Heat and ozone resistance of Bacillus spores isolated from laboratory animals

Heat resistance of free-spores of 78 Bacillus strains isolated from laboratory animals was examined. Spores of 41 out of 78 strains survived for 320 minutes at 70 degrees C, 27 for 160 min, at 100 degrees C, only one for 20 min. at 110 degrees C by autoclaving, and none for 5 min. at 120 degrees C. D-values at 100 degrees C of 9 strains determined were between 5.03 and 30.06 min. Spores of 9 strains from stock cultures were exposed to ozone gas at various conditions. Ozone resistance of spores was closely dependent upon relative humidity. D-values of the spores tested by treatment with 200 ppm ozone at 60% RH were over 200 min., especially over 1,000 min. in 4 strains, indicating that exposure to ozone at a moderate humidity for 6 hours could not sterilize Bacillus spores. At 90% RH, however, treatment with 200 ppm ozone for 6 hr. might be effective for a routine sterilization in laboratory animal facilities.     

A Bacterial Spore Test Piece for the Control of Ethylene Oxide Sterilization

Aluminium foil strips carrying varying numbers of spores of the Camp Detrick strain of Bacillus subtilis dried from water, 90% (v/v) methanol, 5 and 20% (v/v) serum, nutrient broth and isotonic saline were examined for their possible use as test pieces for the control of sterilization by ethylene oxide. Methanolic suspensions of these spores were found to be stable on storage, and foils carrying spores dried from methanol were the most reproducible and stable of those tested. The susceptibility of the test piece system could be controlled by varying the numbers of spores on each foil and the numbers of foils used per test.     

Decontamination of laboratory microbiological waste by steam sterilization.

A steam sterilizer (autoclave) was tested to determine the operating parameters that affected sterilization of microbiological waste. Tests involved standardized loads (5, 10 ad 15 lb [ca. 2.27, 4.54, and 6.80 kg, respectively]) contaminated petri plates in autoclave bags placed in polypropylene or stainless steel containers. Thermal and biological data were obtained by using a digital potentiometer and a biological indicator containing spores of Bacillus stearothermophilus, respectively. The transfer of heat was more efficient when smaller loads of microbiological waste were tested and stainless steel rather than polypropylene containers were used. A single bag with the sides rolled down to expose the top layer of petri plates allowed heat to pass better than did a single bag with the top constricted by a twist-tie. The presence of water in the autoclave bag did not significantly improve heat-up time in stainless steel or polypropylene containers. The results of biological tests substantiated the temperature data. When 10 or 15 lb of microbiological waste was exposed to various test conditions, the only condition that ensured the destruction of B. stearothermophilus involved the use of a stainless steel container (with or without water) for 90 min. Autoclaving for 45 min resulted in the destruction of bacteria included in 10 lb (136 +/- 3 plates) or 15 lb (205 +/- 6 plates) of microbiological waste when stainless steel containers with or without water or polypropylene containers with water used, whereas 60 min was required to kill all bacteria if polypropylene containers without water were used.     

Sterilization of bacteria, yeast, and bacterial endospores by atmospheric-pressure cold plasma using helium and oxygen

Atmospheric-pressure cold plasma (APCP) using helium/oxygen was developed and tested as a suitable sterilization method in a clinical environment. The sterilizing effect of this method is not due to UV light, which is known to be the major sterilization factor of APCP, but instead results from the action of reactive oxygen radicals. Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae deposited on a nitrocellulose filter membrane or Bacillus subtilis spores deposited on polypropylene plates were exposed to helium/oxygen plasma generated with AC input power at 10 kHz, 6 kV. After plasma treatment, nitrocellulose filter membranes were overlaid on fresh solid media and CFUs were counted after incubation overnight. D-values were 18 sec for E. coli, 19 sec for S. aureus, 1 min 55 sec for S. cerevisiae, and 14 min for B. subtilis spores. D-values of bacteria and yeast were dependent on the initial inoculation concentration, while the D-value of B. subtilis spores showed no correlation. When treated cells were observed with a scanning electron microscope, E. coli was more heavily damaged than S. aureus, S. cerevisiae exhibited peeling, and B. subtilis spores exhibited shrunken morphology. Results showed that APCP using helium/oxygen has many advantages as a sterilization method, especially in a clinical environment with conditions such as stable temperature, unlimited sample size, and no harmful gas production.     

Sensitivity of Three Selected Bacterial Species to Ozone

THE MINIMAL LETHAL CONCENTRATION OF OZONE IN WATER WAS DETERMINED FOR THREE BACTERIAL SPECIES: Escherichia coli, Bacillus cereus, and Bacillus megaterium. A contact period of 5 min was selected. The lethal threshold concentration for the cells of B. cereus was 0.12 mg/liter while that for E. coli and B. megaterium was 0.19 mg/liter. Low concentrations of ozone were ineffective when organic matter was present to interfere with the action of ozone on the bacterial cells. Also determined during the study was the sensitivity of spores of B. cereus and B. megaterium to ozone in water. The threshold concentration required to kill the spores of both species was 2.29 mg/liter. The cells and spores of these organisms exhibited the "all-or-none" die-away phenomenon normally associated with ozone treatment.     

Sterilization of various diameter dead-ended tubes

Effect of tube diameter on steam-in-place sterilization of dead-ended tubes was studied by examining temperature profiles and rates of kill of Bacillus stearothermophilus spores. Time required for sterilization was determined for 9.4-cm-long tubes with various inside diameters from 0.4 to 1.7 cm. Sterilization time increased with decreasing tube diameter. Experimentally measured kill kinetics in 1.7-cm tubes were in agreement with those predicted if measured temperatures represented saturated steam. A 12-log spore reduction was achieved in 1.7-cm diameter vertical and horizontal tubes in less than 63 minutes. For smaller diameter tubes, entrapped air remained after 2 hours and rates of kill were very dependent on position within the tube, tube diameter, and tube orientation with respect to the gravitational vector. Times to achieve a 1-log drop in spore population in the smaller tubes were as much as 10 times greater than those expected if measured temperatures represented saturated steam. Sterilization was not achieved throughout the 0.4-cm tubes. Recommendations are made for including steam bleeders or using prevaccum cycles for these smaller diameter tubes. (c) 1993 John Wiley & Sons, Inc.     

Optimizing acidified bleach solutions to improve sporicidal efficacy on building materials

Aims: We evaluated whether lowering pH (with acetic acid) and raising free available chlorine (FAC) levels in bleach solutions would improve efficacy in inactivating Bacillus spores on different materials. We also determined how varying pH and FAC levels affected bleach stability. Methods and Results: Acidified bleach solutions with pH levels of 4·5, 6 and 7·5 and FAC levels between 5000 and 10 000 ppm were evaluated for decontamination efficacy against Bacillus subtilis spores inoculated onto test coupons made from wood, ceramic and galvanized steel. Lowering the pH or increasing the FAC level improved efficacy in some of the tests, but depended on the material, which significantly affected decontamination efficacy. The acidified bleach at pH of 7·5 was significantly less effective than bleach at a pH of 4·5 or 6. The FAC levels in the bleach were the most stable at pH 4·5, and stability at pH 4·5 was not significantly affected by the initial FAC level. Conclusions: It may be advisable to use bleach solutions with lower pH (rather than high FAC levels) in light of both the decontamination efficacy and bleach stability results. For wood materials, use of sporicides other than acidified bleach may be warranted. Significance and Impact of the Study: These results may be useful in preparing acidified bleach solutions for decontamination of materials contaminated with spores such as Bacillus anthracis.     

Mechanisms of Bacillus subtilis spore resistance to and killing by aqueous ozone

AIMS: To determine the mechanisms of Bacillus subtilis spore killing by and resistance to aqueous ozone. METHODS AND RESULTS: Killing of B. subtilis spores by aqueous ozone was not due to damage to the spore's DNA, as wild-type spores were not mutagenized by ozone and wild-type and recA spores exhibited very similar ozone sensitivity. Spores (termed alpha-beta-) lacking the two major DNA protective alpha/beta-type small, acid-soluble spore proteins exhibited decreased ozone resistance but were also not mutagenized by ozone, and alpha-beta- and alpha-beta-recA spores exhibited identical ozone sensitivity. Killing of spores by ozone was greatly increased if spores were chemically decoated or carried a mutation in a gene encoding a protein essential for assembly of the spore coat. Ozone killing did not cause release of the spore core's large depot of dipicolinic acid (DPA), but these killed spores released all of their DPA after a subsequent normally sublethal heat treatment and also released DPA much more readily when germinated in dodecylamine than did untreated spores. However, ozone-killed spores did not germinate with either nutrients or Ca(2+)-DPA and could not be recovered by lysozyme treatment. CONCLUSIONS: Ozone does not kill spores by DNA damage, and the major factor in spore resistance to this agent appears to be the spore coat. Spore killing by ozone seems to render the spores defective in germination, perhaps because of damage to the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide information on the mechanisms of spore killing by and resistance to ozone.     

Decontamination assessment of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surfaces using a hydrogen peroxide gas generator

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using hydrogen peroxide gas. METHODS AND RESULTS: Bacillus anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to > or =1000 ppm hydrogen peroxide gas for 20 min. Hydrogen peroxide exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials except G. stearothermophilus on industrial carpet. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with both surrogates. The effectiveness of gaseous hydrogen peroxide on the growth of biological indicators and spore strips was evaluated in parallel as a qualitative assessment of decontamination. At 1 and 7 days postexposure, decontaminated biological indicators and spore strips exhibited no growth, while the nondecontaminated samples displayed growth. CONCLUSIONS: Significant differences in decontamination efficacy of hydrogen peroxide gas on porous and nonporous surfaces were observed when comparing the mean log reduction in B. anthracis spores with B. subtilis and G. stearothermophilus spores. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using hydrogen peroxide gas.     

Bacillus sporothermodurans and other highly heat-resistant spore formers in milk

A recent example of a micro-organism causing undesired growth in consumer milk is Bacillus sporothermodurans producing highly heat-resistant spores (HRS) which may survive ultra-high temperature (UHT) treatment or industrial sterilization. Molecular typing showed a heterogeneous group of farm isolates (non-HRS strains), but a clonal group of UHT isolates from diverse European countries and other continents (HRS-clone) suggesting a common source. During a survey of Belgian dairy farms for the presence of potentially highly heat-resistant spore formers, high numbers of these spores were detected in filter cloth, green crop and fodder samples. The strain collection showed a high taxonomic diversity with 18 potentially new species and with Bacillus licheniformis and Geobacillus pallidus as predominating species overall. Seventeen B. sporothermodurans isolates were identified, mainly originating from feed concentrate. Heat resistance studies showed the UHT resistance of B. sporothermodurans spores present in industrially contaminated UHT milk, but a lower heat resistance of laboratory-grown strains (HRS and non-HRS). Hydrogen peroxide, used as sanitizer in the dairy industry, was found to induce higher heat resistance of laboratory-grown B. sporothermodurans strains to a certain level. This indicates that sublethal stress conditions may affect the heat resistance. By transmission electron microscopy, structural differences at the spore level were found between HRS and non-HRS strains. The data indicate that the attainment of extreme heat resistance is rather multifactorial.     

臭氧熏气下春小麦叶片脂质过氧化作用的研究

 应用开顶式熏气装置,以低浓度(0.1ppm)的臭氧对春小麦(Triticum aestivum)进行了长时间的熏气,观测到：随着熏气时间的延长,叶片内叶绿素含量下降,丙二醛(MDA)积累增多,膜透性增大。在较高浓度(0.2ppm、0.3ppm、0.4ppm)臭氧熏气下,臭氧浓度越高,叶片内叶绿素降解越快,MDA含量越高,膜透性越大。低浓度和短时间的臭氧熏气可使超氧化物歧化酶(SOD)活性升高,而高浓度和长时间的臭氧熏气则导致SOD活性下降。试验结果表明,由臭氧熏气所引起的植物体内代谢紊乱与诸多伤害是由于脂质过氧化作用的结果。     

The effect of ribose pre-treatment of cortical bone on γ-irradiation sterilization effectiveness

Reconstruction of large skeletal defects is a significant and challenging issue. Tissue banks often use γ-irradiation (15–35 kGy) to sterilize bone allografts, which, however, drastically impairs the post-yield mechanical properties. In previous studies, we reported the development of a method that protects human bone collagen connectivity through ribose crosslinking while still undergoing γ-irradiation. Given these promising results, the next step was to determine if the presence of ribose within the bone tissue would interfere with the effectiveness of the γ-irradiation sterilization against bacteria. This study had two stages. The aim of the first stage was to assess the protective effect of ribose in solution using a Bacillus pumilus spore strip model. The aim of the second stage was to assess the protective effect of ribose (R) on spores within a human cortical bone model in comparison to conventionally irradiated bone (I). Treatment of B. pumilus spore strips with ribose in solution led to temperature-dependent effects on spore viability versus spore strips treated with PBS alone. Ribose solution at 60 °C led to a notable two logs decrease in spore count relative to PBS at 60 °C. In the human bone model, the number of spores in the I and R groups were greatly decreased in comparison to the non-irradiated N group. No spore colonies were detected in the R group (n = 4) whereas two of the four plates of group I formed colonies. This study provides evidence that the method of pre-treating bone with ribose crosslinking prior to irradiation sterilization, while improving irradiation sterilized bone allograft quality, also may improve the effectiveness of the sterilization process.     

Formaldehyde gas inactivation of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials

AIMS: To evaluate the decontamination of Bacillus anthracis, Bacillus subtilis, and Geobacillus stearothermophilus spores on indoor surface materials using formaldehyde gas. METHODS AND RESULTS: B. anthracis, B. subtilis, and G. stearothermophilus spores were dried on seven types of indoor surfaces and exposed to approx. 1100 ppm formaldehyde gas for 10 h. Formaldehyde exposure significantly decreased viable B. anthracis, B. subtilis, and G. stearothermophilus spores on all test materials. Significant differences were observed when comparing the reduction in viable spores of B. anthracis with B. subtilis (galvanized metal and painted wallboard paper) and G. stearothermophilus (industrial carpet and painted wallboard paper). Formaldehyde gas inactivated>or=50% of the biological indicators and spore strips (approx. 1x10(6) CFU) when analyzed after 1 and 7 days. CONCLUSIONS: Formaldehyde gas significantly reduced the number of viable spores on both porous and nonporous materials in which the two surrogates exhibited similar log reductions to that of B. anthracis on most test materials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide new comparative information for the decontamination of B. anthracis spores with surrogates on indoor surfaces using formaldehyde gas.     

过氧化氢干雾对生物安全柜微环境表面消毒效果的研究

生物安全实验室微环境消毒是控制实验室污染的重要环节。过氧化氢广泛用于病原微生物实验室微环境消毒,但其对不同病原微生物的消毒效果有待研究。本文研究了过氧化氢干雾（粒径％10μm）以不同消毒程序对生物安全柜表面常见微生物的消毒效果。结果显示,在生物安全柜内采用优化的消毒程序（发散循环8次,每次1min,达60ppm后,静置消毒2h）,过氧化氢干雾可完全杀灭1×106CFU枯草芽胞杆菌、嗜热脂肪芽胞杆菌、金黄色葡萄球菌、表皮葡萄球菌、耻垢分枝杆菌,以及1×106CFU大肠埃希菌。然而,当金黄色葡萄球菌、表皮葡萄球菌、耻垢分枝杆菌浓度达1×107CFU时,过氧化氢干雾无法完全杀灭。因此,建议在进行过氧化氢干雾消毒时,应先用消毒剂处理,以期彻底杀灭生物安全柜微环境中污染的病原微生物。     

Microcount Method for Petrolatum-Based Topical Ointments Containing Waxes

A practical solvent system for the detection of microorganisms in topical ointments has been developed. The method involves dissolving 0.5 g of topical ointment in 50 ml of a solvent mixture (92 parts isopropyl myristate, 6 parts carbon disulfide, and 2 parts xylene) and filtering it through a 0.45-mum membrane filter. Residual solvent is then washed from the filter pad with 200 ml of sterile 0.5% Brain Heart Infusion broth containing 0.1% Tween 80. The filter pad is then removed and placed on a petri plate containing Trypticase Soy Agar medium. The petri plates thus prepared are then incubated at 37 C for 7 days, and the colonies produced are then counted. The toxicity of the solvent mixture was determined against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella newington, and spores of Bacillus subtilis and was found to be less toxic than the heat-sterilized isopropyl myristate and comparable to the filter-sterilized isopropyl myristate.     

Recovery of bacterial spores dried on aluminium strips

Bacterial spores dried on aluminium strips are used in microbiological validation of packaging and processing systems. Vortex agitation and sonication in Butterfield's buffer, 70% ethanol or 0·1% Tween 80 were evaluated for ease of recovery of bacillus spores dried on aluminium strips to compare the concentration of dried spores to dilutions used to inoculate such strips. The highest recovery for Bacillus subtilis var. globigii spores was observed with sonication in 70% ethanol with average recovery close to the initial inoculum. The highest recovery for B. stearothermophilus spores was with sonication in Butterfield's buffer, averaging 0·8 log less recovery than the initial inoculum. Bacillus subtilis var. globigii spores were recovered from strips in greater numbers than B. stearothermophilus spores for all treatment medium combinations. Scanning electron microscopy revealed unrecovered spores adhering to strips after treatment. Recovery of B. subtilis var. globigii spores decreased with time over the 4 week storage period.     

The Combined Effect of Hydrogen Peroxide and Ultraviolet Irradiation on Bacterial Spores

Ultra-violet (u.v.) light irradiation of spores of Bacillus subtilis in the presence of hydrogen peroxide produced a rapid kill which was up to 2000-fold greater than that produced by irradiation alone. A kill of 99–99% was produced by 30s u.v. irradiation of spores of 6 strains of Bacillus and Clostridium in the presence of hydrogen peroxide 1.0 g/100 ml but with the more resistant spores of 9 further strains, irradiation in the presence of hydrogen peroxide 2–5 g/100 ml followed by mild heating was required.     

Thermal Death of Bacillus subtilis var. niger Spores on Selected Lander Capsule Surfaces

Dry-heat sterilization of planetary lander capsules requires a knowledge of the thermal resistivity of microorganisms in the environment to which they will be subjected during sterilization of the space hardware. The dry-heat resistance of Bacillus subtilis var. niger spores on various lander capsule materials was determined at 125 C. Eight surface materials were evaluated, including a reference material, stainless steel. Survivor curves were computed, and decimal reduction times (D values) were obtained by a linear regression analysis. In four tests on stainless steel, the average value of D at 125 C was 17.07 min. The D values for the other seven materials tested ranged from 18.64 min on magnesium surfaces to 20.83 min on conversion-coated magnesium. Of the materials evaluated, the results indicate that there is only a significant difference in the thermal resistance of B. subtilis var. niger spores on conversion-coated magnesium and conversion-coated aluminum from that on the reference material, stainless steel. The differences in D values for all the test surfaces may be the result of variations in test procedures rather than the effect of the surfaces on the thermal resistivity of the spores.     

Preference for bedding material in Syrian hamsters

This study aimed to determine whether Syrian (golden) hamsters, Mesocricetus auratus, prefer certain bedding materials and whether bedding material can affect paw condition, body weight gain and wheel-running activity. In a first experiment, 26 male hamsters had access to two connected cages, each cage containing a different bedding material (either pine shavings, aspen shavings, corn cob or wood pellets). In a second experiment, 14 male hamsters had access to four connected cages that contained the different bedding materials and also a piece of paper towel to serve as nest material. In a third experiment, 30 male hamsters were each placed in a single cage, 10 of them with pine shavings, 10 with aspen shavings and 10 with corn cob, and they were monitored for 50 days. Significant preferences in the first experiment were: pine shavings over aspen shavings, corn cob over wood pellets, pine shavings over corn cob and aspen shavings over wood pellets (aspen shavings versus corn cob was not tested). However, there was no significant preference expressed in the second experiment, suggesting that the general preference for shavings in the first experiment was based on bedding material suitability as a nesting material. No significant effect of bedding material on paw condition, body weight gain and wheel-running activity was detected. None of the four bedding materials tested in this study can be judged to be inappropriate in the short term if nesting material is added to the cage and if the litter is changed regularly.