Previous Article | Next Article 
Applied and Environmental Microbiology, December 2000, p. 5509-5513, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effect of Calcium in Assay Medium on D
Value of Bacillus stearothermophilus ATCC 7953 Spores
Koichi
Sasaki,1,*
Hideharu
Shintani,2
Junpei
Itoh,3
Takuji
Kamogawa,1 and
Yousei
Kajihara4
Quality Control Department, Misato Plant,
Eisai Co., Ltd., Misato-Machi, Kodama-Gun, Saitama
367-0198,1 Division of Medical Devices,
National Institute of Health Sciences, Setagaya-ku, Tokyo
158-0098,2 PPM Manufacturing, Fukushima
Plant, Nippon Becton Dickinson Company, Ltd., Tsuchifune, Fukushima
City, Fukushima 960-2152,3 and Life
Tech Division, Nihon Pharmaceutical Co., Ltd., Sumiyoshi-cho,
Izumisano City, Osaka 598-8558,4 Japan
Received 24 May 2000/Accepted 29 September 2000
 |
ABSTRACT |
The D value of commercial biological indicator spore
strips using Bacillus stearothermophilus ATCC 7953 was
increased by higher calcium concentrations in assay media. The calcium
concentration in assay media varied among the manufacturers. The
calcium concentration in assay media is an important factor to consider
to minimize the variation of D value.
| |
TEXT |
The effectiveness of sterilization
is examined using biological indicators (BI). Use of a BI provides a
means to directly examine death of microorganisms in the sterilization
process. Therefore, determination of the resistance of BI against the
sterilization method (D value) is an extremely important
parameter for evaluation of the effectiveness of the sterilization
process and equipment. In autoclaving, Bacillus
stearothermophilus ATCC 7953 spores are normally used as BI.
Generally, the D value of BI is determined by using a
soybean casein digest agar medium (SCDA) (15.0 g of casein digest
peptone per liter, 5.0 g of soybean digest peptone per
liter, 5.0 g of NaCl per liter, 15.0 g of agar per liter [pH 7.1 to 7.5]) for the survivor curve method but soybean
casein digest broth medium (SCDB) (17.0 g of casein digest
peptone per liter, 3.0 g of soybean digest peptone per liter,
5.0 g of NaCl per liter, 2.5 g of
K2HPO4 per liter, 2.5 g of glucose per
liter [pH 7.1 to 7.5]) for the fraction negative method.
The D value of B. stearothermophilus ATCC 7953 spores at 121°C was
significantly different when it was determined by using media with the
same composition made by different manufacturers (2).
However, the reason why the D value varies among media of
different manufacturers is not known, and resolution of this problem is
acutely needed to advance validation for scientific assurance of the
sterilization process. We studied the relationship between the calcium
concentration of the medium and the growth of B. stearothermophilus ATCC 7953 spores, because calcium is known to
affect the heat resistance and/or germination of bacterial spores
(1, 3-10).
Abbreviations.
BBL, Becton Dickinson Microbiological Systems;
DAIGO, Nihon Pharmaceutical Co., Ltd.; BIER, biological indicator
evaluation resistometer; NAmSA, North American Science Associates,
Inc., ISO, International Organization for Standardization; BAPTA,
O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid.
Relationship between the D value and the calcium
concentration in the assay medium.
The calcium concentration in
the media was determined by using an atomic absorption
spectrophotometer (AAnalyst 800; Perkin-Elmer, Norwalk, Conn.). The
absorbance was measured at 422.7 nm using a calcium hollow-cathode lamp
as the radiation source and a nitrous oxide-acetylene flame. The ashing
procedure was performed by the method described in United States
Pharmacopeia (9a). The calcium concentrations of the
media are shown in Table 1.
(i) SCDA.
Commercial BI (paper strip type SPORTROL STS-05; lot
no. S53003) was purchased from NAmSA. The BI manufacturer's
specifications were the initial population of 1.8 × 105 spores per strip and the D value of 1.9 min
(at 121°C). Commercial SCDA was obtained from four different
manufacturers (Difco Laboratories [Detroit, Mich.], BBL, Oxoid Ltd.
[Basingstoke, England], and DAIGO). Three separate lots of SCDA from
each manufacturer were used (Difco lot no. 58057JD, 73273JF,
and 100020JB; BBL lot no. E8DFCF, F8DFFV, and J9DHAT; Oxoid lot no. 166 56368, 009 57341, and 219 56605; DAIGO lot no. S105, S109, and S121).
The D values were determined using the survivor curve method
by standard plating techniques (7a). Six replicate strips
were exposed at various time intervals to saturated steam at 121°C in
BIER (Joslyn Sterilizer Corporation). After heating, BI was transferred
immediately to sterile purified water. BI was homogenized to obtain
spore suspensions, if necessary, by serial dilution with sterile
purified water. One milliliter (each) of these spore suspensions was
placed in petri dishes, into which 20 ml of sterilized assay medium was poured. After solidification, they were cultured at 57.5 ± 1°C for 72 h. The logarithmic number of survivors was plotted against exposure time, and the optimal line was determined by regression analysis using the least-squares method. The reciprocal of the slope of
the line obtained was calculated, and the D value was determined.
The calcium concentration in the prepared SCDA was not different among
lots of SCDA from the same manufacturer but differed
significantly
among products from different manufacturers at 1%
level (
= 0.01). The four manufacturers could be divided into
two groups
according to the calcium concentration, which showed
significant
differences at 1% level (
= 0.01). Difco and DAIGO
belonged to
a high-calcium group (mean, 1.41 mM). BBL and Oxoid
belonged to a
low-calcium group (mean, 0.48 mM). The mean calcium
concentrations of
the two groups were 3:1 with a difference of
0.93
mM.
A correlation (
r = 0.921) was observed between the
calcium concentration of SCDA and the
D value (Fig.
1A). The
D value was
highest
at 3.27 min when it was determined using SCDA from DAIGO
(lot no. S121)
and lowest at 1.73 min when it was determined using
SCDA from Oxoid
(lot no. 166 56368), with a difference of 1.54
min. The
D
values obtained showed no significant difference among
the lots of the
same manufacturer but differed significantly among
the manufacturers at
the 1% level (
= 0.01). These results strongly
suggest that
the concentration of calcium in SCDA is largely responsible
for the
variation of the
D value of BI.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 1.
Relationship between the D value of B. stearothermophilus ATCC 7953 spores and the calcium concentration
in assay medium. Commercial SCDA (A) and SCDB (B) were used. Each point
represents the mean of three independent experiments. Symbols:  ,
Difco;  , BBL;  , Oxoid;  , DAIGO. The calcium concentration is
the value in prepared medium.
|
|
(ii) SCDB.
Commercial BI (paper strip type SPORTROL STS-05;
NAmSA lot no. S62705) was used. The BI manufacturer's specifications
were the initial population of 1.9 × 105 spores per
strip and the D value of 1.5 min (at 121°C). Commercial SCDB was obtained from four different manufacturers (Difco, BBL, Oxoid,
and DAIGO). Three separate lots of SCDB from each manufacturer were
used (Difco lot no. 104075JB, 66933JB, and 72198JC; BBL lot no. A8DENZ,
D8DFAZ, and G8DFHH; Oxoid lot no. 018 57329, 073 55886, and 188 56515;
DAIGO lot no. S107, S128, and S130). The D values were
determined using the fraction negative method. The groups of 50 replicate strips were exposed to saturated steam at 121°C in a BIER.
After heating, each strip of BI was immediately transferred to a test
tube that contained 10 ml of SCDB. These test tubes were cultured at
57.5 ± 1°C for 7 days. After incubation, the number of strips
negative for growth was used to calculate the D value
according to the method of limited Stambo-Murphey-Cochran procedure
(7b).
The calcium concentrations in the prepared SCDB were also not
significantly different among lots of the same manufacturer
but showed
significant differences among products of different
manufacturers at
the 1% level (
= 0.01). The manufacturers could
be divided
into two groups: DAIGO in one group and the other three
manufacturers
(Difco, BBL, and Oxoid) in another group. The calcium
concentration was
high in DAIGO (mean, 1.11 mM) but low in the
other group (mean, 0.32 mM) with a significant difference at 1%
(
= 0.01). The mean
calcium concentration in DAIGO was 3.4 times
higher than the mean of
the other three manufacturers, with a
difference of 0.79
mM.
A correlation (
r = 0.934) was observed between the
calcium concentration of SCDB and the
D value (Fig.
1B). The
D value was
highest at 2.36 min when it was determined by
using SCDB from
DAIGO (lot no. S128) but lowest at 1.61 min when it was
determined
by using SCDB from BBL (lot no. A8DENZ), with a difference
of
0.75 min. The
D values obtained showed no significant
difference
among the lots of a manufacturer but differed significantly
among
products of different manufacturers at the 1% level (
= 0.01).
These results strongly suggest that calcium in SCDB is largely
responsible for the variation of the
D value of
BI.
Effect of calcium on the D value.
A 10 mM calcium
solution was prepared by dissolving CaCl2 · 2H2O (Kanto Chemical Co., Inc., lot no. 603E1423) with
purified water and used after sterilization by filtration (pore size,
0.22 µm). The BI were heated at 121°C with a BIER. The initial
population of BI was determined after heat activation at 100°C for 15 min.
(i) SCDA.
Commercial BI (paper strip type SPORTROL STS-05;
NAmSA lot no. S53003) was used. SCDA with different total calcium
concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 mM) were prepared by adding
this calcium solution to sterilized SCDA (BBL lot no. F8DFFV). Changes in the logarithmic mean of the number of surviving spores in
calcium-supplemented SCDA were plotted in Fig.
2. The number of surviving spores in all
heating conditions was increased significantly by adding calcium to the
original SCDA (calcium concentration of 0.21 mM) at the 5% level
( = 0.05). The effect of adding calcium on the number of
surviving spores was more pronounced as the heating time was increased
(Fig. 2). This result suggests that spores are more dependent on
calcium for germination or outgrowth, as a higher percentage of spores
are heat injured. Therefore, calcium is considered to be involved in
the repair of heat-injured spores.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 2.
Effect of calcium on the logarithmic mean of the number
of surviving spores. The calcium concentration is the value in prepared
medium. Each point represents the mean of three independent
experiments. Statistical analysis of data was performed for the
logarithmic mean of surviving spore counts between original SCDA ()
(calcium concentration, 0.21 mM) and calcium-supplemented SCDA.
Statistical significance of differences at the 5% level ( = 0.05) () and the 1% level ( = 0.01) ( ) is indicated.
Non, heat activation only. Heating times involved a temperature of
121°C.
|
|
The change in the
D value was examined with
calcium-supplemented SCDA (Fig.
3A). The
D value increased with the calcium concentration
and nearly
reached a plateau at 3.07 min at calcium concentrations
of 2.0 mM or
above from the value in the original SCDA of 1.87
min. This result
suggests that the optimal calcium concentration
in the assay medium is
required for maximum and consistent
D value
determination. A
calcium concentration of about 2.0 mM was shown
to be necessary to
obtain consistent
D values using SCDA.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 3.
Effect of calcium on the D value of B. stearothermophilus ATCC 7953 spores. The calcium concentration was
changed by the addition of CaCl2 solution to commercial
(original) SCDA (A) and SCDB (B) after autoclaving () or before
autoclaving (). The calcium concentration is the value in prepared
medium. Each point represents the mean of three independent
experiments. Statistical analysis of data was performed for the
D value between the original medium and calcium-supplemented
medium. Statistical significance of differences at the 5% level
( = 0.05) (*, #) and at the 1% level ( = 0.01)
(**, ##) is indicated.
|
|
(ii) SCDB.
SCDB requires a more complex experimental design
than SCDA, because SCDB contains K2HPO4.
Calcium is generally considered to bind with phosphoric acid and to
form insoluble calcium phosphate in the autoclaving process, rendering
it unavailable to microorganisms. Therefore, this assumption is in
conflict with the fact that there was a correlation between the calcium
concentration in the medium and the D value also in SCDB
(Fig. 1B). To answer this question, we conducted two experiments with
SCDB, i.e., the D value was determined for SCDB with added
calcium before autoclaving and for SCDB supplemented with calcium after
autoclaving. SCDB media with different total calcium concentrations
(0.5, 0.75, 1.0, 1.25, 1.75, 2.25, 2.75, and 3.25 mM) were also
prepared by adding the same calcium solution to sterilized SCDB (BBL
lot no. A8DENZ). Other SCDB media were prepared by adding
CaCl2 · 2H2O to SCDB powder, dissolving
the powder, and sterilizing the solution by heating. Commercial BI
(paper strip type SPORTROL STS-05; NAmSA lot no. S62705) was used.
In this experiment, the solution became turbid when calcium solution
was added to SCDB at high calcium concentrations. This
milky
precipitate was washed and centrifuged. After the milky
precipitate was
dried, the elements were analyzed by the energy-dispersive
X-ray
spectroscopy system (scanning electron microscopy [JSM-5400;
JEOL]
and an energy-dispersive X-ray spectrometer [PV9800; EDAX]).
The P/Ca
ratio of milky precipitate was 1.0. This result suggested
that the
precipitates obtained from the turbid SCDB were CaHPO
4.
The
D values determined by the two methods agreed at calcium
concentrations of 1.25 mM or less, and the
D value increased
with the calcium concentration in both experiments (Fig.
3B).
However,
at calcium concentrations exceeding 1.25 mM, the
D value
decreased in SCDB with added calcium before autoclaving, because
of
the formation of CaHPO
4 by
heating.
On the other hand, when calcium was added after autoclaving, the
D value increased to a calcium concentration of 2.25 mM,
a
value 1.43 times the value obtained in the original SCDB. However,
it
decreased at calcium concentrations above 2.25 mM, also because
of the
formation of CaHPO
4. From these results, it may be
concluded
that within certain ranges, the
D value is
correlated with the
calcium concentration. However, why
CaHPO
4 is not formed at a
low calcium concentrations is
unknown at
present.
The important role played by calcium in the assay medium for the
determination of accurate
D values was further demonstrated
by the supplementation
experiments.
Effects of calcium on germination and/or outgrowth.
To test
whether calcium is an indispensable factor in germination and/or
outgrowth of spores of B. stearothermophilus, we used a
specific Ca2+ chelating agent, BAPTA (Dojindo Laboratories
lot no. GN116) (tetrapotassium salt, hydrate). Commercial BI (paper
strip type SPORTROL STS-05; NAmSA lot no. S62705) was used. BAPTA was
added to a final concentration of 0.25 mM to 10 ml of SCDB (BBL lot no.
A8DENZ). To these media, BI that was simply activated by heating
(100°C for 15 min) or BI that was heated at 121°C for 10 min with
BIER was added. Fifty strips of BI were used under each condition. Two
sets of samples were prepared for each condition. The same procedure
was performed with media not containing BAPTA as controls. BI was
cultured in these media at 57.5 ± 1°C for 7 days. After
culturing, a calcium solution was added to the controls and one of the
two sets of the media at 0.25 mM, and BI was cultured for 7 more days.
Each culture was observed daily for the growth of BI after germination.
All spores that were simply heat activated showed outgrowth after 1 day
of culturing in BAPTA-free SCDB (Fig.
4A). However,
no outgrowth was noted in
heat-activated spores in BAPTA-supplemented
SCDB, even after 7 days of
culturing (Fig.
4A). When calcium was
added on day 7 of culturing,
outgrowth occurred in 100% of the
spores, even in BAPTA-supplemented
SCDB (Fig.
4A). On the other
hand, no outgrowth was observed in
calcium-free SCDB even after
14 days of culturing (Fig.
4A). When the
spores were treated at
121°C for 10 min, 30% of the spores showed
outgrowth in BAPTA-free
SCDB (Fig.
4B). However, in BAPTA-supplemented
SCDB, no outgrowth
was noted even after 7 days of culturing (Fig.
4B).
When calcium
was added on day 7 of culturing, outgrowth was noted in
20% of
the spores on the next day and in 30% of the spores after 3 days
(Fig.
4B). In contrast, no outgrowth was observed in calcium-free
SCDB even after culturing for 14 days (Fig.
4B). From these results,
it
appears certain that calcium is an indispensable factor in
the
germination and/or outgrowth of spores regardless of heat
injury.
Whether calcium is needed for germination or outgrowth
could not be
determined.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 4.
Effect of calcium on the outgrowth rate of B. stearothermophilus ATCC 7953 spores. The BI were simply activated
by heating (A) or were heated at 121°C for 10 min (B). The
outgrowth rates were the percentages of positive BI strips/tested BI
strips. Each point represents the mean of three independent
experiments. Symbols: , control (SCDB); , added CaCl2
solution to SCDB containing 0.25 mM BAPTA after 7 days (final calcium
concentration, 0.25 mM);  , SCDB containing 0.25 mM BAPTA.
|
|
In this study we determined that calcium concentration in assay
media plays an important role in the
D value. We concluded
that more-consistent determination of the
D value of
B. stearothermophilus ATCC 7953 spores becomes possible when
the calcium concentration
of the medium is controlled at an appropriate
level. Although
calcium was shown to be important for the germination
or outgrowth
of
B. stearothermophilus, its mode of action
has not been elucidated.
It remains to be determined at which stage of
the germination
calcium is involved. It is also necessary to examine
whether the
importance of calcium in the assay media is limited to
B. stearothermophilus ATCC 7953 spores or whether it applies
to all bacterial spores.
Research concerning these points is expected
to lead to the establishment
of conditions that allow more consistent
determination of the
D value. Since BI of different lots was
used in this study, comparison
between the survivor curve method using
agar media and the fraction
negative method using liquid media was not
possible. In the future,
we will study whether there is a difference
between the two methods
and attempt to develop suitable media for
more-consistent determination
of the
D value of
B. stearothermophilus ATCC 7953
spores.
The official document (
7c) requires that the
D
value of BI be within 0.5 min of the value declared by the
manufacturer.
However, this study showed that this requirement might
not be
met if assay media with different calcium concentrations are
used.
The
D values determined in this study were different
from the
values determined by BI manufacturers because different
manufacturers
use different media of their choice. This particular
point is
important when users of BI select assay media for the
determination
of the
D value.
| |
ACKNOWLEDGMENTS |
This work was supported in part by funds from the Ministry of
Health and Welfare.
We thank Tadayo Hashimoto (Becton Dickinson Company), Kazuhito Watabe
(Setsunan University), Mamoru Kokubo (Shibuya Kogyo Co., Ltd.) and
Masaki Takahashi (Terumo Corporation) for valuable advice. We are
indebted to Yasuhiro Mita, Shigemitu Ohsawa, and Tsunehiko Kataoka
(Eisai Co., Ltd.) who provided the opportunity for this study. We are
also very grateful to Emiko Nakamoto, Koichi Hachimura, Miyako Saito,
Yumi Mori, Kieko Shibasaki, Akemi Yamamoto, Michiko Kishi, Junko
Hagiwara, Masako Sakamoto (Eisai Co., Ltd.) and Atsuko Tanaka (Nihon
Pharmaceutical Co., Ltd.) for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Quality Control
Department, Misato Plant, Eisai Co., Ltd., 950, Hiroki, Misato-Machi, Kodama-Gun, Saitama 367-0198, Japan. Phone: 81-495-76-4365. Fax: 81-495-76-3942. E-mail: k-sasaki{at}hhc.eisai.co.jp.
| |
REFERENCES |
| 1.
|
Amaha, M., and Z. J. Ordal.
1957.
Effect of divalent cations in the sporulation medium on the thermal death rate of Bacillus coagulans var. thermoacidurans.
J. Bacteriol.
74:596-604[Free Full Text].
|
| 2.
|
Boris, C., and G. S. Graham.
1985.
The effect of recovery medium and test methodology on biological indicators.
Med. Device Diagn. Ind.
7:43-48.
|
| 3.
|
Campbell, L. L.,
C. M. Richards, and E. E. Sniff.
1965.
Isolation of strains of Bacillus stearothermophilus with altered requirements for spore germination, p. 55-63.
In
L. L. Campbell, and H. O. Halvorson (ed.), Spores III. American Society for Microbiology, Washington, D.C.
|
| 4.
|
Cook, A. M., and R. J. Gilbert.
1968.
Factors affecting the heat resistance of Bacillus stearothermophilus spores.
J. Food Technol.
3:285-293.
|
| 5.
|
Davis, S. B.,
R. A. Carls, and J. R. Gillis.
1978.
Recovery of sublethal sterilization damaged Bacillus spores in various culture media.
Dev. Ind. Microbiol.
20:427-438.
|
| 6.
|
Edwards, J. L., Jr.,
F. F. Busta, and M. L. Speck.
1965.
Thermal inactivation characteristics of Bacillus subtilis spores at ultrahigh temperatures.
Appl. Microbiol.
13:851-857[Medline].
|
| 7.
|
Hashimoto, T.,
W. R. Frieben, and S. F. Conti.
1969.
Microgermination of Bacillus cereus spores.
J. Bacteriol.
100:1385-1392[Abstract/Free Full Text].
|
| 7a.
|
International Organization for Standardization.
1994.
Sterilization of health care products. Biological indicators. Part 1. General. ISO 11138-1.
International Organization for Standardization, Geneva, Switzerland.
|
| 7b.
|
International Organization for Standardization.
1998.
Biological indicators: guidance for the selection, use and interpretation of results. ISO draft international standard 14161.
International Organization for Standardization, Geneva, Switzerland.
|
| 7c.
|
International Organization for Standardization.
1995.
Sterilization of health care products. Biological indicators. Part 3. Biological indicators for moist heat sterilization.
International Organization for Standardization, Geneva, Switzerland.
|
| 8.
|
Kamat, A. S.,
N. F. Lewis, and D. S. Pradhan.
1985.
Mechanism of Ca2+ and dipicolinic acid requirement for L-alanine induced germination of Bacillus cereus BIS-59 spores.
Microbios
44:33-44[Medline].
|
| 9.
|
Shibata, H.,
S. Adachi,
Y. Hirose,
M. Ike,
I. Tani, and T. Hashimoto.
1993.
Role of calcium in biphasic germination of Bacillus cereus T spores sensitized to lysozyme.
Microbiol. Immunol.
37:187-194[Medline].
|
| 9a.
|
The United States Pharmacopeial Convention.
2000.
United States pharmacopeia., 24th ed.
The United States Pharmacopeial Convention, Inc., Rockville, Md.
|
| 10.
|
Yokoya, F., and G. K. York.
1965.
Effect of several environmental conditions on the "thermal death rate" of endospores of aerobic, thermophilic bacteria.
Appl. Microbiol.
13:993-999[Medline].
|
Applied and Environmental Microbiology, December 2000, p. 5509-5513, Vol. 66, No. 12
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Top
Abstract
Text
