USP-55 生物指示剂-抗性测试.doc

。 精选资料，欢迎下载 USPUSP 5555 BIOLOGICALBIOLOGICAL INDICATORSINDICATORSRESISTANCERESISTANCE PERFORMANCEPERFORMANCE TESTSTESTS 生物指示剂抗性测试 TOTALTOTAL VIABLEVIABLE SPORESPORE COUNTCOUNT 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 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 100 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 timing when the temperature reaches 80 . Cool rapidly in an ice water bath at 0 to 4 . 。 精选资料，欢迎下载 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 selected 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 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 Limit 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。 Incubate 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 recovery 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. 。 精选资料，欢迎下载 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 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 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，根据纸载蒸汽灭菌生物指示剂的指示操 作。 D D VALUEVALUE DETERMINATIONDETERMINATION 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 apparatus of known thermodynamic characteristics that has been validated for compliance with the requirements for safety1 1 and performance,2 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 movement of the air through forced ventilation (by mechanical devices such as blowers), with sensing and control devices for temperature and timing capable of 。 精选资料，欢迎下载 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 1和性能要 求的设备 2 2，此设备带有一个可以加热空气的灭菌腔，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 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 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 temperature 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 ensuring 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 。 精选资料，欢迎下载 enters the test chamber, equipped with temperature control and monitoring, pressure control, humidification, and gas concentration monitoring 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 Gas Vessels (BIER/EO) Gas Vessels.3 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。3 3 For 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, 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 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 their original individual containers, each group consisting of 5 to 10 specimens. 。 精选资料，欢迎下载 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 labeled 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, 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 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 。 精选资料，欢迎下载 holder with the heated specimens, and replace it with another holder with specimens. 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 Oxide 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 30min。 3. Inject a sufficient quantity of temperature-equilibrated ethylene oxide gas to attain the appropriate concentration 30 mg of ethylene oxide per L. 打入足量的已平衡温度的环氧乙烷，使其浓度为30 mgL。 4. Subject a group of specimens to the appropriate temperature, humidification, and gas concentration conditions for the required time. 把一组样本在上述温度、湿度和气体浓度条件下放置一定时间。 5. Evacuate the test chamber to a pressure of 100 3 mm of mercury, and release the vacuum with sterile filtered air. Repeat this until not less than 99% of the remaining gas has been removed, and remove the holder(s) with the exposed specimens. 灭菌腔抽真空使其压力达到 100 3 。 精选资料，欢迎下载 mm 汞柱。然后用灭菌过滤空气释放真空。重复这一操作单元直到不少于 99%的剩 余气体被赶走，然后取出装有样本的容器。 For exposing further groups of specimens to the sterilization conditions, proceed with steps 6 and 7. 其它样本的灭菌程序同第 6 和第 7 步。 6. Flush the test chamber five times with filtered air after evacuation each time to a pressure of not more than 100 3 mm of mercury. 每次抽 真空后用过滤空气冲洗灭菌腔 5 次，使压力不超过 100 3 mm 汞柱。 7. Repeat the entire sterilizing procedure, steps 1 through 6, for other groups of unexposed specimens, but maintain the specified conditions of step 4 for each of the other required times. 对于其它未灭菌的样本组， 重复 1 到 6 步灭菌程序，但第 4 步中的特定条件不随其它时间的改变而变化。 For Biological Indicator for Steam Sterilization, Paper Carrier, exhaust the sterilizing chamber, and within 15 seconds of opening the door, place one of the holders with a group of specimens in the sterilizing chamber, and operate the apparatus to heat up the chamber contents as quickly as possible. 对于纸载的蒸汽灭菌生物指示剂，灭菌腔抽真空，在开门的 15 秒内放入一个装有一组样 本的容器，运行设备，使腔内尽快热起来。 After the contents have been subjected to the sterilizing condition for a predetermined time selected from the series of time increments, exhaust the chamber as quickly as possible. Remove the holder with the heated specimens, and replace it with another group of specimens. 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，尽快排空灭菌腔，取出 装有样本的容器。重复操作其它的样本。 Repeat the sterilizing procedure similarly, but for another predetermined time, and continue with successive groups until all have been appropriately heated. 。 精选资料，欢迎下载 重复同样的灭菌程序，但用另一个预设的灭菌时间，连续做其它的样本组直到所有的样 本都被加热灭菌。 For Biological Indicator for Steam Sterilization, Self-Contained, follow the procedure indicated for Biological Indicator for Steam Sterilization, Paper Carrier, but handle each self-contained unit as a biological indicator system, with the D value determined for the self-contained system. 对于预装的蒸汽灭菌生物指示剂，遵循纸载蒸汽灭菌生物指示剂中的检测程序，但把每 个预装生物指示剂作为一个生物指示剂系统来确定 D 值。 Recovery 回收率 After completion of the sterilizing procedure for Biological Indicator for Dry-Heat Sterilization, Paper Carrier; Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier; or Biological Indicator for Steam Sterilization, Paper Carrier, whichever is applicable, and within a noted time not more than 4 hours, aseptically remove and add each strip to 10 to 30 mL of SoybeanCasein Digest Medium (see Media under Sterility Tests 71 ) to submerge the biological indicator completely in a suitable tube. 完成了纸载干热灭菌生物指示剂、纸载环氧乙烷灭菌生物指示剂或纸载蒸汽灭菌指示剂 的灭菌程序后，无论运用哪个，应在 4 个小时之内，在无菌条件下取出含菌纸条，放入 1030ml 的大豆酪蛋白消化培养基中（参考Sterility Tests 无菌检测 71 中培养基 部分）使之完全浸没。 For each Biological Indicator for Steam Sterilization, Self-Contained specimen, the paper strip is immersed in the self-contained medium according to manufacturers instructions, within a noted time not more than 4 hours. 对于每个预包装的蒸汽灭菌生物指示剂，按供应商的说明，含菌纸条被浸没于自带的培 养基，浸没时间不超过 4 小时。 Incubate each tube at a temperature of 55 to 60 for Biological Indicator for Steam Sterilization, Paper Carrier, and Biological Indicator for Steam Sterilization, Self-Contained, or at 30 to 35 for Biological Indicator for 。 精选资料，欢迎下载 Dry-Heat Sterilization, Paper Carrier, and Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, or in any case at the optimal recovery temperature specified by the manufacturer. 对每个管进行培养，纸载蒸汽灭菌生物指示剂和预包装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。 Observe each inoculated medium-containing tube at 24 and 48 hours, and every 1 or 2 days thereafter for a total of 7 days after inoculation. (Where growth is observed at any particular observation time, further incubation of the specimen(s) concerned may be omitted.) Note the number of specimens showing no evidence of growth at any time. 在 24h 和 48h 观察每个装有培养基的试管，然后每隔 1 到 2 天进行观察，共培养 7 天 （如果在某一观察时间观察到有菌生长，则这个试管就不用再进行培养）。每次观察记 录无菌生长的样品数。 Calculation 计算 This chapter describes the use of the Limited Spearman-Karber Method for determining the D value of biological indicators on spore paper carriers. Use this method in the event of a compendial issue or regulatory referee testing of a biological indicator system. 本章描述使用 Limited Spearman-Karber 方法来确定生物指示剂纸载孢子的 D 值。在生 物指示剂系统的纲要论述或规章仲裁检测中要使用这种方法。 It is recognized that other methods, such as the Survival Curve Method and the Stumbo-Murphy-Cochran procedure, may be routinely used by manufacturers and users of biological indicators to determine D values. 人们公认其它方法如残存曲线法和 Stumbo-Murphy-Cochran 程序可以被生物指示剂厂商 和用户用来确定 D 值。 The calculation of the D value using the Limited Spearman-Karber Method is based on the use of 10 biological indicators per group. NOTEIf less than 10 biological indicators are used (i.e., 5), the formula and the various 。 精选资料，欢迎下载 calculation steps will have to be modified, including the Replacement of Missing Values; however, the requirements of the test remain the same. 用 Limited Spearman-Karber 方法进行 D 值的计算是基于每一组使用 10 个生物指示剂。 注如果所用的生物指示剂少于 10 个（如 5 个），这个公式和各计算步骤进行修正， 包括置换漏测值；但是检测的要求还是一样的。 Designate the number of specimens taken for each group (i.e., 10) by n, and the difference between adjacent times (in minutes) by . Designate for each group of the series the number of specimens showing no growth by: f1, f2, . fk 每组所用的样本数设为n，邻近灭菌时间的差值（以分钟计）设为 。把每组无菌生长的 系列样品以 f1, f2, . fk 来表示。 in which f1 is the response of all 10 specimens showing growth (0/10 inactivated) in the group held for the shortest time for such result that is adjacent to an intermediate mortality; and fk is the response of all 10 specimens of the group showing no growth (10/10 inactivated) in the group held for the longest time for such result that is adjacent to an intermediate mortality. f1代表灭菌时间最短的一组里都长菌的 10 个样本（0/10 灭活），这个结果接近半数死 亡率，fk代表灭菌时间最长的一组里都不长菌的 10 个样本（10/10 灭活），这个结果接 近半数死亡率。 Do not use for the calculations observations for groups beyond the ends of the series, f1 and fk, giving results that are not adjacent to an intermediate mortality. 超出系列两端f1 and fk的实验组不要用于计算观察，因为这样的结果不接近半数死亡率。 The test is valid if there is available a result (0/10) from a group held for a shorter time than that for the selected shortest time result (f1), and there is available a result (10/10) from a group held for a longer time than that for the selected longest ti