Microbiological Safety Evaluation of an Industrial Refuse Incinerator

An industrial refuse incinerator was tested to determine minimal operating temperatures required to prevent release of viable microorganisms into the atmosphere. A liquid suspension of Bacillus subtilis var. niger spores was disseminated into the firebox as an aerosol, and dry spores mixed with animal bedding were dumped into the firebox. The minimal requirement for wet spores was 575 F (302 C) for the firebox air temperature and 385 F (196 C) for the firebrick refractory lining. When dry spores were used, these temperatures were 700 and 385 F (371 and 196 C), respectively.     

Mechanism of microwave sterilization in the dry state.

With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.     

Mechanism of microwave sterilization in the dry state

With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.     

Inactivation of Dried Bacteria and Bacterial Spores by Means of Gamma Irradiation at High Temperatures

Dried preparations with Streptococcus faecium, strain A(2)1, and spores of Bacillus sphaericus, strain C(I)A, normally used for control of the microbiological efficiency of radiation sterilization plants and preparations with spores of Bacillus subtilis, normally used for control of sterilization by dry heat, formalin, and ethylene oxide, as well as similar preparations with Micrococcus radiodurans, strain R(1), and spores of Bacillus globigii (B. subtilis, var. niger) were gamma irradiated with dose rates from 16 to 70 krad/h at temperatures from 60 to 100 C. At 80 C the radiation response of the spore preparations was the same as at room temperature, whereas the radiation resistance of the preparations with the two vegetative strains was reduced. At 100 C the radiation response of preparations with spores of B. subtilis was unaffected by the high temperature, whereas at 16 and and 25 krad/h the radiation resistance of the radiation-resistant sporeformer B. sphaericus, strain C(I)A, was reduced to the level of radiation resistance of preparations with spores of B. subtilis. It is concluded that combinations of heat and gamma irradiation at the temperatures and dose rates tested may have very few practical applications in sterilization of medical equipment.     

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.     

Thermal Inactivation of Aerosolized Bacillus subtilis var. niger Spores

A hot-air sterilizer capable of exposing airborne microorganisms to elevated temperatures with an almost instantaneous heating time was developed and evaluated. With this apparatus, aerosolized Bacillus subtilis var. niger spores were killed in about 0.02 sec when exposed to temperatures above 260 C. This is about 500 times faster than killing times reported by others. Extrapolation and comparison of data on the time and temperature required to klll B. subtilis var. niger spores on surfaces show that approximately the same killing time is required as is necessary for spores in air, if corrections are made for the heating time of the surface.     

100～700 mL液体培养基的微波灭菌效果观察

为研究家用微波炉对少量液体培养基的灭菌效果,对100 -700 mL液体培养基采取微波分别进行灭菌,测定其灭菌时间,并对达到灭菌效果的培养基通过接种不同细菌进行质检.结果显示100、200、300、400、500、600、700 mL液体培养基分别在4.5、5.0、7.0、9.0、12.0、19.0、25.0 min达到最有效的灭菌效果.室温保存15 d仍无细菌生长.枯草杆菌黑色变种芽胞菌测试无菌生长.金黄色葡萄球菌、大肠埃希菌及普通变形杆菌在液体培养基里的生长现象、特种后细菌的菌落和形态染色特征及其生化反应均无明显差异.实验表明2 450 MHz,700 W的微波炉对100 - 700 mL的液体培养基不但能快速达到有效的灭菌效果,且营养成分不会被破坏.     

Laboratory bioassays of entomopathogenic fungi for control of Delia radicum (L.) larvae

Laboratory soil bioassays were performed at economic field rates for in-furrow (3.85 x 10(6)spores/g dry soil) and broadcast (3.85 x 10(5)spores/g dry soil) applications with three isolates of Metarhizium anisopliae (F52, ATCC62176, and ARSEF5520) and one isolate of Beauveria bassiana (GHA). All isolates tested were infective to second instar Delia radicum (L.). The conditionally registered M. anisopliae isolate (F52) performed best killing an average of 85 and 72% of D. radicum larvae at the high and low concentration, respectively. The mean LC50 and LC95 of F52 against second instar D. radicum was 2.7 x 10(6) and 1.8 x 10(8)spores/g dry soil, respectively. The use of F52 in an integrated management program is discussed.     

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.     

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.     

Application of a dense gas technique for sterilizing soft biomaterials

Sterilization of soft biomaterials such as hydrogels is challenging because existing methods such as gamma irradiation, steam sterilization, or ethylene oxide sterilization, while effective at achieving high sterility assurance levels (SAL), may compromise their physicochemical properties and biocompatibility. New methods that effectively sterilize soft biomaterials without compromising their properties are therefore required. In this report, a dense-carbon dioxide (CO(2) )-based technique was used to sterilize soft polyethylene glycol (PEG)-based hydrogels while retaining their structure and physicochemical properties. Conventional sterilization methods such as gamma irradiation and steam sterilization severely compromised the structure of the hydrogels. PEG hydrogels with high water content and low elastic shear modulus (a measure of stiffness) were deliberately inoculated with bacteria and spores and then subjected to dense CO(2) . The dense CO(2) -based methods effectively sterilized the hydrogels achieving a SAL of 10(-7) without compromising the viscoelastic properties, pH, water-content, and structure of the gels. Furthermore, dense CO(2) -treated gels were biocompatible and non-toxic when implanted subcutaneously in ferrets. The application of novel dense CO(2) -based methods to sterilize soft biomaterials has implications in developing safe sterilization methods for soft biomedical implants such as dermal fillers and viscosupplements.     

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.     

Validation of the sterilization procedure of allogeneic avital bone transplants using peracetic acid-ethanol.

Different procedures are available to inactivate bacteria and fungi, including their spores, as well as viruses in human bone transplants. The most efficient methods are considered to be gamma irradiation and thermal inactivation as well as chemical sterilization methods like the peracetic acid-ethanol treatment (PES). Following national and international standards or draft standards, the antimicrobial effectiveness of this procedure was evaluated. Due to the standardizable size as well as the clinical relevance, defatted human spongiosa cuboids (15x15x15 mm) served as model system. After treatment with PES for 2 and 4 hours, respectively, the titre of living micro-organisms was determined in the supernatant and the cuboid. A reduction in the titre of viable micro-organisms below the detection level (reduction factor >5 log10) was already achieved after an incubation time of 2 hours (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Bacillus subtilis, Clostridium sporogenes, Mycobacterium terrae, Candida albicans as well as spores of Bacillus subtilis). No viable micro-organisms could be detected in any of the PES-treated test cuboids. Spores of Aspergillus niger were also completely inactivated. The PES procedure proved to be a reliable method for the sterilization of human bone transplants derived from spongiosa.     

Microbial validation of vent filters

The Upjohn Company uses filtration to remove microorganisms and particulates from air and other gases which may come in contact with sterile products. To validate the microbial retentivity of these filters, they were challenged with an aerosol of Bacillus subtilis var niger spores. An aerosol challenge was used because it more closely simulated the use for which these filters were designed. The test apparatus was constructed of autoclavable components using a jet-type nebulizer and heated air mixing tube. Characterization of the aerosol particle size distribution with a particle size analyzer demonstrated that 80% of the particles had a diameter of x 3.0 times;m and that the particles had a mean mass diameter of 1.9 times;m with a geometric standard deviation of 1.8 times;m. Studies conducted with aerosols of Bacillus subtilis var niger spores demonstrated that the test apparatus could recover ca. 50% of the spores that were aerosolized. Hydrophobic filters from various manufactures were challenged with an aerosol of at least 10(8) spores of Bacillus subtilis. All filters tested could retain at least 10(9) spores when physical integrity of the filter was verfield.     

Resistivity of Spores to Ultraviolet and γ Radiation while Exposed to Ultrahigh Vacuum or at Atmospheric Pressure

Viability studies were conducted on microbial spores subjected to ultrahigh vacuum (UHV) in the 10(-9) to 10(-10) torr range. After 5 to 7 days in vacuum, they were exposed to ultraviolet (UV) or to gamma radiation either while still under vacuum or in the presence of dried air. Among the four test organisms subjected to UHV and ultraviolet radiation, Aspergillus niger was the most resistant; Bacillus megaterium, B. subtilis var. niger, and B. stearothermophilus were about equally less resistant. All four spores were more sensitive to ultraviolet radiation when UHV-dried than when desiccant-dried. Of the four test organisms subjected to UHV and gamma radiation, B. megaterium proved to be the most resistant; A. niger was the least resistant; and the remaining two organisms were of intermediate resistivity. All four organisms were less radiation resistant when UHV-dried than when irradiated in their normally hydrated state, and all showed an increased radiosensitivity after vacuum drying when oxygen was present. In addition, spores of B. subtilis var. niger and A. niger were less radiosensitive when UHV-dried and irradiated in vacuum than when "wet" and irradiated in air, whereas the reverse relationship was observed for the remaining two organisms. Based on the fact that microbial contaminants can be readily shielded from UV light by soils, metal particles, etc., and considering that the levels of ionizing radiations reported to be present in interstellar space are generally lower than those used in these experiments, the decrease in radioresistivity imparted by UHV drying is not of a sufficient magnitude to sterilize dependably portions of a spacecraft while on a mission.     

Sporicidal efficacy of genipin: a potential theoretical alternative for biomaterial and tissue graft sterilization

Terminal sterilization of musculoskeletal allografts by gamma radiation minimizes the risk of disease transmission but impairs allograft mechanical properties. Commonly employed crosslinking agents can sterilize tissues without affecting mechanical properties adversely; however, these agents are toxic. Genipin is reported to be a benign crosslinking agent that strengthens mechanical properties of tissues; however, the antimicrobial capacity of genipin is largely unknown. The present study’s aims were: (1) to assess the sporicidal potential of genipin, (2) to improve antimicrobial capacity by changing chemical and physical treatment conditions. To establish genipin’s sterilization potential Bacillus subtilis var. niger spore strips were treated with 0–10 % genipin in PBS or in 1:1 DMSO:PBS up to 72 h at room temperature (RT). Sterilizing doses and concentrations of genipin were used to treat B. pumilus and Geobacillus stearothermophilus spores to assess broader spectrum sporicidal activity of genipin. Scanning electron microscopy (SEM) was performed to evaluate gross morphological changes after genipin treatment. Optimal sterilization conditions were determined by evaluating the effects of temperature (RT-50 °C), DMSO:PBS ratio (0:100–100:0), and treatment duration (24–72 h) on B. subtilis. Genipin penetration of full thickness bovine patellar tendon and cortical bone specimens was observed to assess the feasibility of the agent for treating grafts. Initial studies showed that after 72 h of treatment at RT with 0.63–10 % genipin/DMSO:PBS B. subtilis spore strips were sterilized; 0.63 % genipin/PBS did not sterilize spore strips at 72 h at RT. Genipin doses and concentrations that sterilized B. subtilis spore strips sterilized B. pumilus and G. stearothermophilus spore strips. SEM revealed no gross morphological differences between untreated and treated spores. Treatment optimization resulted in sterilization within 24 h with 100 % PBS, and DMSO facilitated sporicidal activity. Genipin penetrated full thickness patellar tendon specimens and 3.72 ± 0.58 mm in cortical bone specimens. Genipin sterilizes B. subtilis, B. pumilus, and G. stearothermophilus spore strips. It penetrates soft and hard tissues at doses previously shown to be non-toxic and to improve mechanical strength in collagen-rich soft tissues. Further studies are indicated to assess genipin’s effects on the mechanical properties of genipin-sterilized grafts, the ability of genipin to eradicate infectious species other than spores, and to assess whether sterilant activity persists after penetrating tissues and biomaterials.     

Resistance of Bacillus subtilis var. niger Spores Occluded in Water-insoluble Crystals to Three Sterilization Agents

The resistance to destruction of spores of Bacillus subtilis var. niger occluded in crystals of calcium carbonate and exposed to ethylene oxide and moist and dry heat was determined and compared with the destruction of unoccluded spores. Occluded spores could not be inactivated with ethylene oxide. Resistance to inactivation was approximately 900 and 9 times higher for occluded than for unoccluded spores subjected to moist and dry heat, respectively, at 121 C. The protective effect may be due either to the unavailability of oxygen for destruction by oxidation or to inhibition of the loss of essential cell constituents by vaporization. Evidence also implicates the crystal structure as a thermal conductivity barrier. Occluded spores retained viability over a 3-year period compared with unoccluded spores which decreased over 90% during this period. Occluded spores in insoluble materials are seldom encountered in the technology of sterilization, but could be the most critical factor in the sterilization of interplanetary vehicles. Entrapped spores in insoluble materials are usually difficult to detect, and are very stable as well as extremely resistant to destruction by heat and ethylene oxide.     

Gamma radiation resistance of Bacillus anthracis spores

The aim of the presented study was determined the effectiveness of action the gamma radiation on water suspension B. anthracis spores. The irradiation was performed using a Cobalt 60 (Co 60) source, by using single and fractionary irradiation doses. In the investigations was used B. anthracis stain "Sterne" 34F2. The obtained results show, that gamma radiation effectively inactivates B. anthracis spores. On the efficiency of sterilization process influence the irradiation's method and the number of spores in 1 ml suspension. In the suspension 1.5 x 10(9) spore in 1 ml, sporicidal doses gamma radiation amount to 25.0 kGy (single dose) or 41.5 kGy (fractionary dose). The volume suspension about definite inoculum of spores, subjected working the gamma rays has not influence on sporicidal effectiveness of radiation sterilization.     

Effect of Temperature and Gas Velocity on the Dry-Heat Destruction Rate of Bacterial Spores

Spores of Bacillus subtilis were dried in vacuo for use in dry-heat thermal destruction tests. Survivor curve tests were conducted in a specifically designed dry-heat oven. This oven provided accurate temperature control and permitted air or nitrogen to be passed over the spores during the lethal treatment. Experiments were carried out at various flow rates of the two gases (air and nitrogen) and various temperatures, and the data were expressed as survivor curves from which the decimal reduction time (D value) was obtained. Linear regression analysis methods were used to compute the slope of the survivor curves. The results indicated that as the flow rate of gas is increased, the effect of temperature on the destruction rate of the spores is lessened, the z value becoming very large. It is believed that the higher flow rates of dry gas cause greater dehydration of the spores and that spore moisture loss is one of the major factors in determining the dry-heat thermal destruction rate of bacterial spores.     

Synergistic Effects in Sonochemical Sterilization

The synergistic effects observed during the sterilization of Bacillus subtilis var. niger ATCC 9372 by the combined action of ethylene or propylene oxide with high-intensity airborne sound waves (34.8 kc/sec) were investigated. It has been shown that the number of surviving spores deposited on paper strips decreases exponentially with the sound intensity at sample level. Reductions of the order of one-third of the time required for standard propylene oxide sterilization have been observed by using the combined action of sound waves with gaseous sterilization. At the present time, maximal synergistic effects seem to be achieved for the following experimental conditions: propylene oxide concentration, 500 to 1,000 mg/liter; acoustic intensity, 161 to 162 db; contact time, 80 min; temperature, 60 C; and relative humidity, 40%. The basic mechanism involved in sonochemical sterilization seems to be more of a physical (accelerated gas diffusion) than a chemical nature.