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.

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.

The test is applied to substances, preparations, or articles which, according to the Pharmacopeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating microorganism has been found in the sample examined under the conditions of the test.

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.

Media for the test may be prepared as described below or equivalent commercial media may be used provided that they comply with the requirements of the Growth Promotion Test of Aerobes, Anaerobes, and Fungi.

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.

pH after sterilization: 7.1±0.2.

Mix the l-cystine, agar, 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. Do not use the medium for a longer storage period than has been validated.

Fluid Thioglycollate Medium is to be incubated at 30–35. For products containing a mercurial preservative that cannot be tested by the membrane filtration method, Fluid Thioglycollate Medium incubated at 20–25 may be used instead of Soybean–Casein Digest Medium provided that it has been validated as described in Growth Promotion Test of Aerobes, Anaerobes, and Fungi. Where prescribed or justified and authorized, the following alternative thioglycollate medium might be used. 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. The pH after sterilization is 7.1 ± 0.2. Heat in a water bath prior to use and incubate at 30–35 under anaerobic conditions.

pH after sterilization: 7.3±0.2.

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. Do not use the medium for a longer storage period than has been validated.

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

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

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.

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.

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:

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.

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

When using the technique of direct inoculation of media, use the quantities shown in Table 2, 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.

The minimum number of items to be tested in relation to the size of the batch is given in Table 3.