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
LEVINSON AND HYATT
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J . BACTERIOL.