Journal of Applied Microbiology 1998, 85, 865874

Biological indicators for steam sterilization: characterization

of a rapid biological indicator utilizing Bacillus
stearothermophilus spore-associated alpha-glucosidase
enzyme
H. Albert
1
, D.J.G. Davies
1
, L.P. Woodson
2
and C.J. Soper
1
~
1
School of Pharmacy and Pharmacology, University of Bath, Bath, UK and
2
3M Health Care, Medical Products
Technology Division, St Paul, MN, USA
6430/10/97: received 14 October 1997, revised 15 June 1998 and accepted 17 June 1998
H. ALBERT, D. J. G. DAVI ES, L. P. WOODSON AND C. J. SOPER. 1998. The alpha-glucosidase enzyme
was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953.
Spore-associated enzyme had a molecular weight of approximately 92 700, a temperature
optimum of 60 C, and a pH optimum of 7075. The enzyme in crude aqueous spore extract
was stable for 30 min up to a temperature of 65 C, above which the enzyme was rapidly
denatured. The optimal pH for stability of the enzyme was approximately 72. The alpha-
glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated
enzyme but its molecular weight was 86 700. The vegetative cell and spore-associated enzymes
were cross-reactive. The enzymes are postulated to derive from a single gene product, which
undergoes modication to produce the spore-associated form. The location of alpha-
glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of
most enzymes involved in activation, germination and outgrowth.
I NTRODUCTI ON
Sterilization is the act or process, physical or chemical, that
destroys or eliminates all forms of life, especially micro-
organisms. This term is absolute, i.e. a substance cannot
be partially sterile (Joslyn 1991). Due to the difculty in
conrming sterility, a more practical denition of sterility has
been adopted, dening sterility as the process by which living
organisms are removed or killed to an extent that they are no
longer detectable in standard culture media in which they
have previously been found to proliferate (Joslyn 1991). In
practice, assurance of a low probability of any living micro-
organisms remaining is used as a measure of sterility. Sterility
of a particular item can only be conrmed by destructive
testing of the item, which is not practical for most purposes.
Sterilization monitoring improves the assurance that medi-
cal devices have been adequately sterilized. Biological moni-
toring is accepted as the most effective method. Biological
indicators function by introducing highly resistant bacterial
Correspondence to: Lewis P. Woodson, 3M Health Care, Medical Products
Technology Division, Building 270-3N-02, St Paul, MN 55144-1000, USA
(e-mail: lpwoodson@mmm.com).
~Deceased.
1998 The Society for Applied Microbiology
spores into the sterilization cycle. If these spores are
destroyed, it is assumed that any contaminating organisms in
the load have also been killed, as these organisms have lower
resistance than the spores, and are present in lower numbers
(Pug and Odlaug 1986). Daily biological monitoring (and
monitoring of each load containing implants) is recommended
by the Association of Operating Room Nurses (Association
of Operating Room Nurses 1994), the Joint Commission on
Accreditation of Healthcare Organizations (Joint Commission
on Accreditation of Healthcare Organizations 1994), the
American Hospital Association, and the Association for the
Advancement of Medical Instrumentation (Association for
the Advancement of Medical Instrumentation 1993). The
Centers for Disease Control recommend weekly biological
monitoring (plus each load containing implantable devices).
Biological indicators consist of an inoculated carrier con-
tained within its primary pack ready for use, and providing a
dened resistance to the specied sterilization process. An
inoculated carrier is a carrier on which a dened number
of test organisms have been deposited (Association for the
Advancement of Medical Instrumentation; AAMI ST59).
The test organism used for steam sterilization is B. ste-
arothermophilus because of its high resistance and consistent
866 H. ALBERT ET AL.
inactivation kinetics. The original format of biological indi-
cators was inoculated paper strips inside envelopes, which
were transferred to sterile culture medium following process-
ing, and incubated for 7 d (Rutala et al. 1996). Sterilization
failure was measured by turbidity of the growth medium.
Second generation biological indicators are self-contained
systems which comprise the spore strip and growth medium
required for recovery in a primary pack ready for use. These
biological indicators rely on a pH indicator to measure pro-
duction of acid metabolites in the growth medium by out-
growing spores and replicating cells. These systems remove
the problem of contamination during manipulation enco-
untered with the rst generation indicators, but 24168 h
readout time is still required for detection of surviving spores
(Rutala et al. 1996).
Recently, the third generation biological indicator,
Attest
TM
1291 Rapid Readout Biological Indicator (3M, St
Paul, MN, USA), was developed for use in 132 C gravity
steam sterilization cycles (ash sterilization) in view of the
need for rapid results in emergency sterilization procedures
used in the operating room (Vesley et al. 1992). In 1996,
another third generation biological indicator was developed
for use in 121 C gravity steam sterilization and 132 C vac-
uum-assisted steam sterilization (Attest
TM
1292 Rapid Read-
out Biological Indicator; 3M) (Vesley et al. 1995). These third
generation biological indicators have a dual readout system.
The rapid portion detects active spore-associated a-glu-
cosidase enzyme surviving the sterilization process within 1
3 h. The enzyme is a normal constituent of vegetative cells and
spores of B. stearothermophilus. It is consistently detectable in
viable spores but is destroyed by steam sterilization just after
spore kill (Vesley et al. 1992, 1995). The survival of the spore-
associated a-glucosidase enzyme following exposure to steam
sterilization correlates well with spore survival. The second
readout system comprises a pH indicator which detects acid
metabolites produced by outgrowing spores and replicating
cells, and gives conrmation of the rapid result within 24
168 h (Vesley et al. 1992, 1995).
Alpha-glucosidases catalyse the hydrolysis of terminal,
non-reducing a-glycosidic bonds in short-chain saccharides,
produced by the action of other enzymes, e.g. amylases on
starch releasing D-glucose. Alpha-glucosidases from veg-
etative cells of various bacteria have been isolated and char-
acterized (Suzuki et al. 1976; Urlaub and Wober 1978; Suzuki
et al. 1979, 1982, 1984; Kelly et al. 1986 ). They are found
widely in animals, plants and micro-organisms, and are usu-
ally found in association with amylases (Sata et al. 1987) to
effect the complete degradation of starch and other macro-
molecules (Kelly and Fogarty 1983). The substrate specicity
of the enzyme is dependent on its source(Suzuki et al. 1976,
1979, 1982; Kelly and Fogarty 1983; Suzuki et al. 1984). The
a-glucosidase of B. stearothermophilus ATCC 7953 is most
similar to the less common group of a-glucosidases which
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 865874
have high activity towards p-nitrophenyl-a-D-glucoside
(PNPG), and activity towards maltose and malto-oli-
gosaccharides, but little or no activity towards a-1,6 glycosidic
bonds, i.e. isomaltose and isomaltosaccharides (Kelly et al.
1986). Microbial a-glucosidases can be intracellular, cell-
bound or extracellular enzymes (Urlaub and Wober 1978;
Suzuki et al. 1979; Kelly et al. 1986).
There is little information available regarding the presence
and potential role of bacterial spore-associated a-glucosidase
enzyme. This study was undertaken to characterize the a-
glucosidase enzyme utilized as a rapid monitor of sterilization
efcacy. The extraction and characterization of a-glucosidase
from vegetative cells and spores of B. stearothermophilus
ATCC 7953 are described. Factors affecting the activity of
the enzyme, both during growth of vegetative cell cultures
and spores and during spore germination, will be determined.
The localization of the enzyme within the spore was deter-
mined by a two-step electron microscopic immunolabelling
method using anti-a-glucosidase monoclonal antibody and
colloidal gold-IgG complex, and a role of the enzyme in the
function of the spore was postulated.
MATERI ALS AND METHODS
Bacterial strains and growth conditions
Bacillus stearothermophilus was provided as spore strips (cul-
ture number 213, June 1990) by 3M, St Paul, MN, USA,
derived from ATCC 7953 culture. For culture maintenance,
a spore strip was grown in tryptic soy broth (TSB) overnight
in a shaking incubator. This stock vegetative cell culture was
stored in 1 ml aliquots in liquid nitrogen. For the primary
inoculum, 1 ml of the stock vegetative cell culture was used.
Preparation of crude spore extract
A stationary phase culture was produced in TSB at 60 C
from the stock vegetative cell culture. This culture was used
to inoculate sporulation agar plates consisting of yeast extract
4 g l
1
, nutrient broth 8 g l
1
, manganese chloride 01 g l
1
and agar 20 g l
1
, at a nal pH of 72 201. Sporulation agar
plates were incubated, inverted, at 60 C for 72 h. Spores
were harvested and washed using ice-cold deionized water
and stored at 4 C. Several batches of spore suspensions
were combined; the spores were suspended in 50 mmol l
1
phosphate buffer pH74, and diluted to give a viable count
of approximately 1 10
9
spores ml
1
. The spore suspension
was divided into 20 ml volumes and sonicated on ice for
5 min. The sonicate was centrifuged at 4000 rev min
1
for
20 min. The supernatant uid was stored at 4 C. The spore
pellet was suspended in fresh buffer and sonicated for a 1
min period followed by centrifugation and resuspension in
fresh buffer until no discernible a-glucosidase was present in
RAPI D BI OLOGI CAL I NDI CATOR FOR STEAM STERI LI ZATI ON 867
the supernatant uid (up to three times). The supernatant
uids were combined and concentrated to a nal volume of
5 ml using ultraltration units 8010, 8050 and 8200 (Amicon
Division, W.R. Grace and Company, Danvers, MA, USA)
and Diao ultraltration membranes (10 000 molecular
weight retention). The concentrated spore extract was stored
at 4 C. The crude vegetative cell extract was produced from
a logarithmic phase culture in TSB at 60 C, using the son-
ication method described above.
Chemicals and reagents
Microbiological reagents were obtained from Difco. Other
chemicals were of analytical reagent grade and supplied by
Sigma. Electron microscopic reagents were obtained from
Agar Scientic Ltd unless stated otherwise.
Alpha-glucosidase assay
Alpha-glucosidase activity was measured using two methods.
Method 1. Alpha-glucosidase was determined by the spec-
trophotometric measurement at 410 nm of the release of p-
nitrophenol from the substrate p-nitrophenyl-alpha-D-glu-
coside (PNPG). Assay mixtures consisted of 5 mmol l
1
PNPG with 100 ml of an appropriate dilution of the crude
spore extract in a total volume of 800 ml in 50 mmol l
1
phosphate/EDTA buffer pH74. A Pye-Unicam UV/VIS
kinetics spectrophotometer was used for all absorbance
measurements. The absorbance of the reaction mixture was
recorded at 410 nm at 15 s intervals in a temperature-con-
trolled chamber at 60 C. The linear reaction velocity (change
in absorbance per minute) was calculated from the gradient
of the linear portion of the reaction prole and used to deter-
mine the a-glucosidase activity (mol PNP min
1
mg
1
protein).
Method 2. Alpha-glucosidase activity was determined by the
uorimetric detection of 4-methylumbelliferone (4-MU)
released from 4-methylumbelliferyl-alpha-D-glucoside (4-
MUG). The buffer or culture medium was added to a mic-
rotitre plate (Costar, Cambridge, MA, USA). The appro-
priate volume of 4-MUG solution was added (01 g l
1
). The
spore suspension or crude extract was added, and the reaction
mixture was thoroughly mixed. The microtitre plate was
incubated for 30 min at 60 C. The uorescence (l
em
455 nm,
l
ex
360 nm) was measured immediately at room temperature
using a plate-reading uorimeter (Fluoroskan II, Titertek,
Northbrook, IL, USA).
Protein concentration was assayed by the method of Lowry
(Lowry et al. 1951) using bovine serum albumin (BSA) as
standard.
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 865874
Molecular weight determination
An SDS-PAGE minigel system (Pharmacia Biotech,
Uppsala, Sweden) and a 125% SDS gel were used in con-
junction with low range molecular weight markers (BioRad)
to determine the molecular weights of the spore and veg-
etative cell enzyme sub-units (Suzuki et al. 1976). ASephadex
G-200 gel ltration column was used with molecular weight
markers to separate the protein bands and assay the fractions
for a-glucosidase activity. Gel ltration was also used to
determine the molecular weight of the intact enzyme
molecule.
Immunolabelling
The samples of spore and vegetative cell extract were run on
an SDS-PAGE gel, with molecular weight markers. Fol-
lowing electrophoresis, the gel was rinsed in transfer buffer
for 1 h prior to being electrophoretically transferred to a
nitrocellulose membrane (BioRad) using a Mini Transblot
Cell (BioRad) at 100 V for 1 h. The nitrocellulose membrane
was incubated in blocking buffer (01% Tween-20, 01%
BSA in Tris-buffered saline) for 1 h at room temperature.
The blocking buffer was removed and the membrane was
incubated in a 1 in 50 dilution of mouse antispore a-glu-
cosidase monoclonal antibody in blocking buffer for 4 h at
room temperature. The primary antibody solution was
removed and the membrane was thoroughly washed in block-
ing buffer. A 1 in 50 dilution of the secondary goat antimouse
gold-conjugated antibody (British Biocell International, Car-
diff, UK) was added and incubated at room temperature for
2 h. The membrane was rinsed with TBS. A red colouration
of labelled bands was observed.
Properties of the alpha-glucosidase
The effect of pH on a-glucosidase activity was determined
by assaying the crude vegetative cell extract and crude spore
extract (375 10
4
mg protein) for a-glucosidase activity in
So rensens phosphate buffers of pH between 50 and 82,
with 5 mmol l
1
PNPG at 60 C. The effect of pH on a-
glucosidase activity was measured by diluting the crude veg-
etative cell extract and crude spore extract (3 10
3
mg
protein) 1 in 10 in each of the So rensens phosphate buffers.
The mixtures were incubated at 60 C for 30 min, then buff-
ered PNPG at pH74 was added to give a nal concentration
of 5 mmol l
1
PNPG in the reaction mixture. The a-glu-
cosidase activity was measured at 60 C.
The effect of temperature on a-glucosidase activity was
determined by measuring the a-glucosidase activity of crude
vegetative cell extract and crude spore extract (375 10
4
mg protein) at a series of temperatures with 5 mmol l
1
PNPG at pH74. Alpha-glucosidase stability was determined
868 H. ALBERT ET AL.
by incubating the crude vegetative cell extract and crude
spore extract in phosphate/EDTA buffer pH74 for 30 min
at a series of temperatures. The a-glucosidase activity of the
extract (375 10
4
mg protein) was determined at 62 C at
pH74 with 5 mmol l
1
PNPG.
The substrate specicity of the spore-associated a-glu-
cosidase was assessed using the following method. Solutions
(10 mmol l
1
) of the following carbohydrates were prepared:
maltose, maltotriose, maltotetraose, maltopentaose, mal-
tohexaose, nigerose, trehalose, isomaltose, sucrose and
glucose. A 100 ml aliquot of crude spore extract (003 mg ml
1
protein) was incubated in a 10 mmol l
1
solution of each
carbohydrate in 50 mmol l
1
phosphate buffer pH74 for
30 min at 60 C. A sample of each reaction mixture was
subjected to four to six ascents of ascending paper chro-
matography (Whatman 54 lter paper) using n-buta-
nol/pyridine/water (6:4:3) (Suzuki et al. 1984). The
chromatogram was air-dried. Reducing sugars were vis-
ualized using a modied silver dip method (Suzuki et al.
1976). Solution Awas prepared by diluting 10 ml of saturated
silver nitrate to 60 ml with water and then to 200 ml with
acetone. Solution B consisted of 20% of a solution of 10%
sodium hydroxide and 80% methanol. Solution C was an
aqueous solution of 05 mol l
1
NaS
2
O
3
. The dried chro-
matogram was dipped into solution A and allowed to air dry.
It was then dipped into solution B until black spots appeared.
The chromatogram was next washed with distilled water and
placed in solution C until background colouration disappeared.
The chromatogram was rinsed with distilled water. The glu-
cose solution was used as a standard to compare the position of
the reaction products on the chromatographic paper. Glucose
was produced by reaction of a-glucosidase with various carbo-
hydrates.
Kinetic parameters
The kinetic parameters, K
m
and V
max
, were determined using
the direct linear plot (Eisenthal and Cornish-Bowden 1974;
Cornish-Bowden and Eisnethal 1974). The a-glucosidase
activity was determined by the PNPG assay using a series of
substrate concentrations: 02 mmol l
1
, 05 mmol l
1
,
10 mmol l
1
, 15 mmol l
1
, 20 mmol l
1
, 25 mmol l
1
,
30 mmol l
1
and 50 mmol l
1
. The linear reaction velocity
was calculated from the gradient of the initial portion of the
reaction prole for each substrate concentration. A Hanes
plot (Fig. 6) was initially constructed to inspect the linearity
of the data obtained visually. Pairs of (v, s) values were
generated in the usual manner. The data were analysed using
Enzpack 3 version 30 (published and distributed by
BIOSOFT, Cambridge, UK). This software allowed the cal-
culation of kinetic parameters by the Direct Linear Plot
method and also the Hanes plot, Lineweaver-Burk plot and
the Eadie-Hoftsee plot.
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 865874
Antibody production
Balb/c mice were immunized three to ve times with B.
stearothermophilus ATCC 7953 spores by interperitoneal
injection at 23 week intervals, with a nal injection of
1 10
6
cells given intravenously 3 d prior to hybridization
of the mice spleen cells and Balb/c NS-1 myeloma cells in
35% polyethylene glycol. The treated cells were allowed to
recover for 24 h in Heaps Dulbeccos Modied Eagle Medium
(HDMEM) with 10% foetal calf serum. The cells were
diluted to 75 10
5
ml
1
in hypoxyanthene aminopterin thy-
midine (HAT) medium, and plated in microtitre plates, the
mediumreplaced as required and observed for the appearance
of hybridoma growth. Supernatant uids from the resulting
hybridoma cultures were screened for the production of spore
a-glucosidase-specic antibody by Particle Concentration
Fluorescence Immunoassay (PCFIA) using the IDEXX
Screen Machine (IDEXX Corporation, Portland, OR, USA).
IDEXX carboxylated particles were added to sodium phos-
phate buffer pH70, and incubated at 37 C for 30 min. 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC)
(05 mg ml
1
), a-glucosidase (1 mg ml
1
) from B. ste-
arothermophilus and 2% non-fat dry milk in borate buffer
were added. The beads were centrifuged and the pellet sus-
pended in 005% sodium azide in sodium phosphate buffer
pH70. Binding of the cell culture supernatant uids to the
a-glucosidase-coated beads was measured by the IDEXX
Screen Machine. Balb/c mice primed with pristane and
allowed to rest for 10 d were injected intraperitoneally with
approximately 2 10
6
cells of the cultures showing the high-
est level of binding to spore a-glucosidase. The ascites uid
was collected and the monoclonal antibodies were puried
using the Af-Gel Protein A MAPS 11 system (BioRad).
The immunoglobulin fractions were eluted, pooled, dialysed
overnight and stored in 002% sodium azide in PBS at 4 C.
Electron microscopy and immunolabelling
For preparation of spore sections for examination of ultra-
structure (Fig. 1), spores were xed in 3% glutaraldehyde
and 1% acrolein in 50 mmol l
1
phosphate buffer pH74,
then xed in 1% osmium tetroxide solution at room tem-
perature for 1 h. Following washing in sterile distilled water,
spores were serially dehydrated in 30%, 50%, 70%, 80%,
90%, 95%, 100% acetone and 100% dry acetone. Spores
were suspended in the following series of mixtures of dry
acetone and medium hardness Transmit EM resin (Taab
Laboratories Equipment Ltd, Reading, UK), respectively:
3:1, 1:1 and 1:3, and stored in 100% resin for several days at
room temperature. The samples were polymerized in gelatin
capsules at 70 C for 18 h prior to sectioning.
For immunoelectron microscopic localization, freshly pre-
pared spores were xed using either freeze-drying or incu-
RAPI D BI OLOGI CAL I NDI CATOR FOR STEAM STERI LI ZATI ON 869
12
12
00
0
Time (min)
A
b
s
o
r
b
a
n
c
e

a
t

4
9
0

n
m
5
R
e
l
a
t
i
v
e

f
l
u
o
r
e
s
c
e
n
c
e
5000
0
10
08
06
04
02
6 7 8 9 1 2 3 4
4000
3000
2000
1000
10 11
Fig. 1 Production of a-glucosidase during vegetative cell growth
of B. stearothermophilus, ATCC 7953 in TSB at 60 C. (),
a-glucosidase (relative uorescence); (), absorbance at 490 nm
bation in 3% glutaraldehyde solution in phosphate buffer
pH74 at room temperature for 16 h. Spores were washed
twice in phosphate buffer pH74. Glutaraldehyde-xed
spores were serially dehydrated in 30%, 50%, 70%, 90%
ethanol and 100% dry ethanol at room temperature. Spores
were inltrated with LR White resin (London Resin
Company, London, UK) with 05% w/v benzoin methyl
ether, in the following combinations of dry ethanol and resin
mixture, respectively: 3:1, 1:1 and 1:3. Spores were then
incubated in several changes of 100% resin mixture and
stored for 35 d at 4 C. Samples were polymerized by u.v.
light (l 360 nm) for 3 d in gelatin capsules at 4 C. Ultra-
thin sections were cut with a glass knife on a OMU3 Reichert
ultramicrotome, and mounted on gilded nickel grids (EML
Electron Microscopy Laboratories Ltd, Stansted, UK).
Ultra-thin sections were incubated in blocking buffer (gel-
atin 05%, bovine serum albumin 01%, Tween-20 01% in
Tris buffer pH82) for 5 min at room temperature. The
grids were transferred to appropriate dilutions of monoclonal
antibody in blocking buffer and incubated for 2 h. The grids
were washed in blocking buffer for 30 min, then incubated in
a 1 in 10 dilution of gold-conjugated secondary antibody
(Super EM Grade gold conjugate goat antimouse IgG, 15 nm
gold; British Biocell International, Cardiff, UK) in blocking
buffer for 30 min and washed in sterile distilled water. Sec-
tions were viewed using a Jeol JM 1200EX electron micro-
scope (Tokyo, Japan) at 80 kV.
RESULTS
After an initial lag phase at the start of vegetative cell growth,
the a-glucosidase activity increased during logarithmic
growth of the culture (Fig. 1). Activity reached a plateau at
the onset of stationary phase (45 h) and the a-glucosidase
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 865874
activity rapidly reduced after 8 h growth under these exper-
imental conditions.
The optimum pH for enzyme activity was approximately
pH78 (Fig. 2). The optimum pH for enzyme stability was
approximately 735 (Fig. 3). The activity of the enzyme
increased rapidly from 20 C, reaching an optimum activity
at approximately 63 C in the crude extracts (Fig. 4). The a-
glucosidase activity dropped sharply at temperatures between
63 C and 77 C. The a-glucosidase was stable up to approxi-
mately 65 C, above which its stability rapidly decreased
(Fig. 5). This high thermostability of the enzyme in liquid
extract reects the expected increase in stability of the enzyme
in anhydrous form, and its use as a monitor of steam ster-
ilization. Characteristics of the spore-associated and veg-
85
0016
0000
55
pH
R
e
a
c
t
i
o
n

v
e
l
o
c
i
t
y

(
m
o
l

P
N
P

m
i
n

1

m
g

1

p
r
o
t
e
i
n
)
0010
80
0014
0012
0008
0006
0004
0002
60 65 70 75
Fig. 2 Effect of pH on the reaction velocity of a-glucosidase.
Reaction was carried out using crude cell or spore extract of
Bacillus stearothermophilus, ATCC 7953 (375 10
4
mg ml
1
protein), 5 mmol l
1
PNPG at 60 C. (), Vegetative cell enzyme;
(), spore enzyme
84
0014
0000
64
pH
R
e
a
c
t
i
o
n

v
e
l
o
c
i
t
y
(
m
o
l

P
N
P

m
i
n

1

m
g

1

p
r
o
t
e
i
n
)
0010
74
0012
0008
0006
0004
0002
66 68 70 72 76 78 80 82
Fig. 3 Effect of pH on the stability of a-glucosidase. Reaction
velocity (at pH 74, 60 C, 5 mmol l
1
PNPG) was determined after
incubation for 30 min at 60 C at a series of pH values. Crude
vegetative cell and spore extracts of Bacillus stearothermophilus,
ATCC 7953 were used (375 10
4
mg ml
1
protein). (),
Spore enzyme; (), vegetative cell enzyme
870 H. ALBERT ET AL.
80
0020
0000
20
Temperature (C)
R
e
a
c
t
i
o
n

v
e
l
o
c
i
t
y
(
m
o
l

P
N
P

m
i
n

1

m
g

1

p
r
o
t
e
i
n
)
0015
70
0010
0005
30 40 50 60
Fig. 4 Effect of temperature on the reaction velocity of a-
glucosidase. Reaction was carried out with crude vegetative
cell or spore extract of Bacillus stearothermophilus, ATCC 7953
(375 10
4
mg ml
1
protein), 5 mmol l
1
PNPG at pH 74. (),
Vegetative cell enzyme; (), spore enzyme
80
0025
0000
20
Temperature (C)
R
e
a
c
t
i
o
n

v
e
l
o
c
i
t
y
(
m
o
l

P
N
P

m
i
n

1

m
g

1

p
r
o
t
e
i
n
)
0015
70
0010
0005
30 40 50 60
0020
Fig. 5 Effect of temperature on stability of a-glucosidase.
Reaction velocity (pH 74, 60 C, 5 mmol l
1
PNPG) was
determined after incubation for 30 min at a series of
temperatures. Crude extracts of vegetative cells and spores
of Bacillus stearothermophilus, ATCC 7953 (375 10
4
mg ml
1
protein) were used. (), Vegetative cell enzyme; (), spore enzyme
etative cell forms were very similar, with the spore-associated
enzyme generally having a broader optimum range. The kin-
etic parameters of the cell and spore-associated forms were
similar, K
m
values 1405 mmol l
1
and 144 mmol l
1
PNPG,
respectively (Table 1).
Glucose was produced by reaction of spore-associated a-
glucosidase with maltose and the malto-oligosaccharides (a-
1,4). Asmall amount of glucose was detected following 30 min
incubation at 60 C of a-glucosidase with nigerose (a-1,3)
and trehalose (a-1,1). However, no glucose was formed by
incubation with sucrose (a-1,2) or isomaltose (a-1,6). The
molecular weight of the spore a-glucosidase was 92 700, and
that of the vegetative cell enzyme was 86 700. The properties
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 865874
2
5
12
0
s
s
/
v
6
8
4
1 3 4
10
2
slope = 1/V
max
K
m
/V
max
Fig. 6 Hanes plot for determination of kinetic parameters, K
m
and V
max
. Reaction was carried out with crude vegetative cell extract
in 5 mmol l
1
PNPG in phosphate/EDTA buffer pH 74 at
60 C
Table 1 Kinetic parameters, K
m
and V
max
, for vegetative cell and
spore-associated a-glucosidase in crude extract (375 10
4
mg
ml
1
protein) in PNPG at pH 74, 60 C. Parameters are stated
as the mean 2S.D.

V
max
(mol PNP min
1
K
m
(mmol l
1
) mg
1
protein)

Vegetative cell 1405 20095 001119 2000003

Spore 144 2009 000046 2000004

of the a-glucosidase enzyme from vegetative cells and spores

of B. stearothermophilus ATCC 7953 were compared with
a-glucosidases from other Bacillus species (Table 2). These
enzymes vary in size, optimum conditions for activity and
catalytic properties.
Figure 7 shows the correlation between a-glucosidase
activity (measured by uorescence after 1 h incubation) and
24 h growth readout of Attest
TM
1291 Rapid Readout Bio-
logical Indicators following exposure to 132 C gravity ster-
ilization. Very good correlation existed between 1 h
uorescence and 24 h growth readout results. The enzyme
remained viable longer than the outgrowth of the spores,
giving a margin of safety for the use of the uorescence
readout result.
Figure 8 shows the effect of the length of sporulation on
the activity of a-glucosidase during the germination and out-
growth of spores with varying lengths of sporulation. Ger-
mination of the spore suspension was measured by a reduction
in absorbance at 490 nm. Samples of the germinating spores
were assayed for a-glucosidase activity by uorimetric detec-
tion. There was no increase in a-glucosidase activity above
RAPI D BI OLOGI CAL I NDI CATOR FOR STEAM STERI LI ZATI ON 871
Table 2 Properties of several a-glucosidases from the genus Bacillus

Temperature
optimum pH
Organism MW (C) optimum Specicity of bond cleavage Ref.

Bacillus stearothermophilus ATCC 7953 86 700 (c) 63 77

92 700 (s) 5663 7077 a-1,4 a-1,3, a-1,1. No a-1,2 or a-1,6
Bacillus stearothermophilus ATCC 12016 47 000 70 63 a-1,4. No a-1.6 Hunter 1993
Bacillus thermoglucosidius KP1006 60 000 60 68 a-1,4 a-1,6 Cornish-Bowden
and Eisenthal
1974
Bacillus cereus ATCC 7064 60 000 41 67 exo a-1,6 Eisenthal and
Cornish-Bowden
1974
Bacillus licheniformis 66 000 40 60 a-1,4 a-1,6 Kelly and Fogarty
1983
Bacillus amyloliquefaciens 27 000 39 68 a-1,4 a-1,2 a1,6 Kelly et al. 1986

(c), Cellular form; (s), spore form.

Data from this study.
Data not obtained.
100
0
Exposure time (min)
P
e
r
c
e
n
t
a
g
e

p
o
s
i
t
i
v
e

r
e
s
u
l
t
s
80
60
40
20
1 15 175 2 225 25 3
Fig. 7 Correlation of spore and a-glucosidase survival following
exposure of 3M Attest
TM
1291 Rapid Readout Biological
Indicators to 132 C gravity steam sterilization. (), 60 min
uorescent result; ( ), 24 h growth result (colour change)
the basal level during the rst 2 h of incubation in any of the
spore crops. After this period, the spores started to produce
increasing amounts of enzyme. The highest levels of a-glu-
cosidase were detected in the spores with a 1 d sporulation
period. The activity of a-glucosidase decreased as the length
of sporulation of the spore crops increased.
The general appearance of sections of B. stearothermophilus
spores was highly variable due to inherent heterogeneity of
spore populations and alignment of spores in the resin during
sectioning. Figure 9 shows a typical electron micrograph of
the ultrastructure of a dormant B. stearothermophilus spore.
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 865874
6
700
0
0
Time (h)
F
l
u
o
r
e
s
c
e
n
c
e

u
n
i
t
s
1
600
200
100
2 3 4 5
300
400
500
Fig. 8 Effect of length of sporulation period on the a-glucosidase
activity of spore suspensions during germination in TSB at
60 C. Length of sporulation period: , 1 d; , 2 d; R, 3 d; T,
4 d; E, 5 d
Sections of freeze-dried or glutaraldehyde-xed spores were
reacted with anti-a-glucosidase monoclonal antibodies and
labelled with gold-conjugated secondary antibodies. The col-
loidal gold particles were localized almost exclusively in the
outer coats of dormant spores. Very few gold particles were
associated with spores that had undergone control treatments.
Similar results were obtained with freeze drying or glu-
taraldehyde xation. As the conditions used for glu-
taraldehyde xation were shown to reduce the activity of a-
glucosidase signicantly(unpublished data), it is probable that
the antibody binds at a site distinct from the active site.
872 H. ALBERT ET AL.
Fig. 9 A typical electron micrograph of the ultrastructure of a
dormant Bacillus stearothermophilus ATCC 7953 spore
A high level of background labelling was initially enco-
untered when standard blocking buffer was used (Naish 1989;
Hunter 1993), due to binding of the gold particles to sulphur
present in cysteine-rich proteins of the spore coats. This
background labelling was particularly troublesome in this
situation as the spore coat was a likely site of a-glucosidase
activity. Addition of 05% gelatin to the blocking buffer
eliminated this source of background labelling due to com-
petitive binding of the gold and gelatin.
DI SCUSSI ON
The location of the a-glucosidase outside the spore protoplast
is in common with most enzymes involved in activation,
germination and outgrowth. Immunoelectron microscopic
localization of the Ger A proteins (Ger AA, Ger AB and
Ger AC), putative receptor proteins for the spore germinant,
L-alanine, found these proteins to be located on the inner
surface of cortex-less spore coat fragments (Sakae et al. 1995).
Ger AB protein was also localized just inside the spore coat
layer of B. subtilis spores using transmission electron
microscopy (Yasuda et al. 1996). Although bacterial spores
are metabolically dormant, they retain an alert sensory mech-
anism which allows them to detect germinants in their exter-
nal environment and trigger germination. The systems
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 865874
Fig. 10 Immunoelectron microscopic localization of a-
glucosidase enzyme in dormant spores of Bacillus stearothermophilus
ATCC 7953 xed in 3% glutaraldehyde in phosphate buffer pH
74. Ultrathin sections were labelled with normal mouse
serum and colloidal gold (15 nm)-IgG complex. Bar, 200 nm
responsible for germination triggering must escape the mech-
anisms of stabilization which result in spore dormancy, at the
same time retaining the resistance properties of the whole
spore (Foster and Johnstone 1989). The location of the a-
glucosidase in the outer spore coats would also correlate with
high inherent thermostability of the enzyme, as it would not
be inuenced by stabilizing factors present in the spore core.
The properties of the spore-associated and vegetative cell
enzymes were very similar and the antispore a-glucosidase
antibody bound to both the spore-associated and vegetative
forms of the enzyme. The molecular weight difference is
equivalent to approximately 4550 amino acid residues. It is
likely that post-translation modication of the vegetative cell
enzyme leads to the increased size of the spore form. This
modication is presumed to allow specic targeting of the a-
glucosidase to its site within the spore.
The a-glucosidase proved to be a useful predictor of spore
survival as it is consistently present in viable spores and
vegetative cells, and it survives just longer than the spore
following moist heat sterilization (Vesley et al. 1992). Its
location in the spore coat suggests that it is necessary in the
early stages of germination or outgrowth, prior to newenzyme
RAPI D BI OLOGI CAL I NDI CATOR FOR STEAM STERI LI ZATI ON 873
Fig. 11 Immunoelectron microscopic localization of a-
glucosidase enzyme in dormant spores of Bacillus stearothermophilus
ATCC 7953. Ultrathin sections were labelled with anti-a-
glucosidase monoclonal antibodies and colloidal gold (15 nm)-
IgG complex. (a) Spores xed with 3% glutaraldehyde in
phosphate buffer pH 74 for 16 h at 4 C. (b) Spores xed by
freeze drying in nitrogen slush. Bars, 200 nm
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85, 865874
synthesis. Enzymes not required during germination and
outgrowth of spores can be synthesized at a later stage during
vegetative growth.
Synthesis of a-glucosidase, L-alanine dehydrogenase and
alkaline phosphatase during spore outgrowth were studied in
B. cereus strain T spores (Kobayashi et al. 1965). Synthesis
of these enzymes began a few minutes after the start of
germination; DNA synthesis began 80120 min later. The
dormant spore is essentially lacking in stable, functional
mRNA, and its conversion to vegetative growth is dependent
on mRNA transcription. These data further suggest that the
a-glucosidase is required for spore outgrowth and normal
vegetative cell function.
ACKNOWLEDGEMENTS
The authors would like to express their sincere gratitude to
Ursula Potter, Department of Electron Optics, University of
Bath, for her invaluable assistance. They also thank Chris
Binsfeld for monoclonal antibody production. The electron
microscope was funded by the Science and Engineering
Research Council. H. Albert was in receipt of a PhD stud-
entship, funded by 3M Healthcare, St Paul, MN, USA.
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