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1、detecting Mycoplasmas. Test the effectiveness of the cells to be used by applying the procedure shown below and inocu- lating not more than 100 cfu or ccu microorganisms of suit- able reference strains of M. hyorhinis and M. orale. The cells are suitable if both reference strains are detected. The i
2、ndi- cator cells must be subcultured without an antibiotic before use in the test. Test Method NOTEThe following is provided for information. SOLUTIONS Phosphate Buffered Saline 2.0 M Monobasic Potassium PhosphateDissolve 13.61 g of anhydrous monobasic potassium phosphate in 50 mL of water. 2.0 M Di
3、basic Potassium PhosphateDissolve 17.42 g of anhydrous dibasic potassium phosphate in 50 mL of water. Phosphate Buffered Saline Solution (pH 7.4)Combine 3.6 mL of 2.0 M Monobasic Potassium Phosphate, 16.4 mL of 2.0 M Dibasic Potassium Phosphate, 8 g of sodium chloride, and 1 L of water. Mix thorough
4、ly. Adjust the pH if necessary. Bisbenzimide Stock SolutionDissolve 5 mg of bisben- zimide in water, and dilute with the same solvent to 100 mL. Store in the dark. Bisbenzimide Working SolutionImmediately before use, dilute 100 mL of Bisbenzimide Stock Solution with Phos- phate Buffered Saline Solut
5、ion (pH 7.4) to 100 mL. Phosphate-Citrate Buffer Solution pH 5.5Mix 56.85 mL of a 28.4-g/L solution of anhydrous disodium hydrogen phosphate and 43.15 mL of a 21-g/L solution of citric acid. METHOD 1. Seed the indicator cell culture at a suitable density (for example, 2 104 to 2 105 cells/mL, 4 103
6、to 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 36 1. 2. After at least 3 days of incubation, when the cells have grown to confluence, make a subculture on cover slips in suitable cont
7、ainers or on some other surface (for ex- ample, chambered slides) suitable for the test proce- dure. Seed the cells at low density so that they reach 50% confluence after 35 days of incubation. Com- plete confluence impairs visualization of Mycoplasmas after staining and must be avoided. 3. Remove t
8、he medium and rinse the indicator cells with phosphate buffered saline, pH 7.4, then add a suitable 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 staining). 4. Remove the fixing solution and wa
9、sh the cells with ster- ile Purified Water. Dry the slides completely if they are to be stained more than 1 hour later (particular care is needed for staining of slides after drying owing to arti- facts that may be produced). 5. Add a suitable DNA stain and allow standing for a suit- able time (bisb
10、enzimide working solution and a stand- ing time of 10 minutes are suitable). 6. Remove the stain and rinse the monolayer with Purified Water. 7. Mount each coverslip, where applicable (a mixture of equal volumes of glycerol and Phosphate-Citrate Buffer Solution pH 5.5 is suitable for mounting). Exam
11、ine by fluorescence (for bisbenzimide stain a 330 nm/380 nm excitation filter and an LP 440 nm barrier filter are suit- able) at 400 magnification or greater. 8. Compare the microscopic appearance of the test cul- tures with that of the negative and positive controls, examining for extranuclear fluo
12、rescence. Mycoplasmas produce pinpoints or filaments over the indicator cell cytoplasm. They may also produce pinpoints and fila- ments in the intercellular spaces. Multiple microscopic fields are examined according to the protocol establish- ed during validation. Interpretation of Results The produ
13、ct to be examined complies with the test if flu- orescence typical of Mycoplasmas is not present. The test is invalid if the positive controls do not show fluorescence typ- ical of Mycoplasmas. The test is invalid if the negative con- trols show fluorescence typical of Mycoplasmas. 71 STERILITYTESTS
14、 uPortions 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 (uu) to specify this fact.u These Pharmacopeial procedures are not by themselves designed
15、 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 pro- cedures. The test is applied to substances, preparations, or articles which, according to the Pharmacopeia, are required t
16、o be ster- ile. However, a satisfactory result only indicates that no con- taminating microorganism has been found in the sample ex- amined under the conditions of the test. PRECAUTIONS AGAINST MICROBIAL CONTAMINATION The test for sterility is carried out under aseptic condi- tions. In order to achieve such conditions, the test environ- ment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they
