Limitations of Thioglycolate Broth as a Sterility Test Medium for Materials Exposed to Gaseous Ethylene Oxide

Although ethylene oxide is a reliable sterilizer, the process may be limited by diffusion. Thus, situations may exist where microorganisms are protected from the sterilizing gas. It is possible that the exterior of a substance may be sterilized, whereas the interior is not. We investigated three general types of materials in which this limitation of diffusion could occur: the bore of glass and plastic tubing, the center of cotton balls, and plastic adhesive film/paper backing interface. These materials were contaminated as close to their geometric center as possible with Bacillus subtilis var. niger spores occluded in crystals of sodium chloride. After exposure of the contaminated materials (except aluminum foil) to ethylene oxide, thioglycolate broth (a standard sterility-test medium) indicated sterility, whereas Trypticase Soy Broth indicated nonsterility. It is likewise possible that aerobic microorganisms, surviving in or on material after exposure to dry heat or steam sterilization processes, would not be recovered by thioglycollate broth. Entrapped aerobic organisms will probably not grow out in the low oxygen tension zone of an anaerobic medium such as thioglycollate broth. It is recommended than an aerobic medium such as Trypticase Soy Broth be used concurrently with thioglycolate broth for sterility testing.     

Ethylene Oxide Resistance of Nondesiccated and Desiccated Spores of Bacillus subtilis var. niger Hermetically Sealed in Various Polymeric Films

The resistance to destruction of spores of Bacillus subtilis var. niger hermetically sealed in various polymeric films and exposed to ethylene oxide with and without relative humidity was determined. The effect of desiccation was also determined. The order of increased resistance to sterilization with regard to type of polymeric film was found to be: polyethylene equal to polyvinyl chloride, less than nylon, less than cellophane/polyethylene laminate, less than phenoxy, less than mylar/polyethylene laminate. Desiccated spores sealed in various polymeric films were much more resistant to ethylene oxide sterilization than nondesiccated spores. Relative humidity was an important factor in ethylene oxide sterilization with spores not sealed in polymeric films. However, with spores hermetically sealed in polyethylene, added relative humidity was an insignificant factor in the sterilization process.     

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.     

A method for the assessment of safe rehydration regimes for pasta

It is well-known that bacterial spores are more resistant to dry heat than moist heat. Therefore, in order to ensure the safe application of thermal processes aimed at the destruction of Clostridium botulinum it is essential that all the ingredients of a food product are fully rehydrated. A simple microbiological method, based on the immobilization of spores of known moist heat resistance in a product, has been developed to evaluate the processing conditions required to give full rehydration of pasta. The method is compared to traditional approaches.     

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.     

The influence of water on the resistance of spores to inactivation by gaseous ethylene oxide

Standardized conditions for exposure to ethylene oxide were used to evaluate the resistance of spores dried for various times at different relative humidities and temperatures. Spores dried under conditions of high humidity exhibited low resistance to the sterilant, the resistance increasing as the relative humidity (RH) was decreased. Increasing the temperature of drying amplified this effect by reducing the time required for equilibration to a specific RH. Spores dried over a desiccant at 37 degrees C showed a slight rise followed by a fall in resistance. Spores maintained under these conditions for a long period of time increased in resistance. Spores rapidly dried by exposure to low RH, over a desiccant or at elevated temperature, dried unevenly resulting in a heterogeneous population with respect to ethylene oxide resistance. This was expressed as non-logarithmic survivor curves. The initial vacuum drawn influences resistance. The resistance of spores dried on aluminium foil increased as the pressure was reduced. The rate at which the pressure was reduced had little effect on resistance, except with highly desiccated spores. Dried spores held at different reduced pressures with humidification, showed no differences in resistance. The implications of these findings in relation to the operation of ethylene oxide sterilization cycles and the preparation and use of biological monitors is discussed.     

The influence of water on the resistance of spores to inactivation by gaseous ethylene oxide

Standardized conditions for exposure to ethylene oxide were used to evaluate the resistance of spores dried for various times at different relative humidities and temperatures. Spores dried under conditions of high humidity exhibited low resistance to the sterilant, the resistance increasing as the relative humidity (RH) was decreased. Increasing the temperature of drying amplified this effect by reducing the time required for equilibration to a specific RH. Spores dried over a desiccant at 37°C showed a slight rise followed by a fall in resistance. Spores maintained under these conditions for a long period of time increased in resistance. Spores rapidly dried by exposure to low RH, over a desiccant or at elevated temperature, dried unevenly resulting in a heterogeneous population with respect to ethylene oxide resistance. This was expressed as non-logarithmic survivor curves. The initial vacuum drawn influences resistance. The resistance of spores dried on aluminium foil increased as the pressure was reduced. The rate at which the pressure was reduced had little effect on resistance, except with highly desiccated spores. Dried spores held at different reduced pressures with humidification, showed no differences in resistance. The implications of these findings in relation to the operation of ethylene oxide sterilization cycles and the preparation and use of biological monitors is discussed.     

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.     

Improved Sterilization of Sensitive Biomaterials with Supercritical Carbon Dioxide at Low Temperature

The development of bio-resorbable implant materials is rapidly going on. Sterilization of those materials is inevitable to assure the hygienic requirements for critical medical devices according to the medical device directive (MDD, 93/42/EG). Biopolymer-containing biomaterials are often highly sensitive towards classical sterilization procedures like steam, ethylene oxide treatment or gamma irradiation. Supercritical CO₂ (scCO₂) treatment is a promising strategy for the terminal sterilization of sensitive biomaterials at low temperature. In combination with low amounts of additives scCO₂ treatment effectively inactivates microorganisms including bacterial spores. We established a scCO₂ sterilization procedure under addition of 0.25% water, 0.15% hydrogen peroxide and 0.5% acetic anhydride. The procedure was successfully tested for the inactivation of a wide panel of microorganisms including endospores of different bacterial species, vegetative cells of gram positive and negative bacteria including mycobacteria, fungi including yeast, and bacteriophages. For robust testing of the sterilization effect with regard to later application of implant materials sterilization all microorganisms were embedded in alginate/agarose cylinders that were used as Process Challenge Devices (PCD). These PCD served as surrogate models for bioresorbable 3D scaffolds. Furthermore, the impact of scCO₂ sterilization on mechanical properties of polysaccharide-based hydrogels and collagen-based scaffolds was analyzed. The procedure was shown to be less compromising on mechanical and rheological properties compared to established low-temperature sterilization methods like gamma irradiation and ethylene oxide exposure as well as conventional steam sterilization. Cytocompatibility of alginate gels and scaffolds from mineralized collagen was compared after sterilization with ethylene oxide, gamma irradiation, steam sterilization and scCO₂ treatment. Human mesenchymal stem cell viability and proliferation were not compromised by scCO₂ treatment of these materials and scaffolds. We conclude that scCO₂ sterilization under addition of water, hydrogen peroxide and acetic anhydride is a very effective, gentle, non-cytotoxic and thus a promising alternative sterilization method especially for biomaterials.     

Microbiological Aspects of Ethylene Oxide Sterilization: I. Experimental Apparatus and Methods

A specially built thermochemical death-rate apparatus is described which can be used to determine the resistance of microorganisms to ethylene oxide under controlled conditions. The apparatus was designed to provide instantaneous exposure of microorganisms to ethylene oxide and to eliminate variables that could result in errors when death kinetic reaction rates are calculated. The apparatus is used to obtain ethylene oxide resistance data which are useful in evaluating and developing sterilizing cycles for materials with known bacterial concentrations, as well as for calculating probability factors on which a given test condition can be expected to provide sterilization.     

Recovery Patterns of Spores of Putrefactive Anaerobe 3679 in Various Subculture Media after Heat Treatment

A comparative study was made of the heat resistance of spores of putrefactive anaerobe 3679 grown in two different sporulation media and of the recovery pattern of these spores in several subculturing media after treatment with moist and dry heat. The heat resistance of the spores was characterized in the form of D and z values. The D values were determined by the modified Schmidt method. The z values were established by the graphic method. The results revealed significant differences in D and z values, depending on the type of heat and sporulation and subculture media. Spores grown in beef heart infusion showed higher heat resistance than those grown in Trypticase. Among the seven subculture media used, the largest number of spores was recovered in beef infusion. The magnitude of the D values at 121.1 C obtained with spores heated in moist heat decreased, depending on the subculture medium used, in the following order: beef infusion, pea infusion, yeast extract, liver infusion, Eugonbroth, Trypticase, synthetic medium. With spores subjected to dry heat, D values at 148.9 C decreased with the subculture medium in the following order: beef infusion, yeast extract, pea infusion and liver infusion, Trypticase, Eugonbroth, synthetic medium. The z values obtained with spores subjected to dry heat were approximately double those obtained with moist heat. Their relative magnitude varied slightly, depending on the type of subculture medium used. However, the relative magnitudes of the D values and z values with reference to the subculture media used were different with moist heat from those obtained with dry heat. Two theories are discussed as possible explanations for the logarithmic order of death of bacterial spores. The results obtained in these experiments, together with the findings of other workers, are most compatible with the theory that heat treatment of spores results in an increased rate of random injury to the genetic material of the spores.     

Heat, hydrogen peroxide, and UV resistance of Bacillus subtilis spores with increased core water content and with or without major DNA-binding proteins.

Spores of a Bacillus subtilis strain with an insertion mutation in the dacB gene, which codes for an enzyme involved in spore cortex biosynthesis, have a higher core water content than wild-type spores. Spores lacking the two major alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha-beta- spores) have the same core water content as do wild-type spores, but alpha-beta- dacB spores had more core water than did dacB spores. The resistance of alpha-beta-, alpha-beta- dacB, dacB, and wild-type spores to dry and moist heat, hydrogen peroxide, and UV radiation has been determined, as has the role of DNA damage in spore killing by moist heat and hydrogen peroxide. These data (i) suggest that core water content has little if any role in spore UV resistance and are consistent with binding of alpha/beta-type SASP to DNA being the major mechanism providing protection to spores from UV radiation; (ii) suggest that binding of alpha/beta-type SASP to DNA is the major mechanism unique to spores providing protection from dry heat; (iii) suggest that spore resistance to moist heat and hydrogen peroxide is affected to a large degree by the core water content, as increased core water resulted in large decreases in spore resistance to these agents; and (iv) indicate that since this decreased resistance (i.e., in dacB spores) is not associated with increased spore killing by DNA damage, spore DNA must normally be extremely well protected against such damage, presumably by the saturation of spore DNA by alpha/beta-type SASP.     

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.     

Bioindicator production with <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis>’ thermal-resistant spores cultivated by solid-state fermentation

Bacillus atrophaeus’ spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost–productivity relation. Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental designs and response surface methodology in which the influence of different variables—particle size, moisture content, incubation time, pH, inoculum size, calcium sources, and medium composition—was studied. The results have suggested that soybean molasses and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time. Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%), initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the control and no significant difference in dry heat resistance was observed.     

Portable Ethylene Oxide Sterilization Chamber

A portable ethylene oxide sterilization chamber was designed, constructed, and tested for use in the sterilization of embolectomy catheters. The unit can accommodate catheters up to 40 inches (101.6 cm) in length and can be operated for less than 4 cents per cycle. A constant concentration of 500 mg of ethylene oxide per liter of space and holding periods of 4 and 6 hr at 43 and 22 C, respectively, were adequate when tested with B. subtilis spores. The estimated cost of construction was $165.00. If temperature control is unnecessary, the cost is approximately $80.00.     

Antisense-RNA-mediated decreased synthesis of small, acid-soluble spore proteins leads to decreased resistance of clostridium perfringens spores to moist heat and UV radiation

Previous work has suggested that a group of alpha/beta-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding alpha/beta-type SASP in this organism. We have now used antisense RNA to decrease levels of alpha/beta-type SASP in C. perfringens spores by approximately 90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of alpha/beta-type SASP in the resistance of C. perfringens spores.     

Sterilization of oils: Data to support the use of a continuous point-of-use sterilizer

The continuous slow feed of animal and vegetable oils is frequently found to be an economically attractive method for maintaining antibiotic fermentations at a highly productive rate. The sterilization of oil feeds can present a major problem in the production plant. In this article, we present data and methodology on the kinetics of the thermal sterilization of No. 2 lard oil and soybean oil. It was also determined that dry heat sterilization condition could occur if dry spores were added to dry oil. This condition could only be partially alleviated by the addition of water to the oil. The theory and design of a small tubular coil continuous sterilizer for sterilizing oils at the point of use is also presented. This type of sterilizer is economical and versatile and simplifies control and instrument requirements. It provides a realistic alternative to the use of sterile feed tanks and sterile headers.     

Resistance of Bacillus Spores to Combined Sporicidal Treatments

S ummary . Moist heat at 82° (100° for Bacillus stearothermophilus ) and solutions of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride at 24° separately showed no sporicidal activity against B. pumilis, B. stearothermophilus, B. subtilis and B. subtilis var. niger . Spores of the last organism were the most sensitive to γ radiation, the D value being 0.16 Mrad. Prior irradiation with a dose of 0.16 Mrad brought about only a slight increase in the sensitivity of the spores to moist heat. The presence of bactericide during irradiation did not affect radiation resistance. Inactivation rates were greater when the spores were heated in the presence of a bactericide than in aqueous suspension and benzalkonium chloride was more active than chlorocresol. Chlorocresol enhanced the heat activation of B. stearothermophilus at 100°. Irradiation in the presence of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride had no effect on the subsequent resistance of the spores when heated in the presence of these bactericides. It is concluded that it is unlikely that combinations of moist heat, radiation and bactericides, each less severe than when used in an accepted sterilization process, will lead to an alternative process which, while less damaging to the materials being sterilized, would still maintain the accepted standards of freedom from contamination.     

Glycerol-based sterilization bioindicator system from Bacillus atrophaeus: development, performance evaluation, and cost analysis

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D _160 °C value of 6.6?±?0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3?±?1.2?×?10⁸?CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.     

Quantitative assessment of sterilization efficiency using lyophilized calcium alginate biological indicators

Biological indicators were prepared using either Bacillus stearothermophilus or Bacillus subtilis incorporated into lyophilized calcium alginate beads. These were subjected to a variety of moist heat and dry heat treatments as appropriate and then dissolved in buffer to allow enumeration of survivors. The utility of these spore carriers as biological indicators for the quantitative assessment of heat sterilization processes is discussed.