生物指示劑－抗性測試

1、USP 55 BIOLOGICAL INDICATORSRESISTANCE PERFORMANCE TESTS生物指示劑抗性測試TOTAL VIABLE SPORE COUNT活孢子數(shù)總數(shù)計(jì)數(shù)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

2、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.從生物指示劑的原包裝袋中取出3支作為樣本。把待測樣品放入一個(gè)已滅菌的250mL有攪拌器的杯子里，杯子內(nèi)裝100 mL放冷的滅菌純化水。把杯子里的

3、紙片磨碎成纖維狀，并混合35min使之成為均一的懸浮液。轉(zhuǎn)移10ml的整數(shù)倍的懸浮液至一個(gè)已滅菌的16 125mm的螺蓋管中。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.如果是紙載的蒸汽滅菌生物指示劑，則把裝有懸浮

4、液的管放入95100水浴15min（熱休克），在懸浮液溫度達(dá)到95時(shí)開始計(jì)時(shí)。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 te

5、mperature reaches 80. Cool rapidly in an ice water bath at 0 to 4. 如果是紙載的干熱滅菌生物指示劑及環(huán)氧乙烷滅菌生物指示劑，則把裝有懸浮液的管放入8085水浴10min，在懸浮液溫度達(dá)到80時(shí)開始計(jì)時(shí)。在04冰浴中迅速冷卻。Transfer two 1-mL aliquots to suitable tubes, and make appropriate serial dilutions in sterilized Purified Water, the dilutions being selected as calculate

6、d to yield preferably 30 to 300 colonies, but not less than 6, on each of a pair of plates when treated as described below. 轉(zhuǎn)移2個(gè)1ml的懸浮液到合適的試管，用滅菌純化水進(jìn)行適宜的系列稀釋，所選稀釋液要求在進(jìn)行下述操作后能達(dá)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 dilutio

7、n 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 ha

8、s been melted and cooled to 45 to 50. Swirl to attain a homogeneous suspension, and allow to solidify.如果此生物指示劑的孢子濃度較低，則有必要改變稀釋系列，在每個(gè)稀釋級(jí)增加更多的平皿。每一取整數(shù)倍制備一個(gè)單獨(dú)的系列平皿。分別取1.0 mL所選的稀釋液加入兩個(gè)15 100mm的平皿中。在20min內(nèi)，向每個(gè)平皿中倒入20 mL溶解并冷卻到4550的大豆酪蛋白消化物瓊脂（參考Microbial Limit Tests 61）?；旌暇鶆?，待其凝固。Incubate the plates in an

9、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 spec

10、ified 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. 倒扣平皿進(jìn)行培養(yǎng)，紙載蒸汽滅菌生物指示劑：5560培養(yǎng)，紙載的干熱滅菌生物指示劑及環(huán)氧乙烷滅菌生物指示劑：3035培養(yǎng)，或在供應(yīng)商指定的有最佳回收率的溫度下培養(yǎng)，24h和48h后檢查平皿，記錄各皿的菌落數(shù)，用

11、48h后的菌落數(shù)進(jìn)行結(jié)果計(jì)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

12、 Sterilization, Self-Contained, aseptically remove the spore strip from the container, and proceed as directed for Biological Indicator for Steam Sterilization, Paper Carrier.由上述結(jié)果計(jì)算每個(gè)樣本的平均孢子數(shù)，使用恰當(dāng)?shù)南♂屢蜃?。如果每個(gè)懸浮液48h的孢子數(shù)的對(duì)數(shù)等于或大于其24h的孢子數(shù)的對(duì)數(shù)，則實(shí)驗(yàn)成立。對(duì)于預(yù)裝式蒸汽滅菌生物指示劑，在無菌條件下從容器中取出孢子條，根據(jù)紙載蒸汽滅菌生物指示劑的指示操作。D VALUE

13、 DETERMINATIOND值的確定For all tests described in this section, handle each test specimen with aseptic precautions, using sterilized equipment where applicable.在本部分的所有實(shí)驗(yàn)中，要在無菌的條件下處理各檢測樣品，并使用無菌的器具。Apparatus 設(shè)備For Biological Indicator for Dry-Heat Sterilization, Paper Carrier, use an apparatus of known th

14、ermodynamic 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 t

15、hrough 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. 對(duì)于紙載的干熱滅菌生物指示劑，使用已知熱力學(xué)特性的、經(jīng)過驗(yàn)證符合安全1和性能要求的設(shè)備2，此設(shè)備帶有一個(gè)可以加熱空氣的滅菌腔，最好是電加熱而不是煤氣加

16、熱，而且通過強(qiáng)制通風(fēng)有足夠的空氣對(duì)流（通過如鼓風(fēng)機(jī)這樣的機(jī)械設(shè)備），此滅菌室還帶有溫度和時(shí)間的探測和控制裝置能適時(shí)調(diào)節(jié)使溫度精度不超過0.5，時(shí)間精度不超過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 sp

17、ots, hot spots, and slow heat zones identified. 熱源的幾何模式是用來確保受測的生物指示劑能在特定的條件下受熱均勻。腔內(nèi)的溫度曲線圖是已知的，并且冷點(diǎn)、熱點(diǎn)和慢熱區(qū)域已被確定。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 addi

18、tional 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.滅

19、菌腔能夠在40300的溫度范圍內(nèi)運(yùn)行，在任何特定設(shè)置點(diǎn)的精度不少于2。設(shè)備還應(yīng)配備一個(gè)額外的通道門或艙，可以使樣品的插入（或取出）在6秒鐘之內(nèi)，并且若設(shè)定溫度在120190時(shí)能使溫度在0.5min內(nèi)恢復(fù)到設(shè)定溫度；若設(shè)定溫度在220時(shí)能使溫度在1.0min內(nèi)恢復(fù)到設(shè)定溫度。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

20、 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. 對(duì)于紙載環(huán)氧乙烷滅菌生物

21、指示劑，使用帶有測試腔的設(shè)備，此測試腔可以確保滅菌氣體充分混勻，并且可以把滅菌氣體加熱到不低于預(yù)設(shè)的運(yùn)行溫度從而沒有液體進(jìn)入這個(gè)測試腔。設(shè)備帶有溫度控制和監(jiān)測裝置、壓力控制裝置、潮濕裝置和氣體濃度監(jiān)測裝置。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) G

22、as Vessels.3設(shè)備的詳細(xì)說明和和運(yùn)行參數(shù)發(fā)表載在生物指示劑標(biāo)準(zhǔn)環(huán)氧乙烷氣體管道(BIER/EO)氣體管道鎳鉻表評(píng)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, and steam control and

23、 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(duì)于紙載的蒸汽滅菌生物指示劑和預(yù)裝式蒸汽滅菌生物指示劑，使用的設(shè)備需帶有一個(gè)有加熱、溫度及蒸汽控制和監(jiān)測裝置的腔室。設(shè)備的詳細(xì)說明和和運(yùn)行參數(shù)發(fā)表載在生物指示劑標(biāo)準(zhǔn)

24、飽和蒸汽 (BIER/蒸汽管道)鎳鉻表評(píng)估。Procedure 測試程序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 individ

25、ual containers, each group consisting of 5 to 10 specimens. 對(duì)每一個(gè)運(yùn)用了生物指示劑的滅菌條件進(jìn)行D值測試。從生物指示劑的原包裝盒里取出足量的一組生物指示劑樣本，每組包含510個(gè)樣本。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 l

26、abeled 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. 每組樣本的數(shù)量提供了從不少于一個(gè)標(biāo)示D值低于標(biāo)示存活時(shí)間到不少于一個(gè)標(biāo)示D值高于標(biāo)示滅活時(shí)間的觀察范圍。把每一組置于一個(gè)適宜的樣本容器中，使滅菌腔中特定位置的每個(gè)樣本都能暴露于以上所述的滅

27、菌條件。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 be

28、tween adjacent times is no greater than 75% of the labeled D value.用沒有樣本的樣本容器來檢查設(shè)備的運(yùn)行參數(shù)。選擇一系列以樣本檢測用最短時(shí)間為基礎(chǔ)不斷增加的滅菌時(shí)間。系列滅菌時(shí)間的差異要盡可能恒定，鄰近滅菌時(shí)間的差異不大于標(biāo)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, p

29、lace 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. 對(duì)于紙載的干熱滅菌生物指示劑，滅菌腔需預(yù)熱30min。打開通道門或艙，放入裝有一組樣本

30、的容器，關(guān)閉通道門或艙，持續(xù)運(yùn)行滅菌設(shè)備。當(dāng)滅菌腔溫度回升至設(shè)定溫度以下2時(shí)開始計(jì)算曝?zé)釙r(shí)間。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 sterilizin

31、g procedure similarly, but for another predetermined time, and continue with successive groups until all have been heated appropriately.腔內(nèi)的樣本接受了系列遞增預(yù)設(shè)時(shí)間中某個(gè)特定時(shí)間的滅菌后，取出裝有樣本的容器，然后再將另一個(gè)容器進(jìn)行滅菌。重復(fù)相似的滅菌程序，但用的是另一個(gè)預(yù)設(shè)時(shí)間，用同樣的方法做其它的樣本組直到所有的樣本都被加熱滅菌。For Biological Indicator for Ethylene Oxide Sterilization, Pape

32、r Carrier, proceed as follows:對(duì)于紙載的環(huán)氧乙烷滅菌生物指示劑，檢測程序如下：1. Evacuate the test chamber to a pressure of not more than 100 3 mm of mercury.滅菌腔抽真空使其壓力不大于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 humidif

33、ication condition, and allow the chamber to equilibrate with moisture and to temperature for about 30 minutes. 打入足量的水氣（如飽和蒸汽）使腔內(nèi)含有10以下的相對(duì)濕度，讓腔內(nèi)濕度和溫度平衡30min。3. Inject a sufficient quantity of temperature-equilibrated ethylene oxide gas to attain the appropriate concentration 30 mg of ethylene oxide p

34、er L. 打入足量的已平衡溫度的環(huán)氧乙烷，使其濃度為30 mgL。4. Subject a group of specimens to the appropriate temperature, humidification, and gas concentration conditions for the required time. 把一組樣本在上述溫度、濕度和氣體濃度條件下放置一定時(shí)間。5. Evacuate the test chamber to a pressure of 100 3 mm of mercury, and release the vacuum with sterile

35、 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. 滅菌腔抽真空使其壓力達(dá)到100 3 mm汞柱。然后用滅菌過濾空氣釋放真空。重復(fù)這一操作單元直到不少于99%的剩余氣體被趕走，然后取出裝有樣本的容器。For exposing further groups of specimens to the sterilization conditions, proceed w

36、ith steps 6 and 7. 其它樣本的滅菌程序同第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. 每次抽真空后用過濾空氣沖洗滅菌腔5次，使壓力不超過100 3 mm汞柱。7. Repeat the entire sterilizing procedure, steps 1 through 6, for other groups of unexposed s

37、pecimens, but maintain the specified conditions of step 4 for each of the other required times. 對(duì)于其它未滅菌的樣本組，重復(fù)1到6步滅菌程序，但第4步中的特定條件不隨其它時(shí)間的改變而變化。For Biological Indicator for Steam Sterilization, Paper Carrier, exhaust the sterilizing chamber, and within 15 seconds of opening the door, place one of the

38、holders with a group of specimens in the sterilizing chamber, and operate the apparatus to heat up the chamber contents as quickly as possible. 對(duì)于紙載的蒸汽滅菌生物指示劑，滅菌腔抽真空，在開門的15秒內(nèi)放入一個(gè)裝有一組樣本的容器，運(yùn)行設(shè)備，使腔內(nèi)盡快熱起來。After the contents have been subjected to the sterilizing condition for a predetermined time selec

39、ted 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. 腔內(nèi)的樣本接受了系列遞增預(yù)設(shè)時(shí)間中某個(gè)特定時(shí)間的滅菌后，盡快排空滅菌腔，取出裝有樣本的容器。重復(fù)操作其它的樣本。Repeat the sterilizing procedure similarly, but for another predete

40、rmined time, and continue with successive groups until all have been appropriately heated.重復(fù)同樣的滅菌程序，但用另一個(gè)預(yù)設(shè)的滅菌時(shí)間，連續(xù)做其它的樣本組直到所有的樣本都被加熱滅菌。For Biological Indicator for Steam Sterilization, Self-Contained, follow the procedure indicated for Biological Indicator for Steam Sterilization, Paper Carrier, bu

41、t handle each self-contained unit as a biological indicator system, with the D value determined for the self-contained system.對(duì)于預(yù)裝的蒸汽滅菌生物指示劑，遵循紙載蒸汽滅菌生物指示劑中的檢測程序，但把每個(gè)預(yù)裝生物指示劑作為一個(gè)生物指示劑系統(tǒng)來確定D值。Recovery 回收率After completion of the sterilizing procedure for Biological Indicator for Dry-Heat Sterilization,

42、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 Me

43、dium (see Media under Sterility Tests 71) to submerge the biological indicator completely in a suitable tube. 完成了紙載干熱滅菌生物指示劑、紙載環(huán)氧乙烷滅菌生物指示劑或紙載蒸汽滅菌指示劑的滅菌程序后，無論運(yùn)用哪個(gè)，應(yīng)在4個(gè)小時(shí)之內(nèi)，在無菌條件下取出含菌紙條，放入1030ml的大豆酪蛋白消化培養(yǎng)基中（參考Sterility Tests無菌檢測71中培養(yǎng)基部分）使之完全浸沒。For each Biological Indicator for Steam Sterilization, Sel

44、f-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. 對(duì)于每個(gè)預(yù)包裝的蒸汽滅菌生物指示劑，按供應(yīng)商的說明，含菌紙條被浸沒于自帶的培養(yǎng)基，浸沒時(shí)間不超過4小時(shí)。Incubate each tube at a temperature of 55 to 60 for Biological Indicator for Steam St

45、erilization, 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 temperatur

46、e specified by the manufacturer. 對(duì)每個(gè)管進(jìn)行培養(yǎng)，紙載蒸汽滅菌生物指示劑和預(yù)包裝蒸汽滅菌生物指示劑：5560培養(yǎng)；紙載的干熱滅菌生物指示劑及環(huán)氧乙烷滅菌生物指示劑：3035培養(yǎng)，或在供應(yīng)商指定的有最佳回收率的溫度下培養(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

47、 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觀察每個(gè)裝有培養(yǎng)基的試管，然后每隔1到2天進(jìn)行觀察，共培養(yǎng)7天（如果在某一觀察時(shí)間觀察到有菌生長，則這個(gè)試管就不用再進(jìn)行培養(yǎng)）。每次觀察記錄無菌生長的樣品數(shù)。Calculation 計(jì)算This chapter describes the use of

48、 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方法來確定生物指示劑紙載孢子的D值。在生物指示劑系統(tǒng)的綱要論述或規(guī)章仲裁檢測中要使用

49、這種方法。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.人們公認(rèn)其它方法如殘存曲線法和Stumbo-Murphy-Cochran程序可以被生物指示劑廠商和用戶用來確定D值。The calculation of the D valu

50、e 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 calculation steps will have to be modified, including the Replacement of Missing Values; however, the requir

51、ements of the test remain the same. 用Limited Spearman-Karber方法進(jìn)行D值的計(jì)算是基于每一組使用10個(gè)生物指示劑。注如果所用的生物指示劑少于10個(gè)（如5個(gè)），這個(gè)公式和各計(jì)算步驟進(jìn)行修正，包括置換漏測值；但是檢測的要求還是一樣的。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

52、 of the series the number of specimens showing no growth by: f1, f2, . fk每組所用的樣本數(shù)設(shè)為n，鄰近滅菌時(shí)間的差值（以分鐘計(jì)）設(shè)為。把每組無菌生長的系列樣品以 f1, f2, . fk來表示。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 interm

53、ediate 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代表滅菌時(shí)間最短的一組里都長菌的10個(gè)樣本（0/10滅活），這個(gè)結(jié)果接近半數(shù)死亡率，fk代表滅菌時(shí)間最長的一組里都不長菌的10個(gè)樣本（10/10滅活），這個(gè)結(jié)果接近半數(shù)

54、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. 超出系列兩端f1 and fk的實(shí)驗(yàn)組不要用于計(jì)算觀察，因?yàn)檫@樣的結(jié)果不接近半數(shù)死亡率。The test is valid if there is available a result (0/10) from a group held for a shorter

55、 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). 如果一個(gè)組里有個(gè)結(jié)果（0/10）所用時(shí)間比所選最短時(shí)間的結(jié)果(f1)還要短，并且組里有個(gè)結(jié)果（10/10）所用時(shí)間比所選最長時(shí)間的結(jié)果(f1)還要長的話，則實(shí)驗(yàn)是有效的。Calculate the mean heating

56、 time, T, for achieving complete kill by the equation: in which Tk is the time for achieving the result fk. Calculate the D value by the equation: 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. 用以下公式計(jì)算達(dá)到完全滅活的平均加熱

57、時(shí)間，T：Tk是達(dá)到fk結(jié)果所用的時(shí)間。D值的計(jì)算公式如下：N0是用活孢子總數(shù)計(jì)數(shù)方法得到的每個(gè)載片的平均孢子數(shù)。Calculate the variance of T, VT , by the equation: in which represents a constant interval between successive exposures, as defined above.The standard deviation, sT , is the square root of the variance: Calculate the lower and upper 95% confid

58、ence limits (approximate CL) for the D value by the equation: approximate CL for D = (T 2sT/log N0 + 0.2507)用以下公式計(jì)算T值的變異VT：表示相鄰曝?zé)岬暮愣ㄩg隔，定義將前文。標(biāo)準(zhǔn)偏差sT是VT的平方根：Replacement of Missing Values 漏測值的置換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 nine spec