美國藥典USP31-NF26無菌檢查法《71》.doc

71 STERILITY TESTS 無菌檢查法

Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopeia and/or the Japanese Pharmacopeia. Those portions that are not harmonized are marked with symbols ( ) to specify this fact.

此通則的各部分已經與歐洲藥典和/或日本藥典的對應部分做了協調。不一致的部分用符號（ ）來標明。

The following procedures are applicable for determining whether a Pharmacopeial article purporting to be sterile complies with the requirements set forth in the individual monograph with respect to the test for sterility. Pharmacopeial articles are to be tested by the Membrane Filtration method under Test for Sterility of the Product to be Examined where the nature of the product permits. If the membrane filtration technique is unsuitable, use the Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined. All devices, with the exception of Devices with Pathways Labeled Sterile, are tested using the Direct Inoculation of the Culture Medium method. Provisions for retesting are included under Observation and Interpretation of Results.

下面這些步驟適用于測定是否某個用于無菌用途的藥品是否符合其具體的各論中關于無菌檢查的要求。只要其性質許可，這些藥品將使用供試產品無菌檢查法項下的膜過濾法來檢測。如果膜過濾技術是不適合的，則使用在供試產品無菌檢查法項下的培養基直接接種法。除了具有標記為無菌通道的設備之外，所有的設備均須使用培養基直接接種法進行檢測。在結果的觀測與理解項下包含了復驗的規定。

Because sterility testing is a very exacting procedure, where asepsis of the procedure must be ensured for a correct interpretation of results, it is important that personnel be properly trained and qualified. The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any microorganisms that are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.

由于無菌檢查法是一個非常精確的程序，在此過程中程序的無菌狀態必須得到確保以實現對結果的正確理解，因此人員經過適當的培訓并取得資質是非常重要的。無菌檢查在無菌條件下進行。為了實現這樣的條件，試驗環境必須調整到適合進行無菌檢查的方式。為避免污染而采取的特定預防措施應不會對任何試圖在檢查中發現的微生物產生影響。通過在工作區域作適當取樣并進行適當控制，來定期監測進行此試驗的工作條件。

These Pharmacopeial procedures are not by themselves designed to ensure that a batch of product is sterile or has been sterilized. This is accomplished primarily by validation of the sterilization process or of the aseptic processing procedures.

這些藥典規定程序自身的設計不能確保一批產品無菌或已經滅菌。這主要是通過滅菌工藝或者無菌操作程序的驗證來完成。

When evidence of microbial contamination in the article is obtained by the appropriate Pharmacopeial method, the result so obtained is conclusive evidence of failure of the article to meet the requirements of the test for sterility, even if a different result is obtained by an alternative procedure. For additional information on sterility testing, see Sterilization and Sterility Assurance of Compendial Articles 1211 .

當通過適當的藥典方法獲得了某物品中微生物污染的證據，這樣獲得的結果是該物品未能達到無菌檢驗要求的結論性證據，即便使用替代程序得到了不同的結果也無法否定此結果。 如要獲得關于無菌檢驗的其他信息，見藥品的滅菌和無菌保證<1211>

MEDIA 培養基

Prepare media for the tests as described below, or dehydrated formulations may be used provided that, when reconstituted as directed by the manufacturer or distributor, they meet the requirements of the Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Media are sterilized using a validated process.

按照下面描述的方法配制實驗用培養基；或者使用脫水培養基，只要根據其制造商或者分銷商說明進行恢復之后，其能夠符合好氧菌、厭氧菌、霉菌生長促進試驗的要求即可。使用經過驗證的工藝對培養基進行滅菌操作。

The following culture media have been found to be suitable for the test for sterility. Fluid Thioglycollate Medium is primarily intended for the culture of anaerobic bacteria. However, it will also detect aerobic bacteria. Soybean–Casein Digest Medium is suitable for the culture of both fungi and aerobic bacteria.

下面的培養基已經被證實適合進行無菌檢查。巰基醋酸鹽液體培養基主要用于厭氧菌的培養。但其也用于檢測好氧菌。大豆酪蛋白消化物培養基適合于培養霉菌和好氧菌。

Fluid Thioglycollate Medium 巰基醋酸鹽液體培養基

Mix the L-cystine, sodium chloride, dextrose, yeast extract, and pancreatic digest of casein with the purified water, and heat until solution is effected. Dissolve the sodium thioglycollate or thioglycolic acid in the solution and, if necessary, add 1 N sodium hydroxide so that, after sterilization, the solution will have a pH of 7.1 ± 0.2. If filtration is necessary, heat the solution again without boiling, and filter while hot through moistened filter paper. Add the resazurin sodium solution, mix, and place the medium in suitable vessels that provide a ratio of surface to depth of medium such that not more than the upper half of the medium has undergone a color change indicative of oxygen uptake at the end of the incubation period. Sterilize using a validated process. If the medium is stored, store at a temperature between 2 and 25 in a sterile, airtight container. If more than the upper one-third of the medium has acquired a pink color, the medium may be restored once by heating the containers in a water-bath or in free-flowing steam until the pink color disappears and by cooling quickly, taking care to prevent the introduction of nonsterile air into the container.

將L-胱氨酸、氯化鈉、葡萄糖、酵母提取物、酪蛋白胰酶消化物與純凈水混合，并加熱至實現溶解。將巰基乙酸鈉或者巰基乙酸溶解于該溶液，如果需要可再加入1N氫氧化鈉，以便在滅菌后該溶液呈pH值7.1 ± 0.2。如需要則過濾，再次加熱該溶液但不得煮沸，并趁熱以濕潤濾紙將該溶液過濾。加入刃天青鈉溶液，混勻，并將該培養基置于適當容器中，該容器應為培養基提供特定的面積－深度比，以使在培養期末表明氧氣攝入的變色部分不超過培養基的上半部分。使用經過驗證的工藝進行滅菌。如果需要儲存該培養基，將其置于無菌、氣密容器中，在2 至25 之間儲藏。如果超過上部三分之一的培養基已經呈粉色，可以用以下方法恢復該培養基一次：在水浴鍋中或者自由流動蒸氣中加熱該容器，直至粉色消失，并迅速放涼，須小心防止非無菌空氣進入到容器中。

Fluid Thioglycollate Medium is to be incubated at 32.5 ± 2.5 .

巰基醋酸鹽液體培養基將在32.5 ± 2.5 條件下進行培養。

Alternative Thioglycollate Medium 替代巰基醋酸鹽培養基

Prepare a mixture having the same composition as that of the Fluid Thioglycollate Medium, but omitting the agar and the resazurin sodium solution, sterilize as directed above, and allow to cool prior to use. The pH after sterilization is 7.1 ± 0.2. Incubate under anaerobic conditions for the duration of the incubation period.

配制與巰基醋酸鹽液體培養基成分相同，但省略了瓊脂和刃天青鈉溶液的混合物，按上述方法滅菌，并在使用前靜置至涼。滅菌后pH值為7.1 ± 0.2。在厭氧條件下培養，培養時間同培養期。

Alternative Fluid Thioglycollate Medium is to be incubated at 32.5 ± 2.5 .

替代性巰基醋酸鹽培養基將在32.5 ± 2.5 條件下進行培養。

Soybean–Casein Digest Medium 大豆-酪蛋白消化物培養基

Dissolve the solids in the Purified Water, heating slightly to effect a solution. Cool the solution to room temperature, and adjust the pH with 1 N sodium hydroxide so that, after sterilization, it will have a pH of 7.3 ± 0.2. Filter, if necessary to clarify, dispense into suitable containers, and sterilize using a validated procedure. Store at a temperature between 2 and 25 in a sterile well-closed container, unless it is intended for immediate use.

將固體物質溶解于純凈水，輕微加熱以實現溶解。放涼溶液至室溫，并用1N氫氧化鈉調整pH值，以便在滅菌后其pH值呈7.3 ± 0.2。過濾，如需要則使之澄清，分裝入適合的容器，并用經過驗證的程序消毒。如果不立刻使用，則在2 到25 之間以無菌且密閉良好的容器保存。

Soybean–Casein Digest Medium is to be incubated at 22.5 ± 2.5 .

大豆-酪蛋白消化物培養基將在22.5 ± 2.5 條件下培養。

Media for Penicillins or Cephalosporins 用于青霉素和頭孢菌素的培養基

Where sterility test media are to be used in the Direct Inoculation of the Culture Medium method under Test for Sterility of the Product to be Examined, modify the preparation of Fluid Thioglycollate Medium and the Soybean–Casein Digest Medium as follows. To the containers of each medium, transfer aseptically a quantity of -lactamase sufficient to inactivate the amount of antibiotic in the specimen under test. Determine the quantity of -lactamase required to inactivate the antibiotic by using a -lactamase preparation that has been assayed previously for its penicillin- or cephalosporin-inactivating power. [NOTE—Supplemented -lactamase media can also be used in the membrane filtration test.]

當無菌檢查培養基用于供試產品無菌檢查項下的培養基直接接種法時，按如下內容變更巰基醋酸鹽液體培養基和大豆-酪蛋白消化物培養基的制備方法。向每一種培養基的容器中，以無菌操作轉移足夠滅活供試樣品中所存在抗生素的 -內酰胺酶。使用此前已經對其青霉素或頭孢菌素滅活能力進行了測定的 -內酰胺酶配制品，來測定滅活該抗生素所必需的 -內酰胺酶數量。[注意：補充的 -內酰胺酶培養基也可以用于膜過濾試驗]

Alternatively (in an area completely separate from that used for sterility testing), confirm that an appropriate amount of -lactamase is incorporated into the medium, following either method under Validation Test, using less than 100 colony-forming units (cfu) of Staphylococcus aureus (see Table 1) as the challenge. Typical microbial growth of the inoculated culture must be observed as a confirmation that the -lactamase concentration is appropriate.

或者（在與無菌試驗所用場所徹底隔離的區域中），按照驗證試驗項下的任意一種方法，使用少于100個菌落（cfu）的金黃色葡萄球菌（見表1）作為驗證菌，來確認適當數量的 -內酰胺酶已經被整合到該培養基中。必須觀測到接種后培養物中出現典型微生物生長，才能確認 -內酰胺酶濃度是適當的。

Table 1. Strains of the Test Microorganisms Suitable for Use in the Growth Promotion Test and the
Validation Test

表1 適合用于生長促進試驗和驗證試驗中的試驗微生物的菌株

Suitability Tests 適合性試驗

The media used comply with the following tests, carried out before, or in parallel, with the test on the product to be examined.

所使用的培養基須符合下列試驗，這些試驗應在檢驗供試產品之前或者同時進行。

STERILITY 無菌狀態

Confirm the sterility of each sterilized batch of medium by incubating a portion of the media at the specified incubation temperature for 14 days. No growth of microorganisms occurs.

通過在指定培養溫度下將一部分培養基培養14天，來確認每一批已滅菌培養基的無菌狀態。不得出現微生物生長。

GROWTH PROMOTION TEST OF AEROBES, ANAEROBES, and FUNGI

好氧菌、厭氧菌、霉菌的生長促進試驗

Test each lot of ready-prepared medium and each batch of medium prepared either from dehydrated medium or from ingredients ¹ . Suitable strains of microorganisms are indicated in Table 1.

檢查每一批已經配制好的培養基和每一批用脫水培養基或配料制備的培養基 ¹ 。適當微生物菌株見表1。

Inoculate portions of Fluid Thioglycollate Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Clostridium sporogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. Inoculate portions of Alternative Fluid Thioglycollate Medium with a small number (not more than 100 cfu) of Clostridium sporogenes. Inoculate portions of Soybean–Casein Digest Medium with a small number (not more than 100 cfu) of the following microorganisms, using a separate portion of medium for each of the following species of microorganism: Aspergillus niger, Bacillus subtilis, and Candida albicans. Incubate for not more than 3 days in the case of bacteria and not more than 5 days in the case of fungi.

在部分巰基醋酸鹽液體培養基上接種少量（不超過100cfu）下列微生物，每一種微生物均使用單獨一部分培養基：產芽胞梭狀芽胞桿菌、綠膿桿菌、金黃色葡萄球菌。 在部分替代巰基醋酸鹽液體培養基上接種少量（不超過100cfu）產芽胞梭狀芽胞桿菌。 在部分大豆-酪蛋白消化物培養基上接種少量（不超過100cfu）下列微生物，每一種微生物均使用單獨一部分的培養基：黑曲霉、枯草芽孢桿菌、白色念珠菌。細菌培養時間不超過3天，霉菌培養時間不超過5天。

The media are suitable if a clearly visible growth of the microorganisms occurs.

如果出現清晰可見的微生物生長，則該培養基是適合的。

STORAGE 保存

If prepared media are stored in unsealed containers, they can be used for 1 month, provided that they are tested for growth promotion within 2 weeks of the time of use and that color indicator requirements are met. If stored in tight containers, the media can be used for 1 year, provided that they are tested for growth promotion within 3 months of the time of use and that the color indicator requirements are met.

如果配制好的培養基保存于未密閉的容器中，只要在使用時間的2周內對其進行了生長促進試驗并且符合顏色指示劑的要求，它們就可以使用1個月。如果保存在密閉的容器中，只要在使用時間的3個月內對其進行了生長促進試驗并且符合顏色指示劑的要求，則該培養基可以使用1年。

DILUTING AND RINSING FLUIDS FOR MEMBRANE FILTRATION

用于膜過濾的稀釋和沖洗液

Fluid A 液體A

PREPARATION 配制品

Dissolve 1 g of peptic digest of animal tissue in water to make 1 L, filter or centrifuge to clarify, if necessary, and adjust to a pH of 7.1 ± 0.2. Dispense into containers, and sterilize using a validated process.

將1g動物組織胃蛋白酶消化物溶于1L水中，如果需要則通過濾或離心使其澄清，再調節pH值至7.1 ± 0.2。分裝入容器中，并用經過驗證的工藝滅菌。

PREPARATION FOR PENICILLINS OR CEPHALOSPORINS

用于青霉素或頭孢菌素的配制品

Aseptically add to the above Preparation, if necessary, a quantity of sterile -lactamase sufficient to inactivate any residual antibiotic activity on the membranes after the solution of the test specimen has been filtered (see Media for Penicillins or Cephalosporins).

在供試樣品溶液已經過濾（見用于青霉素或頭孢菌素的培養基）之后，如果需要，向上述配制品中，以無菌操作加入數量足夠滅活濾膜上殘余抗生素活性的 -內酰胺酶。

Fluid D 液體D

To each L of Fluid A add 1 mL of polysorbate 80, adjust to a pH of 7.1 ± 0.2, dispense into containers, and sterilize using a validated process. Use this fluid for articles containing lecithin or oil, or for devices labeled as “sterile pathway.”

向每升液體A中，加入1mL聚山梨酯80，調節pH值至7.1 ± 0.2，分裝入容器中，并使用經過驗證的工藝滅菌。此液體用于含有卵磷脂或油脂的物品，或用于標為 “無菌通道”的設備。

Fluid K 液體K

Dissolve 5.0 g of peptic digest of animal tissue, 3.0 g of beef extract, and 10.0 g of polysorbate 80 in water to make 1 L. Adjust the pH to obtain, after sterilization, a pH of 6.9 ± 0.2. Dispense into containers, and sterilize using a validated process.

將5.0g動物組織胃蛋白酶消化物、3.0g牛肉提取物、10.0g聚山梨酯80溶解于1L水中。調節pH值，以便使pH值在滅菌后呈6.9 ± 0.2。分裝入容器中，并使用經過驗證的工藝滅菌。

VALIDATION TEST 驗證試驗

Carry out a test as described below under Test for Sterility of the Product to be Examined using exactly the same methods, except for the following modifications.

按照下面供試產品無菌檢查項下的描述，使用除了下面變更之外完全相同的方法，進行試驗。

Membrane Filtration 膜過濾

After transferring the content of the container or containers to be tested to the membrane, add an inoculum of a small number of viable microorganisms (not more than 100 cfu) to the final portion of sterile diluent used to rinse the filter.

在將一個或多個供試容器中的內容物轉移到濾膜之后，在最后一次的沖洗液中加入少量（不超過100cfu）試驗菌.

Direct Inoculation 直接接種

After transferring the contents of the container or containers to be tested (for catgut and other surgical sutures for veterinary use: strands) to the culture medium, add an inoculum of a small number of viable microorganisms (not more than 100 cfu) to the medium.

在將一個或多個供試容器（對于獸醫的腸線和其他外科縫合用線：若干股線）中的內容物轉移至培養基之后，將少量試驗菌（不超過100cfu）加入至培養基中。

In both cases use the same microorganisms as those described above under Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Perform a growth promotion test as a positive control. Incubate all the containers containing medium for not more than 5 days.

在這兩種情況中，均按照上述好氧菌、厭氧菌、霉菌生長促進試驗項下的描述，使用同樣的微生物。進行一個生長促進試驗作為陽性對照。培養所有含有培養基的容器，培養時間不超過5天。

If clearly visible growth of microorganisms is obtained after the incubation, visually comparable to that in the control vessel without product, either the product possesses no antimicrobial activity under the conditions of the test or such activity has been satisfactorily eliminated. The test for sterility may then be carried out without further modification.

如果在培養后得到清晰可見的微生物生長，看起來與沒有產品的對照容器中的生長類似，則該產品在此試驗條件下沒有任何抗微生物活性，或者此活性已經被令人滿意地消除了。然后，無菌試驗可以進行，而無需進一步的變更。

If clearly visible growth is not obtained in the presence of the product to be tested, visually comparable to that in the control vessels without product, the product possesses antimicrobial activity that has not been satisfactorily eliminated under the conditions of the test. Modify the conditions in order to eliminate the antimicrobial activity, and repeat the validation test.

如果用肉眼與沒有產品的對照容器比較，無法在存在供試產品的情況下得到清晰可見的生長，則該產品在試驗條件下所具有的抗微生物活性尚未令人滿意地消除。變更條件以便消除抗微生物活性，并重復驗證試驗。

This validation is performed (a) when the test for sterility has to be carried out on a new product; and (b) whenever there is a change in the experimental conditions of the test. The validation may be performed simultaneously with the Test for Sterility of the Product to be Examined.

當(a)一個新產品必須進行無菌試驗時，和(b)無論何時該試驗的試驗條件發生改變時，則需進行此驗證試驗。該驗證可以與供試產品的無菌試驗同時進行。

TEST FOR STERILITY OF THE PRODUCT TO BE EXAMINED

供試產品的無菌檢查

Number of Articles to Be Tested 供試物品數量

Unless otherwise specified elsewhere in this chapter or in the individual monograph, test the number of articles specified in Table 3. If the contents of each article are of sufficient quantity (see Table 2), they may be divided so that equal appropriate portions are added to each of the specified media. [NOTE—Perform sterility testing employing two or more of the specified media.] If each article does not contain sufficient quantities for each medium, use twice the number of articles indicated in Table 3.

除非在此章節的其他位置或在具體的各論中另有規定，供試物品的數量遵照表3中的規定。如果每個物品的內容物有足夠數量（見表2），可以將其分成若干等份，將適當的等份加入到每個指定的培養基。[注意：使用兩個或更多指定培養基，來進行無菌試驗。]如果每個物品內容物的數量不夠每個培養基的用量，使用表3中所規定物品數量的2倍。

Table 2. Minimum Quantity to be Used for Each Medium

表2：用于每個培養基的最小數量

Table 3. Minimum Number of Articles to be Tested in Relation to the Number of Articles in the Batch 表3：與物品批量相關的最小供試物品數量

The test may be carried out using the technique of Membrane Filtration or by Direct Inoculation of the Culture Medium with the product to be examined. Appropriate negative controls are included. The technique of membrane filtration is used whenever the nature of the product permits; that is, for filterable aqueous preparations, for alcoholic or oily preparations, and for preparations miscible with, or soluble in, aqueous or oily solvents, provided these solvents do not have an antimicrobial effect in the conditions of the test.

此試驗可以使用膜過濾法或培養基直接接種法進行。應包括多個適當的陰性對照。只要該產品的性質許可，就應使用膜過濾法；這些性質是，可過濾的水溶性配制品、酒精或油性配制品、易混合或溶解于水或油性溶劑的配制品，只要這些溶劑在試驗條件下沒有抗生素效果。

Membrane Filtration 膜過濾

Use membrane filters having a nominal pore size not greater than 0.45 µm whose effectiveness to retain microorganisms has been established. Cellulose nitrate filters, for example, are used for aqueous, oily, and weakly alcoholic solutions; and cellulose acetate filters, for example, are used for strongly alcoholic solutions. Specially adapted filters may be needed for certain products (e.g., for antibiotics).

使用標稱孔徑不大于0.45 µm的膜過濾器，此孔徑已知能夠有效截留微生物。例如，硝酸纖維素過濾器可用于水、油、稀醇溶液；而醋酸纖維素可用于濃醇溶液。特定產品（例如，抗生素）可能需要特別改造過的過濾器。

The technique described below assumes that membranes about 50 mm in diameter will be used. If filters of a different diameter are used, the volumes of the dilutions and the washings should be adjusted accordingly. The filtration apparatus and membrane are sterilized by appropriate means. The apparatus is designed so that the solution to be examined can be introduced and filtered under aseptic conditions: it permits the aseptic removal of the membrane for transfer to the medium, or it is suitable for carrying out the incubation after adding the medium to the apparatus itself.

下面描述的方法所使用直徑約50mm的濾膜。如果使用不同直徑的過濾器，稀釋液和洗液的體積應當作相應調節。以適當方法將過濾設備和濾膜滅菌。該設備設計用于在無菌條件下加入和過濾供試溶液：其使得在無菌狀態下將濾膜摘掉轉移至培養基成為可能，或者其適合于將培養基加入該設備自身之中，并進行培養。

AQUEOUS SOLUTIONS 水性溶液

If appropriate, transfer a small quantity of a suitable, sterile diluent such as Fluid A (see Diluting and Rinsing Fluids for Membrane Filtration) onto the membrane in the apparatus and filter. The diluent may contain suitable neutralizing substances and/or appropriate inactivating substances, for example, in the case of antibiotics.

如果適當，將少量適當的無菌稀釋劑，例如液體A（見用于膜過濾的稀釋和沖洗液），轉移至設備中的濾膜上并過濾。該稀釋劑可以含有適當的中和物質和/或適當的滅活物質，例如針對抗生素。

Transfer the contents of the container or containers to be tested to the membrane or membranes, if necessary, after diluting to the volume used in the Validation Test with the chosen sterile diluent, but using not less than the quantities of the product to be examined prescribed in Tables 2 and 3. Filter immediately. If the product has antimicrobial properties, wash the membrane not less than three times by filtering through it each time the volume of the chosen sterile diluent used in the Validation Test. Do not exceed a washing cycle of 5 times 200 mL, even if during validation it has been demonstrated that such a cycle does not fully eliminate the antimicrobial activity. Transfer the whole membrane to the culture medium or cut it aseptically into two equal parts, and transfer one half to each of two suitable media. Use the same volume of each medium as in the Validation Test. Alternatively, transfer the medium onto the membrane in the apparatus. Incubate the media for not less than 14 days.

將一個或多個供試容器的內容物轉移到濾膜，如需要可先使用選定的無菌稀釋劑稀釋至驗證試驗中所用體積，但須使用不少于表2和3中規定的供試產品數量。立即過濾。如果該產品具有抗微生物特性，沖洗濾膜不少于3次，每次均將驗證試驗中所使用的無菌稀釋劑體積濾過該濾膜。即便驗證中顯示5次200mL的沖洗循環沒有完全消除抗微生物活性，也不要超越這樣一個循環。轉移整個濾膜至培養基，或以無菌操作將其切開至相等的2部分，并將每一部分轉移至適當的培養基中。每個培養基的體積與驗證試驗所用的一樣。或者，將培養基轉移至設備中的濾膜上。培養該培養基，不少于14天。

SOLUBLE SOLIDS (other than antibiotics) 可溶固體（非抗生素）

Use for each medium not less than the quantity prescribed in Tables 2 and 3 of the product dissolved in a suitable solvent, such as Fluid A (Diluting and Rinsing Fluids for Membrane Filtration), and proceed with the test as described above for Aqueous Solutions using a membrane appropriate to the chosen solvent.

在每個培養基中，使用不少于表2和3規定的產品數量溶于適當溶劑，例如溶液A（用于膜過濾的稀釋和沖洗液），并按照上述關于水性溶液的描述，使用適合所選溶劑的濾膜，繼續進行該試驗。

OILS and OILY SOLUTIONS 油和油性溶液

Use for each medium not less than the quantity of the product prescribed in Tables 2 and 3. Oils and oily solutions of sufficiently low viscosity may be filtered without dilution through a dry membrane. Viscous oils may be diluted as necessary with a suitable sterile diluent such as isopropyl myristate shown not to have antimicrobial activity in the conditions of the test. Allow the oil to penetrate the membrane by its own weight, and then filter, applying the pressure or suction gradually. Wash the membrane at least three times by filtering through it each time about 100 mL of a suitable sterile solution such as Fluid A (see Diluting and Rinsing Fluids for Membrane Filtration) containing a suitable emulsifying agent at a concentration shown to be appropriate in the validation of the test, for example polysorbate 80 at a concentration of 10 g per L (Fluid K) . Transfer the membrane or membranes to the culture medium or media, or vice versa, as described above for Aqueous Solutions, and incubate at the same temperatures and for the same times.

在每個培養基中，使用不少于表2和3中描述的產品用量。粘性足夠低的油和油性溶液可能在不經稀釋的情況下濾過干燥濾膜。如需要，粘稠油質可以用適合的無菌稀釋劑進行稀釋，例如已證實在該試驗條件下不具有抗微生物活性的豆蔻酸異丙酯。使該油質依靠其自身的重量穿過濾膜，然后逐漸應用壓力或抽吸過濾。每次過濾約100mL適當的無菌溶液，例如液體A（見用于膜過濾的稀釋和沖洗液），并含有適當乳化劑且其濃度已證實適用于該試驗的驗證，例如濃度為10克每升的聚山梨酯80（液體K）。將一個或多個濾膜轉移到一個或多個培養基，或反之，如上面關于水性溶液的描述，并在相同溫度下培養同樣的時間。

OINTMENTS and CREAMS 油膏和乳膏

Use for each medium not less than the quantities of the product prescribed in Tables 2 and 3. Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 1% in isopropyl myristate as described above, by heating, if necessary, to not more than 40 . In exceptional cases it may be necessary to heat to not more than 44 . Filter as rapidly as possible, and proceed as described above for Oils and Oily Solutions.

在每個培養基中，使用不少于表2和3中描述的產品用量。脂肪狀的油膏和水在油中形態乳化劑可以按照上面所述，在豆蔻酸異丙酯中稀釋至1%，如需要可加熱至不高于40 。在特別情況下，其可能必須加熱到不超過44 。盡可能迅速地過濾，并按照上面針對油和油性溶液所述內容繼續操作。

PREFILLED SYRINGES 預裝填的注射器

For prefilled syringes without attached sterile needles, expel the contents of each syringe into one or two separate membrane filter funnels or into separate pooling vessels prior to transfer. If a separate sterile needle is attached, directly expel the syringe contents as indicated above, and proceed as directed for Aqueous Solutions. Test the sterility of the needle, using Direct Inoculation under Validation Test.

對于沒有附無菌針頭的預裝填注射器，在轉移之前，將每個注射器的內容物排出至一個或兩個單獨的膜過濾器漏斗，或至若干單獨的合并容器。如果附了單獨的滅菌針頭，直接按照上面的規定將注射器內容物直接排出，并按照關于水性溶液的規定繼續進行。使用驗證試驗項下的直接接種，檢查針頭的無菌情況。

SOLIDS FOR INJECTION OTHER THAN ANTIBIOTICS 除了抗生素之外的注射用固體

Constitute the test articles as directed on the label, and proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies. [NOTE—If necessary, excess diluent can be added to aid in the constitution and filtration of the constituted test article.]

按照其標簽上的規定配制供試物品，并按照適用的關于水性溶液或油和油性溶液的規定繼續進行。[注意：如需要，可以加入額外的稀釋劑以幫助對已配制的供試物品進行再配制和過濾]

ANTIBIOTIC SOLIDS FOR INJECTION 用于注射的抗生素

Pharmacy Bulk Packages, < 5 g— From each of 20 containers, aseptically transfer about 300 mg of solids, into a sterile 500-mL conical flask, dissolve in about 200 mL of Fluid A (see Diluting and Rinsing Fluids for Membrane Filtration), and mix; or constitute, as directed in the labeling, each of 20 containers and transfer a quantity of liquid or suspension, equivalent to about 300 mg of solids, into a sterile 500-mL conical flask, dissolve in about 200 mL of Fluid A, and mix. Proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies.

藥房散裝< 5 g：取20個容器，每個容器均以無菌操作將約300mg固體轉移至一個無菌的500mL錐形燒瓶，溶解于約200mL液體A中（見用于膜過濾的稀釋和沖洗液），并混勻；或取20個容器，每個容器均按照標簽上的規定配制，并將相當于大約300mg固體的液體或懸浮液轉移至一個無菌的500mL錐形燒瓶，溶解于約200mL液體A中，并混勻。按照適合的關于水性溶液或油和油性溶液的規定，繼續操作。

Pharmacy Bulk Packages, 5 g— From each of 6 containers, aseptically transfer about 1 g of solids into a sterile 500-mL conical flask, dissolve in about 200 mL of Fluid A, and mix; or constitute, as directed in the labeling, each of 6 containers and transfer a quantity of liquid, equivalent to about 1 g of solids, into a sterile 500-mL conical flask, dissolve in about 200 mL of Fluid A, and mix. Proceed as directed for Aqueous Solutions.

藥方散裝 5 g：取6個容器，每個均以無菌操作將大約1克的固體轉移至一個無菌的500mL錐形燒瓶，溶解于約200mL液體A中，并混勻；或取6個容器，每個均按照標簽的規定配制，將相當于1g固體的液體轉移至一個無菌的500mL錐形燒瓶，溶解于200mL液體A中，并混勻。按照關于水性溶液的規定，繼續操作。

ANTIBIOTIC SOLIDS, BULKS, and BLENDS 抗生素固體、散裝品、混合品

Aseptically remove a sufficient quantity of solids from the appropriate amount of containers (see Table 2), mix to obtain a composite, equivalent to about 6 g of solids, and transfer to a sterile 500-mL conical flask. Dissolve in about 200 mL of Fluid A, and mix. Proceed as directed for Aqueous Solutions.

以無菌操作從適當數量的容器中（見表2）取出足夠數量的固體，混勻以獲得等同于6g固體的混合物，轉移至一個無菌的500mL錐形燒瓶。溶解于約200mL液體A中，并混勻。按照關于水性溶液的規定，繼續進行。

STERILE AEROSOL PRODUCTS 無菌氣(噴)霧劑產品

For fluid products in pressurized aerosol form, freeze the containers in an alcohol-dry ice mixture at least at –20 for about 1 hour. If feasible, allow the propellant to escape before aseptically opening the container, and transfer the contents to a sterile pooling vessel. Add 100 mL of Fluid D to the pooling vessel, and mix gently. Proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies.

對于以加壓氣(噴)霧劑形式存在的液體產品，在大約–20 的酒精－干冰混合物中冷凍容器約1小時。如果可行，在以無菌操作打開容器之前使推進劑散發掉，并將內容物轉移至一個無菌的合并容器。加入100mL液體D至該合并容器，并輕輕混勻。按照適合的關于水性溶液或油和油性溶液的規定，繼續進行。

DEVICES WITH PATHWAYS LABELED STERILE 具有導管的醫療器具供試品

Aseptically pass not less than 10 pathway volumes of Fluid D through each device tested. Collect the fluids in an appropriate sterile vessel, and proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies.

以無菌操作用10個通道體積的液體D通過供試設備。在適當的無菌容器中收集沖洗液，并按照適合的關于水性溶液或油和油性溶液的規定，繼續進行。

In the case of sterile, empty syringes, draw sterile diluent into the barrel through the sterile needle, if attached, or th, rough a sterile needle attached for the purpose of the test, and express the contents into a sterile pooling vessel. Proceed as directed above.

對于無菌的空注射器，如果附有無菌針頭，通過其將無菌稀釋劑吸取至管中，或者使用一個專為此試驗準備的無菌針頭，并將內容物壓出至合并容器中。按照上述內容繼續進行。

Direct Inoculation of the Culture Medium 培養基的直接接種

Transfer the quantity of the preparation to be examined prescribed in Tables 2 and 3 directly into the culture medium so that the volume of the product is not more than 10% of the volume of the medium, unless otherwise prescribed.

If the product to be examined has antimicrobial activity, carry out the test after neutralizing this with a suitable neutralizing substance or by dilution in a sufficient quantity of culture medium. When it is necessary to use a large volume of the product, it may be preferable to use a concentrated culture medium prepared in such a way that it takes into account the subsequent dilution. Where appropriate, the concentrated medium may be added directly to the product in its container.

按照表2和3規定的供試配制品的數量，將該配制品直接轉移到培養基中，除非另有規定，產品體積不得超過該培養基體積的10%。如果該供試產品具有抗微生物活性，通過使用適當的中和物質或在充足數量的培養基中稀釋以中和其抗菌活性之后，進行該試驗。當必須使用大量產品時，最好使用在配制時考慮了后續的稀釋需要的濃縮培養基。在適當時，該濃縮培養基可以直接加入到放在其容器中的產品。

OILY LIQUIDS 油性液體

Use media to which have been added a suitable emulsifying agent at a concentration shown to be appropriate in the validation of the test, for example polysorbate 80 at a concentration of 10 g per L.

使用已經加入了適當乳化劑的培養基，乳化劑濃度需已經證實適于該試驗的驗證，例如濃度為10克每升的聚山梨酯80。

OINTMENTS and CREAMS 油膏和乳膏

Prepare by diluting to about 1 in 10 by emulsifying with the chosen emulsifying agent in a suitable sterile diluent such as Fluid A (see Diluting and Rinsing Fluids for Membrane Filtration). Transfer the diluted product to a medium not containing an emulsifying agent.

通過將選定的乳化劑在適當的無菌稀釋液中乳化，例如液體A（見用于膜過濾的稀釋和沖洗液），稀釋至約1比10，來配制該產品。轉移稀釋后的產品至不含乳化劑的一個培養基中。

Incubate the inoculated media for not less than 14 days. Observe the cultures several times during the incubation period. Shake cultures containing oily products gently each day. However, when thioglycollate medium or other similar medium is used for the detection of anaerobic microorganisms, keep shaking or mixing to a minimum in order to maintain anaerobic conditions.

將接種后的培養基培養不少于14天。在培養期中觀察培養基若干次。每天輕輕搖動含有油性產品的培養基一次。但是，當使用巰基醋酸鹽培養基或其他相似培養基檢測厭氧微生物時，將搖動或混合保持到最少，以維持厭氧條件。

CATGUT and OTHER SURGICAL SUTURES FOR VETERINARIAN USE

獸醫用腸線和其他外科縫合線

Use for each medium not less than the quantities of the product prescribed in Tables 2 and 3. Open the sealed package using aseptic precautions, and remove three sections of the strand for each culture medium. Carry out the test on three sections, each 30-cm long, which have been cut off from the beginning, the center, and the end of the strand. Use whole strands from freshly opened cassette packs. Transfer each section of the strand to the selected medium. Use sufficient medium to cover adequately the material to be tested (20 mL to 150 mL).

在每個培養基中，使用不少于表2和3中所規定數量的產品。采取無菌預防措施，打開封閉的包裝，并取該股線的3個部分，分別至每個培養基。使用三節，每節30cm長，并分別從該股線的前端、中間、末端截取的產品，進行該試驗。使用從剛剛打開的包裝盒中取出的整股線。將該股線的每個部分轉移至選定的培養基。使用充足的培養基，以充分覆蓋該供試物料（20mL至150mL）

SOLIDS 固體

Transfer a quantity of the product in the form of a dry solid (or prepare a suspension of the product by adding sterile diluent to the immediate container), corresponding to not less than the quantity indicated in Tables 2 and 3. Transfer the material so obtained to 200 mL of Fluid Thioglycollate Medium, and mix. Similarly, transfer the same quantity to 200 mL of Soybean–Casein Digest Medium, and mix. Proceed as directed above.

轉移干燥固體形態的產品（或通過加入無菌稀釋劑至中間容器中配制該產品的懸浮液），數量不少于表2和3中的規定。轉移如此獲得的物料至200mL巰基醋酸鹽液體培養基中，并混勻。同法轉移同樣數量的物料至200mL大豆－酪蛋白消化物培養基，并混勻。按照上述規定繼續進行。

PURIFIED COTTON, GAUZE, SURGICAL DRESSINGS, and RELATED ARTICLES

脫脂棉花、紗布、外科敷料、相關物品

From each package of cotton, rolled gauze bandage, or large surgical dressings being tested, aseptically remove two or more portions of 100- to 500-mg each from the innermost part of the sample. From individually packaged, single-use materials, aseptically remove the entire article. Immerse the portions or article in each medium, and proceed as directed above.

對于待檢的每個包裝中的棉花、卷狀紗布繃帶、大塊外科敷料，以無菌操作從該樣品最核心的部位，取出2個或更多部分，每個部分100-500mg。從單個包裝、一次性使用的物料中，以無菌操作取出整個物品。將這些部分或單個物品浸沒在每個培養基中，并繼續按照上述內容操作。

STERILE DEVICES 無菌設備

Articles can be immersed intact or disassembled. To ensure that device pathways are also in contact with the media, immerse the appropriate number of units per medium in a volume of medium sufficient to immerse the device completely, and proceed as directed above. For extremely large devices, immerse those portions of the device that are to come into contact with the patient in a volume of medium sufficient to achieve complete immersion of those portions.

若干物品可以完整地或在拆開后浸沒在培養基中。為確保設備通道與培養基接觸，使用體積足夠浸沒整個設備的培養基，在每個培養基中浸入適當數量的部件，并按照上述內容繼續操作。對于極大的設備，將該設備中將要與患者接觸的那些部分，浸入到體積足夠完全浸沒這些部分的培養基中。

For catheters where the inside lumen and outside are required to be sterile, either cut them into pieces such that the medium is in contact with the entire lumen or fill the lumen with medium, and then immerse the intact unit.

對于內部的腔體和外部均要求無菌的導尿管，將它們切成片，這樣培養基就可以接觸整個腔體，或者用培養基填充腔體，然后浸沒整個設備。

OBSERVATION AND INTERPRETATION OF RESULTS 結果的觀測和理解

At intervals during the incubation period and at its conclusion, examine the media for macroscopic evidence of microbial growth. If the material being tested renders the medium turbid so that the presence or absence of microbial growth cannot be readily determined by visual examination, 14 days after the beginning of incubation transfer portions (each not less than 1 mL) of the medium to fresh vessels of the same medium, and then incubate the original and transfer vessels for not less than 4 days.

在培養期中間的觀測點和作結論時，檢查該培養基是否有肉眼可見的微生物生長的證據。如果供試物料導致培養基混濁，從而使得無法通過肉眼觀察來確定是否存在微生物生長，在開始培養14天之后，將該培養基的若干部分（每個部分不少于1mL）轉移至裝有相同培養基的新鮮容器中，然后將原有和轉移的容器培養不少于4天。

If no evidence of microbial growth is found, the product to be examined complies with the test for sterility. If evidence of microbial growth is found, the product to be examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product to be examined. The test may be considered invalid only if one or more of the following conditions are fulfilled:

如果沒有找到微生物生長的證據，則該供試產品符合無菌檢查。如果找到了微生物生長的證據，則該供試產品不符合無菌檢查，除非能夠清楚地證實此次試驗無效且無效原因與該供試產品無關。該試驗僅可能在下面一個或多個條件被滿足的情況下，才有可能認為無效：

a. The data of the microbiological monitoring of the sterility testing facility show a fault.該無菌試驗設施的微生物監控數據顯示有缺陷。

b. A review of the testing procedure used during the test in question reveals a fault.對該試驗過程中存有疑問的試驗步驟進行審核后揭示出缺陷。

c. Microbial growth is found in the negative controls.在陰性對照中發現微生物生長。

d. After determination of the identity of the microorganisms isolated from the test, the growth of this species (or these species) may be ascribed unequivocally to faults with respect to the material and or the technique used in conducting the sterility test procedure.在從試驗中分離出的微生物確定之后，此種（或這些種）微生物的生長可以毫不含糊地歸咎于與物料和/或者進行無菌試驗過程中所使用的方法。

If the test is declared to be invalid, it is repeated with the same number of units as in the original test. If no evidence of microbial growth is found in the repeat test, the product examined complies with the test for sterility. If microbial growth is found in the repeat test, the product examined does not comply with the test for sterility.

如果該試驗證明無效，應用與原試驗同樣數量的產品進行復檢。如果在復檢中未發現微生物生長的證據，則該供試產品符合無菌試驗的要求。如果在復檢中發現了微生物生長，則該產品不符合無菌試驗。

APPLICATION OF THE TEST TO PARENTERAL PREPARATIONS, OPHTHALMIC, AND OTHER NONINJECTABLE PREPARATIONS REQUIRED TO COMPLY WITH THE TEST FOR STERILITY

此試驗在注射藥品、眼科和其他必須符合無菌試驗的非注射藥品中的應用

When using the technique of membrane filtration, use, whenever possible, the whole contents of the container, but not less than the quantities indicated in Tables 2 and 3, diluting where necessary to about 100 mL with a suitable sterile solution, such as Fluid A (see Diluting and Rinsing Fluids for Membrane Filtration).

當使用膜過濾法時，只要可能，就要使用該容器的全部內容物，但不少于表2和3中規定的數量，必需時以適當的無菌溶液將其稀釋至約100mL，例如液體A（見用于膜過濾的稀釋和沖洗液）。

When using the technique of direct inoculation of media, use the quantities shown in Tables 2 and 3, unless otherwise justified and authorized. The tests for bacterial and fungal sterility are carried out on the same sample of the product to be examined. When the volume or the quantity in a single container is insufficient to carry out the tests, the contents of two or more containers are used to inoculate the different media.

當使用培養基直接接種法時，除非另有證據或授權，使用表2和3中顯示的數量。用于檢驗細菌和霉菌的試驗使用供試產品的同一個樣品。當單一容器中的產品體積或數量不足以進行該試驗時，適用2個或更多容器的內容物來接種不同的培養基。

¹  In appropriate cases, periodic testing of the different batches prepared from the same lot of dehydrated medium is acceptable.

在適當的情況下，用同一個批次的脫水培養基配制的不同批次的定期試驗是可以接受的。

Auxiliary Information— Staff Liaison : Radhakrishna S Tirumalai, Scientist

Expert Committee : (MSA05) Microbiology and Sterility Assurance

USP30–NF25 Page 97

Phone Number : 1-301-816-8339