaCl2 and spore
inoculum; samples of the mixture were added
to the autoclaved solution of the remaining salts
in 1-liter flasks. The final pH of this synthetic
medium was 6.8. Incubation and harvesting con-
ditions were the same as for Liver "B" media,
and the yield from the synthetic medium was
approximately 100 mg of spores per liter.
Although we are cognizant of the incongruity
of the terms, we occasionally, for the sake of
brevity, refer to spores grown on the "basal"
medium as "basal spores";to thosegrownon the
L-glutamate, etc., as "CaCl2 spores," "L-gluta-
mate spores," etc.; and to spores produced on
the synthetic medium as "synthetic spores."
Spores produced on the Liver "B" and on
synthetic media were tested for their content of
Ca, Mn, P, and dipicolinic acid; heat resistance;
germination in glucose, L-alanine, L-leucine, or
LiNO3, and the effect of "heat shock" on ger-
mination in these compounds; and oxygen con-
sumption during germination and postgermina-
Spore digests were prepared in 10-ml micro-
Kjeldahl flasks by a wet ashing procedure (Sle-
pecky and Foster, 1959); 10 mg of lyophilized
spores were digested with HN03, H2SO4, and
H202 ; the digestate, made to 10 ml with water,
was used for subsequent colorimetric analyses
for total P, Ca, and Mln. Analysis for P was
carried out by the method of Fiske and Subba-
Row (1925); Ca analysis was done according to
Roe and Kahn (1929), with the use of 4 ml of
digestate; and Mn was determined colorimet-
rically (560 m,u) as KMnO4 after oxidation of 4
ml of digestate with 0.1 g of K104 (Coleman and
Gilbert, 1939; Slepecky, personal communication).
In addition, supernatant fluids of spores sus-
pended in 0.1 N H2SO4 (1 mg of spores per ml)
were assayed for "acid-released" P and Ca. Di-
picolinic acid was determined by the colorimetric
assay of Janssen, Lund, and Anderson (1958),
with the use of 4 ml of the supernatant solution
from an autoclaved water suspension of spores
(1 mg of spores per ml).
Aqueous suspensions of spores (0.5 mg/ml)
were heated at 90 C for various times, and the
number of surviving spores was estimated by
colony counts on nutrient agar (with 0.1%7 yeast
extract). Heat resistance is expressed as per cent
For studies on germination, aqueous suspen-
sions of unheated spores, or spores heated for 10
min at specified temperatures between 50 and
60 C, were diluted with an equal volume of phos-
phate buffer (pH 7). To 50-ml Erlenmeyer flasks
containing 0.6 ml of glucose, L-alanine, L-leucine,
or KNO3 were added 2.4 ml of spore suspension to
give concentrations of substrates as indicated; of
phosphate, 50 mM; and of spores, 1.0 mg (ca.
5 X 108 spores) per ml. The reaction mixture
was incubated on a reciprocal shaker at 30 C,
and slides for microscopic observation of ger-
mination (staining with 0.5%,7, aqueous methylene
blue) were made at intervals to 2 hr.
Oxygen uptake was measured by standard
Warburg techniques. The total volume of re-
action mixture was 1.5 ml, containing heated
(10 min at 60 C) or unheated spores (1.0 mg/ml)
in phosphate buffer, pH7 (50 mM); (NH4)2 S04 (10
mM); and glucose (25 mM). The center well con-
tained 0.2 ml of 10% KOH. After 7 hr of incu-
bation at 30 C, slides wvere made from the con-
tents of each vessel for determination of the
VOL. 87, 1964
TABLE 1. Phosphorus, dipicolinic acid, and calcium content of Bacillus megaterium spores
produced on variously supplemented media
Supplement to medium
Liver "B" (0.5%)
*Concentrations given as jAg/mg of spores.
t "Basal" medium contains Liver "B," 0.5%, to which KH2PO4, 10 mM, has been added. See text for
composition of synthetic medium.
Regardless of the concentration of phosphate
added to the "basal" medium, the total P con-
centration is about 20 Mg/mg of spores (Table 1).
However, the P content of spores grown on the
"basal" or synthetic medium increased with in-
creasing CaCl2 content of the medium. The addi-
tion of 12.5 mM L-glutamate or L-proline to the
"basal" medium resulted in the production of
spores with decreased P content. Supplementa-
tion of "basal" medium with other compounds
did not affect the P concentration of the spores.
Appreciable amounts of P were released from
spores by acid treatment (0.1 N H2SO4) only when
the spores had been derived from the glutamate-
from the synthetic medium containing 1.0 or 2.0
mM CaCl2. It may be that much of the total P
of these spores is accumulated as inorganic ortho-
phosphate in the spore outer layers (as high as
50% of the total phosphate in the case of spores
derived from the synthetic medium containing
2.0 mM CaCl2).
Calcium and manganese. The Ca content of
of spores: phosphorus.
CaCl2 had been added to the sporulation medium
(Table 1, Fig. 1). Increasing the MnCl2 content
of the medium yielded spores with increased Mn
(Fig. 1). Furthermore, the Ca content of spores
decreased with increasing MnCl2 in the sporula-
tion medium, and the Mn content of spores de-
creased with increasing CaCl2 in the sporulation
As with P, spore Ca may not be firmly bound
to the spore (Table 1). The ratio of "acid re-
leased" Ca to total Ca varies with growth con-
ditions, but appears to be highest with "L-glu-
tamate spores," "L-proline spores," or "synthetic
Dipicolinic acid. Generally, spores contained
from 90 to 110 ,g of dipicolinic acid per mg of
spores. Spores produced on media with high
CaCI2 concentrations or with glucose supple-
mentation had somewhat less dipicolinic acid,
and spores produced on the medium supple-
mented with L-proline had a higher level of di-
Heat resistance. Spores produced on CaCIa-
supplemented "basal" medium ("CaCl2 spores")
relatively unchanged unless
LEVINSON AND HYATT
SPORULATION MEDIUNI AND SPORE GERMINATION
and on the synthetic medium with 1.0 mm CaC12
were more heat resistant (Fig. 2) than were those
grown on the unsupplemented "basal" medium.
This is perhaps attributable to the higher Ca-
dipicolinic acid molar ratio in the "CaCl2 spores"
(Levinson, Hyatt, and Moore, 1961). "MnCI2
spores" were no more resistant than were "basal
sIores." Increasing the phosphate concentration
of the sporulation medium to 100 mm yielded
spores with decreased heat resistance. Of the
amino acids with which we supplemented the
"basal" medium, only L-glutamic acid and L-pro-
line had an effect, and these greatly reduced heat
resistance. "Synthetic spores" were much more
heat resistant than were spores produced on the
"basal" medium supplemented with L-glutamate,
in spite of the synthetic medium's content of
L-glutamic acid. In all probability, then, L-glu-
tamic acid in the sporulation medium does not,
per se, control heat resistance.
Spore germination: CaCl2 and M1InCl2. The total
extent (Fig. 3) and early rate (data not shown)
of germination on glucose, L-leucine, and KN03
increased with increasing CaC12sup)plementation
of the "basal" sporulation medium. On the other
Ca OR Mn ADDED TO SPORULATION
FIG. 1. Effect of CaCl2 or MnCl2 supplementation
of the "basal" sporulation medium on the Ca and. Mn
content of Bacillus megaterium spores. Ca content of
spores grown with various concentrations of CaCl2,
0; Ca contentof spores grownwith 0.25
and various concentrations of MnCl2, 0;Mn con-
tent of spores grown with various concentrations of
tit-iI MnCl2 and various concentrationsof CaCl2, *.
L1; MIn content of spores grown with 0.25
FIG. 2. Heat resistance of Bacillus megaterium
spores produced on variously supplemented media.
Spores were produced on "basal" (containing 10 mM
phosphate) medium, 0; or on "basal" medium
supplemented with phosphate so as to contain 100
mM phosphate (0), or with 0.5 mM CaCl2 (Ol),
0.5 mM MnCl2 (C), 12.5 mM L-glutamic acid (c),
or 12.5 mM L-proline (*); or on synthetic medium
containing 0.5 mM CaCl2 (A) or 1.0mM CaCl2 (-).
Aqueous suspensions of spores (0.5 mg/ml) were
heated at 90 Cfor indicated times, and the number of
survivors was estimated by plating on nutrient agar
with 0.1% yeast extract.
hand, on L-alanine, the total extent (Fig. 3) of
germination was decreased, and the initial rate
(data not shown) wAas unaltered by addition of
CaCl2 . MnCl2 in the growth medium had a more
complex effect. Germination induced by L-ala-
nine was optimal when spores were produced on
media supplemented with 0.1 mm MnCl2; but
germination on glucose or on L-leucine was min-
imal when the spores were grown on this con-
centration of MnCl2. Germination on KNO3
increased with higher concentrations of MnCl2
in the sporulation medium (as with CaCl2-sup-
"CaCl2 spores" had a reduced heat-shock re-
quirement for germination on glucose, L-leucine,
and KN03 (Fig. 4); that is, their "dormancy"
VoL. 87, 1964
LEVINSON AND HYATT
80_ 1.0 mM
Ca OR Mn ADDED TO SPORULATION MEDIUM (mM)
FIG. 3. Effect of concentration of CaCi
added to sporulation medium on germination of
Bacillus megaterium spores in glucose, L-alanine,
L-leucine, and KNO3
for 10 min and incubated for 2 hr at 30 C with the
indicated concentration of germination agent.
. Spores were heated at 60 C
RTc0o5 5 55
before incubation at 30 C for- 2 hr with the various
at 30 C for 2Jrwt
on heat activation of Bacillus
spore germination in
L-leucine, and KlO.
with no added Ca (0 Ca), or sutpplemented with
0.5 mm Ca.
weie heated at the indicated teoperatures
medium was used
germination agents. RT
(LKeynan et al., 1961la) was reduced. Forexample,
on 10 mm glucose, unheated "basal spores" ger-
minated 2 to 3%, but205%/Oof t.he "CaC12 spores"
germinated. The heat-activation requirement for
50% germination of "basal spores" on 10 mm
glucose (10 min at 58 C) decreased with supple-
mentation of the "basal" sporulation medium
sporulation medium had
effect on the "dormancy" of spores in germina-
tion induced by L-alanine. "CaC12 spores" re-
quired more heat activation than did "basal
Phosphate concentration. The phosphate con-
centration of the sporulation medium also in-
(Fig. 5). On glucose, and less markedly on L-leu-
cine, spores from the medium supplemented with
100 mm phosphate eventually germinated to
almost as high a percentage as did those from
the "basal" medium (10 mm phosphate), but
their rate of germination ov er the first 30 min of
incubation was substantially lower. With KNO3
as substrate, both the initial rate of germination
and the total amount of germination were higher
with spores grown in the lower concentration of
phosphate. In contrast to these, neither the total
extent nor the rate of germination on L-alanine
| I O~~~~~~'mMPO)4
FIG. 5. Effect
sporulation medium on rate and extent of germina-
tion of Bacillus megaterium
sions of spores produced on "basal" medium, con-
taining either 10 mm
heated at 60 Cfor 10 min and incubated at 30 C with
the indicated concentration of germination agent.
spores in glucose,
a function of time. Aqueous
or 100 mM phosphate, were
SPORULATION MEDIUM AND SPORE GERMINATION
appeared to be affected by the phosphate com-
position of the sporulation medium.
Heat shock increased germination of "basal
spores" (10mmphosphate) on all four germinants
(Fig. 6). However, spores grown with 100 mm
phosphate, while retaining the stimulatory pat-
tern of heat shock for germination on glucose,
L-leucine, and KNO3, had no heat-activation
requirement for germination induced by L-ala-
Other supplements. B. megaterium spores with
altered susceptibility to heat activation of ger-
mination on the four germinants (Table 2) were
produced on the "basal" medium supplemented
with 12.5 mm concentrations of various nitrog-
enous compounds or of glucose, or on the syn-
thetic medium. The requirement for heat activa-
tion ("dormancy") depends on both the chemical
composition of the sporulation medium and the
nature of the germination agent. "Basal spores,"
or spores grown with L-alanine, glucose, or KN03
supplements, did not germinate on glucose to
extent unless heat shocked.
Heated "L-valine spores" and "L-leucine spores"
had somewhat lower levels of germination on
glucose than did "basal spores." "L-glutamate
spores," "L-proline spores," or "synthetic spores"
(Table 2) had reduced heat-activation require-
example, approximately 50% of unheated "L-
glutamate spores" germinated on glucose, as
compared with only 3% of unheated "basal
spores." The patterns of heat activation of "syn-
thetic spores" could, in general, be attributed to
the calcium or glutamate content of the sporula-
The influence of the sporulation medium on
germination induced by L-alanine is in marked
contrast with that for glucose-induced germina-
tion (Table 2). "L-glutamate spores" and "L-
proline spores" were not heat activatable for
germination induced by
spores, grown on KNO3-supplemented medium
germinated to a greater extent than did "basal
spores," and such "KN03 spores," after heating,
had a higher percentage of germination on L-
alanine than did any others we tested. "Glucose
spores" had a moderately increased (over "basal
spores") percentage of germination on L-alanine,
but this was not substantially increased by heat-
ing. L-Alanine-induced germination
grown in the "basal" medium supplemented with
L-alanine or with L-valine was actually depressed
30 ~ ~~~~~
HEAT ACTIVATION (0C)
_ = =n
FIG. 6. Effect of phosphate supplementation of the
sporulation medium on heat activation of Bacillus
megaterium spore germination in glucose, L-alanine,
L-leucine, and KNO3 . Aqueous suspensions of
spores derived from "basal" medium containing 10
or 100 mM phosphate were heated for 10 min at the
by heating at 52.5 or 55 C, but increased when
the spores were heated at 60 C.
At heat-activation temperatures giving essen-
tially no germination of "basal spores," "L-glu-
tamate spores" did germinate on L-leucine or on
KNO3 (i.e., 1% of unheated "basal" and 11% of
L-leucine; 57% of "basal" and 28% of "L-glu-
tamate spores," heated at 55 C, germinated on
We explored the possibility that spores grown
on the medium supplemented with L-glutamate
release substances which are capable of increas-
ing glucose-induced germination. For example,
traces of L-alanine released into the suspending
medium could allow unheated spores to ger-
minate in glucose (Levinson and Hyatt, 1956;
Hyatt and Levinson, 1961). Supernatant fluids
from water suspensions of "basal spores" (2.5
mg of spores per ml) and from spores produced
on the "basal" medium supplemented with 12.5
mM L-glutamic acid, L-proline, D-glucose, L-ala-
nine, L-valine, L-leucine, or KN03 were tested
for ninhydrin reactivity (Moore and Stein, 1948).
The supernatant solutions from "L-glutamate
spores" and from "L-proline spores" contained
0.3 mm and 0.24 mm amino nitrogen, respectively;
that is, five to ten times as much ninhydrin-re-
active material as did sul)ernatant fluids from
VOL. 87, 1964
LEVINSON AND HYATT
TABLE 2. Effect of "heat shock" on germination of Bacillus megaterium spores grown
in various media*
"Basal" sporulation medium +
*Suspensions of spores, heated for 10 min at indicated temperature and incubated in phosphate
buffer, 50mM (pH 7), for 2 hr at 30 C with the indicated germinant. Supplements to "basal" sporulation
medium were at 12.5 mm.
the other spores (0.03 to 0.06 inum). The tempting
theory that this excretion of amino nitrogen was
somehow the basis for increased germination of
unheated "L-glutamate spores" was not sub-
stantiated experimentallv. The addition of super-
natant fluids from "L-glutamate spores" to sus-
pensions of "basal spores" did not increase the
germination of the latter on glucose.
Ionic germination. Unheated spores, produced
on "basal" medium supplemented with either
L-glutamic acid or CaCl2, or produced on the
synthetic medium, were more sensitive to ger-
mnination in salt solutions such as KI (Rode and
Foster, 1962a) than weere spores produced on any
of the other media (Table 3). The differences in
effects of the various media were not so apparent
when heated (30 min at 60 C) spores were used.
W0vith such heat activatioin, spores from all the
media, with the notable exception of the "basal"
medium stupplemente(l with 100 mwlphosphlate,
produced spores which were sensitive to "ionic
Oxygen consumption. The increases in oxygen-
consumption rates associated with germination
and postgerminative development of spores on
glucose (Levinson and Hyatt, 1956) are shown
in Fig. 7. The reduced respiratory rate and the
delay in respiratory-rate changes associated with
postgerminative development of unheated
compared with heated "basal spores" reflect the
necessity for heat activation of germination of
such spores (Fig. 7A). On the other hand, re-
spiratory activities of "CaCl2 spores" (Fig. 7B)
and of "L-glutamate spores" (Fig. 7C) are similar
for heated and unheated spores, reflecting the
reduction in necessity for heat activation (de-
creased "dormancy") of such spores. Unheated
"L-proline spores" (data not shown) also have a
high initial oxygen-uptake rate approaching that
of heated spores. UJnheated spores grown in
MIEDIUMI AND SPORE GERMINATION
TABLE 3. "Ionic germlination" of Bacillus megateriumii spores
Supplement to medium
Concn of supplement
Liver "B" (0.5%)
*"Basal" medium contains Liver "B", 0.5%, to which KH2PO4, 10 mM, has been added.
7. Effect of heat activation and of sporulation
medium on the rate of oxygen consumption of Bacil-
unheated (0) or heated at 60Csfor
incubated at 30 C with phosphate buffer (pH 7.0),
Spores produced on "basal" medium containingA,
CaCl2 ; C,
12.5 MMt L-glutamic acid; or on D, syn-
thetic medium with 0.5 m
or 100 mm (e) phosphate; B,
"basal" medium with increased phosphate (100
(10 mm phosphate),
longer delay in inception of respiratory chnnges
(Fig. 7A). The
of oxygen consumption
heated "basal spores" increased
150 min, corresponding
TABLE 4. Germination and cell division of
Bacillus megateriumn spores produced
on various media*
22 18 (82) 93 81 (87)
71 62 (87)
94 75 (80) 99 66 (67)
99 83 (84)
40 13 (33)
97 29 (30)
were made from contents of Warburg flasks (Fig.
7) after 400 min of incubation with 25 mm glucose,
10 mM (NH4) 2SO4, and 50 mm KH2PO4. G =
germinates; CD = percentage of original spore
inoculum which divides (numbers in parentheses
indicate percentage of germinated spores which
t "Basal" medium contains Liver "B," 0.5%,
to which KH2PO4, 10 mm, has been added.
spore inoculum which
emergence and cell-division stages of postger-
minative development. Cell division continued
for about 1 hr before the rate decreased at ca.
210 min. On the other hand, spores produced on
the synthetic medium appeared to have some
VOL. 87, 1964
LEVINSON AND HYATT
deficiency incapacitating them for full develop-
ment, the respiratory-rate changes accompanying
cell division being depressed (Fig. 7D). The per-
cedtages of spores germinating and dividing in
these media are shown in Table 4. Only a small
percentage of "synthetic spores," as compared
w-ith "basal spores," undergo cell division.
Chemical composition and heat resistance of
enclogenous respiration, and dormancy of fungal
spores (Darby and Mandels, 1955) are influenced
by sporulation conditions. Curran, Brunstetter,
and Myers (1943) were among the first to dem-
onst rate the high calcium content of bacterial
sp)oies and to suggest a relationship between
calcium content and heat resistance. Many other
importance of the observations of Curran and
his associates. We confirm that the calcium con-
tent of spores is important in determining heat
resistance. We also confirm the observations of
Slepeckv and Foster (1959) that the calcium and
mangainese content of spores varies with the
concentration of the metal ion in the sporulation
medium, and that manganese and calcium ions
compete for accumulation in spores. Lechowich
and Ordal (1962) and Levinson et al. (1961)
relportedvirtually simultaneously that the molar
ratio of cations to dipicolinic acid was higher in
slporeswith increased resistance. It would be of
(loosely bound) calcium is involved in heat re-
sistance. Perhaps a situation analogous to that
described by El-Bisi et al. (1962) is operative.
These authors postulated that, of the two forms
of B. subtilis dipicolinic acid (loosely and firmlv
bound), only the firmly bound was determinant
of heat resistance. El-Bisi and Ordal
showed that increasing the phosphate concen-
tration of the sporulation medium yielded B.
coaguilans var. thern2oacidorans spores with low-
ered heat resistance, which they postulated was
owing to decreased availability of calcium in the
growth medium. The heat sensitivity of B. miega-
terium spores produced on the "basal" medium
also increased with increased phosphate
mar), but there wN-as no significant decrease in
calcium incorporation in these spores.
However, we have been mainly concerned with
germination characteristics of spores grown under
various conditions. There was some basis for
believing that different growth conditions would
yield spores with different germination character-
istics. Heat activation was ineffective with B.
media, and oxygen consumption on glucose wNras
stimulated by KN03 only wNhen spores were
produced on glucose-deficient media
Spore dormancy is not an inherent attribute
of spores, but is a function of the germination
medium (Morrison and Rettger, 1930; Foster
and Wynne, 1948). We agree with Keynan et al.
(1961a) that the effect of the sporulation medium
on the state of a given spore cannot be generalized
but must be considered relative to a particular
germination agent. These authors found that
spores of B. cereus strain T, grown so as to con-
tain low levels of dipicolinic acid, did not require
heat shock for germination induced by L-alanine,
but heat shock was required for germination of
spores with high levels of endogenous dipicolinic
acid. In contrast, the rate of germination in cal-
cium-dipicolinic acid was not influenced by either
heat-shock treatment or the endogenous level of
dipicolinic acid. We think it is significant that
L-alanine and calcium-dipicolinic
germination of B. cereus strain T spores tlhrough
different metabolic pathways (Keynan, Murrell,
and Halvorson, 1961b).
With B. niegateriuin, too, the nature and extent
(i.e., alteration of dormancy) depend on both
the germination agent and the sporulation me-
dium. Spores produced on medium with 100 mm
phosphate are less dormant than those produced
with 10 mri phosphate ("basal spores") when
germinated in L-alanine (but not in glucose);
"CaCl2 spores" and "L-glutamate spores" are
less dormant than are "basal spores" if glucose
(but not L-alanine)
cantly, the influence of the sporulation medium
on the physiological state of B. niegaterium spores
is readily demonstrable with agents (glucose and
inducing germination via different
metabolic pathways (Hyatt and Levinson, 1962).
The effectiveness of the synthetic medium in
altering spore dormancy on glucose (Table 2)
may depend on
content. Heated spores of B. cere?is strain T,
grown on G medium with 10 mg of CaCI2 per
liter (Stewart and Halvorson, 1953), actively
spores grown on
is the germinant. Signifi-
its calcium and L-glutamate
SPORULATION AIEDIUM AND SPORE GERMINATION
oxidize glucose without germination (Church and
Halvorson, 1957). On the other hand, Goldman
and Blumenthal (1960) reported that glucose
oxidation by these spores, grown on G medium
with 100 mg of CaCl2 per liter, was always ac-
companied by spore germination. Perhaps the
discrepancy is attributable to different calcium
concentrations in the sporulation medium.
Enzyme patterns and physiological properties
of bacterial endospores are subject to metabolic
Halvorson, 1963). B. cereus strain T spores pro-
duced on media with increased levels of L-alanine
and having increased levels of L-alanine dehy-
drogenase show a significantly decreased rate of
germination on L-alanine and are more dormant
than are spores with the "normal" amount of
L-alanine dehydrogenase. In B.megaterium,too,
physiological differences among spores produced
on different media may be related to the spore's
enzymatic content. Analyses of such spores are
currently being undertaken in our laboratory.
Rode and Foster (1962a, b) proposed that ions
are the primary germination agents ("ionic ger-
mination") and that organic germination agents
such as glucose or L-alanine act as augmenters
of ions. However, we found that B. megaterium
spores do germinate in "deionized glucose" solu-
tions if there is a high enough concentration of
glucose (i.e., 25 mM; unpublished data), and B.
megaterium spores germinate in the absence of
organic "germinants" only after rather extensive
heating of the spores, except when the spores
were grown in the presence of added CaC12 or
glutamate. These are the very sporulation-me-
dium supplements which permit germination on
glucose without heat shock. Perhaps the available
concentration of organic materials in spores is
increased by growth in media supplemented with
CaC12 or with glutamate, and added germinative
ions (such as I-) act by augmentation of the
materials. [Levinson and Sevag (1953) demon-
strated Cl- stimulation of glucose-induced ger-
mination.] In those cases where heating is neces-
sary to demonstrate "ionic germination," perhaps
during heating, and these interact with added
ions (Hyatt and Levinson, 1961). In any case,
the whole question of "ionic germination" war-
rants more intensive investigation.
We cannot but reaffirm the conclusions of
effect of these
are released endogenously
Grelet (1957): "Spores can be formed in a variety
of environments.... These environments may
influence the chemical constitution and enzyme
equipment of the spores.... We think that it is
useful to know the exact conditions which pro-
moted the formation of a crop of spores when
studying the factors which influence the ger-
mination of these spores."
The authors are grateful to H. M. El-Bisi,
G. R. Mandels, and E. T. Reese for their critical
reviews of the manuscript, and to D. R. Linna-
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