An evaluation of the sporicidal activity of ozone

This study was undertaken to determine the feasibility of sterilizing surfaces with ozone-saturated water by the methods of the Association of Official Analytical Chemists (AOAC). Initially, it was determined that there was no apparent difference in ozone resistance between spores of Bacillus subtilis and Clostridium sporogenes when they are suspended in water. Both species were inactivated by a 10-min exposure at ambient temperature. Resistance was increased when the spores were dried on AOAC carriers. Viable organisms were recovered after an exposure of 40 min at ambient temperature. An increase in the reactor water temperature to 60 degrees C did not improve the effectiveness of the ozone in sterilizing AOAC carriers. Dried spores of C. sporogenes were more resistant than B. subtilis spores because of a greater accumulation of organic matter on the carriers. No significant sporicidal activity was demonstrated after 40 min for spores of either species when they were inoculated on silk suture loops. The data suggest that organic loading and poor ozone penetrability are key factors in effecting the ability of ozone to sterilize surfaces rapidly.     

Effect of Various Gas Atmospheres on Destruction of Microorganisms in Dry Heat

The heat resistance of dry bacterial spores was tested in various gases at temperatures ranging from 121.1 to 160 C (250 to 320 F). Spores of Clostridium sporogenes (PA 3679) were heated in air, carbon dioxide, and helium; spores of Bacillus subtilis 5230 were heated in these gases and also in oxygen and in nitrogen. The surrounding gas influenced the heat resistance, but the differences among gases were small. D values were about 7 min at 148.9 C (300 F); z values were about 18.3 C (33 F) for B. subtilis, and about 21.7 C (39 F) for C. sporogenes. The resistance of B. subtilis in carbon dioxide was about the same as in air, but lower than in all other gases; resistance in helium and nitrogen was about the same, and was higher than in all other gases. C. sporogenes had the least resistance in air; the resistance was about the same in carbon dioxide and helium. For B. subtilis, the gases in order of increasing heat resistance were carbon dioxide, air, oxygen, helium, and nitrogen, and for C. sporogenes, air, carbon dioxide, and helium. Neither oxygen content nor molecular weight of the gas appeared to have a marked influence on dry-heat resistance of the spores, whereas the more inert gases seemed to yield larger D values.     

Microbiological Aspects of Ethylene Oxide Sterilization: II. Microbial Resistance to Ethylene Oxide

The death rate kinetics of several sporeforming and nonsporeforming microorganisms, including radiation-resistant cocci, were determined by exposing them to a mixture of ethylene oxide and dichlorodifluoromethane (500 mg of ethylene oxide per liter, 30 to 50% relative humidity, and 54.4 C). Spore survivor curves obtained from tests of inoculated and exposed hygroscopic and nonhygroscopic carriers showed that the spores of Bacillus subtilis var. niger are more resistant to ethylene oxide than are spores of Clostridium sporogenes, B. stearothermophilus, and B. pumilus. The decimal reduction times (expressed as D values at 54.4 C-500 mg of ethylene oxide per liter) obtained under the test conditions for B. subtilis var. niger spores on hygroscopic and nonhygroscopic carriers exceeded the values obtained for the other organisms considered, both sporeformers and nonsporeformers. The decimal reduction times for the vegetative cells of the radiation-resistant organisms (Micrococcus radiodurans and two strains of Streptococcus faecalis) and the ATCC strain of S. faecalis demonstrated comparable resistance to ethylene oxide with the spores of C. sporogenes, B. stearothermophilus, and B. pumilus, but not those of B. subtilis var. niger.     

Effects of inoculum level and pressure pulse on the inactivation of Clostridium sporogenes spores by pressure-assisted thermal processing

The effects of initial concentration and pulsed pressurization on the inactivation of Clostridium sporogenes spores suspended in deionized water were determined during thermal processing (TP; 105 degrees C, 0.1 MPa) and pressure-assisted thermal processing (PATP; 105 degrees C and 700 MPa) treatments for 40 min and 5 min holding times, respectively. Different inoculum levels (10(4), 10(6), and 10(8) CFU/ml) of C. sporogenes spores suspended in deionized water were treated at 105 degrees C under 700 MPa with single, double, and triple pulses. Thermally treated samples served as control. No statistical significances (p > 0.05) were observed among all different inoculum levels during the thermal treatment, whereas the inactivation rates (k1 and k2) were decreased with increasing the initial concentrations of C. sporogenes spores during the PATP treatments. Double- and triple-pulsed pressurization reduced more effectively the number of C. sporogenes spores than single-pulse pressurization. The study shows that the spore clumps formed during the PATP may lead to an increase in pressure-thermal resistance, and multiple-pulsed pressurization can be more effective in inactivating bacterial spores. The results provide an interesting insight on the spore inactivation mechanisms with regard to inoculum level and pulsed pressurization.     

Sterilization by means of Hydrogen Chloride: Influence of Process Parameters on Biocidal Activity

At ambient temperatures, hydrogen chloride is a strong bactericide. At a partial hydrogen chloride pressure of 30 mm Hg, the number of spore-bearing colony forming units present in straw dust particles of 0–32 μm was reduced by a factor-of more than 50 000 within 60 s. Naked spores of Bacillus subtilis were reduced in viability by a factor of 100 000 within only 10 s. Viability of vegetative cells of various kinds of bacteria was reduced by a factor of more than 10⁷ within 10 s. The method of administration of the gas influenced the sterilization and had to be adapted to the geometry of the surfaces to be sterilized. The smaller the dust particles, the better the sterilization, seemingly due to the lower degree of inhibition of diffusion of gas to the microorganisms enclosed in the particles. Increases in treatment time and concentration of hydrogen chloride enhanced the sterilizing effect to a lesser extent when large dust particles were involved. The partial water vapour pressure did not influence the sterilization between 0.3 and 6 mm Hg. An increase in temperature decreased the sterilizing effect.     

Monoclonal antibodies for use in detection of Bacillus and Clostridium spores.

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.     

Effect of heat and ultrasonic waves on the survival of two strains of Bacillus subtilis

The combined effect of ultrasonic (20 KHz, 150 W) and heat treatment on the survival of two strains of Bacillus subtilis in three suspending media (distilled water, glycerol and milk) has been studied. When spores suspended in water or milk were subjected to ultrasonic waves before heat treatments a little or no decrease of the heat resistance was observed. However, both sporicidal agents applied simultaneously (thermo-ultrasonication) decreased by 63% (B. subtilis, var. niger-40) and 74% (B. subtilis ATCC 6051) the decimal reduction times for the heat treatment when the spores were suspended in glycerol and by 79% and 40%, respectively when suspended in milk. The thermo-ultrasonication of spores in water markedly reduced the heat resistance of them (between 99.9% and 70%) in the range 70-95 degrees C but the effect of the thermo-ultrasonication significantly diminished as the temperature of the treatment was approached to the boiling point of the water.     

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.     

SOME OBSERVATIONS ON BACTERICIDAL PAINT FILMS

SUMMARY: A method is described for testing the self-sterilizing activity of surfaces coated with paints containing bactericidal agents. The most active coating consisted of 10% (w/w) Cetavlon dissolved directly in urea-formaldehyde:alkyd resin. The recovery of viable organisms from such surfaces infected with Streptococcus faecalis, Staphylococcus aureus, Aerobacter aerogenes and Chromobacterium prodigiosum was less than 10% of that from painted control surfaces after exposure for 1 hr at atmospheric relative humidity of 70% and temperature of 20°. Bacillus subtilis spores were not affected until they became heat-sensitive. The self-sterilizing activity was markedly diminished at 40% r.h. and the inclusion of blood in the deposited bacterial particle was then completely protective. Repeated washing in water with gentle rubbing quickly inactivated Cetavlon paint films.     

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.     

Heat sensitization of bacterial spores after exposure to ethidium bromide, acriflavine, or daunomycin.

A 20-min exposure of 10(7) unmodified spores of either Bacillus subtilis NCTC 3610 (harvested from potato-dextrose agar plus manganese) or Bacillus megaterium ATCC 19213 (harvested from nutrient agar plus manganese) per ml to 5 microgram of ethidium bromide per ml did not kill the spores (recovered on TAM [thermoacidurans agar modified]-plus thymidine medium). However, in both cases, the ability to survive various heat treatments was reduced after exposure of the spores to ethidium bromide. With B. subtilis, a 10-min heat treatment at 85 degrees C of unexposed spores resulted in an 85% survival rate, whereas only 50% of the ethidium bromide-exposed spores survived. With B. megaterium similar results were obtained at 75 degrees C; 77% of the unexposed spores survived, whereas only 31% of the ethidium bromide-exposed spores survived. Similarly, a 10-min exposure of B. subtilis spores to 0.005 microgram of acriflavine per ml did not kill unheated spores; however, the ability of the spores to survive exposure at 85 degrees C for 10 min was reduced to 40%. After exposure to 10 microgram of daunomycin per ml, the survival rate was 35%. Binding studies with ethidium bromide showed strong binding to spores, but as yet, the site of binding is unknown.     

Synergism between Ultrasonic Waves and Hydrogen Peroxide in the Killing of Micro-organisms

The lethal effect on different micro-organisms of ultrasonic waves and hydrogen peroxide separately and in combination was examined. Ultrasonic waves were able to disintegrate Fusobacterium nucleatum within 3 min and to kill Veillonella parvula after 15 min and Streptoccus sanguis after 20 min; 20 vols H₂O₂ (6% w/v) killed V. parvula, Strep. sanguis and Staphylococcus aureus after 5 min treatment, and Clostridium sporogenes spores after 25 min. Sonication of Cl. sporogenes spores, Bacillus cereus spores and Candida albicans in 20 vols H₂O₂, using an ultrasonic probe, was lethal to the organisms after 15, 10 and 10 min, respectively. The latter 2 organisms were not killed by 30 min exposure to either agent separately. Similar results were obtained when an ultrasonic tank was used for sonication.     

Gas discharge plasmas are effective in inactivating <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Clostridium</Emphasis> spores

Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance.     

Resistance of Micro-organisms to Inactivation by Gaseous Ethylene Oxide

A simple method for the exposure of micro-organisms to ethylene oxide on membrane filters in a modified desiccator has been devised and used to study microbial resistance to the gaseous sterilant and the term ' R -value' is suggested to express this. The resistance of many known species and isolates has been assessed and compared. Several species of Bacillus were isolated from natural habitats and their spores were found to be more resistant than the strain of Bacillus subtilis var. niger (NCTC 10073) frequently used to monitor ethylene oxide sterilization. However, endospores of some bacterial species exhibited little resistance. Fungal spores and vegetative bacteria exhibited low resistance to the sterilant except after drying in organic material when they appeared more resistant than spores of B. subtilis var. niger. It was concluded that resistance to ethylene oxide did not correlate with resistance to heat, irradiation or other chemical disinfectants, or to the existence in the endospore form per se.     

The effect of transition metal ions on the resistance of bacterial spores to hydrogen peroxide and to heat.

The presence of 10 microM-Cu2+ increased the lethal effect of hydrogen peroxide on spores of Clostridium bifermentans but not on those of Clostridium sporogenes PA 3679, Clostridium perfringens, Bacillus cereus or Bacillus subtilis var. niger. Cu2+ at 100 muM also increased the lethal effect of heat on spores of C. bifermentans but not on those of B. sutilis var. niger. The rate and extent of Cu2+ uptake by spores of C. bifermentans and B. subtilis var. niger were similar, but examination of unstained sections of spores by electron microscopy suggested that Cu2+ is bound by the protoplasts of spores of C. bifermentans but not of B. subtilis var. niger.     

Dry Heat or Gaseous Chemical Resistance of Bacillus subtilis var. niger Spores Included Within Water-soluble Crystals

Inclusion of spores of Bacillus subtilis var. niger in water-soluble crystals increased the resistance of the spores to dry heat and to a gaseous mixture of methyl bromide and ethylene oxide. Resistance of spores in glycine crystals to dry heat at 125 C was increased 5 to 24 times compared to unprotected spores. There appeared to be a positive correlation between the size of the crystal and the degree of resistance. The resistance to dry heat of spores included in sodium chloride crystals was about six times greater than unprotected spores. A gaseous mixture of methyl bromide (964 mg/liter) and ethylene oxide (642 mg/liter) at 37% relative humidity was ineffective in sterilizing spores enclosed within these water-soluble crystals, as was ethylene oxide alone. However, if the relative humidity was sufficiently high to dissolve the crystals during exposure to the vapor, viable-spore counts were drastically reduced or were negative. The surfaces of crystals grossly contaminated with dry spores were sterilized by exposure to gaseous ethylene oxide. Sterilization of heat-labile or moisture-labile materials with a critical requirement for sterility, as in planetary probes or drugs, may be complicated by the presence of spores in naturally occurring water-soluble crystals. This phenomenon is similar to the protection afforded spores entrapped in solid plastics.     

METHODS OF ASSESSING THE SPORICIDAL EFFICIENCY OF AN ULTRA-HIGH-TEMPERATURE MILK STERILIZING PLANT I. EXPERIMENTS WITH SUSPENSIONS OF SPORES IN WATER

SUMMARY: A method of assessing the sporicidal efficiency of a UHT milk sterilizing plant operating on water is described. Water heavily contaminated with spores of a strain of Bacillus subtilis was filtered, after treatment in the plant, through membrane filters and the surviving spores estimated by incubation of the membranes in nutrient agar. With this plant a temperature of c . 135° caused a 99·99999% kill of B. subtilis spores. Confirmation of the lethal effects of temperatures above 135° was obtained by passing treated water into 10 gal churns containing sterile concentrated nutrient broth and incubating the churns.     

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.     

Effect of the osmotic conditions during sporulation on the subsequent resistance of bacterial spores

The causes of Bacillus spore resistance remain unclear. Many structures including a highly compact envelope, low hydration of the protoplast, high concentrations of Ca-chelated dipicolinic acid, and the presence of small acid-soluble spore proteins seem to contribute to resistance. To evaluate the role of internal protoplast composition and hydration, spores of Bacillus subtilis were produced at different osmotic pressures corresponding to water activities of 0.993 (standard), 0.970, and 0.950, using the two depressors (glycerol or NaCl). Sporulation of Bacillus subtilis was slower and reduced in quantity when the water activity was low, taking 4, 10, and 17 days for 0.993, 0.970, and 0.950 water activity, respectively. The spores produced at lower water activity were smaller and could germinate on agar medium at lower water activity than on standard spores. They were also more sensitive to heat (97 degrees C for 5-60 min) than the standard spores but their resistance to high hydrostatic pressure (350 MPa at 40 degrees C for 20 min to 4 h) was not altered. Our results showed that the water activity of the sporulation medium significantly affects spore properties including size, germination capacity, and resistance to heat but has no role in bacterial spore resistance to high hydrostatic pressure.     

Initiation of Bacillus spore germination by hydrostatic pressure: effect of temperature.

Suspensions of Bacillus cereus T, B. subtilis, and B. pumilus spores in water or potassium phosphate buffer were germinated by hydrostatic pressures of between 325 and 975 atm. Kinetics of germination at temperatures within the range of 25 to 44 degrees C were determined, and thermodynamic parameters were calculated. The optimum temperature for germination was dependent on pressure, species, suspending medium, and storage time after heat activation. Germination rates increased significantly with small increments of pressure, as indicated by high negative deltaV values of -230 +/- 5 cm3/mol for buffered B. subtilis (500 to 700 atm) and B. pumilus (500 atm) spores and -254 +/- 18 cm3/mol for aqueous B. subtilis (400 to 550 atm) spores at 40 degrees C and -612 +/- 41 cm3/mol for B. cereus (500 to 700 atm) spores at 25 degrees C. The ranges of thermodynamic constants calculated at 40 degrees C for buffered B. pumilus and B. subtilis spores at 500 and 600 atm and for aqueous B. subtilis spores at 500 atm were: Ea = 181,000 to 267,000 J/mol; deltaH = 178,000 to 264,000 J/mol; deltaG = 94,000 to 98,300 J/mol; deltaS = 264 to 544 J/mol per degree K. These values are consistent with the concept that the transformation of a dormant to a germinating spore induced by hydrostatic pressure involves either hydration or a reduction in the visocosity of the spore core and a conformational change of an enzyme.