55BI抵抗力測試

1、á55ñ BIOLOGICAL INDICATORSRESISTANCE PERFORMANCE TESTS 生物指示劑抵抗性能測試TOTAL VIABLE SPORE COUNT 活孢子總數計算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-m

2、L 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. 從各個原始容器中取出相關生物指示劑的三份樣品。將試樣置于一個裝有100毫升已冷卻滅菌純化水的容積為250毫升的滅菌攪拌杯中，攪拌3至5分鐘獲得均勻混懸液，使試紙分散到成分纖維內。Transfer a 10-mL aliquot of the suspension to a sterile, s

3、crew-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 to 100 for 15 minutes (heat shock), starting the timing when the temperature reaches 95. 將10毫升的等份混懸液試樣置于一支16- × 125-mm的帶螺旋蓋的滅菌試管內。對于

4、蒸汽滅菌生物指示劑試紙載體，在95100水浴器里加熱含有混懸液的試管15分鐘（熱震），溫度達到95時開始計時。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 ti

5、ming when the temperature reaches 80. Cool rapidly in an ice water bath at 0 to 4. 對于干熱滅菌生物指示劑試紙載體與環(huán)氧乙烷滅菌生物指示劑試紙載體，在8085水浴器里加熱含有混懸液的試管10分鐘，溫度達到80時開始計時。在0 4冰水浴器里迅速冷卻。Transfer two 1-mL aliquots to suitable tubes, and make appropriate serial dilutions in sterilized Purified Water, the dilutions being s

6、elected 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. 將兩份

7、1毫升等份試樣置于合適的試管內，并在已滅菌純化水中進行適當的連續(xù)稀釋，按照以下方法進行稀釋，根據計算結果選擇的適宜稀釋液能夠在一對平板的每個上面產生30300個菌落，但不少于6個。生物指示劑孢子濃度較低時，有必要更改稀釋次數，并且對每次稀釋使用更多的平板。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 p

8、late 20 mL of SoybeanCasein Digest Agar Medium (see Microbial Limit Tests á61ñ) that has been melted and cooled to 45 to 50. 分別為每份等份試樣準備一組平板。在兩個15- × 100-mm 的培養(yǎng)皿內分別放置1.0毫升所選稀釋液。在20分鐘內，往每個平板添加20毫升已融化并冷卻至4550的大豆酪蛋白消化瓊脂培養(yǎng)基（見微生物限度試驗<61>）。Swirl to attain a homogeneous suspension, and

9、 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 Carrie

10、r, 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. 攪拌得到均勻混懸液并使其凝固。對于蒸汽滅菌生物指示劑試紙載體，在55 到 60，對于環(huán)氧乙烷滅菌生物指示劑試紙載體與

11、干熱滅菌生物指示劑試紙載體，在30 到 35或者在由廠商指定的最佳恢復溫度倒置培養(yǎng)培養(yǎng)皿，并在24小時和48小時后檢查培養(yǎng)皿，記錄每個平板的菌落數量，并采用48小時后的菌落數量來計算結果。 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 tha

12、n 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. 根據結果使用適當的稀釋因子來計算每份樣品的平均孢子數量。若在每種情形下在48小時時每個載

13、體的孢子記錄數量等于或多于24小時之后的記錄數量，則測試有效。對于獨立的蒸汽滅菌生物指示劑，在無菌條件下從容器中取出孢子條，并按照蒸汽滅菌生物指示劑試紙載體的指定方法繼續(xù)進行。D VALUE DETERMINATION D值測定For all tests described in this section, handle each test specimen with aseptic precautions, using sterilized equipment where applicable.對本部分描述的所有試驗，按照無菌方式對每個供試品進行處理，并在適當時候使用滅菌設備。Apparat

14、us 儀器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 cont

15、ained 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

16、 intervals, respectively. 對于干熱滅菌生物指示劑試紙載體，使用的設備要具有已知熱力學特征，安全與性能經驗證符合要求，帶有電力而非煤氣加熱封閉氣體的滅菌艙室，能夠通過強制通風（使用機械裝置如送風機）使空氣充分流動，并配備指示準確度分別不超過0.5和1秒的溫度與時間傳感控制裝置。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 co

17、nditions. 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. 熱源的幾何模式是能夠在指定條件下均勻加熱測試用生物指示劑。清楚艙室

18、的溫度分布，并標識出冷點、熱點與慢熱區(qū)。艙室能夠在40 到 300的溫度范圍內工作，并且在任何特定的溫度設置下準確度不超出±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

19、minute where the specified temperature is 120 to 190 and within 1.0 minute where such temperature is 220 and above. 設備另外裝有一個合適的通道門或通道口，能夠在6秒鐘內放進（或移出）樣品，當規(guī)定溫度為120190時，能夠在0.5分鐘內恢復至設定溫度，當規(guī)定溫度為220或以上時，能夠在1.0分鐘內恢復至設定溫度。For Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, use an appar

20、atus 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 co

21、ntrol, humidification, and gas concentration monitoring devices. 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 對于環(huán)氧乙烷滅菌生物指示劑試紙載體，使用的設備包括

22、一個測試艙室，能夠充分混合滅菌氣體，并加熱滅菌氣體至不低于預定操作溫度，以免液體進入測試艙室，艙室配有溫度監(jiān)控、壓力控制、增濕與氣體濃度監(jiān)控裝置。設備的詳細規(guī)格與操作參數詳見已出版的生物指示劑標準環(huán)氧乙烷器皿(BIER/EO)抗性評估儀。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 eq

23、uipped 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 對于蒸汽滅菌生物指示劑試紙載體和獨立的蒸汽滅菌生物指

24、示劑，使用的設備包括一個艙室，艙室內裝有加熱、溫度和蒸汽監(jiān)控裝置。設備的詳細規(guī)格與操作參數詳見已出版的生物指示劑標準飽和水蒸汽抗性評估儀（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 spec

25、imens 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

26、 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 seri

27、es 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.在每種規(guī)定適用的滅菌條件下進行D值測試，使用標明待用的已包裝測試用生物指示

28、劑。從各個原始生物指示劑容器取出足夠多組的樣品，每組樣品有5至10份樣品。樣品組的數量提供的觀察范圍從低于標記存活時間不少于一個標記D值到高于標記滅殺時間不少于一個標記D值。將每組樣品置于滅菌室指定位置的單獨樣品托盤上，使樣品暴露在指定的滅菌條件下。使用空的樣品托盤來檢查設備的操作參數。以供試品的最短滅菌時間開始，按照遞增順序選擇一組滅菌時間。組間滅菌時間的差異在可行的前提下盡量保持不變，而相鄰時間的差異不大于標記D值的75%。For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, preheat the ster

29、ilizing 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 spe

30、cified 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

31、for another predetermined time, and continue with successive groups until all have been heated appropriately.對于干熱滅菌生物指示劑試紙載體，對滅菌艙室預熱30分鐘。打開通道門或通道口，將其中一個載有一組樣品的托盤置于滅菌艙室內，關閉通道門或通道口，然后繼續(xù)運行設備。艙室溫度恢復至低于指定溫度2時開始對熱輻射計時。樣品在滅菌條件下持續(xù)一段預定時間（從遞增時間組中選出）后，取出裝有已加熱樣品的托盤，并放置另一個裝有樣品的托盤。以同樣方法重復滅菌程序，但滅菌時間為不同的預定時間，并對其它組樣

32、品繼續(xù)滅菌直到所有樣品都被適當加熱。For Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier, proceed as follows:對于環(huán)氧乙烷滅菌生物指示劑試紙載體，按以下步驟進行：1. Evacuate the test chamber to a pressure of not more than 100 ± 3 mm of mercury.對測試艙室抽氣，使氣壓不高于100 ± 3毫米汞柱。2. Inject sufficient water vapor (e.g., satur

33、ated 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（如飽和水蒸汽），使艙室內容物的相對濕度在規(guī)定增濕條件的10%以內，并使艙室濕度與溫度保持平衡30分鐘。3. Inject a sufficient quantity of

34、 temperature-equilibrated ethylene oxide gas to attain the appropriate concentration ±30 mg of ethylene oxide per liter.注入足量保持溫度平衡的環(huán)氧乙烷氣體，使?jié)舛冗_到每公升±30 mg環(huán)氧乙烷。4. Subject a group of specimens to the appropriate temperature, humidification, and gas concentration conditions for the required tim

35、e.將一組樣品置于適宜溫度、濕度與氣體濃度條件下保持一段規(guī)定時間。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 fur

36、ther groups of specimens to the sterilization conditions, proceed with steps 6 and 7.對測試艙室抽氣，使氣壓為100 ± 3毫米汞柱，并用無菌過濾空氣釋放真空。重復此操作直至排出不少于99%的剩余氣體，并取出裝有樣品的托盤。將其它組的樣品暴露于滅菌條件下，按第6步和第7步繼續(xù)下去。6. Flush the test chamber five times with filtered air after evacuation each time to a pressure of not more than

37、 100 ± 3 mm of mercury.每次抽氣至壓力不高于100 ± 3毫米汞柱后，分五次讓過濾空氣充滿測試艙室。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.對其它組未暴露的樣品重復第1步到第6步的整個滅菌程序，但在第4步規(guī)定條件下保持不

38、同的規(guī)定時間。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 p

39、ossible. 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 steri

40、lizing procedure similarly, but for another predetermined time, and continue with successive groups until all have been appropriately heated.對于蒸汽滅菌生物指示劑試紙載體，排出滅菌艙室氣體，并在打開艙室門的15秒內，將其中一個裝有一組樣品的托盤置于滅菌艙室內，然后運轉設備盡快加熱滅菌艙室內容物。當內容物在無菌條件下保持預定的時間（從一組遞增時間中選出）后，盡快排出滅菌艙室內氣體。取出裝有已加熱樣品的托盤，放入另一組樣品。以同樣方法重復滅菌程序，但是滅菌時

41、間為不同的預定時間，并對其它組樣品繼續(xù)滅菌直到所有樣品都被適當加熱。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 th

42、e self-contained system.對于獨立的蒸汽滅菌生物指示劑，遵循蒸汽滅菌生物指示劑試紙載體的指定程序，但將每個獨立單元作為生物指示劑系統進行處理，并測定獨立系統的D值。Recovery 恢復After completion of the sterilizing procedure for Biological Indicator for Dry-Heat Sterilization, Paper Carrier; Biological Indicator for Ethylene Oxide Sterilization, Paper Carrier; or Biologica

43、l 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 á71ñ) to submerge the biological indicator comple

44、tely in a suitable tube. 在干熱滅菌生物指示劑試紙載體、環(huán)氧乙烷滅菌生物指示劑試紙載體和蒸汽滅菌生物指示劑試紙載體均完成滅菌以后，在不超出4小時的指定時間內，以無菌操作取出所有試紙條，并將其放入10至30毫升的大豆酪蛋白消化液培養(yǎng)基（見無菌試驗71中的培養(yǎng)基）中，使生物指示劑完全浸沒在試管內。For each Biological Indicator for Steam Sterilization, Self-Contained specimen, the paper strip is immersed in the self-contained medium acco

45、rding 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 Indica

46、tor 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. 對于每份獨立的蒸汽滅菌生物指示劑樣品，按照廠商的說明，在不超出4小時的指定時間內，使試紙浸沒在獨立介質中。在55 到60的溫度下培養(yǎng)蒸汽滅菌生物指示劑試紙載體與獨立的蒸汽滅菌生物指示劑，在30

47、到35的溫度下培養(yǎng)干熱滅菌生物指示劑試紙載體與環(huán)氧乙烷滅菌生物指示劑試紙載體，或無論如何按照廠商指定的最佳恢復溫度進行培養(yǎng)。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(

48、s) concerned may be omitted.) Note the number of specimens showing no evidence of growth at any time. 在24小時與48小時后觀察每支裝有介質的試管，此后每1天或2天觀察一次，共觀察7天。（若在任何特定觀察時間都觀察到生長情況，則可停止相關樣品的進一步培養(yǎng)。）注意在任何時間都未顯示生長跡象的樣品數量。Calculation 計算This chapter describes the use of the Limited Spearman-Karber Method for determining

49、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 rout

50、inely 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. NOTEIf less than 10 biological indicators are used (i.e., 5), the formula and the v

51、arious 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法測定孢子試紙載體上生物指示劑的D值。本方法應用于藥典規(guī)定的事件或生物指示劑系統的法定測試。其它方法被公認由生物指示劑廠商和用戶例行用來測定D值，如存活曲線法與Stumbo-Murphy-Cochran程序。用限度Spearman-Karb

52、er法計算D值以每組10份生物指示劑的使用為基礎。注意若使用少于10份生物指示劑，則公式與各個計算步驟需修改，包括遺漏值的替換；但測試要求仍相同。Designate the number of specimens taken for each group (i.e., 10) by n, and the difference between adjacent times (in minutes) by d. Designate for each group of the series the number of specimens showing no growth by: 指定每組使用的樣品

53、數量（如10）為n，相鄰時間的差異（單位：分鐘）為d。指定每組未顯示生長樣品的數量為：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 sho

54、wing 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

55、. 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 hea

56、ting time, T, for achieving complete kill by the equation: 其中f1表示對接近中間死亡率的結果來說，存放時間最短的組所有10份樣品都顯示生長（0/10未存活），fk表示對接近中間死亡率的結果來說，存放時間最長的組所有10份樣品都未顯示生長（10/10未存活）。不要用超過范圍的樣品組的觀察結果f1 與fk來計算，因為給出的結果不接近中間死亡率。若存放時間比所選最短時間更短的一組樣品結果為(0/10)，并且存放時間比所選最長時間更長的一組樣品結果為(10/10)，則測試有效。用以下公式計算達到完全殺滅的平均加熱時間T：in which Tk

57、 is the time for achieving the result fk. Calculate the D value by the equation: 其中Tk為達到結果fk的時間。用以下公式計算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為進行此

58、測試時由活孢子總數（見以上內容）決定的每個載體的平均孢子計數。用以下公式計算T的方差VT：in which d represents a constant interval between successive exposures, as defined above.The standard deviation, sT , is the square root of the variance: 其中d表示連續(xù)輻射之間的固定時間，上文已作定義。標準差sT為方差的平方根：Calculate the lower and upper 95% confidence limits (approximate

59、 CL) for the D value by the equation: approximate CL for D = (T ± 2sT/log N0 + 0.2507).用以下公式計算D值的95%可信限（近似CL）低限與高限：D值近似CL= (T ± 2sT/log N0 + 0.2507)Replacement of Missing Values 遺漏值的替換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 n