Sporicidal Test Research Articles

Inhibition of bacillus spores germination by cinnamon bark, fingerroot, and moringa leaves extract.

This study was intended to discover the natural food preservatives by comparing the antibacterial effect of the ethanolic extract of cinnamon bark, finger root, and moringa leaves toward Bacillus cereus both the vegetative cells and spores. The antibacterial activities of the investigated extracts were assessed against cells using the agar diffusion method. Whereas the sporicidal test was performed by observing the colony growth, after various times of incubation (1, 3, and 5 h). The investigated extracts produced inhibition in a diameter ranging from 10.6 to 35.3 mm, and it can be classified that the extract of cinnamon bark was the most potent extract to inhibit the vegetative cells form, followed by fingerroot and the moringa leaves extract. Consistently, the ethanolic extract of cinnamon bark and fingerroot significantly yielded sporicidal activities higher than the moringa leaves extract. Both extracts exerted sporicidal activity within 1 h of contact time at the lowest test concentration of 5% w/v, whereas moringa leaves extract required a longer contact time (5 h) at higher concentration of 20% w/v. It can be concluded that cinnamon bark and fingerroot extract have great potential as effective food preservative candidates to inhibit the B. cereus growth than moringa leaves extract.

Comparative evaluation of hydrogen peroxide sporicidal efficacy by different standard test methods

There are different sporicidal standard tests with various specifications to deal with products that are claimed for sporicidal activity. The aim of this study was to compare the 7% H2O2 sporicidal efficacy against Bacillus subtilis spores using different standard test methods. The 7% H2O2 sporicidal efficacy against Bacillus subtilis spores was determined according to the AOAC MB-15-04 standard of carrier test and two standard suspension tests (BS EN 13704, AFNOR NF 72-230) in both clean and dirty conditions and by using different interfering substances including bovine serum albumin, yeast extract and skimmed milk. The results of suspension tests with 3 × 105 and 2 × 107 CFU/ml of B. subtilis spore concentration demonstrated that the higher spore counts lead to lower efficacy of 7% H2O2. Also, the sporicidal activity of 7% H2O2 was reduced in the presence of interfering substances. Bovine serum albumin, yeast, and skimmed milk showed similar interfering effects in suspension test with 3 × 105 CFU/ml. While, in suspension tests with higher initial spore count (2 × 107 CFU/ml) severity of interfering effects were intensified and distinct. Our results indicated that the carrier sporicidal test in comparison with suspension tests required more contact time to kill B. subtilis spores. The results of this study showed that it is reasonable to use interfering substances and inoculated carriers in accordance with actual conditions of product usage in a sporicidal test. Interfering substances may reduce the contact surface between H2O2 and test spores; therefore, the sporicidal efficacy of H2O2 was diminished. So applying suspension test in clean condition to verify the claim of sporicidal activity is strongly discouraged.

Impact of standard test protocols on sporicidal efficacy

There has been an increase in the availability of commercial sporicidal formulations. Any comparison of sporicidal data from the literature is hampered by the number of different standard tests available and the use of diverse test conditions including bacterial strains and endospore preparation. To evaluate the effect of sporicidal standard tests on the apparent activity of eight biocides against Clostridium difficile and Bacillus subtilis. The activity of eight biocidal formulations including two oxidizing agents, two aldehydes, three didecyldimethylammonium chloride (DDAC) and amine formulations, and sodium hypochlorite were evaluated using four standard sporicidal tests (BS EN 14347, BS EN13704, ASTM E2197-11, and AOAC MB-15-03) against B.subtilis (ACTC 19659) and C.difficile (NCTC 11209) spores. C.difficile spores were more susceptible to the sporicides than were B.subtilis spores, regardless of the method used. There were differences in sporicidal activity between methods at 5min but not at 60min exposure. DDAC and amine-based products were not sporicidal when neutralized appropriately. Neutralization validation was confirmed for these biocides using the reporting format described in the BS EN standard tests, although the raw data appear to indicate that neutralization failed. The different methods, whether based on suspension or carrier tests, produced similar sporicidal inactivation data. This study suggests that detailed neutralization validation data should be reported to ensure that neutralization of active spores is effective. Failure to do so may lead to erroneous sporicidal claims.

Synergistic sporicidal effect of ethanol on a combination of orthophthalaldehyde and didecyldimethylammonium chloride.

The objective of this study was to evaluate the potential synergistic effect of ethanol on a combination of orthophthalaldehyde (OPA) and didecyldimethylammonium chloride (DDAC) against the spores of Bacillus subtilis var. Niger. The quantitative carrier test for sporicidal testing of high-level disinfectants according to the guideline of China (Technical Standard for Disinfection 2002) was used as method. Considerable synergistic effect was observed after a 30-min treatment at 20°C. There was an augment in mean log reduction as the concentration of DDAC was increased ranging from 0·2 to 3gl(-1) in combination with 6gl(-1) OPA. Ten and 20% ethanol in combination with 6gl(-1) OPA and 2gl(-1) DDAC caused more than a 3-log reduction while either 6gl(-1) OPA, 2gl(-1) DDAC and 20% ethanol alone or a combination of two of the three agents produced less than a 1-log reduction. Further, 40-min exposure time of combination of OPA, DDAC and 20% ethanol led to greater than a 5-log reduction in spores, and no spore growth was observed following 60- and 90-min exposures. Orthophthalaldehyde (OPA) is very effective at concentrations far lower than its recommended in-use concentration of 0·5% (w/v) and is equally effective against both the gram-negative and gram-positive bacteria. However, it shows lower activity against spores. The synergistic sporicidal effect exhibited by ethanol on a combination of OPA and DDAC can be considered to enhance sporicidal activity for using in situations of sterilization, to reduce in-use concentration of OPA used alone, which may minimize its side effect. OPA may be a more satisfactory and the first-choice agent to replace glutaraldehyde (GTA) as a high-level disinfectant for medical devices.

Aldahol high-level disinfectant

Glutaraldehyde and ortho-phthalaldehyde (OPA) are widely used as the active ingredients of high level disinfectants for heat-sensitive, semicritical medical instruments. However, both of these chemicals have limitations in their spectrum of antimicrobial activity. Glutaraldehyde disinfectants are poorly mycobactericidal, and require impractically long exposure times to kill spore-forming bacteria. OPA disinfectants kill many types of mycobacteria in practical exposure times, but require 32 hours to pass the Association of Official Analytical Chemists (AOAC) sporicidal test, and do not claim to be sterilants. These could be serious limitations that contribute to the formation of biofilms in endoscopes, after which the endoscopes are difficult to disinfect. The objective of our research was to discover a disinfectant formulation, based on aldehydes, that killed mycobacteria and spore-forming bacteria in a practical exposure time and temperature. Solutions of glutaraldehyde or OPA were prepared with various concentrations of alcohols, sodium and potassium salts, chelating agents, and detergents at alkaline pH values, and tested against cultures of mycobacteria and spore-forming bacteria to find a formulation that would kill these bacteria in practical exposure times at 20 degrees C or 25 degrees C. Concentrations of < or =20% w/w isopropanol and < or =8% potassium acetate in combination with < or =3.5% w/w glutaraldehyde at alkaline pH values killed 6 log(10) of mycobacteria within 10 minutes at 20 degrees C. Similar combinations killed 6 log(10) of Bacillus subtilis in suspension within 30 minutes at 25 degrees C, and B subtilis within 60 minutes at 20 degrees C. The sporicidal activity of OPA was not increased by combination with isopropanol and potassium acetate salts. Aldahol high-level disinfectant (US FDA K041360), a formulation of 3.5% glutaraldehyde in combination with 20% w/w isopropanol and 8% potassium acetate, kills mycobacteria within 10 minutes at 20 degrees C and kills 6 log(10) of cultures of the spore-forming bacteria B subtilis within 60 minutes at 20 degrees C.

METODOLOGIA PARA LIMPEZA NO LOCAL EM EQUIPAMENTO DE ALTA PRESSÃO DINÂMICA

Summary Cleaning-in-place (CIP) is an important procedure for the correct cleaning and sanitization of equipment, thus avoiding contamination amongst batches and the formation of biofilms. The purpose of this research was to determine CIP methodology for dynamic high pressure equipment contaminated with Aspergillus niger conidia. In vitro tests were carried out with aqueous suspensions of conidia to determine the minimum inhibitory concentration (MIC), and sporicidal tests of the surface to determine the efficiency of the alkaline detergent and sanitation agent (based on peracetic acid) against A. niger conidia. The efficiency of the peracetic acid was tested at concentrations of 0.0025 to 0.2% (v/v) for 20 and 30 min contact time in the in vitro MIC trials,, and at 0.01 to 0.03% (v/v) for 30 min in the sporicidal trials. The detergent was tested at concentrations of 1.5 to 2.5% (v/v) for 30 and 40 min in both trials. The pilot test was carried out in an ultra-high pressure homogenizer after treatment with mango nectar that had been previously contaminated with 10

Efficacy of Amphoteric Surfactant– and Peracetic Acid–Based Disinfectants on Spores of Bacillus cereus In Vitro and on Food Premises of the German Armed Forces

In mass catering facilities of the German Armed Forces, foodborne outbreaks are commonly attributed to Bacillus cereus, and spores of this organism are regularly found on equipment surfaces. B. cereus is the causative agent of foodborne emetic or diarrheic disease. Hence, the use of sporicidal disinfectants may provide a starting point for successful risk mitigation of diseases associated with B. cereus. In this study, the amphoteric surfactant–based disinfectant (product A) that has been routinely used in catering facilities of the German Armed Forces and a peracetic acid–based disinfectant (product B) were compared for their efficacy against B. cereus spores in laboratory tests and under field conditions. In a carrier test for B. cereus spores, product A displayed no efficacy against spores of the test strain. In contrast, a substantial reduction in spore concentration (>5-log reduction) was achieved with product B. In a controlled trial conducted in seven Army catering facilities, the reduction in the number of B. cereus–positive samples was significantly greater (P < 0.001) for product B (from 28% initial contamination to 3% after application) than for product A (from 33 to 21%). Product B was estimated to be 6.25 times more efficacious than product A. Field trials confirmed the higher efficacy of the peracetic acid–based disinfectant to reduce B. cereus contamination on surfaces as found for the in vitro experiments. The tests used to assess the sporicidal effects of disinfectants were adequate under practice conditions and could be used for sporicidal testing of disinfectants for catering and food production establishments.

Studies of Polyester Fiber as Carrier for Microbes in a Quantitative Test Method for Disinfectants

Tests were conducted by a Task Force on Disinfectant Test Methods that was appointed to investigate controversies regarding the accuracy of AOAC test methods for disinfectants as presented in AOAC's Official Methods of Analysis, Chapter 6. The general principles for new and improved AOAC tests are discussed, and a disinfectant test using microbes labeled onto a polyester fiber surface is described. The quantitative test measures the survival of test microbes as a function of exposure time as well as the exposure conditions required to kill 6 log10 of the test microbes. The time required was similar to that for the kinetics of the kill of Bacillus subtilis-labeled cylinders as tested by methods of the AOAC Sporicidal Test 966.04.

Efficacy Testing of Disinfectants

The U.S. Environmental Protection Agency's (EPA) Office of Pesticide Programs (OPP) has the responsibility for regulating antimicrobial products, including sporicides, used to treat and decontaminate inanimate surfaces. In response to the anthrax (Bacillus anthracis) attacks of 2001 and the associated need for verifying the performance of chemicals for building decontamination, the EPA initiated research in late 2003 to evaluate and improve efficacy test methods for sporicides. The OPP Microbiology Laboratory located at the Environmental Science Center, Ft. Meade, MD is the lead laboratory. Through funding provided by EPA's Office of Research and Development (Safe Buildings Program), a collaborative research plan has been established to address several key issues. Research is currently being conducted on 2 fronts: (1) the evaluation of quantitative methodology for assessing the efficacy of sporicides, and (2) the development and comparative testing of selected modifications to improve the AOAC Sporicidal Activity Test (AOAC Method 966.04). Future studies will include the evaluation of candidate surrogates of B. anthracis using a quantitative method, and a multilaboratory validation study of a quantitative method-surrogate combination. The General Referee is serving as the Principal Investigator for all research described in this report, and has the overall responsibility for the technical conduct of the projects. In cases where the General Referee has oversight of projects that involve official collaborative studies and validation support from AOAC INTERNATIONAL, AOAC officials and the Committee Chair will determine the appropriate mechanism for formal study review. The 2003 General Referee report provides the background on the development and direction of the research projects. The preliminary data, general conclusions, next steps, and recommendations are provided in this report.

Alternative AOAC Sporicidal Test Carrier for Evaluating Peracetic Acid-Based Sterilants (Modification of AOAC Official Method 966.04)

Dacron suture loops were demonstrated to be inert, consistent carriers in the presence of peracetic acid-based sterilants, whereas black silk sutures had a variable preparation process and interacted with peracetic acid. In addition, Dacron suture loops provided comparable spore loading to black silk suture loops and an HCI resistance of > or = 2 min. These results indicate that black silk suture loops are not appropriate carriers for assessing peracetic acid-based sterilants, and Dacron loops are an acceptable alternative. This finding is consistent with the Office of Science and Technology Laboratory (Center for Devices and Radiological Health) study which determined that "polyester suture material is a viable alternative to silk for the AOAC sporicidal test for liquid disinfectants."

Comparison of the disinfectant efficacy of Perasafe® and 2% glutaraldehyde in in vitro tests

Two percent glutaraldehyde has been the reference disinfectant for high-level disinfection, but its frequent association with adverse effects has stimulated a search for newer disinfectants. We compared the efficacy of 2% glutaraldehyde with that of a new disinfectant formulation, Perasafe® (0.2% peracetic acid) in six in vitro tests: germicidal effect in a suspension of micro-organisms; using a microbe-contaminated metallic endodoncy file; an endoscope model; a modified capacity test; a sporicidal test, and corrosiveness on metallic instruments. Both products were effective germicides in 10–20min, completely destroying microbial inocula except for Mycobacterium and spores. Internal irrigation of endoscopes with 100mL of either was totally effective. Both substances resisted inactivation after repeated inoculation and did not corrode clean instruments; however, when organic matter was added the 0.2% peracetic acid formulation cleaned without corrosion, while 2% glutaraldehyde fixed the matter to the scalpel, causing corrosion within 2h. In summary, Perasafe® is a good substitute for 2% glutaraldehyde for high-level disinfection.

Culture Age and Drying Time as Variables of the AOAC Sporicidal Test

In the United States, the AOAC Sporicidal Activity of Disinfectants Method 966.04 is the standard for identifying a liquid chemical germicide as a sterilant. Furthermore, the highest level of a disinfectant must also be a sterilant as defined by Method 966.04, when used in its sterilant mode for a longer exposure time. The AOAC Sporicidal Test is also used as a part of the standard test methods to define a sterilant for Australia and the European Union. Many laboratories have identified variables of this test that can affect the sterilization exposure time for sterilants, or even the ability to classify a chemical as a sterilant. Method 966.04 requires spore-labeled porcelain penicylinders (cylinders) and silk suture loops, collectively referred to as carriers, to be dried for 24 h, but allows these carriers to be used for at least 7 days, in effect allowing a drying time of 24 h to at least 7 days. We tested the resistance of cylinders that had been labeled with Bacillus subtilis spores cultured for 72, 96, and 120 h, and dried for 24, 48, and 72 h against a 60 min exposure to 2.0% alkaline glutaraldehyde, and 2, 5, 10, 15, and 20 min exposures to 2.5N HCl. All the culture incubation and drying times met the standard of resistance to 2.5N HCI for at least 2.0 min at 20 degrees C, and all carriers contained at least 10(5) colony-forming units (CFU) of B. subtilis per carrier. However, for 3 repeated tests, regardless of incubation time, an average of 96% of the carriers were sterilized by the 2.0% glutaraldehyde after drying for 24 h, and an average of 61 % were sterilized after drying for 48 or 72 h. We propose that the variable of drying time be eliminated from Method 966.04.

Use of Dacron as an Alternative Carrier for Evaluating Oxidizing Sterilants in the AOAC Sporicidal Test

The AOAC sporicidal method (966.04) recommends the use of porcelain penicylinders and black waxed silk sutures as carriers for demonstrating the sporicidal activity of sterilants. However, the silk carriers are not suitable for evaluating the sporicidal efficacy of oxidizing agents, and an inert polyester material (Dacron) is recommended as an alternative. Dacron provides an equivalent microbial and physical challenge to silk. Microbiologically, both materials demonstrated similar HCI resistance, which is required by the AOAC test, as well as equivalent spore loading and spore wash-off. Electron microscopy showed that both materials present the same braided microstructure, providing an equivalent physical challenge to the test sterilant. Dacron was more consistent than silk, and did not require extraction prior to spore loading. The extraction method for black waxed silk was variable and incomplete, which may compromise the activity of oxidizing sterilants and add to method variability. Silk was also structurally altered in the presence of oxidizing sterilants and increased sterilant degradation. Dacron did not affect the sterilant and was inert in the presence of oxidizing agents. Dacron sutures are proposed as inert alternatives to silk for evaluating the sporicidal efficacy of oxidizing agents.

Recovery and Sporicidal Resistance of Various B. subtilis Spore Preparations on Porcelain Penicylinders Compared with Results from AOAC Test Methods

Sporicidal test results obtained from carriers inoculated with 4 types of defined Bacillus subtilis spore preparations were compared with the standard AOAC sporicidal test using soil extract nutrient broth (SENB) B. subtilis 19659 spores. Recoveries of spores inoculated on penicylinders from B. subtilis clean spores (washed and suspended in water) and B. subtilis 19659 spores inoculated from culture filtrates according to the AOAC method were compared. Spores were exposed to 6 concentrations (0.5-3.0% w/v) of glutaraldehyde in phosphate buffer (pH 7.5) for 10 h. Concentrations were established by titrimetry and liquid chromatography. Recoveries of surviving spores were determined for 3 types of clean B. subtilis var. niger preparations, one clean B. subtilis 19659 preparation, and the SENB B. subtilis 19659 filtrates. Spore carriers, inoculated by the standard AOAC protocol, resulted in as much as a 2-log number difference in runs 1-12, but not more than 0.5 log number for each clean spore preparation. The SENB spores varied most in resistance to glutaraldehyde, with no growth in recovery media from 3 different batches of 1, 1.5, and 2% glutaraldehyde. Separate batches of SENB preparations of B. subtilis 19659 were resistant and destroyed by 1.0% glutaraldehyde, with 3.98 and 6.0 log numbers of spores on penicylinders, respectively. Clean spore preparations of B. subtilis 19659 on porcelain penicylinders were more resistant to glutaraldehyde than were SENB spores. Nutrient agar/Mg/Ca and nutrient agar/Mg spore preparations of B. subtilis var. niger showed the most uniform resistance to glutaraldehyde. Spores with calcium added showed increased resistance to glutaraldehyde. B. subtilis 19659 spores from the Columbia broth spore preparation were the most resistant and were recovered after exposure to 3.0% glutaraldehyde.

Sporicidal Testing of Commercial Germicides Using a Chemical Standard and a Calibrated Bioindicator

The AOAC sporicidal method uses as a standard the resistance of spores on carriers to 2.5N HCl. This resistance is variable at exposure times ranging from 2 to 20 min. The method described in this paper uses a glutaraldehyde standard and distinguishes various levels of sporicidal activity in the presence of 1-5% glutaraldehyde by using appropriate spore strains, spore preparations, and spore levels. The resistances of 2 Bacillus subtilis 19659 spore preparations cultured in 10% Columbia broth plus manganese and nutrient agar plus minerals, as well as that of B. subtilis var. niger cultured on Lab-Lemco agar, were tested. T-soy broth was a better recovery medium than fluid thioglycollate or modified fluid thioglycollate for B. subtilis 19659 spores exposed to HCl. Sporicidal tests were done on B. subtilis 19659 spores with 2 types of spore preparations. A commercial glutaraldehyde germicide was used for comparison of the sporicidal activity of the glutaraldehyde standard. Two strains of B. subtilis spores and 4 levels of spores (20,000-80,000, 100,000-400,000, 500,000-800,000, and 1,000,000 and up) were removed from check penicylinders from the same batches used for sporicidal tests. B. subtilis var. niger spores were the most resistant to HCl, while B. subtilis 19659 spores were more resistant to glutaraldehyde. Sporicidal activities of a commercial germicide containing 2.5% glutaraldehyde with additives and another containing 5% glutaraldehyde in phosphate buffer were similar. Both totally destroyed high levels of B. subtilis 19659 spores cultured in 10% Columbia broth plus manganese. Results indicate that use of a glutaraldehyde standard, calibrated numbers of spores on penicylinders (bioindicators), and appropriate spore strains and preparations can reduce the variability of sporicidal testing of commercial germicides.

Modified quantitative association of official analytical chemists sporicidal test for liquid chemical germicides

The Association of Official Analytical Chemists (AOAC) test for sporicidal activity of disinfectants (966.04) is used in the United States as the legal criteria for classifying a liquid as a chemical sterilant and also as the indicator of the highest level of disinfectant. This qualitative test contains procedures that may cause inaccurate results. A modified, quantitative version of the AOAC sporicidal test has been developed that uses a specified minimum number of spores (2.0 x 10(sup5)) of Bacillus subtilis or Clostridium sporogenes dried onto porcelain penicylinders. This modified test was validated with three commercial sterilant chemicals tested on three separate groups of spore-labeled cylinders.

Comparative sporicidal effect of liquid chemical germicides on three medical devices contaminated with spores of Bacillus subtilis

Background: The relatively limited variety of surfaces and geometries challenged in current sporicidal testing reduces the predictive value of these analyses when extrapolated to the wide variety of medical devices. The unknown spore load being challenged and the qualitative nature (growth/no growth) of those tests further prevent precise comparison among liquid chemical disinfectants. Hence, the relative activity of different chemical substances has not been clearly established, hindering selection of the best agent for each clinical situation. Methods: A micromethod was developed to assess sporicidal activity against Bacillus subtilis spores deposited on three different medical devices: carbon-steel dental burs silicone-rubber medical catheters, and titanium-alloy dental abutment screws. The spore load on each device and the recovery after three analytical steps were quantitatively assessed with spores radiolabeled with carbon 14 menthionine. Results: The killing of 2 to 7 × 10 6 spores loaded on three different devices and exposed to glutaraldehyde, fomaldehyde, copper ascorbate, hydrogen peroxide, peracetic acid, sodium hypochlorite, or phenol for 30 minutes at 20° C ranged from a 10 3-fold decrease for 10% hydrogen peroxide to zero decrease for 5% phenol. Our results suggest that the nature of the surface being challenged may affect the sporicidal activity of some chemical agents. Conclusion: The quantitative data presented allow comparison of the sporicidal effect of different liquid chemical agents. These findings may help prevent an overestimation of sporicidal activity and possible transmission of pathogens from the surface of improperly decontaminated medical devices.

Identification of possible artifacts in the association of official analytical chemists sporicidal test

Two laboratories tested four different brands of alkaline 2% glutaraldehyde sterilants by the Association of Official Analytical Chemists sporicidal test. Each laboratory found survival of Clostridium sporogenes spores on spore-labeled unglazed porcelain penicylinders (cylinders) to vary from test to test, and survival did not always correlate with increasing sterilant exposure time. These results were consistent with a theory that there may be random conditions within the test that prevent the sterilant from contacting all spores. Further studies indicated that the prior history of the unglazed porcelain cylinders and whether the C. sporogenes culture grown in egg-meat media had been processed (homogenized) to eliminate visible pieces of egg-meat media were important factors affecting the results and repeatability of this test.

Sporicidal activity of chemical sterilants used in hospitals.

The current study was designed to assess the sporicidal activity of chemical sterilants using the Association of Official Analytical Chemists (AOAC) sporicidal test. Chemical sterilants are used most commonly in the healthcare setting to disinfect medical instruments such as endoscopes. This study was undertaken to evaluate the efficacy of several newer formulations of glutaraldehydes and hydrogen peroxide as chemical sterilants. Using the AOAC test the following agents were tested: two 2% alkaline glutaraldehydes (2% Alk-Glut), 2% acid glutaraldehyde (2% Acid Glut), 2% glutaraldehyde-7.05% phenol-1.20% sodium phenate, 10% glutaraldehyde-0.5% phenylphenol-0.1% tertiary amylphenol, and 6% hydrogen peroxide (H2O2). Growth in > 1 of 60 seeded penicylinders is considered by the AOAC to indicate failure of sporicidal activity. Test results of the six disinfectants against Bacillus subtilis using the manufacturers' specified use-dilution and exposure time were: 0/60 with 2% Alk-Glut (product 1) at 10 hours, 0/60 with 2% Alk-Glut (product 2) at 8 hours, 0/60 with 2% Acid Glut at 10 hours, 2/60 with 2% glutaraldehyde-7.05% phenol-1.20% sodium phenate at 6.75 hours, 0/60 with a 1:5 dilution (2.0% Glut) of 10% glutaraldehyde-0.5% phenylphenol-0.1% amylphenol at 6 hours, 59/60 with a 1:20 dilution (0.5% Glut) of 10% glutaraldehyde-0.5% phenylphenol-0.1% tertiary amylphenol at 12 hours and 0/60 with 6% H2O2 at 6 hours. Test results against Clostridium sporogenes were: 2/60 with 2% Alk-Glut (product 1) at 10 hours, 1/60 with 2% Alk-Glut (product 2) at 8 hours, 1/60 with 2% Acid Glut at 10 hours, 2/60 with undiluted 2% glutaraldehyde-7.05% phenol-1.20% sodium phenate at 6.75 hours, 6/60 with a 1:5 dilution (2% Glut) of 10% glutaraldehyde-0.5% phenylphenol-0.1% amylpenol at 6 hours, 60/60 with a 1:20 dilution (0.5% Glut) of 10% glutaraldehyde-0.5% phenylphenol-0.1% amylpenol at 12 hours, and 0/60 with 6% H2O2 at 6 hours. Dilutions of 2% glutaraldehyde-7.05% phenol-1.20% sodium phenate yielded the following results against C sporogenes: 59/60 with a 1:8 dilution (0.25% Glut) at 6.75 hours, 60/60 with a 1:16 dilution (0.125% Glut) at 6.75 hours, 11/90 with a 1:8 (0.25% Glut) dilution at 12 hours, and 60/60 with a 1:16 dilution (0.125% Glut) at 12 hours. Both 2% acid and alkaline glutaraldehydes are effective chemical sterilants. Diluting glutaraldehyde-based sterilants below 2% glutaraldehyde resulted in failure to kill spores of B subtilis and C sporogenes.

Evaluation of Microbial Loads of Bacillus subtilis Spores on Penicylinders

Three types of penicylinders were compared for their retention of spores of Bacillus subtilis testing for sporicidal activity of disinfectants. Glass, porcelain, and stainless steel penicylinders are inoculated with a water suspension of B. subtilis var. niger (ATCC 9372) spores and dried. One set of each type of penicylinder is submerged 1 h in 0.9% saline. One set of porcelain penicylinders is submerged 15 h in a neutralized chemical germicide, and one set is also inoculated with a culture filtrate of B. subtilis (ATCC 19659), dried according to the AOAC method, and submerged 1 h in 0.9% saline. Microbial loads simulate those held on carriers used to test sporicidal activity of disinfectants. Carriers are immersed in chemical germicide, transferred to a neutralizer, and placed in a culture medium. Average percentages of B. subtilis var. niger spores retained on 10 carriers after 1 h submersion in saline and in water were as follows: glass, 93.6%; porcelain, 99.9%; and stainless steel, 99.5%. Retention of spores after 15 h submersion in a neutralized chemical germicide and in water was 98.9%. Porcelain penicylinders inoculated from a culture filtrate of B. subtilis (ATCC 19659) retained 26% of the spores after being submerged 1 h in saline and placed in water. Glass penicylinders, which retained the lowest and most variable number of spores, were the least suitable for sporicidal activity testing. B. subtilis (ATCC 19659) spores tested on porcelain penicylinders met only the minimum HCl resistance requirements of > or = 2 min. On porcelain penicylinders, the resistance of B. subtilis var. niger spores to 2.5N HCl was relative to the number of spores inoculated.