Culture Media and Incubation

The test is applied to substances, preparations, or articles METHOD which, according to the Pharmacopeia, are required to be sterile. However, a satisfactory result only indicates that no con1. Seed the indicator cell culture at a suitable density taminating microorganism has been found in the sample ex(for example, 2 × 104 to 2 × 105 cells/mL, 4 × 103 to amined under the conditions of the test. 2.5 × 104 cells/cm2) that will yield confluence after 3 days of growth. Inoculate 1 mL of the product to be examined into the cell culture vessel, and incubate at PRECAUTIONS AGAINST MICROBIAL 36 ± 1°. CONTAMINATION 2. After at least 3 days of incubation, when the cells have grown to confluence, make a subculture on The test for sterility is carried out under aseptic condicover slips in suitable containers or on some other tions. In order to achieve such conditions, the test environsurface (for example, chambered slides) suitable for ment has to be adapted to the way in which the sterility the test procedure. Seed the cells at low density so test is performed. The precautions taken to avoid contamithat they reach 50% confluence after 3–5 days of innation are such that they do not affect any microorganisms cubation. Complete confluence impairs visualization that are to be revealed in the test. The working conditions of Mycoplasmas after staining and must be avoided. in which the tests are performed are monitored regularly by 3. Remove the medium and rinse the indicator cells with appropriate sampling of the working area and by carrying phosphate buffered saline, pH 7.4, then add a suitaout appropriate controls. ble fixing solution (a freshly prepared mixture of 1 volume of acetic acid, glacial, TS and 3 volumes of methanol, is suitable when bisbenzimide is used for CULTURE MEDIA AND INCUBATION staining). TEMPERATURES 4. Remove the fixing solution and wash the cells with sterile Purified Water. Dry the slides completely if they Media for the test may be prepared as described below or are to be stained more than 1 hour later (particular equivalent commercial media may be used provided that care is needed for staining of slides after drying owthey comply with the requirements of the Growth Promotion ing to artifacts that may be produced). Test of Aerobes, Anaerobes, and Fungi. 5. Add a suitable DNA stain and allow standing for a The following culture media have been found to be suitasuitable time (bisbenzimide working solution and a ble for the test for sterility. Fluid Thioglycollate Medium is standing time of 10 minutes are suitable). primarily intended for the culture of anaerobic bacteria. 6. Remove the stain and rinse the monolayer with PuriHowever, it will also detect aerobic bacteria. Soybean–Casein fied Water. Digest Medium is suitable for the culture of both fungi and 7. Mount each coverslip, where applicable (a mixture of aerobic bacteria. equal volumes of glycerol and Phosphate-Citrate Buffer Solution pH 5.5 is suitable for mounting). Examine by fluorescence (for bisbenzimide stain a 330 nm/380 Fluid Thioglycollate Medium nm excitation filter and an LP 440 nm barrier filter L-Cystine 0.5 g are suitable) at 400× magnification or greater. Sodium Chloride 2.5 g 8. Compare the microscopic appearance of the test cultures with that of the negative and positive controls, Dextrose Monohydrate/Anhydrous 5.5/5.0 g examining for extranuclear fluorescence. MycoplasAgar 0.75 g mas produce pinpoints or filaments over the indicator Yeast Extract (water-soluble) 5.0 g cell cytoplasm. They may also produce pinpoints and Pancreatic Digest of Casein 15.0 g filaments in the intercellular spaces. Multiple microSodium Thioglycollate 0.5 g scopic fields are examined according to the protocol or Thioglycolic Acid 0.3 mL established during validation.