[55]BI抵抗力测试

55 BIOLOGICAL INDICATORS—RESISTANCE PERFORMANCE TESTS 生物指示剂——抵抗性能测试 TOTAL VIABLE SPORE COUNT 活孢子总数计算 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. 从各个原始容器中取出相关生物指示剂的三份样品。将试样置于一个装有100毫升已冷却灭菌纯化水的容积为250毫升的灭菌搅拌杯中，搅拌3至5分钟获得均匀混悬液，使试纸分散到成分纤维内。Transfer a 10-mL aliquot of the suspension to a sterile, screw-capped 16- × 125-mm tube. For Biological Indicator for Steam Sterilization, Paper Carrier, heat the tube containing the suspension in a water bath at 95 INCLUDEPICTURE "http://218.1.68.118/USP28/v28230/uspnf/pub/images/chars/deg.gif" \* MERGEFORMATINET to 100 for 15 minutes (heat shock), starting the timing when the temperature reaches 95. 将10毫升的等份混悬液试样置于一支16- × 125-mm的带螺旋盖的灭菌试管内。对于蒸汽灭菌生物指示剂试纸载体，在95~100水浴器里加热含有混悬液的试管15分钟（热震），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. 对于干热灭菌生物指示剂试纸载体与环氧乙烷灭菌生物指示剂试纸载体，在80~85水浴器里加热含有混悬液的试管10分钟，温度达到80时开始计时。在0~ 4冰水浴器里迅速冷却。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. 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. 将两份1毫升等份试样置于合适的试管内，并在已灭菌纯化水中进行适当的连续稀释，按照以下进行稀释，根据计算结果选择的适宜稀释液能够在一对平板的每个上面产生30~300个菌落，但不少于6个。生物指示剂孢子浓度较低时，有必要更改稀释次数，并且对每次稀释使用更多的平板。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 Soybean–Casein Digest Agar Medium (see Microbial Limit Tests á61ñ) that has been melted and cooled to 45 to 50. 分别为每份等份试样准备一组平板。在两个15- × 100-mm 的培养皿内分别放置1.0毫升所选稀释液。在20分钟内，往每个平板添加20毫升已融化并冷却至45~50的大豆酪蛋白消化琼脂培养基（见微生物限度试验<61>）。Swirl to attain a homogeneous suspension, and allow to solidify. 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. 搅拌得到均匀混悬液并使其凝固。对于蒸汽灭菌生物指示剂试纸载体，在55 到 60，对于环氧乙烷灭菌生物指示剂试纸载体与干热灭菌生物指示剂试纸载体，在30 到 35或者在由厂商指定的最佳恢复温度倒置培养培养皿，并在24小时和48小时后检查培养皿，记录每个平板的菌落数量，并采用48小时后的菌落数量来计算结果。 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. 根据结果使用适当的稀释因子来计算每份样品的平均孢子数量。若在每种情形下在48小时时每个载体的孢子记录数量等于或多于24小时之后的记录数量，则测试有效。对于独立的蒸汽灭菌生物指示剂，在无菌条件下从容器中取出孢子条，并按照蒸汽灭菌生物指示剂试纸载体的指定方法继续进行。 D VALUE DETERMINATION D值测定 For all tests described in this section, handle each test specimen with aseptic precautions, using sterilized equipment where applicable. 对本部分描述的所有试验，按照无菌方式对每个供试品进行处理，并在适当时候使用灭菌设备。 Apparatus 仪器 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 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 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. 对于干热灭菌生物指示剂试纸载体，使用的设备要具有已知热力学特征，安全与性能经验证符合要求，带有电力而非煤气加热封闭气体的灭菌舱室，能够通过强制通风（使用机械装置如送风机）使空气充分流动，并配备指示准确度分别不超过0.5和1秒的温度与时间传感控制装置。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. 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. 热源的几何模式是能够在指定条件下均匀加热测试用生物指示剂。清楚舱室的温度分布，并出冷点、热点与慢热区。舱室能够在40 到 300的温度范围内工作，并且在任何特定的温度设置下准确度不超出±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. 设备另外装有一个合适的通道门或通道口，能够在6秒钟内放进（或移出）样品，当规定温度为120~190时，能够在0.5分钟内恢复至设定温度，当规定温度为220或以上时，能够在1.0分钟内恢复至设定温度。 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 enters the test chamber, equipped with temperature control and monitoring, pressure control, humidification, and gas concentration monitoring devices. Detailed specifications and operational parameters for suitable apparatus are those published in Standard for a Biological Indicator—Evaluator Resistometer for Ethylene Oxide Gas Vessels (BIER/EO) Gas Vessels.3 对于环氧乙烷灭菌生物指示剂试纸载体，使用的设备包括一个测试舱室，能够充分混合灭菌气体，并加热灭菌气体至不低于预定操作温度，以免液体进入测试舱室，舱室配有温度监控、压力控制、增湿与气体浓度监控装置。设备的详细规格与操作参数详见已出版的生物指示剂——环氧乙烷器皿(BIER/EO)抗性评估仪。 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 Indicator—Evaluator Resistometer for Saturated Steam (BIER/Steam Vessels).4 对于蒸汽灭菌生物指示剂试纸载体和独立的蒸汽灭菌生物指示剂，使用的设备包括一个舱室，舱室内装有加热、温度和蒸汽监控装置。设备的详细规格与操作参数详见已出版的生物指示剂标准——饱和水蒸汽抗性评估仪（BIER/蒸汽容器）。 Procedure 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. 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. 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. 在每种规定适用的灭菌条件下进行D值测试，使用标明待用的已包装测试用生物指示剂。从各个原始生物指示剂容器取出足够多组的样品，每组样品有5至10份样品。样品组的数量提供的观察范围从低于标记存活时间不少于一个标记D值到高于标记灭杀时间不少于一个标记D值。将每组样品置于灭菌室指定位置的单独样品托盘上，使样品暴露在指定的灭菌条件下。使用空的样品托盘来检查设备的操作参数。以供试品的最短灭菌时间开始，按照递增顺序选择一组灭菌时间。组间灭菌时间的差异在可行的前提下尽量保持不变，而相邻时间的差异不大于标记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. 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. Repeat the sterilizing procedure similarly, but for another predetermined time, and continue with successive groups until all have been heated appropriately. 对于干热灭菌生物指示剂试纸载体，对灭菌舱室预热30分钟。打开通道门或通道口，将其中一个载有一组样品的托盘置于灭菌舱室内，关闭通道门或通道口，然后继续运行设备。舱室温度恢复至低于指定温度2时开始对热辐射计时。样品在灭菌条件下持续一段预定时间（从递增时间组中选出）后，取出装有已加热样品的托盘，并放置另一个装有样品的托盘。以同样方法重复灭菌程序，但灭菌时间为不同的预定时间，并对其它组样品继续灭菌直到所有样品都被适当加热。 For Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, proceed as follows: 对于环氧乙烷灭菌生物指示剂试纸载体，按以下步骤进行： 1. Evacuate the test chamber to a pressure of not more than 100 ± 3 mm of mercury. 对测试舱室抽气，使气压不高于100 ± 3毫米汞柱。 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. 注入足量水蒸汽（如饱和水蒸汽），使舱室物的相对湿度在规定增湿条件的10%以内，并使舱室湿度与温度保持平衡30分钟。 3. Inject a sufficient quantity of temperature-equilibrated ethylene oxide gas to attain the appropriate concentration ±30 mg of ethylene oxide per liter. 注入足量保持温度平衡的环氧乙烷气体，使浓度达到每公升±30 mg环氧乙烷。 4. Subject a group of specimens to the appropriate temperature, humidification, and gas concentration conditions for the required time. 将一组样品置于适宜温度、湿度与气体浓度条件下保持一段规定时间。 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. For exposing further groups of specimens to the sterilization conditions, proceed with steps 6 and 7. 对测试舱室抽气，使气压为100 ± 3毫米汞柱，并用无菌过滤空气释放真空。重复此操作直至排出不少于99%的剩余气体，并取出装有样品的托盘。 将其它组的样品暴露于灭菌条件下，按第6步和第7步继续下去。 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. 每次抽气至压力不高于100 ± 3毫米汞柱后，分五次让过滤空气充满测试舱室。 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. 对其它组未暴露的样品重复第1步到第6步的整个灭菌程序，但在第4步规定条件下保持不同的规定时间。 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. 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. Repeat the sterilizing procedure similarly, but for another predetermined time, and continue with successive groups until all have been appropriately heated. 对于蒸汽灭菌生物指示剂试纸载体，排出灭菌舱室气体，并在打开舱室门的15秒内，将其中一个装有一组样品的托盘置于灭菌舱室内，然后运转设备尽快加热灭菌舱室内容物。当内容物在无菌条件下保持预定的时间（从一组递增时间中选出）后，尽快排出灭菌舱室内气体。取出装有已加热样品的托盘，放入另一组样品。以同样方法重复灭菌程序，但是灭菌时间为不同的预定时间，并对其它组样品继续灭菌直到所有样品都被适当加热。 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. 对于独立的蒸汽灭菌生物指示剂，遵循蒸汽灭菌生物指示剂试纸载体的指定程序，但将每个独立单元作为生物指示剂系统进行处理，并测定独立系统的D值。 Recovery 恢复 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 Soybean–Casein Digest Medium (see Media under Sterility Tests á71ñ) to submerge the biological indicator completely in a suitable tube. 在干热灭菌生物指示剂试纸载体、环氧乙烷灭菌生物指示剂试纸载体和蒸汽灭菌生物指示剂试纸载体均完成灭菌以后，在不超出4小时的指定时间内，以无菌操作取出所有试纸条，并将其放入10至30毫升的大豆酪蛋白消化液培养基（见无菌试验71中的培养基）中，使生物指示剂完全浸没在试管内。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. 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 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. 对于每份独立的蒸汽灭菌生物指示剂样品，按照厂商的说明，在不超出4小时的指定时间内，使试纸浸没在独立介质中。在55 到60的温度下培养蒸汽灭菌生物指示剂试纸载体与独立的蒸汽灭菌生物指示剂，在30 到35的温度下培养干热灭菌生物指示剂试纸载体与环氧乙烷灭菌生物指示剂试纸载体，或无论如何按照厂商指定的最佳恢复温度进行培养。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24小时与48小时后观察每支装有介质的试管，此后每1天或2天观察一次，共观察7天。（若在任何特定观察时间都观察到生长情况，则可停止相关样品的进一步培养。）注意在任何时间都未显示生长迹象的样品数量。 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. 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. The calculation of the D value using the Limited Spearman-Karber Method is based on the use of 10 biological indicators per group. [NOTE—If less than 10 biological indicators are used (i.e., 5), the formula and the various calculation steps will have to be modified, including the Replacement of Missing Values; however, the requirements of the test remain the same.] 本章描述使用Limited Spearman-Karber法测定孢子试纸载体上生物指示剂的D值。本方法应用于药典规定的事件或生物指示剂系统的法定测试。其它方法被公认由生物指示剂厂商和用户例行用来测定D值，如存活曲线法与Stumbo-Murphy-Cochran程序。用限度Spearman-Karber法计算D值以每组10份生物指示剂的使用为基础。[注意——若使用少于10份生物指示剂，则公式与各个计算步骤需修改，包括遗漏值的替换；但测试要求仍相同。] 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: 指定每组使用的样品数量（如10）为n，相邻时间的差异（单位：分钟）为。指定每组未显示生长样品的数量为： f1, f2, ... fk, 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. 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. 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 time result (fk). Calculate the mean heating time, T, for achieving complete kill by the equation: 其中f1示对接近中间死亡率的结果来说，存放时间最短的组所有10份样品都显示生长（0/10未存活），fk表示对接近中间死亡率的结果来说，存放时间最长的组所有10份样品都未显示生长（10/10未存活）。不要用超过范围的样品组的观察结果f1 与fk来计算，因为给出的结果不接近中间死亡率。若存放时间比所选最短时间更短的一组样品结果为(0/10)，并且存放时间比所选最长时间更长的一组样品结果为(10/10)，则测试有效。用以下公式计算达到完全杀灭的平均加热时间T： in which Tk is the time for achieving the result fk. Calculate the D value by the equation: 其中Tk为达到结果fk的时间。用以下公式计算D值： in which N0 is the average spore count per carrier determined by Total Viable Spore Count (see above) at the time of making this test. Calculate the variance of T, VT , by the equation: 其中N0为进行此测试时由活孢子总数（见以上内容）决定的每个载体的平均孢子计数。用以下公式计算T的方差VT： in which  represents a constant interval between successive exposures, as defined above. The standard deviation, sT , is the square root of the variance: 其中表示连续辐射之间的固定时间，上文已作定义。 标准差sT为方差的平方根： Calculate the lower and upper 95% confidence limits (approximate CL) for the D value by the equation: approximate CL for D = (T ± 2sT/log N0 + 0.2507). 用以下公式计算D值的95%可信限（近似CL）低限与高限： D值近似CL= (T ± 2sT/log N0 + 0.2507) Replacement of Missing Values 遗漏值的替换 If not more than one specimen from a group and not more than two specimens from all of the groups giving the results f1 through fk are missing, replace each missing value by adding 0 to the number showing no growth, if the number showing no growth in the remaining nine specimens of that group is 4 or less, and adding 1 if the number showing no growth in the remaining nine specimens of that group is 5 or more. 若一组中不多于一份样品且所有组中不多于两份样品给出从f1 到 fk的结果遗漏，应替换每个遗漏值，若该组剩余9份样品中未显示生长的数量为4或小于4，则在未显示生长的数量基础上加0，若该组剩余9份样品中未显示生长的数量为5或大于5，则在未显示生长的数量基础上加1。 Survival Time and Kill Time 存活时间与杀灭时间 Take two groups, each consisting of 10 specimens of the relevant biological indicator, in their original, individual containers. Place the specimens of a group in suitable specimen holders that permit each specimen to be exposed to the sterilizing conditions at a specific location in the sterilizing chamber. Check the chamber for operating parameters by preheating it to the selected temperature ±2 in the cases of Biological Indicator for Dry-Heat Sterilization, Paper Carrier, and Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, or ±0.5 in the cases of Biological Indicator for Steam Sterilization, Paper Carrier, and Biological Indicator for Steam Sterilization, Self-Contained. 从单独的原始容器取每组含有相关生物指示剂10份样品的两组样品。将一组样品置于灭菌室特定位置的合适的样品托盘上，使每份样品暴露于灭菌条件下。对灭菌室预热以检查操作参数，干热灭菌生物指示剂试纸载体与环氧乙烷生物指示剂试纸载体预热至所选温度±2，蒸汽灭菌生物指示剂试纸载体与独立的蒸汽灭菌生物指示剂预热至所选温度±0.5。 For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, preheat the unit to temperature, and equilibrate the heat chamber. Open the access door or port, and place the holder(s) in the chamber, close the access door or port, and continue to operate the apparatus. Commence timing the heat exposure when the chamber temperature returns to the lower limit of the selected temperature. Expose the specimens for the required survival time, enter the chamber, and remove the holder(s) containing the 10 specimens. Repeat the above procedure immediately, or preheat if a substantial interval has elapsed, so as to subject the second holder(s) containing 10 specimens similarly to the first conditions, but for the required kill time. 对于蒸汽灭菌生物指示剂试纸载体，预热装置并使热室温度保持平衡。打开通道门或通道口，将托盘置于灭菌室内，关闭通道门或通道口，并继续运行设备。热室温度恢复至所选温度的低限时开始对热辐射计时。使样品辐射达所需存活时间，进入灭菌室，并取出装有10份样品的托盘。立