Effect of Environmental Conditions During Heating on Commercial Spore Strip Performancet

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1982, p Vol. 44, No /82/ $02.00/0 Effect of Environmental Conditions During Heating on Commercial Spore Strip Performancet GERALDINE M. SMITH, MAGGY KOPELMAN,t ANGELA JONES, AND I. J. PFLUG* Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota Received 29 May 1981/Accepted 30 November 1981 Commercial biological indicator spore strips in glassine envelopes, produced by three manufacturers, were evaluated by fraction-negative procedures after being heated at ± 0.05 C. Only one type of spore strip met the manufacturer's specifications. The strips of one manufacturer were further evaluated by fractionnegative and survivor curve-plate count procedures after being heated under several conditions (enclosed in glassine envelopes, in trypticase soy broth plus % bromocresol purple, in Trypticase soy broth alone, in Water for Injection, directly); Trypticase soy broth plus bromocresol purple and tryptic soy agar, respectively, were used as recovery media. The heating condition affected the D-value of the spore strip. Recovery procedures also had an effect; in all cases, the D-values obtained from the survivor curve tests were larger than those obtained from fraction-negative tests carried out under the same conditions. To determine if the differences in D-values between the two evaluation procedures were caused by the recovery media, we evaluated, by both methods, one type of spore strip heated directly and in glassine envelopes, using tryptic soy agar plus bromocresol purple and Trypticase soy broth plus 1.5% agar, respectively, as the recovery media. The survivor curve results showed that for both enclosed and unenclosed spore strips, there was a marked difference between the two recovery media; however, there was no difference when fraction-negative tests were used. Downloaded from Bacterial spores, placed on or in suitable carriers and calibrated in a specific stress system, can be used to quantitatively measure the stress conditions imposed by a sterilization process (1). The spores, carriers, and production and use procedures comprise a biological indicator (BI) system. BIs have been used for more than 80 years in designing, validating, and monitoring the delivery of sterilization processes (6, 8, 16, 18). BIs, as a measuring tool, were originally developed for in-house control purposes, each organization generating both BIs and use protocol appropriate to plant conditions. The use of BIs to validate the performance of hospital autoclaves brought a degree of standardization'to paper strip BIs containing Bacillus stearothermophilus spores. Later, paper strips containing B. subtilis subsp. niger spores came into wide use for monitoring ethylene oxide sterilization processes. In recent years, BIs have been steadily moving from in-house use to governmental regulatory requirement satisfaction. The use of BIs for legal purposes imposes special requirements; Mascoli (11) has cited the t Scientific journal series no. 11,793 of the Minnesota Agricultural Experiment Station, St. Paul. t Present address: Ministry of Health, Haifa, Israel Present address: Medtronic Inc., Minneapolis, MN need for performance, use, and test condition standards for BIs. Unfortunately, the response of these devices to lethal stress under a wide range of conditions is not entirely understood, as evidenced by discrepancies between specifications of some BIs and actual performance (2, 9, 10, 12). We studied the effect of environmental conditions on commercial paper spore strip BIs used to validate and monitor wet-heat sterilization processes by determining the agreement between performance and manufacturers' specifications, the effect of several environmental conditions during heating, and the differences in performance when fraction-negative and survivor curve methods of calibration were used. MATERIALS AND METHODS Sterile techniques were used throughout. Spore strip handling and recovery procedures were carried out in a class 100 laminar flow hood or a clean room. The same lots of Trypticase soy broth (TSB; BBL Microbiology Systems) and tryptic soy agar (TSA; Difco Laboratories) were used throughout. Spore strips. Paper spore strips enclosed in glassine envelopes, produced by three commercial manufacturers and designated A, B, and C, were evaluated. All were designed to monitor steam sterilization processes and used B. stearothermophilus spores as the indicator on October 28, 2017 by guest

2 VOL. 44, 1982 TABLE 1. Spore strip manufacturers' specifications for steam sterilization at 121.0'C Specifications Response time at 121 C Spore strip No. of spores/ (min) strip All positive All negative A 1.0x B Not given 5 12 C 1.5 X organism. The manufacturers' specifications are listed in Table 1. Upon receipt in this laboratory, the strips were stored at 4'C. Determination of No. Two procedures were used to determine the initial number of spores per strip (No). We prefer to use the Waring blender method, but we have found that the paper used in some spore strips will not always disintegrate in a blender; grinding with a mortar and pestle assured disintegration. For both No determination methods, appropriate dilutions of the spore strip suspension were made, and portions were plated in duplicate, TSA being used as the recovery medium. The plates were incubated in a humidified incubator at 55'C for 48 h. The number of colony-forming units was determined with a Bactronic colony counter. (i) Mortar and pestle. Each unheated spore strip was placed in a mortar. Butterfield phosphate buffer (3) was added, and the spore strip was ground with the pestle for 2 min. (ii) Waring blender. Each spore strip and the liquid in which the spores were heated (for strips heated in envelopes or directly, we used the strips plus 10 ml of Butterfield buffer) were placed in a Waring blender jar. We added 40 ml of Butterfield buffer and blended the contents for 2 min. Soaking the strip in liquid for at least 10 min before blending facilitates disintegration. Heating procedures. All heating was carried out at ± 0.05'C in a custom-built miniature steam retort (14). For enclosed heating, the strip enclosed in a glassine envelope was heated with steam. For heating in liquid, the spore strip was removed from the envelope and placed in a screw cap test tube (18 by 150 mm) containing (i) 10 ml of TSB plus bromocresol purple (BCP), (ii) 10 ml of TSB, or (iii) 10 ml of Water for Injection (United States Pharmacopeial Convention, Inc.). For unenclosed heating, the spore strip was removed from the envelope and heated directly in steam. (i) Strips heated in glassine envelopes. For fractionnegative tests, the strips in envelopes were heated in a rack where each envelope was in its own compartment and surrounded by steam. For survivor curve tests, we heated the glassine envelope after laying it on a screen in an open metal container (2.5-in. [6.47-cm] diameter, 0.25-in. [0.635-cm] depth; same dimensions as those of the container for heating unenclosed strips, thus giving results that could be compared). (Hi) Strips heated unenclosed. Immediately before being heated, the strip was removed from the glassine envelope and placed on a screen in an open metal container. The metal container was placed in a rack and heated. SPORE STRIP PERFORMANCE VARIATIONS 13 (iii) Strips heated in liquids. Immediately before being heated, the strip was removed from the glassine envelope and placed in a test tube (18 by 150 mm) containing 10 ml of the test liquid substrate. The tubes were heated in the miniature retort. After being heated, the tubes were cooled and held in an ice water bath until spore recovery procedures were started. Lags in heating. When the spore strips were heated directly or in envelopes, they were considered to be at the temperature of the retort. Therefore, no lag correction factor was required. However, when the spore strips were heated in test tubes containing 10 ml of liquid, the experimentally determined lag correction factor of 2 min was used. Fraction-negative test recovery procedures. Inoculated tubes were incubated at 55'C for 2 weeks and then scored for growth. (i) Strips heated directly or in glassine envelopes. Each heated spore strip was transferred to a culture tube containing 10 ml of TSB plus BCP or 10 ml of melted TSA plus BCP equilibrated to 55'C. (The TSA plus BCP remained molten during the incubation period.) (ii) Strips heated in TSB or TSB plus BCP. The heated tubes containing the strips and medium were cooled in an ice water bath for at least 2 min and then incubated. (iii) Strips heated in Water for Injection. The heated tubes containing the strips in Water for Injection were cooled in an ice water bath for at least 2 min; then 10 ml of double-strength TSB plus BCP was added to each tube. Survivor curve test recovery procedures. All heated strips were ground with a Waring blender. The recovery medium was TSA or TSB plus 1.5% agar. The plates were incubated in a humidified incubator at 55'C for 48 h. The number of colony-forming units was determined with a Bactronic colony counter. Analysis of data. The fraction-negative or quantal data were analyzed by the Spearman-Karber method described by Holcomb and Pflug (7). Spearman- Karber times (USK) (the expected heating time before a sample of No organisms becomes sterile [as we used it, the expected or mean time needed to sterilize a spore strip]) were calculated. At the Spearman-Karber time, the number of organisms is 0.57/U. Using the No of the experiment and the USK, we calculated the D- value and statistical confidence limits (14). For survivor curve tests, the number of surviving organisms per spore strip as a function of heating time was analyzed with a survivor curve computer program. The D-value, 95% confidence interval of the D- value, and the intercept ratio were calculated for all experiments. The intercept ratio value is defined as the log of the zero time intercept of the regression line divided by the log of the No value. RESULTS Confirmation of manufacturers' specifications for spore strips A, B, and C. (i) No specification. Table 2 shows the mean and range of the number of spores recovered per unheated spore strip; the manufacturers' No specifications are also included. The No of spore strip A averaged more than a log greater than the manufacturer's specifica-

3 14 SMITH ET AL. APPL. ENVIRON. MICROBIOL. TABLE 2. Number of spores per strip (No) Spore Specification No. of strips Method of No. of spores/strip strip (No) evaluated evaluation" Mean Range A 1.0 x MP 1.1 x l x x 105 B Not given 6 MP -2.2 x 105 C 1.5 x MP 1.7 x x x 106 C 1.5 x B-WFI 1.5 x x 106 C 1.5 x B-TSB + BCP 6.5 x x x 105 C 1.5 x B-TSB 7.2 x X x 106 a MP, Mortar and pestle plus Butterfield phosphate buffer; B-WFI, blending with Water for Injection; B-TSB, blending with TSB. tion. The manufacturer of spore strip B did not give an No specification. The No of spore strip C, when evaluated by the mortar-and-pestle method with Butterfield buffer and by the blender and mortar-and-pestle methods with Water for Injection, was very close to the manufacturer's specification. When TSB or TSB plus BCP was used, the blender method produced an average No about one-half log lower than that produced when Butterfield buffer or Water for Injection was used, and the variation among replicate strips increased. (ii) Performance specifications. The combined raw data for the fraction-negative tests in which spore strips A, B, and C were heated in glassine envelopes are shown in Table 3. A comparison TABLE 3. Combined raw data for fraction-negative tests of spore strips A, B, and C heated in glassine envelopes No. of negative strips/no. of strips Heating time heated (min) Strip A Strip B Strip C 3.5 0/6 4 1/ /16 5 0/9 12/ /6 6 1/9 12/12 7 0/12 3/3 8 10/ / / /3 12 6/6 13 0/8 14 0/ /6 7/ / / / / / / / /8 of the manufacturer's specifications (Table 1) with our results (Table 3) indicated that only spore strip A performed within the designated specifications. Spore strip B had a lower heat resistance than that specified: 14 of 49 strips heated for 5 min or less produced no growth. Comparison of performances of spore strips A and B when fraction-negative procedures were used: heating in glassine envelopes versus TSB plus BCP. USK and D-value estimates are shown in Table 4. When spore strips A and B were heated in TSB plus BCP, the USK and D-values increased by a factor of >2 over the values for the same strips heated in glassine envelopes. Comparison of performances of spore strip C heated by each method and analyzed by the fraction-negative and survivor curve procedures. (i) Fraction-negative tests in which TSB plus BCP or TSB was used. When spore strip C was heated in TSB plus BCP, the USK and D-values (Table 4) increased by a factor of 1.5 over the values for the same strip heated in glassine envelopes. The USK and D-values (Table 4) for strips heated in Water for Injection were very close to those for strips heated in glassine envelopes. The values for the unenclosed spore strips were about the same as those for strips heated in TSB plus BCP. In Experiment 136C, we heated the spore strips and recovered the spores in TSB containing no BCP to determine the effect of BCP on the results. The USK and D-values (Table 4) were comparable to those for experiments in which TSB plus BCP was used. (ii) Survivor curve tests in which TSA was used as the recovery medium. Because of the differences in the fraction-negative test results for spore strips heated under different conditions, survivor curve tests were carried out to verify these results and to determine the shapes of the survivor curves. Spore strip C was chosen because a large supply was available. D- and intercept ratio values obtained from the survivor curve tests of strip C heated under different conditions are summarized in Table 5. The D-values for strips heated in glassine envelopes and those for strips heated in 10 ml of

4 VOL. 44, 1982 Water for Injection were very close. However, the survivor curves for strips heated in glassine envelopes were close to straight lines through No, whereas the survivor curves for strips heated in Water for Injection were concave and downward. SPORE STRIP PERFORMANCE VARIATIONS 15 As determined by survivor curve evaluation, the mean D-value for strips heated directly was 25% higher than that for strips heated in TSB plus BCP; as determined by fraction-negative procedures, the D-values were similar. The survivor curves for unenclosed strips were close to TABLE 4. USK and D-values calculated from fraction-negative data for spore strips heated under various conditions Strip type No. of Recovery No used to USK value 95% CI USK D-value and expt Heating condition strips/ calculate (min)' value (min)b (min)a no. heating D-value Strip A 297A 312AC 339Ad 339B 321A 332A 353A Strip B 303A 312B 339C 339Dd 312D 332D 353B Strip C 004AC 123A 249A 298A 282A 114A 120B 136B 136C 092A 102A 136A 205Ad 248A 249B 315A 10 ml of TSB 10 ml of WFJf 10 ml of WFI 10 ml of WFI 1.2 x TSB + BCP 1.2 X X X x 10i 1.2 X X TSB + BCP 8 TSB + BCP 8 TSB + BCP 1.2 x x x } J 8 TSB 1.8 x TSA + BCP 1.8 X TSB 8 TSB + BCP 1.9 X x lo5 5.6 x x x J 6.8 x x J J J J e TSA + BCP 1.8 x I Means are indicated by braces. b CI, Confidence interval. c For calculation purposes, a heating time was arbitrarily added where all tubes were negative. d For calculation purposes, a heating time was arbitrarily added where all tubes were positive. ' -, Data insufficient to calculate 95% confidence interval. f WFI, Water for Injection } J ( J J } J

5 16 SMITH ET AL. APPL. ENVIRON. MICROBIOL. TABLE 5. Results of survivor curve tests of spore strip C heated under various conditions Expt No. of Recovery D-value 95% CI D-value I" no. Heating condition strips/ medium Nostrip (min)' (min)b 09B 2 TSA 1.2 x B 2 TSA 1.2 x ' A 3 TSA 4.49J J 281A 3 TSB + agar 1.3 X B 2 TSA 5.7 x B 2 TSA 5.9 x A 2 TSA 4.0 x A 3 TSA 7.1 x J 073B 10 ml of TSB 2 TSA 6.6 x B 10 ml of TSB 3 TSA 6.9 x A 10 mlofwfid 2 TSA 1.7 x A 10 ml of WFI 2 TSA 3.05 j j A 10 ml of WFI 2 TSA 1.5 X J J 096A 2 TSA 1.3 X A 2 TSA 1.4 X A 3 TSA 2.1 X J J 303A 3 TSB + agar 1.7 x a Means are indicated by braces. b CI, Confidence interval. c IR, Intercept ratio. d WFI, Water for Injection. Downloaded from TABLE 6. Mean D-values for spore strip C heated by various methods straight lines through No, whereas the survivor curves for strips heated in TSB plus BCP varied from straight lines to lines that were concave and downward to sigmoidal. A comparison of the D-values for the fractionnegative tests (Table 4) in which TSB plus BCP was used as the recovery medium with those for survivor curve tests (Table 5) in which TSA was used as the recovery medium indicate that under all conditions, the D-values determined by survivor curve-plate count techniques were higher. The results were similar for all conditions evalu- Mean D value (min) Survivor curve D- Heating value/fractioncondition Survivor Fraction- negative D-value curvea negativeb ratio TSB + BCP TSB c 1.5 WFId a TSA was the recovery b medium. Except when noted otherwise, TSB plus BCP was the recovery medium. c TSB was the recovery medium. d WFI, Water for Injection. ated. The mean D-values and the ratios of the means for the two test methods are shown in Table 6. Overall, it appears that the differences among the results of the various heating conditions were larger when the results were determined by survivor curve-plate count procedures. (iii) Effect of recovery media. The effects of the recovery media on spore survival on strips heated by each method, as determined by fractionnegative and survivor curve tests, are presented in Tables 4 and 5, respectively. The results of the survivor curve (points of data and leastsquares regression lines) and fraction-negative (quantal area points of data) tests of the spore strips heated in glassine envelopes and recovered with each medium are shown in Fig. 1. For both heating conditions, there was a marked difference in the survivor curve test results between TSA and TSB plus 1.5% agar. However, when fraction-negative test procedures were used, both media produced D-values comparable to those obtained when survivor curve analysis of strips recovered with TSB plus 1.5% agar was performed. DISCUSSION The large differences in D-values between strips heated directly and in glassine envelopes on October 28, 2017 by guest

6 VOL. 44, 1982 are unexplainable at this time. The differences in heat resistance of spore strip C heated in Water for Injection, TSB, and TSB plus BCP were expected: other investigators (5, 13, 15) have reported that the response of spores varies according to the chemical formulation of the solution used for heating. Several investigators (4, 17, 19) have reported that the formulation of the recovery medium used for heated spores influences the number of recoverable survivors. TSA and TSB differ in formulation. In addition to tryptone, soy peptone, and sodium chloride, TSB contains dex- C _0 a C 1._ o E SPORE STRIP PERFORMANCE VARIATIONS 17 trose and dipotassium phosphate. When survivor curve tests of enclosed and unenclosed strips recovered on each medium were performed, there were differences (Table 5) that could be explained by medium formulation. However, when fraction-negative tests of the two recovery media were used, there were no differences in D-value (Table 4). Further study of the effects of the recovery medium on heated spores may lead to an explanation of this result. Differences between the D-values obtained by the survivor curve method and those obtained by the fraction-negative method are expected ' Heating time U, minutes FIG. 1. Results of survivor curve and fraction-negative tests of spore strip C heated in glassine envelopes.

7 18 SMITH ET AL. when the survivor curve is not a straight line. In our laboratory, we calculate the D-value for survivor curve data from the linear regression line fitted to the points of data, excluding the No data. The D-value, therefore, will be influenced by the shape of the survivor curve. Survivor data should extend from No to 102 survivors per U. When calculating a D-value from fractionnegative data, one assumes that the destruction of spores occurs logarithmically (survivor curve is a straight line), starting from No. In this study, the survivor curves for both enclosed and unenclosed spore strips were close to straight lines through No. Therefore, the survivor curve and fraction-negative D-values should have been approximately the same. Calibration of BIs must be carried out with accurate, temperature-measuring equipment that gives reproducible results and is itself accurately calibrated. The same spore carrier test system and environmental conditions that will be used in the final application must be used in calibration. When calibrating BIs by survivor curve procedures, extrapolating the survivor curve line beyond the experimentally determined points of data is not recommended. The results of this study suggest that more research is needed on the effects of environmental factors on bacterial spore strip performance before standards for calibration and use are established. ACKNOWLEDGMENTS The technical assistance of Yvonne Heisserer, John Kao, and Janet Rowles is greatly appreciated. We acknowledge with appreciation the cooperation of the staff at the University of Minnesota hospitals, who supplied some spore strips. LITERATURE CITED 1. Bruch, C. W Biological indicators and degrees (probabilities of sterilization). Dev. Ind. Microbiol. 14: Buhlmann, X., M. Gay, and I. Shiller Test objects containing Bacillus stearothermophilus spores for the monitoring of antimicrobial treatment in steam autoclaves. Pharm. Acta Helv. 48: Butterfield, C. T The selection of dilution waters for bacteriological examinations. Public Health Rep. 48: Cook, A. M., and R. J. Gilbert Factors affecting the APPL. ENVIRON. MICROBIOL. heat resistance of Bacillus stearothermophilus spores. I. The effect of recovery conditions on the colony count of unheated and heated spores. J. Food Technol. 3: Cook, A. M., and R. J. Gilbert Factors affecting the heat resistance of Bacillus stearothermophilus spores. II. The effect of sporulating conditions and nature of the heating medium. J. Food Technol. 3: Holcomb, R. G., and I. J. Pflug Performance of biological indicators designed for monitoring wet-heat sterilization processes, p In Sterile disposable devices update. '73 Technical Symposium, October Health Industries Association, Washington, D.C. 7. Holcomb, R. G., and I. J. Pflug The Spearman- Karber method for analyzing quantal assay microbial destruction data, p In I. J. Pflug (ed.), Microbiology and engineering of sterilization processes-a collection of reports dealing with specific aspects of the design, delivery, and monitoring of sterilization processes, 3rd ed. Environmental Sterilization Services, St. Paul, Minn. 8. Kelsey, J. C The testing of sterilizers. 2. Thermophilic spore papers. J. Clin. Pathol. 14: Kereluk, D., and R. Gammon A comparative study of biological indicators for steam sterilization. Dev. Ind. Microbiol. 15: Lashais, T., and A. L. Chaney Reliability of biologic autoclave sterilization indicators. Am. J. Clin. Pathol. 52: Mascoli, C. C Should end-protect sterility testing continue? Med. Devices Diagn. Ind. 4: Mayernik, J. J Biological indicators for steam sterilization-a USP collaborative study. Bull. Parenter. Drug Assoc. 26: Ordal, Z. J., and R. V. Lechowich Some factors influencing thermal resistance of bacterial spores, p In Proceedings of the 10th Research Conference. American Meat Institute Foundation, Chicago. 14. Pflug, I. J Syllabus for an introductory course in the microbiology and engineering of sterilization processes, 4th ed. Environmental Sterilization Services, St. Paul, Minn. 15. Pflug, I. J., and G. M. Smith Survivor curves of bacterial spores heated in parenteral solutions, p In A. N. Barker, J. Wolf, D. J. Ellar, G. J. Dring, and G. W. Gould (ed.), Spore research Academic Press, Inc., London. 16. Pflug, I. J., G. Smith, R. Holcomb, and R. Blanchett Measuring sterilizing values in containers of food using thermocouples and biological indicator units. J. Food Prot. 43: Roberts, T. A Recovering spores damaged by heat, ionizing radiation or ethylene oxide. J. Appl. Bacteriol. 33: Selkon, J. B., P. R. Sisson, and H. R. Ingham The use of spore strips for monitoring the sterilization of bottled fluids. J. Hyg. 83: Yokoya, F., and G. K. York Effect of several environmental conditions on the "thermal death rate" of endospores of aerobic, thermophilic bacteria. AppI. Microbiol. 13: