USP55 生物指示剂抗性测试.docx

1、USP55 生物指示剂抗性测试USP 55 BIOLOGICAL INDICATORSRESISTANCE PERFORMANCE TESTS 生物指示剂抗性测试TOTAL VIABLE SPORE COUNT 活孢子数总数计数Remove three specimens of the relevant biological indicator from their original individual containers. Disperse the paper into component fibers by placing the test specimens in a sterile

2、 250-mL cup of a suitable blender containing 100 mL of chilled, sterilized Purified Water and blending for 3 to 5 minutes to achieve a homogeneous suspension. Transfer a 10-mL aliquot of the suspension to a sterile, screw-capped 16- 125-mm tube. 从生物指示剂的原包装袋中取出3支作为样本。把待测样品放入一个已灭菌的250mL有搅拌器的杯子里，杯子内装10

3、0 mL放冷的灭菌纯化水。把杯子里的纸片磨碎成纤维状，并混合35min使之成为均一的悬浮液。转移10ml的整数倍的悬浮液至一个已灭菌的16 125mm的螺盖管中。For Biological Indicator for Steam Sterilization, Paper Carrier, heat the tube containing the suspension in a water bath at 95 to 100 for 15 minutes (heat shock), starting the timing when the temperature reaches 95. 如果是

4、纸载的蒸汽灭菌生物指示剂，则把装有悬浮液的管放入95100水浴15min（热休克），在悬浮液温度达到95时开始计时。For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, and for Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, heat the tube containing the suspension in a water bath at 80 to 85 for 10 minutes, starting the

5、 timing when the temperature reaches 80. Cool rapidly in an ice water bath at 0 to 4. 如果是纸载的干热灭菌生物指示剂及环氧乙烷灭菌生物指示剂，则把装有悬浮液的管放入8085水浴10min，在悬浮液温度达到80时开始计时。在04冰浴中迅速冷却。Transfer two 1-mL aliquots to suitable tubes, and make appropriate serial dilutions in sterilized Purified Water, the dilutions being se

6、lected as calculated to yield preferably 30 to 300 colonies, but not less than 6, on each of a pair of plates when treated as described below. 转移2个1ml的悬浮液到合适的试管，用灭菌纯化水进行适宜的系列稀释，所选稀释液要求在进行下述操作后能达到30300CFU皿，但不少于6 CFU皿。Where the biological indicator has a low spore concentration, it may be necessary to

7、 modify the dilution series and to use more plates at each dilution. Prepare a separate series of plates for each aliquot. Place 1.0 mL of each selected dilution in each of two 15- 100-mm Petri dishes. Within 20 minutes, add to each plate 20 mL of SoybeanCasein Digest Agar Medium (see Microbial Limi

8、t Tests 61) that has been melted and cooled to 45 to 50. Swirl to attain a homogeneous suspension, and allow to solidify. 如果此生物指示剂的孢子浓度较低，则有必要改变稀释系列，在每个稀释级增加更多的平皿。每一取整数倍制备一个单独的系列平皿。分别取1.0 mL所选的稀释液加入两个15 100mm的平皿中。在20min内，向每个平皿中倒入20 mL溶解并冷却到4550的大豆酪蛋白消化物琼脂（参考Microbial Limit Tests 61）。混合均匀，待其凝固。Incuba

9、te the plates in an inverted position at 55 to 60 for Biological Indicator for Steam Sterilization, Paper Carrier, and at 30 to 35 for Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, and for Biological Indicator for Dry-Heat Sterilization, Paper Carrier, or at the optimal recov

10、ery temperature specified by the manufacturer, and examine the plates after 24 and 48 hours, recording for each plate the number of colonies, and using the number of colonies after 48 hours to calculate the results. 倒扣平皿进行培养，纸载蒸汽灭菌生物指示剂：5560培养，纸载的干热灭菌生物指示剂及环氧乙烷灭菌生物指示剂：3035培养，或在供应商指定的有最佳回收率的温度下培养，24h

11、和48h后检查平皿，记录各皿的菌落数，用48h后的菌落数进行结果计算。Calculate the average number of spores per specimen from the results, using the appropriate dilution factor. The test is valid if the log number of spores per Carrier at 48 hours is equal to or greater than the log number after 24 hours in each case. For Biological

12、 Indicator for Steam Sterilization, Self-Contained, aseptically remove the spore strip from the container, and proceed as directed for Biological Indicator for Steam Sterilization, Paper Carrier.由上述结果计算每个样本的平均孢子数，使用恰当的稀释因子。如果每个悬浮液48h的孢子数的对数等于或大于其24h的孢子数的对数，则实验成立。对于预装式蒸汽灭菌生物指示剂，在无菌条件下从容器中取出孢子条，根据纸载蒸汽

13、灭菌生物指示剂的指示操作。D VALUE DETERMINATION D值的确定For all tests described in this section, handle each test specimen with aseptic precautions, using sterilized equipment where applicable.在本部分的所有实验中，要在无菌的条件下处理各检测样品，并使用无菌的器具。Apparatus 设备For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, use an

14、apparatus of known thermodynamic characteristics that has been validated for compliance with the requirements for safety1 and performance,2 that consists of a sterilizing chamber equipped with a means of heating the contained air, preferably electrically rather than gas fired, and that has adequate

15、movement of the air through forced ventilation (by mechanical devices such as blowers), with sensing and control devices for temperature and timing capable of indicating with an accuracy of not more than 0.5 and 1-second intervals, respectively. 对于纸载的干热灭菌生物指示剂，使用已知热力学特性的、经过验证符合安全1和性能要求的设备2，此设备带有一个可以

16、加热空气的灭菌腔，最好是电加热而不是煤气加热，而且通过强制通风有足够的空气对流（通过如鼓风机这样的机械设备），此灭菌室还带有温度和时间的探测和控制装置能适时调节使温度精度不超过0.5，时间精度不超过1秒。The geometrical pattern of the heat source(s) is such as to enable the biological indicators under test to be uniformly heated under the specified conditions. The temperature profile in the chamber

17、is known, and cold spots, hot spots, and slow heat zones identified. 热源的几何模式是用来确保受测的生物指示剂能在特定的条件下受热均匀。腔内的温度曲线图是已知的，并且冷点、热点和慢热区域已被确定。The chamber has the capability to work within a temperature range of 40 to 300, with an accuracy at any particular setting of not less than 2. The apparatus is equipped

18、 with a suitable additional access door or port so as to enable the entry and insertion (or removal) of specimens within 6 seconds and to enable the temperature to return to the set temperature within 0.5 minute where the specified temperature is 120 to 190 and within 1.0 minute where such temperatu

19、re is 220 and above.灭菌腔能够在40300的温度范围内运行，在任何特定设置点的精度不少于2。设备还应配备一个额外的通道门或舱，可以使样品的插入（或取出）在6秒钟之内，并且若设定温度在120190时能使温度在0.5min内恢复到设定温度；若设定温度在220时能使温度在1.0min内恢复到设定温度。For Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, use an apparatus that consists of a test chamber with a means of ens

20、uring adequate mixing of the sterilant gas and a means of heating the sterilant gas to not lower than the preselected operating temperature so that no liquid enters the test chamber, equipped with temperature control and monitoring, pressure control, humidification, and gas concentration monitoring

21、devices. 对于纸载环氧乙烷灭菌生物指示剂，使用带有测试腔的设备，此测试腔可以确保灭菌气体充分混匀，并且可以把灭菌气体加热到不低于预设的运行温度从而没有液体进入这个测试腔。设备带有温度控制和监测装置、压力控制装置、潮湿装置和气体浓度监测装置。Detailed specifications and operational parameters for suitable apparatus are those published in Standard for a Biological IndicatorEvaluator Resistometer for Ethylene Oxide Ga

22、s Vessels (BIER/EO) Gas Vessels.3 设备的详细说明和和运行参数发表载在生物指示剂标准环氧乙烷气体管道(BIER/EO)气体管道镍铬表评估。3For Biological Indicator for Steam Sterilization, Paper Carrier, and for Biological Indicator for Steam Sterilization, Self-Contained, use an apparatus that consists of a chamber equipped with heating, temperature,

23、 and steam control and monitoring devices. Detailed specifications and operational parameters for suitable apparatus are those published in Standard for a Biological IndicatorEvaluator Resistometer for Saturated Steam (BIER/Steam Vessels).4 对于纸载的蒸汽灭菌生物指示剂和预装式蒸汽灭菌生物指示剂，使用的设备需带有一个有加热、温度及蒸汽控制和监测装置的腔室。设

24、备的详细说明和和运行参数发表载在生物指示剂标准饱和蒸汽 (BIER/蒸汽管道)镍铬表评估。Procedure 测试程序Carry out the tests for D value at each of the applicable sets of sterilization conditions for which the packaged biological indicator under test is labeled for use. Take a sufficient number of groups of specimens of biological indicators in

25、 their original individual containers, each group consisting of 5 to 10 specimens. 对每一个运用了生物指示剂的灭菌条件进行D值测试。从生物指示剂的原包装盒里取出足量的一组生物指示剂样本，每组包含510个样本。The number of groups provides a range of observations from not less than one labeled D value below the labeled survival time through not less than one labe

26、led D value above the labeled kill time. Place each group on a separate suitable specimen holder that permits each specimen to be exposed to the prescribed sterilizing condition at a specific location in the sterilizing chamber. 每组样本的数量提供了从不少于一个标示D值低于标示存活时间到不少于一个标示D值高于标示灭活时间的观察范围。把每一组置于一个适宜的样本容器中，使灭

27、菌腔中特定位置的每个样本都能暴露于以上所述的灭菌条件。Check the apparatus for operating parameters using specimen holders without specimens. Select a series of sterilizing times in increments from the shortest time for the specimens to be tested. The differences in sterilizing times over the series are as constant as feasible

28、, and the difference between adjacent times is no greater than 75% of the labeled D value.用没有样本的样本容器来检查设备的运行参数。选择一系列以样本检测用最短时间为基础不断增加的灭菌时间。系列灭菌时间的差异要尽可能恒定，邻近灭菌时间的差异不大于标示D值的75%。For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, preheat the sterilizing chamber for 30 minutes. Open the

29、 access door or port, place one of the holders with a group of specimens in the sterilizing chamber, close the access door or port, and continue to operate the apparatus. Commence timing the heat exposure when the chamber temperature returns to 2 below the specified temperature. 对于纸载的干热灭菌生物指示剂，灭菌腔需预

30、热30min。打开通道门或舱，放入装有一组样本的容器，关闭通道门或舱，持续运行灭菌设备。当灭菌腔温度回升至设定温度以下2时开始计算曝热时间。After the contents have been subjected to the sterilizing condition for a predetermined time selected from a series of time increments, remove the holder with the heated specimens, and replace it with another holder with specimens

31、. Repeat the sterilizing procedure similarly, but for another predetermined time, and continue with successive groups until all have been heated appropriately.腔内的样本接受了系列递增预设时间中某个特定时间的灭菌后，取出装有样本的容器，然后再将另一个容器进行灭菌。重复相似的灭菌程序，但用的是另一个预设时间，用同样的方法做其它的样本组直到所有的样本都被加热灭菌。For Biological Indicator for Ethylene Ox

32、ide Sterilization, Paper Carrier, proceed as follows:对于纸载的环氧乙烷灭菌生物指示剂，检测程序如下：1. Evacuate the test chamber to a pressure of not more than 100 3 mm of mercury.灭菌腔抽真空使其压力不大于100 3 mm汞柱。2. Inject sufficient water vapor (e.g., saturated steam) to bring the chamber contents to within 10% relative humidity of the required humidification condition, and allow the chamber to equilibrate with moisture and to temperature for about 30 minutes. 打入足量的水气（如饱和蒸汽）使腔内含有10以下的相对湿度，让腔内湿度和温度平衡