Biological indicators for low temperature steam and formaldehyde sterilization: the effect of defined media on sporulation, growth index and formaldehyde resistance of spores of Bacillus stearothermophilus strains

Preliminary screening was carried out on spores of 29 strains of Bacillus stearothermophilus to determine their potential as biological indicator organisms for low temperature steam and formaldehyde sterilization. Each strain was sporulated on four chemically defined media. Fourteen strains produced satisfactory sporulation on one or more of the media but there was considerable variation in the extent of sporulation. The growth index of the spores, which was dependent on both the strain of organism and the sporulation medium, ranged from 1% to 90%. The spores were appraised on the basis of their resistance to inactivation by 0.5% w/v formaldehyde in aqueous solution at 70°C. The survivor curves obtained could be characterized into five types on the basis of the shape of the curve. Only five strains of Bacillus stearothermophilus produced spores with the characteristics of high resistance, linear semi-logarithmic survivor curve and high growth index that would be required of a potential biological indicator organism.     

Effects of superheated steam on Geobacillus stearothermophilus spore viability

Aims: To examine the effect of processing with superheated steam (SS) on Geobacillus stearothermophilus ATCC 10149 spores. Methods and Results: Two inoculum levels of spores of G. stearothermophilus were mixed with sterile sand and exposed to SS at 105–175°C. The decimal reduction time (D‐value) and the thermal resistance constant (z‐value) were calculated. The effect of cooling of spores between periods of exposure to SS was also examined. A mean z‐value of 25·4°C was calculated for both inoculum levels for SS processing temperatures between 130°C and 175°C. Conclusions: Spore response to SS treatment depends on inoculum size. SS treatment may be effective for reduction in viability of thermally resistant bacterial spores provided treatments are separated by intermittent cooling periods. Significance and Impact of the Study: There is a need for technologies that require short thermal processing times to eliminate bacterial spores in foods. The SS processing technique has the potential to reduce microbial load and to modify food texture with less energy in comparison to commonly used hot air treatment. This work provides information on the effect of SS processing parameters on the viability of G. stearothermophilus spores.     

Use of soybean vinasses as a germinant medium for a Geobacillus stearothermophilus ATCC 7953 sterilization biological indicator

A novel low-cost medium was developed from by-products and wastes from the ethanol agro-industry to replace commercial media in the production of a steam sterilization biological indicator (BI). Various recovery media were developed using soybean or sugarcane molasses and vinasse to prepare a self-contained BI. Media performance was evaluated by viability and heat resistance (D _121 °C value) according to regulatory standards. A medium produced with a soybean vinasse ratio of 1:70 (1.4%) (w/v) produced the results, with D _121 °C = 2.9 ± 0.5 min and Usk = 12.7 ± 2.1 min. The addition of 0.8% (w/v) yeast extract improved the germination of heat-damaged spores. The pH variation from 6.0 to 7.3 resulted in a gradual increase in the D _121 °C value. The absence of calcium chloride resulted in a decrease in germination, while no significant differences were observed with starch addition. Soybean vinasses may thus be used as the main component of a culture medium to substitute for commercial media in the production of self-contained biological indicators. The use of ethanol production waste in this biotechnological process realized a reliable performance, minimized the environmental impact, and decreased BI production costs while producing a high quality product.     

Biological indicators for low temperature steam formaldehyde sterilization: effect of defined media on sporulation, germination index and moist heat resistance at 110dEC of Bacillus strains

Choice of a biological indicator depends upon selecting a strain with the optimum balance of desirable properties. Screening 20 strains of Bacillus spp. for sporulation on three defined media has shown the wide variation that occurs in requirements for sporulation and properties of the resultant spores. Comparison of germination index and moist heat resistance of resultant spores suggest that a combination of high germination index, high heat resistance and linear inactivation may not be possible.     

Biological indicators for low temperature steam formaldehyde sterilization: effect of defined media on sporulation, germination index and moist heat resistance at 110 degrees C of Bacillus strains

Choice of a biological indicator depends upon selecting a strain with the optimum balance of desirable properties. Screening 20 strains of Bacillus spp. for sporulation on three defined media has shown the wide variation that occurs in requirements for sporulation and properties of the resultant spores. Comparison of germination index and moist heat resistance of resultant spores suggest that a combination of high germination index, high heat resistance and linear inactivation may not be possible.     

Biological indicators for low temperature steam and formaldehyde sterilization: effect of variations in recovery conditions on the response of spores of Bacillus stearothermophilus NCIMB 8224 to low temperature steam and formaldehyde

Spores of a Bacillus stearothermophilus strain thought to be a good biological monitor for low temperature steam and formaldehyde (LTSF) sterilization have been subjected to different recovery environments following exposure to relevant LTSF treatments and prior to enumerating the numbers of surviving spores. The recovery treatments essentially consisted of holding samples at a temperature of 90°C for different time periods prior to plating out following exposure to 12 μg ml^-1 formaldehyde at temperatures between 63° and 83°C The data indicate that LTSF-exposed spores show a marked recovery when kept at 90°C prior to plating out; the extent of the recovery increases with increasing inactivation temperature between 73° and 83°C. The data bring into question the sporicidal activity of LTSF.     

Effects of temperature and relative humidity on biological indicators used for ethylene oxide sterilization.

A study was made to determine the effects of temperature and moisture on the D-value of a common biological indicator. Relative humidity (RH) was varied between 10 and 70% in increments of 10%, and temperature was varied between 30 and 70 degrees C in increments of 10 degrees C. Temperature was found to have a pronounced effect on the D-value. At 60% RH, the D-value varied from 15.0 min at 30 degrees C to 1.1 min at 70 degrees C. When RH was plotted against the average D-value at the various temperatures, the temperature curves at or above 50 degrees C were more erratic and the RH had a significant effect. The study showed that temperature and RH must be controlled if biological indicators are to be properly calibrated for use in ethylene oxide sterilization.     

Comparison of the efficacy of steam sterilization indicators.

Twenty-one commercially available chemical steam sterilization indicators were processed in an empty autoclave for various times at temperatures between 240 and 270 degrees F (ca. 116 and 132 degrees C). The time required to reach a sterilized reading at each temperature was plotted on a semilogarithmic time-temperature plot and compared with the time-temperature sterilization curve for Bacillus stearothermophilus. Five of the indicators had time-temperature kinetics similar to those of B. stearothermophilus, but three of these overestimated the effect of processing. Two of the indicators overestimated the effect of processing and were less sensitive to temperature changes when was B. stearothermophilus. Thirteen of the indicators had time-temperature curves that crossed the B. stearothermophilus plot. One indicator produced such ambiguous results that no determinations could be made with it. Out of 21 indicators tested, only 2 appear to be capable of accurately integrating the time-temperature effect at temperatures between 240 and 270 degrees F. The other indicators should be used only after careful analysis of their suitability for use at a given temperature.     

Comparison of the efficacy of steam sterilization indicators.

Twenty-one commercially available chemical steam sterilization indicators were processed in an empty autoclave for various times at temperatures between 240 and 270 degrees F (ca. 116 and 132 degrees C). The time required to reach a sterilized reading at each temperature was plotted on a semilogarithmic time-temperature plot and compared with the time-temperature sterilization curve for Bacillus stearothermophilus. Five of the indicators had time-temperature kinetics similar to those of B. stearothermophilus, but three of these overestimated the effect of processing. Two of the indicators overestimated the effect of processing and were less sensitive to temperature changes when was B. stearothermophilus. Thirteen of the indicators had time-temperature curves that crossed the B. stearothermophilus plot. One indicator produced such ambiguous results that no determinations could be made with it. Out of 21 indicators tested, only 2 appear to be capable of accurately integrating the time-temperature effect at temperatures between 240 and 270 degrees F. The other indicators should be used only after careful analysis of their suitability for use at a given temperature.     

压力蒸汽灭菌指示剂的筛选和效果验证

利用结晶物质在一定温度下熔融的特性,通过对几种灭菌指示剂内容物进行熔点测定,选出苯甲酸化学试剂和升华硫化学试剂作为高压灭菌指示剂和灭菌参考指示剂。试验表明,这两种指示剂终点明确,简便快速,价格便宜,能满足121℃高压灭菌的温度要求,可以对高压灭菌的日常监测提供依据。     

Effects of sporulation pH on the heat resistance and the sporulation of Bacillus cereus

Spores of Bacillus cereus ATCC 7004, 4342 and 9818 were obtained in nutrient agar at several pH from 5·9 to 8·3. The optimum pH for sporulation was around 7, but good production of spores was obtained in the range 6·5–8·3. With all three strains, D -values clearly dropped with sporulation pH, decreasing by about 65% per pH unit. z -Values were not significantly modified ( P > 0·05) by this factor. Mean z -values of 7·13 °C ± 0·16 for strain 7004, 7·67 °C ± 0·04 for 4342 and 8·80 °C ± 0·64 for 9818 were obtained.     

Biological indicators for low temperature steam and formaldehyde sterilization: investigation of the effect of change in temperature and formaldehyde concentration on spores of Bacillus stearothermophilus NCIMB 8224

A.M. WRIGHT, E.V. HOXEY, C.J. SOPER AND D.J.G. DAVIES. 1996. Five strains of Bacillus stearothermophilus have been studied to identify a spore strain to be used as a biological indicator organism for low temperature steam and formaldehyde sterilization. Three strains gave poor reproducibility of batch size and growth index and were discarded. The other two strains gave good reproducibility with a high growth index and gave rise to linear survivor curves when exposed to 5% aqueous formaldehyde. However, only NCIMB 8224 sporulates on a simpler medium and as it was the most resistant to formaldehyde, it was further studied. Tests were carried out in a modified miniclave and factors studied included temperature of the steam and formaldehyde concentration. All studies confirmed the suitability of this strain as a biological indicator organism.     

嗜热脂肪土芽孢杆菌CHB1生长特性与培养条件研究

目的:研究嗜热脂肪土芽孢杆菌CHB1的生长特性与培养条件。方法:以菌体生长量为主要评价指标,利用单因子试验与正交试验相结合的方法对影响CHB1生长的主要因素进行分析。结果:CHB1最低和最高生长温度分别为45℃和74℃,最佳培养温度为60℃;最低和最高起始生长pH值分别为6.5和9.0,最适起始pH为8.0;菌体生长到达对数期的时间为15~18h;接种量2%,装液量40mL,转速180r/min。结论:CHB1为高温菌,生长pH范围偏碱性,条件优化后总菌体浓度可达1.1×109CFU/mL。     

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.     

A note on the effect of sporulation conditions on the resistance of Bacillus spores to heat and chemicals

Spores of Bacillus subtilis SA22 harvested after 22 d incubation on nutrient agar at 30°C were more resistant to 0–04% peracetic acid at 20°C than spores harvested following 2 d incubation. Similarly, spores of B. subtilis globigii B17, harvested after 7 d incubation on a sporulation agar were up to 10 times less resistant to 0.04% peracetic acid at 20°C than spores harvested after 35 d incubation. An increase in resistance to heating at 100°C and to exposure to 17.7% hydrogen peroxide at 20°C occurred as the age of B. subtilis SA22 spores prior to harvesting increased, whereas differences in resistance were not observed with spores of B. subtilis globigii B17.     

Comparison of European and US biological indicators for ethylene oxide sterilization

Summary Biological indicators (BIs) are used to monitor ethylene oxide (EO) gas sterilization processes for medical devices. Several European and United States BIs for EO sterilization were evaluated for resistance according to both United States Pharmacopeia (USP) XXI and United Kingdom's (UK) tests for D-values. US BIs areB. subtilis var. niger spores on paper strips or disc carriers while European BIs use aluminum strips, quartz sand, or cotton yarn. Numerous BIs per run and runs per lot, as well as 2–3 different lots of BIs from each manufacturer, were examined. Both British and US BIs met their respective label claims for rates of inactivation when tested against British and USP EO test parameters, respectively. However, Danish BIs, on cotton yarn or quartz sand, were not inactivated following USP specifications during the exposure dwell times tested (600 mg L^–1 EO, 54°C, 60% RH, 0–110 min). The Danish BIs will require further testing in order for us to determine if theirB. subtilis spores are unusually resistant to EO or if the spore carrier substrates protect the spores from the sterilizing gas. In conclusion, the British and American BIs for EO sterilization are equivalent in resistance despite differences in carrier substrate, recovery conditions, calculation methods for D-values, and the labeled sterilization conditions for use.     

Engineering of the alpha-amylase from Geobacillus stearothermophilus US100 for detergent incorporation

AmyUS100DeltaIG is a variant of the most thermoactive and thermostable maltohexaose forming alpha-amylase produced by Geobacillus stearothermophilus sp.US100. This enzyme which was designed to improve the thermostability of the wild-type enzyme has acquired a very high resistance to chelator agents. According to modeling structural studies and with the aim of enhancing its resistance towards chemical oxidation, a mutant (AmyUS100DeltaIG/M197A) was created by substituting methionine 197 to alanine. The catalytic proprieties of the resulting mutant show alterations in the specific activity and the profile of starch hydrolysis. Interestingly, AmyUS100DeltaIG/M197A displayed the highest resistance to oxidation compared to the AmyUS100DeltaIG and to Termamyl300, the well-known commercial amylase used in detergent. Further, performance of the engineered alpha-amylase was estimated in the presence of commonly used detergent compounds and a wide range of commercial detergent (liquid and solid). These studies indicated a high compatibility and performance of AmyUS100DeltaIG/M197A, suggesting its potential application in detergent industry.     

应用响应面法优化嗜热脂肪土芽孢杆菌培养基

目的：筛选并优化嗜热脂肪芽孢杆菌CHB1液体发酵培养基。方法：通过Plackett-Burman试验和响应面分析方法,确定培养基的主要影响因素和最佳浓度。结果：利用SAS软件进行分析,确定对响应值影响最大的3个因素为豆粕、酵母粉、K2HPO4。最佳培养基组成为0.51%、豆粕浓度为0.425%、K2HPO4浓度为0.994%。根据模型预测得到的理论最大菌数为2.94×10^8cfu。在初始条件下实验,菌数为2.40×10^8cfu,在优化的最佳培养基条件下,实际的菌数为3.06×10^8cfu。菌数比优化前提高了27.3%。试验值与预测值的误差为4.08%。结论：实验值与模型预测值拟合良好。