DIFFERENTIAL RESPONSE OF BACILLUS ANTHRACIS AND OTHER SPORE

SPECIES TO SPORICIDAL DISINFECTANTS

Department of Microbiology and Molecular Biology

BRIGHAM YOUNG UNIVERSITY

In the fall of 2001, letters laced with anthrax spores were delivered to various news

organizations in New York and Florida, as well as to two Senators in Washington, D.C.

Over 22 anthrax infections and five deaths resulted from exposure to these spores, and

decontamination of the affected buildings was both time consuming and costly. Since these

attacks, interest in sporicidal disinfectants has increased greatly. Many chemical sporicidal

disinfectants are available commercially, but the exposure time required to sterilize can be

relatively long. In addition, some spores are simply injured or inhibited by chemical

disinfectants, but not necessarily killed. Studies have shown that heat shocking spores after

exposure to some disinfectants can aid in the recovery of injured spores, but these studies

have not evaluated this effect on spores exposed to peracetic acid-based disinfectants.

Recently, our lab has evaluated two novel peracetic acid-based chemical disinfectants,

STERIPLEX™ HC and STERIPLEX™ Ultra for their activity against a variety of bacterial

agents. Results indicated that the STERIPLEX™ HC solutions had extremely rapid cidal

activity on a wide variety of microorganisms, especially those with innate germicide

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resistance, such as bacterial endospores. However, possible recovery of these spores after

heat shock was not evaluated. The purpose of this study was to compare the sporicidal

activity of three disinfectants: CIDEX™, STERIPLEX™ HC, and STERIPLEX™ Ultra on

three species of spores (Bacillus subtilis, Bacillus anthracis, and Clostridium sporogenes)

in suspension, with and without heat shocking. Spores in suspension were exposed to

disinfectants for specified times and assayed for viable spores. These spore suspensions

were then heat shocked (80º C for 20 min) and assayed again. After exposure to peracetic

acid-based disinfectants and subsequent heat shock, some B. subtilis spores recovered,

resulting in up to a one log difference in viable spores. Other species and disinfectants did

not show this effect. In addition, the activity of these disinfectants on spores dried onto a

surface was evaluated using the standard AOAC sporicidal test. The current AOAC test

specifies heat shocking after three weeks of incubation. In this study, we evaluated the

AOAC sporicidal test by heat shocking immediately following disinfection and after three

weeks of incubation as prescribed. Carrier tests showed a greater number of positive B.

subtilis carriers when heat shocked immediately following STERIPLEX™ HC, and

STERIPLEX™ Ultra treatment, than when carriers were heat shocked after three weeks. In

summary, results showed that heat shocking increases resuscitation of spores treated with

some disinfectants, but not others. Spores in suspension and those dried onto carriers

responded similarly to heat shocking. Finally, STERIPLEX™ HC formulations had

surprisingly rapid sporicidal kinetics.

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Table of Contents

Table of Contents .................................................................................................................. iii

List of Tables ......................................................................................................................... iv
List of Figures ......................................................................................................................... v
Introduction ............................................................................................................................. 1
Sporulation and Germination .............................................................................................. 1
Disinfectants ....................................................................................................................... 3
CIDEX™ ...................................................................... Error! Bookmark not defined.
STERIPLEX™ HC Formulations................................................................................... 3
Heat Shock .......................................................................................................................... 4
Specific Aims of This Study ............................................................................................... 5
Aim 1: Suspension Tests................................................................................................. 5
Aim 2: Carrier Tests ....................................................................................................... 5
Materials and Methods............................................................................................................ 6
Laboratory Conditions .................................................................................................... 6
Bacteria ........................................................................................................................... 6
Spore Suspensions .......................................................................................................... 6
Disinfectants ................................................................................................................... 7
Suspension Tests ............................................................................................................. 7
Carriers............................................................................................................................ 8
Carrier Tests .................................................................................................................... 9
Statistical Methods ........................................................................................................ 10
Results ................................................................................................................................... 10
Suspension Tests ............................................................................................................... 10
Carrier Tests ...................................................................................................................... 13
Discussion ............................................................................................................................. 14
Aim 1: Suspension Tests................................................................................................... 14
Aim 2: Carrier Tests ......................................................................................................... 17
References ............................................................................................................................. 29

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List of Tables

Table 1. Various Sporicidal Disinfectants and Their Properties .......................................... 19

Table 2. Concentrations of Spores Dried Onto Carriers From Recovery Experiments ........ 20
Table 3. Effect of Heat Shock on Log Reduction Values of Disinfectant-treated Spores in
Suspension ................................................................................................................... 21
Table 4. Mean Differences in Log Reduction Values of Disinfectant-Treated Spores in
Suspension Following Heat Shock (From Statistical Analysis*) ................................ 22
Table 5. Effect of Immediate versus Delayed Heat Shock on the Resuscitation of
Disinfectant-treated Spores Dried onto Carriers ......................................................... 23

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List of Figures

Figure 1. Kill kinetics of STERIPLEX™ HC on spores of (A) B. anthracis and (B) B.

subtilis in suspension ................................................................................................... 24
Figure 2. Kill kinetics of STERIPLEX™ Ultra on spores of (A) B. anthracis and (B) B.
subtilis in suspension ................................................................................................... 25
Figure 3. Kill kinetics of CIDEX™ on spores of (A) B. anthracis, (B) B. subtilis, and (C) C.
sporogenes in suspension ............................................................................................ 26
Figure 4. Kill kinetics of (A) STERIPLEX™ HC, (B) STERIPLEX™ Ultra, and (C)
CIDEX™ on B. anthracis, B. subtilis, and C. sporogenes in suspension ................... 27
Figure 5. Kill kinetics of STERIPLEX™ HC, STERIPLEX™ Ultra, and CIDEX™ on
spores of (A) B. anthracis and (B) B. subtilis in suspension ....................................... 28

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Introduction

One week after the terrorist attacks of September 11, 2001, two waves of bioterrorist

attacks occurred in the form of letters laced with anthrax spores sent through the U.S.

Postal Service. These spores were the virulent Ames strain of Bacillus anthracis. Over 22

anthrax infections resulted from these attacks, including 11 cases of inhalational anthrax

and five deaths. Decontamination of the buildings involved in the attacks was both time

consuming and expensive. The Brentwood postal facility in Washington, D.C. alone took

26 months to decontaminate, at a cost of over $130 million. As a result of these attacks,

interest in sporicidal disinfectants has increased greatly. Bacteria of the genera Bacillus and

Clostridium form endospores in response to external stress. Bacterial endospores are

extremely resilient and can withstand extremes in temperature and pH. They are also very

resistant to ionizing and nonionizing (UV) radiation, chemical germicides, dessication, and

the vacuum of outer space (27). Upon returning to a favorable environment, spores can

readily convert back to a vegetative state through the process of germination (28).

Disinfection of surfaces contaminated with bacterial endospores is not only an issue of bio-

security, but also an issue faced repeatedly in clinical settings (32). For example, surgical

instruments and endoscopes require sterilization or high-level disinfection between uses

(33). Dental instruments must be treated similarly to prevent cross-contamination (1).

Sporulation and Germination

When bacterial cells of the genera Bacillus and Clostridium are challenged by factors such

as starvation or environmental extremes, they will undergo the process of sporulation (8).

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The bacterial spore is a complex structure composed of several layers, typically including

the core, the plasma membrane, the germ cell wall, the cortex, the inner spore coat, the

outer spore coat, and the exosporium. During spore formation, the spore becomes

progressively dehydrated, with a final water concentration of about 15%.

Sporogenesis is a complex process which, once initiated, takes approximately six to

eight hr to complete (12). Under conditions of starvation, or upon other cell signals,

sporulation begins, bringing about a rapid series of morphological changes (13). First, an

asymmetrically placed septum forms inside the vegetative cell. The smaller compartment,

which is soon engulfed by the mother cell, is known as the forespore and will later become

the spore itself (20). This core typically includes the DNA and RNA, carbohydrates,

dipicolinic acid, Ca2+, K+, Mn2+, and some proteins (31). While inside the vegetative cell,

the forespore is enclosed in two layers of cell wall. The inner layer is known as the germ

cell wall and, after germination, will become the cell wall of the vegetative cell. The outer

layer, known as the cortex, is composed mainly of peptidoglycan (31) and contributes to the

dehydrated state of the spore (8). The spore coat is then formed around the cortex. This

layer, which will make up the bulk of the spore, consists mainly of protein, along with

some complex carbohydrates and lipids (31). Some species, including B. anthracis, posses

an exosporium, a loose-fitting layer around the spore (4). Finally, the mother cell lyses and

releases the fully formed spore (8).

Germination, or the return of a spore to a vegetative state, is generally triggered by

the presence of nutrients, including amino acids, sugars, and nucleosides (9). It has

previously been reported that germination can be activated by heat (41). For some spores,

heat activation is not essential but increases the frequency of germination (26). Other

factors, such as ions (9), and high pressure (29) also act as germinants. During germination,

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the spore undergoes the following changes: loss of heat resistance, Zn2+, Ca2+, and

dipicolinic acid from the core, hydrolysis of the cortex peptidoglycan, rehydration of the

core protoplast, and the resumption of metabolic activity (15, 26).

Disinfectants

Destruction of spores by chemical agents can be difficult and expensive due to the inherent

resistance of bacterial spores to chemical attack. Many sporicidal disinfectants, including

aldehydes (glutaraldehyde), chlorine-releasing agents (hypochlorite), peroxygens (peracetic

acid and hydrogen peroxide), and ethylene oxide are commercially available and effective

(31, 36). Some of these are listed in Table 1. However, most require long contact times, are

toxic to humans, and are corrosive to various materials.

CIDEX™. Glutaraldehyde has been shown to be an effective sporicide (1, 5, 30, 37) and

CIDEX™ has been in use for many years (18). CIDEX™ Activated Dialdehyde Solution is

a 2.4% activated gultaraldehyde solution available commercially. CIDEX™ was chosen for

use in this study because of its common use and effectiveness as a sporicide. Aqueous

solutions of glutaraldehyde are acidic and must be buffered (or activated) by alkalinating

agents to a pH of 8.2 to 9.2 in order to have optimal antimicrobial activity (31).

Glutaraldehyde inactivates spores by cross-linking the spore’s outer protein layers and by

blocking normal germination events prior to dipicolinic acid release (37).

STERIPLEX™ HC Formulations. Novel disinfectants, STERIPLEX™ HC and

STERIPLEX™ Ultra, (sBioMed, LLC) have been shown to have very rapid sporicidal

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activity (6) with very few toxic or corrosive properties (sBioMed LLC, personal

communication).

STERIPLEX™ HC formulations are peracetic acid (also known as peroxyacetic

acid, or PAA)-based disinfectants with additional proprietary active ingredients that work

synergistically. Peracetic acid has previously been shown to be an effective disinfectant on

a variety of microbes, including vegetative bacterial cells, fungi, viruses, and bacterial

spores, including those of B. anthracis (3, 14, 16, 19, 21, 34). The primary mode of action

of peracetic acid is the oxidation of the double bonds of carbohydrates, nucleic acids, lipids,

and the outer cell membrane proteins of vegetative bacterial cells, endospores, yeasts, and

mold spores (21). Oxidation by peracetic acid usually occurs through the generation of free

radicals. Carbon-centered free radicals such as CH3C(=O)O· and CH3C(=O)· have been

implicated in the sporicidal action of peracetic acid, as has the hydroxyl radical (·OH) (21).

Peracetic acid may also increase cell wall permeability by disrupting sulfhydryl and sulfur

bonds (23). Two STERIPLEX™ HC formulations were used in this study, one with 0.25%

peracetic acid (STERIPLEX™ HC) and one with 1.3% peracetic acid (STERIPLEX™

Ultra).

Heat Shock

When exposed to chemical disinfectants, spores may be inhibited, injured, or killed. The

possibility exists that if spores sub-lethally injured by chemical disinfectants find a

favorable resuscitation environment, they may then germinate and cause disease. Several

factors have been shown to aid in the resuscitation of spores injured, but not killed, by

chemical disinfectants, and it has been proposed that this could be a matter of clinical

importance (23). Some treatments that have been shown to aid in this resuscitation include

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lysozyme (11), sodium hydroxide (7), and heat (43). Heat shock, or exposing spores to high

heat for a specific period of time (usually 80ºC for 20 min), has been shown previously to

aid in the resuscitation of spores treated with certain biocides (35, 43). However, no

previous studies have evaluated the effect of heat-shock on spores exposed to peracetic

acid-based disinfectants, nor were spores of virulent B. anthracis evaluated in such studies.

While heat shock following exposure to chemical disinfectants is not a typical practice

during the clinical use of these agents, it can be a valuable research tool to determine the

actual sporicidal efficacy of chemical disinfectants.

Specific Aims of This Study

The purpose of this study was to examine the differential response of various spore species

to sporicidal disinfectants, as determined by both suspension and carrier-type tests. There

were two specific aims to this study:

Aim 1: Suspension Tests. The first aim was to show the differences in heat shock-induced

resuscitation between disinfectant-treated bacterial spores of B. subtilis, virulent B.

anthracis, and C. sporogenes in suspension, and to compare sporicidal kill kinetics of the

disinfectants.

Aim 2: Carrier Tests. The second aim was to compare the effect of heat shock on the

resuscitation of bacterial spores of B. subtilis and C. sporogenes which have been dried

onto porcelain penicylinders and polyester suture loops, as specified in the AOAC Official

Method 966.04 – Sporicidal Activity of Disinfectants. The effect of heat shocking

5
immediately after disinfectant exposure versus following three weeks of incubation (as

specified by the AOAC method) was also investigated.

Materials and Methods

Laboratory Conditions. Experiments with B. anthracis were performed under Biosafety

Level 3 (BSL-3) conditions. All other experiments were performed under BSL-2

conditions.

Bacteria. Bacterial strains used were Bacillus subtilis ATCC 19659, Clostridium

sporogenes ATCC 3584, and virulent Bacillus anthracis A0462 (Ames strain). B. subtilis

and B. anthracis were grown on Columbia Agar (CA, Becton, Dickinson, and Company,

Sparks, MD). C. sporogenes was grown on Reinforced Clostridial Agar (RCA, Becton,

Dickinson, and Company) and incubated under anaerobic conditions.

Spore Suspensions. Two-hundred-twenty-five ml of Nutrient Broth (Becton, Dickinson,

and Company) were added to each of two 2-L Erlenmeyer flasks and autoclaved. To each,

25 ml of filter-sterilized dextrose-salts solution (1.6% KCl, 0.3% CaCl2, 0.9% dextrose,

0.025% MgSO4•7H2O, 0.002% MnSO4•H2O, 0.0003% FeSO4•7H2O) was added to make

complete Leighton-Doi Broth (LDB). One hundred μl each of B. subtilis and B. anthracis

freezer stocks (with a titer of approximately 1x108 cfu/ml) were used to inoculate two

flasks of LDB. The two Erlenmeyer flasks were placed in a Lab-Line 3525 shaking

incubator at 32°C and 100 rpm for three days. The suspensions were then placed in conical

vials, heated for 30 min at 65°C to kill any remaining vegetative cells, and centrifuged.

6
Spores were then resuspended in 20 ml sterile HPLC water and refrigerated overnight. This

washing procedure was repeated three times. Spore content was monitored with phase-

contrast microscopy. The spore suspensions were quantified using serial dilution and

triplicate plating by membrane filtration. The B. anthracis spore suspension had a titer

between 1.80x109 and 1.98x109 spores/ml. The B. subtilis spore suspension had an initial

titer of between 4.16x109 and 7.02x109 spores/ml. A spore suspension of C. sporogenes

was purchased from Presque Isle Cultures (Presque Isle, PA), and was quantified using

serial dilution and triplicate plating by membrane filtration. This suspension had a titer of

between 1.24x107 and 3.12x107 spores/ml.

Disinfectants. All three spore suspensions were tested against three disinfectants:

CIDEX™ Activated Dialdehyde Solution, 2.4% gultaraldehyde (Advanced Sterilization

Products, Irvine, CA), STERIPLEX™ HC, 0.25% peracetic acid (sBioMed, LLC, Orem,

UT), and STERIPLEX™ Ultra, 1.3% peracetic acid (sBioMed, LLC). All disinfectants

were activated immediately prior to use, according to manufacturer’s instructions.

Suspension Tests. A kill-time suspension test performed at 20°C was used to evaluate each

disinfectant. Briefly, 0.1 ml of spore suspension was added to 9.9 ml of disinfectant at time

zero. The suspension was mixed thoroughly and returned to the water bath. At various

times, the spore mixture was sampled. This involved removal of one ml of the

spore/disinfectant solution, which was then added to nine ml of an appropriate neutralizer.

To neutralize the aldehyde-based disinfectant, a freshly-prepared glycine solution (1%) was

used, while a freshly-prepared neutralizer containing tris buffer (500mM, pH 8.0), Tween

80 (12.72%), tamol (6%), lecithin (1.7%), catalase (1.1%), cysteine (1%), and peptone (1%)

7
was used for the peracetic acid-based disinfectants. The neutralized sample was then mixed

thoroughly and serially diluted in physiological saline solution (0.9% NaCl). Neutralization

controls were performed for each assay to ensure that adequate neutralization of the

disinfectants was achieved.

The number of viable spores in each dilution was quantified by membrane filtration

of one ml samples in triplicate. Millipore E-Z Pak 0.45 μm membranes were used. Filter

membranes were placed onto CA plates in the case of B. subtilis and B. anthracis, while

pre-reduced RCA plates were used for C. sporogenes. Once samples were taken from each

time point/dilution, all dilution tubes were then heat shocked in a water bath (80ºC) for 20

min, and the tubes were re-sampled and tested, following the same protocol as above. The

plates were incubated at 37°C, and counted after approximately 24 and 48 hr of incubation.

Plates of C. sporogenes were counted only after 48 hr to ensure that anaerobic conditions

were maintained.

Carriers. Porcelain penicylinders and polyester suture loops inoculated with B. subtilis

ATCC 19659 and C. sporogenes ATCC 3584 were purchased from Presque Isle Cultures.

Recent studies showed that polyester suture loops are preferred to silk suture loops, as the

latter interact with peracetic acid (22). All carriers were prepared according to the AOAC

Official Method 966.04 (15th ed.) and were tested for acid resistance by the manufacturer

according to the AOAC Official Method. Spore titers were documented by the

manufacturer to exceed 1x106 spores/carrier.

These titers were confirmed in our lab using the following method: carriers were

placed in tubes containing modified nutrient broth (2% Tween) and sonicated for ten min in

a Sonicor SC-200 ultrasonic cleaner to remove the spores from the carrier. After serial

8
dilution, the number of viable spores in each dilution was quantified by membrane filtration

of one ml samples. Millipore E-Z Pak 0.45 μm membranes were used. Filter membranes

were placed onto CA plates in the case of B. subtilis, while pre-reduced RCA was used for

the C. sporogenes. Samples from each dilution were plated in triplicate. Counts were

averaged for the three replications. The carrier titers are listed in Table 2.

Carrier Tests. Carriers were tested against the three disinfectants used in the suspension

tests. Experiments were performed according to the AOAC Official Method 966.04 –

Sporicidal Activity of Disinfectants. Briefly, ten ml of each disinfectant was placed into 50

ml conical centrifuge tubes and brought to 20º C. Two suture loops or penicylinders were

placed into each tube of disinfectant using a flamed metal hook. After the specified contact

time, the carriers were removed using flamed metal hooks and placed individually into

tubes containing Sodium Thioglycolate Broth (STB, Becton, Dickinson, and Company).

Using sterile metal hooks, each carrier was retransferred into a second tube of STB. Tubes

were incubated at 37 ºC for 21 days, and then heat shocked at 80 ºC for 20 min and

reincubated at 37 ºC for 72 hr and checked for growth. This test was performed on 30 of

each carrier per species.

Exposure time for STERIPLEX™ HC was 25 min, the exposure time for

STERIPLEX™ Ultra was 15 min, and the exposure times for CIDEX™ were one hr for C.

sporogenes and four hr for B. subtilis. These contact times were determined experimentally

by previous testing (data not shown) to be at the end of the kill curve for each

organism/disinfectant combination.

A repeat of the above testing was performed, which differed from the AOAC

Official Method in the following respect: instead of incubating for 21 days before heat

9
shock, these tubes were heat shocked at 80 ºC for 20 min immediately following retransfer

to the second tube of STB. They were not heat shocked after 21 days. Again, the test was

performed on 30 of each carrier per species. In all, 60 carriers of each type were tested for

each species, for each of the three disinfectants.

Statistical Methods. For suspension tests, the susceptibilities of three species of bacterial

spores, to three disinfectants were evaluated. On each test day, both B. subtilis and C.

sporogenes were tested with different disinfectants selected at random until each

disinfectant was repeated three times. For B. anthracis, on each test day, all three

disinfectants were tested in random order. Each dilution assayed for viable spores was

plated in triplicate and these counts were averaged to obtain the estimate for each dilution.

Statistical analysis was performed using the GLIMMIX procedure in SAS. An F-test

adjusted for multiple replications was used. Data from all three dilutions plated were used

in this analysis.

For carrier tests, the number of positive tests after delayed heat shock was compared

to the number of positive tests after immediate heat shock using the FREQ procedure in

SAS. For this data, Fisher’s exact test was used.

Results
Suspension Tests

The first aim of this study was to show the differences in heat shock-induced resuscitation

between disinfectant-treated bacterial spores of B. subtilis, virulent B. anthracis, and C.

sporogenes in suspension. To test the hypothesis that spores treated with heat after

disinfection had different recoveries than those not heat shocked, the spore suspensions

10
were exposed to a disinfectant, assayed for viable spores, then heat shocked and assayed

again.

Upon exposure to STERIPLEX™ HC, B. anthracis showed an average log

reduction of 4.99 in 3.5 min among three replications, with an additional average log

reduction of 1.68 caused by heat shocking. The kill kinetics of STERIPLEX™ HC on B.

anthracis spores are shown in Figure 1A. STERIPLEX™ HC produced an average log

reduction of B. subtilis spores of 6.12 after one min, but an average log reduction of only

5.30 after heat shock. The kill kinetics of STERIPLEX™ HC on B. subtilis spores are

shown in Figure 1B. C. sporogenes showed a log reduction of greater than 5.89 after 15

sec. This data represents complete kill. No increase was seen after heat shock. Figure 4A

shows a comparison of the kill kinetics of STERIPLEX™ HC on B. anthracis and B.

subtilis spores. Note that B. anthracis spores are significantly more resistant to

STERIPLEX™ HC than B. subtilis spores. Statistical analysis confirmed that the heat-

shock treatment resulted in a significant difference in both species (p < 0.0001), with spores

of B. anthracis showing potentiated kill and B. subtilis spores showing increased

resuscitation.

Upon exposure to STERIPLEX™ Ultra, B. anthracis showed an average log

reduction of 5.18 in 45 sec, with an additional log reduction of 1.56 following heat

shocking. The kill kinetics of STERIPLEX™ Ultra on B. anthracis spores are shown in

Figure 2A. B. subtilis showed an average log reduction of 6.04 in 15 sec, but this value

dropped to 4.82 following heat shocking. See Figure 2B for kill kinetics of STERIPLEX™

Ultra on B. subtilis spores. C. sporogenes again showed a log reduction of more than 5.67

in 15 sec due to complete kill. A comparison of the kill kinetics of STERIPLEX™ Ultra on

B. anthracis and B. subtilis spores can be seen in Figure 4B. As was seen with

11
STERIPLEX™ HC, B. anthracis spores are more resistant to STERIPLEX™ Ultra than B.

subtilis spores. Again, the difference in log reduction due to heat shock was significant (p <

0.0001), with species of B. anthracis showing potentiated kill and B. subtilis spores

showing increased resuscitation.

After exposure to CIDEX™, B. anthracis showed an average log reduction of 4.59

after 4 min, with an additional average log reduction of 0.63 following heat shocking. The

kill kinetics of CIDEX™ on B. anthracis spores are shown in Figure 3A. B. subtilis showed

a log reduction of 4.74 after 190 min, with an additional average log reduction of 0.90 after

heat shocking. Figure 3B shows the kill kinetics of CIDEX™ on B. subtilis spores. C.

sporogenes showed an average log reduction of 3.23 after 20 min, with an additional

average log reduction of 0.69 after heat shock. See Figure 3C for kill kinetics of CIDEX™

on spores of C. sporogenes. Figure 4C shows a comparison of the kill kinetics of CIDEX™

on all three species. Note the dramatic resistance of B. subtilis spores to CIDEX™.

Statistical analysis again showed heat shock to significantly potentiate CIDEX™-mediated

killing with higher log reductions following heat-shock treatment (p < 0.0001).

Figure 5A shows a comparison of the kill kinetics of all three disinfectants on

spores of B. anthracis. Note the extremely rapid kill of STERIPLEX™ Ultra on these

spores. Figure 5B shows the same comparison for spores of B. subtilis. Again, the extreme

speed of kill of the STERIPLEX™ HC formulations is evident. All suspension test results

are summarized in Table 3. The statistical analysis, using data from all three dilutions

plated, produced the data inTable 4. Mean differences in log reductions before and after

heat shock for each bacterial species and disinfectant, along with standard errors and p

values, are listed.

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Carrier Tests

The second aim of this study was to compare the effect of delayed or immediate heat shock

on the recovery of disinfectant-treated bacterial spores of B. subtilis and C. sporogenes

dried onto porcelain penicylinders and polyester suture loops. B. anthracis was not used in

these experiments because it is not mandated by the AOAC Official Method. Again, in one

experiment, the carriers were heat shocked immediately after disinfectant exposure and in

the other, the carriers were heat shocked following three weeks of incubation, as specified

in the AOAC procedure. Each carrier possessed at least 1x106 viable spores. Any viable

spores remaining on the carrier after exposure to disinfectant will result in growth, or a

positive test. Carriers with no remaining viable spores after exposure to disinfectant will

result in a negative test (no growth). The results from the carrier tests are shown in Table 5.

In the AOAC Official Method, carrier test results are based on a combined total of carriers

(porcelain penicylinders and suture loops) that yielded growth. The results in Table 5 are

reported similarly.

For STERIPLEX™ HC, a total of three B. subtilis carriers were positive when the

test was performed according to the AOAC guidelines (heat shock after three weeks of

incubation). However, the group subjected to immediate heat shock had nine positive

carriers, a three-fold increase (p=0.0627). For C. sporogenes, only one carrier was positive

in each group (p=0.7521).

For STERIPLEX™ Ultra, two B. subtilis carriers were positive under AOAC

guidelines, as opposed to seven after immediate heat shock, a 3.5-fold increase (p=0.0815).

Two C. sporogenes carriers were positive under AOAC guidelines, and four were positive

when subjected to immediate heat shock, a two-fold difference (p=0.3397).

13
 For CIDEX™, three B. subtilis carriers were positive under AOAC guidelines, with

five positives when carriers were exposed to immediate heat shock, a 1.67-fold increase

(p=0.3585). Three C. sporogenes carriers were positive under AOAC guidelines, with four

positives produced when carriers were immediately heat shocked, a 1.33-fold increase

(p=0.5000).

Discussion
Aim 1: Suspension Tests

Results show that STERIPLEX™ HC and STERIPLEX™ Ultra have extremely rapid

sporicidal activity (see Figures 1 and 2). Overall, STERIPLEX™ Ultra had the most rapid

sporicidal activity across all spore species (see Figures 5A and 5B), resulting in complete

kill in as little as 15-45 sec (see Table 3). While CIDEX™ had comparable activity to

STERIPLEX™ HC on spores of B. anthracis, it was generally much slower than the two

peracetic acid-based disinfectants on the other spore species (see Figure 5). One possible

reason for this disparity relates to differences in mechanisms of action of the two types of

disinfectants. Glutaraldehyde, the active ingredient of CIDEX™, specifically targets

proteins, which are found primarily in the spore coat. Peracetic acid, the active ingredient

of STERIPLEX™ HC and STERIPLEX™ Ultra, oxidizes a variety of molecules, including

nucleic acids, proteins, lipids, carbohydrates, and any other molecule containing a double

bond. Thus, the peracetic acid-based disinfectants have conceivably more cellular targets

on which to act.

These results also showed important differences between the two Bacillus species

with respect to sporicide resistance. B. subtilis proved to be less resistant than B. anthracis

to STERIPLEX™ HC and STERIPLEX™ Ultra. Greater exposure times (up to 3.5 times

14
greater) were required for B. anthracis, to achieve the level of disinfection seen in B.

subtilis (see Figures 4A and 4B). However, the opposite is true of CIDEX™. In this case,

B. subtilis was significantly more resistant than B. anthracis, requiring a 186 min longer

exposure time to achieve a similar inactivation of spores (see Figure 4C).

Differences in spore coat proteins between B. subtilis and B. anthracis may be

responsible for these differences in resistance. Genome analysis of the two species has

shown that spore coat proteins with key roles in spore morphogenesis are present in the

genomes of both species (17). This may indicate that the sporulation process is similar for

B. subtilis and B. anthracis. However, this same genomic analysis showed several

differences in the proteins that make up the spore coat, which may be responsible for

differences in their susceptibilities to disinfectants (17). Under electron microscopy, the

spore coats of B. subtilis and B. anthracis are visibly different. B. subtilis has a thick coat

with two major layers, while the B. anthracis coat is thinner and more compact (2, 10, 42).

Another possibility is that resistance or susceptibility to disinfection may be the result of

interactions between multiple coat proteins (17). The exosporium, a structure which

surrounds the entire spore, including the spore coat, is absent in B. subtilis but present in B.

anthracis. This may also affect resistance to disinfectants (17). Differences between species

obviously exist, and the identification of which proteins or combinations of proteins are

affected by glutaraldehyde-based disinfectants is a question for future research.

C. sporogenes proved to be extremely susceptible to disinfection with

STERIPLEX™ HC and STERIPLEX™ Ultra, but less so to CIDEX™. Complete kill was

always observed in 15 sec with the two peracetic acid-based disinfectants (a >5-log

reduction), while a three-log reduction took 20 minutes with CIDEX™. It is not surprising,

however, that C. sporogenes, an anaerobic bacterium, is more susceptible to oxidative

15
damage than aerobic bacteria such as B. subtilis and B. anthracis. The similarities in kill

kinetics of CIDEX™ on C. sporogenes to those of B. anthracis (see Figure 4C), support

this line of thought.

Perhaps most interesting is the resuscitation observed in B. subtilis spores when heat

shocked immediately following exposure to peracetic acid-based disinfectants. After

disinfection with STERIPLEX™ HC and STERIPLEX™ Ultra, heat shock of B. subtilis

spores resulted in statistically significant (p < 0.0001) resuscitation of spores (see Table 4).

Heat shocking after disinfection with all three disinfectants resulted in further killing of the

remaining viable spores in all other species, including B. subtilis when treated with

CIDEX™ (see Table 3). These results indicate that while peracetic acid causes rapid injury

to bacterial spores, heat shock aids in the repair of at least some of this damage. On the

other hand, no resuscitation was seen on any of the species exposed to CIDEX™,

indicating that heat shock does little to reverse the damage mediated by glutaraldehyde.

Interestingly, the resuscitation observed when B. subtilis spores were immediately

heat shocked after exposure to STERIPLEX™ HC and STERIPLEX™ Ultra was not

observed in B. anthracis (Figure 4). B. subtilis is often used as a surrogate organism for B.

anthracis when BSL-3 conditions are not available. Thus, care should be taken when

extrapolating data from surrogate organisms.

Taken together, these data indicate that, while STERIPLEX™ HC and

STERIPLEX™ Ultra show a much faster kill rate than CIDEX™ for spores in suspension,

some of these spores are resuscitated upon heat shock. In a clinical setting, it would be

advisable to increase the STERIPLEX™ HC exposure times toward the end-points tested

here to ensure complete kill. For disinfection of B. anthracis, however, these peracetic acid-

16
based disinfectants are as effective as the glutaraldehyde-based disinfectants, and, in the

case of STERIPLEX™ Ultra, much more so.

Aim 2: Carrier Tests

The second aim of this study was to determine if the resuscitation of spores in suspension

after heat shock was similar to that of spores dried onto solid surfaces. It was expected that

this may be the case, however, the rate of diffusion of the disinfectants into dried films

containing spores and other unknown factors may affect this phenomenon.

With STERIPLEX™ HC, the B. subtilis carriers heat shocked immediately

following exposure to disinfectant showed a three-fold increase in positives over the

carriers tested according to AOAC guidelines. With STERIPLEX™ Ultra, the B. subtilis

carriers immediately heat shocked after disinfectant exposure showed a 3.5-fold increase in

positives over the carriers tested according to the AOAC method. These results are

significant at a 90% confidence level, and approached significance at the 95% confidence

level (p=0.0627 and 0.0815 for STERIPLEX™ HC and STERIPLEX™ Ultra, respectively

– see Table 5). The C. sporogenes carriers showed no heat-shock mediated increase with

STERIPLEX™ HC and only a two-fold increase with STERIPLEX™ Ultra. These results

were not statistically significant (p=0.7521 and 0.3397 for STERIPLEX™ HC and

STERIPLEX™ Ultra, respectively – see Table 5). All spores treated with CIDEX™

showed almost no increase in the number of positive carriers when immediately exposed to

heat shock vs. the group tested according to AOAC guidelines (p=0.3585 and 0.5000 for B.

subtilis and C. sporogenes, respectively – see Table 5).

Taken together, these results indicate that, when testing STERIPLEX™ HC,

STERIPLEX™ Ultra, and perhaps other peracetic acid-based disinfectants with B. subtilis,

immediate heat shock after disinfection may be a better indicator of the effectiveness of the

sporicidal activity of a disinfectant than the current AOAC guidelines. In 2003, the

17
Environmental Protection Agency initiated research to improve efficacy test methods for

sporicides (38). Since then, the AOAC Official Method 966.04 has been reevaluated

several times (23-25, 38-40), but none of these evaluations addressed the effect of

immediate heat shock. The findings of this study may warrant further evaluation of the

AOAC Official Method 966.04 in this respect.

In conclusion, spore species differ widely in their susceptibility to disinfectants and

their response to heat shock following disinfectant treatment. These data showed a

significant spore species-disinfectant-heat shock interaction. Because these interactions are

complex and unpredictable, tests with and without heat shocking should be performed

when evaluating the sporicidal properties of a disinfectant. In addition, the reported

sporicidal efficacy of peracetic acid-based disinfectants may be overrepresented when heat

shock is not performed immediately following disinfection treatment. Therefore, the current

AOAC Official Method 966.04 – Sporicidal Activity of Disinfectants may be suboptimal

for determining the activity of peracetic acid-based disinfectants on B. subtilis spores.

Lastly, STERIPLEX™ Ultra has exceptionally fast sporicidal kinetics, being able to kill B.

subtilis spores in suspension about 1,000 times faster than CIDEX™. Further confirmation

of the reported lack of toxicity and corrosion of the STERIPLEX™ HC formulations will

likely open the door to countless practical uses of these products in infection control.

18
Table 1. Various Sporicidal Disinfectants and Their Properties

Sporicidal Disinfection Shelf-life

Active ingredient(s)
time time
8.3% Hydrogen Peroxide
5 hours 5 minutes 5 days
7.0 % Peracetic Acid
1.0% hydrogen peroxide
8 hours 25 minutes 14 days
0.08% peracetic acid
3.4% glutaraldehyde
10 hours 10 minutes 14 days
26% isopropanol

2.4% glutaraldehyde 10 hours 45 minutes 14 days

Hypochlorite and
Hypochlorous acid 650-675 ppm Active free 24 hours 10 minutes Single -use
chlorine

0.55% ortho-phthaldehyde 32 hours 12 minutes 14 days

19
Table 2. Concentrations of Spores Dried Onto Carriers
From Recovery Experiments

B. subtilis
Lot 032107* Carrier Average Titer (cfu)
Porcelain Penicylinder 1.05x106
Polyester Suture Loop 1.46x106
Lot 052507† Carrier Average Titer (cfu)
Porcelain Penicylinder 1.12x106
Polyester Suture Loop 1.00x106
C. sporogenes
Lot 040207* Carrier Average Titer (cfu)
Porcelain Penicylinder 1.21 x106
Polyester Suture Loop 3.05 x106
Lot 052607† Carrier Average Titer (cfu)
Porcelain Penicylinder 1.61 x106
Polyester Suture Loop 5.27 x106

* Lots used for CIDEX™ and STERIPLEX™ HC Ultra tests.

† Lots used for STERIPLEX™ HC tests.

20
Table 3. Effect of Heat Shock on Log Reduction Values of Disinfectant-treated Spores in Suspension

Replication 1 Replication 2 Replication 3

Log Log Log Log Log Log
STERIPLEX™ Exposure Mean
Reduction Reduction Difference Reduction Reduction Difference Reduction Reduction Difference
HC Time Difference†
Before HS After HS Before HS After HS Before HS After HS
B. anthracis 3.5 min 5.02 6.43 -1.41 4.92 6.64 -1.72 5.02 6.93 -1.91 -1.68
B. subtilis 1 min 6.01 5.36 0.65 5.97 5.20 0.77 6.38 5.35 1.03 0.82
C. sporogenes 15 sec >5.85* >5.85* 0.00 >5.85* >5.85* 0.00 >5.97* >5.97* 0.00 0.00

Replication 1 Replication 2 Replication 3

Log Log Log Log Log Log
STERIPLEX™ Exposure Mean
Reduction Reduction Difference Reduction Reduction Difference Reduction Reduction Difference
Ultra Time Difference†
Before HS After HS Before HS After HS Before HS After HS
B. anthracis 45 sec 5.32 6.89 -1.57 5.19 6.50 -1.31 5.02 6.82 -1.80 -1.56
B. subtilis 15 sec 5.91 4.70 1.21 5.90 4.85 1.05 6.31 4.91 1.40 1.22
C. sporogenes 15 sec >5.77* >5.77* 0.00 >5.57* >5.57* 0.00 >5.68* >5.68* 0.00 0.00

Replication 1 Replication 2 Replication 3

Log Log Log
Exposure Log Reduction Log Reduction Log Reduction Mean
CIDEX™ Reduction Difference Reduction Difference Reduction Difference
Time Before HS Before HS Before HS Difference†
After HS After HS After HS
B. anthracis 4 min 4.54 5.20 -0.66 4.59 5.23 -0.64 4.63 5.23 -0.60 -0.63
B. subtilis 190 min 4.67 5.53 -0.86 4.63 5.88 -1.25 4.93 5.53 -0.60 -0.90
C.
20 min 3.15 4.02 -0.86 3.33 3.94 -0.61 3.22 3.83 -0.61 -0.69
sporogenes

Abbreviations: HS = Heat Shock. All calculations were made using the most reliable dilution plated.
*Data represents complete kill. Actual log reduction may be greater.
† Negative values indicate further inactivation of spores by heat shock. Positive values indicate resuscitation.

21
Table 4. Mean Differences in Log Reduction Values of Disinfectant-Treated
Spores in Suspension Following Heat Shock (From Statistical Analysis*)

STERIPLEX™ HC† Exposure Time Mean Difference‡ Standard Error p§

B. anthracis 3.5 min -1.3338 0.07157 < 0.0001
B. subtilis 1 min 0.8897 0.1061 < 0.0001

STERIPLEX™ Ultra† Exposure Time Mean Difference Standard Error p

B. anthracis 45 sec -1.5452 0.09206 <0.0001
B. subtilis 15 sec 0.8699 0.09445 <0.0001

CIDEX™ Exposure Time Mean Difference Standard Error p

B. anthracis 4 min -0.5981 0.09725 <0.0001
B. subtilis 190 min -0.3238 0.06632 <0.0001
C. sporogenes 20 min -0.4425 0.06569 <0.0001

* Data from all three dilutions was used in the analysis.

† C. sporogenes was not calculated because complete kill was observed.
‡ Negative values indicate further inactivation of spores. Positive values indicate
resuscitation.
§ F-test adjusted for multiple replications used.

22
Table 5. Effect of Immediate versus Delayed Heat Shock on the Resuscitation of
Disinfectant-treated Spores Dried onto Carriers

Exposure Delayed Immediate

Species Disinfectant p*
Time HS HS
B. subtilis STERIPLEX™ HC 25 min 3/60 9/60 0.0627
C. sporogenes STERIPLEX™ HC 25 min 1/60 1/60 0.7521
B. subtilis STERIPLEX™ Ultra 15 min 2/60 7/60 0.0815
C. sporogenes STERIPLEX™ Ultra 15 min 2/60 4/60 0.3397
B. subtilis CIDEX™ 4 hr 3/60 5/60 0.3585
C. sporogenes CIDEX™ 1 hr 3/60 4/60 0.5

Abbreviations: HS = Heat Shock. Results are based on the combined number of positive
carriers
(porcelain penicylinders and polyester suture loops).
* Fisher’s exact test used.

23
Figure 1. Kill kinetics of STERIPLEX™ HC on spores of (A) B. anthracis and (B) B.
subtilis in suspension. Kinetics of C. sporogenes kill are not represented because complete
kill was observed in 15 sec. Values are means from three replications. Error bars indicate
standard errors.

24
Figure 2. Kill kinetics of STERIPLEX™ Ultra on spores of (A) B. anthracis and (B) B.
subtilis in suspension. Kinetics of C. sporogenes kill are not represented because complete
kill was observed in 15 sec. Values are means from three replications. Error bars indicate
standard errors.

25
Figure 3. Kill kinetics of CIDEX™ on spores of (A) B. anthracis, (B) B. subtilis, and
(C) C. sporogenes in suspension. Values are means from three replications. Error bars
indicate standard errors.

26
Figure 4. Kill kinetics of (A) STERIPLEX™ HC, (B) STERIPLEX™ Ultra, and (C)
CIDEX™ on B. anthracis, B. subtilis, and C. sporogenes in suspension. Kinetics of C.
sporogenes is not represented in (A) and (B) because complete kill was observed in 15 sec.
Values are means from three replications.

27
Figure 5. Kill kinetics of STERIPLEX™ HC, STERIPLEX™ Ultra, and CIDEX™ on
spores of (A) B. anthracis and (B) B. subtilis in suspension. Complete kill was observed
in C. sporogenes in 15 sec for both STERIPLEX™ HC and STERIPLEX™ Ultra. Values
are means from three replications.

28
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