<71> STERILITY TESTS

1 USP 35 Microbiological TESTS / 71 STERILITY Tests69 The test is applied to substances, preparations, or articlesMETHOD which, according to the Pharmacopeia, are required to be ster-ile. However, a satisfactory result only indicates that no the indicator cell culture at a suitable densitytaminating microorganism has been found in the sample ex-(for example, 2 104 to 2 105 cells/mL, 4 103 toamined under the conditions of the 104 cells/cm2) that will yield confluence after 3days of growth. Inoculate 1 mL of the product to beexamined into the cell culture vessel, and incubate atPRECAUTIONS AGAINST MICROBIAL36 1 . at least 3 days of incubation, when the cellshave grown to confluence, make a subculture onThe test for STERILITY is carried out under aseptic condi-cover slips in suitable containers or on some othertions.

2 In order to achieve such conditions, the test environ-surface (for example, chambered slides) suitable forment has to be adapted to the way in which the sterilitythe test procedure. Seed the cells at low density sotest is performed. The precautions taken to avoid contami-that they reach 50% confluence after 3 5 days of in-nation are such that they do not affect any microorganismscubation. Complete confluence impairs visualizationthat are to be revealed in the test. The working conditionsof Mycoplasmas after staining and must be which the TESTS are performed are monitored regularly the medium and rinse the indicator cells withappropriate sampling of the working area and by carryingphosphate buffered saline, pH , then add a suita-out appropriate fixing solution (a freshly prepared mixture of 1volume of acetic acid, glacial, TS and 3 volumes ofmethanol, is suitable when bisbenzimide is used forCULTURE MEDIA AND INCUBATION staining).

3 The fixing solution and wash the cells withsterile Purified Water. Dry the slides completely if theyMedia for the test may be prepared as described below orare to be stained more than 1 hour later (particularequivalent commercial media may be used provided thatcare is needed for staining of slides after drying ow-they comply with the requirements of the Growth Promotioning to artifacts that may be produced).Test of Aerobes, Anaerobes, and a suitable DNA stain and allow standing for aThe following culture media have been found to be suita-suitable time (bisbenzimide working solution and able for the test for STERILITY . Fluid thioglycollate Medium isstanding time of 10 minutes are suitable).

4 Primarily intended for the culture of anaerobic the stain and rinse the monolayer with Puri-However, it will also detect aerobic bacteria. Soybean Caseinfied Medium is suitable for the culture of both fungi each coverslip, where applicable (a mixture ofaerobic volumes of glycerol and Phosphate-Citrate BufferSolution pH is suitable for mounting). Examine byfluorescence (for bisbenzimide stain a 330 nm/380 Fluid thioglycollate Mediumnm excitation filter and an LP 440 nm barrier gare suitable) at 400 magnification or the microscopic appearance of the test cul-tures with that of the negative and positive controls,Dextrose gexamining for extranuclear fluorescence.

5 Gmas produce pinpoints or filaments over the indicatorYeast Extract (water-soluble) gcell cytoplasm. They may also produce pinpoints andPancreatic Digest of gfilaments in the intercellular spaces. Multiple micro-Sodium gscopic fields are examined according to the protocolor Thioglycolic mLestablished during Sodium Solution (1 in 1000),freshly mLInterpretation of ResultsPurified Water1000 mLThe product to be examined complies with the test ifpH after sterilization: typical of Mycoplasmas is not present. The testMix the L-cystine, agar, sodium chloride, dextrose, yeastis invalid if the positive controls do not show fluorescenceextract, and pancreatic digest of casein with the purifiedtypical of Mycoplasmas.

6 The test is invalid if the negativewater, and heat until solution is effected. Dissolve the so-controls show fluorescence typical of thioglycollate or thioglycolic acid in the solution and,if necessary, add 1N sodium hydroxide so that, after sterili-zation, the solution will have a pH of If filtration isnecessary, heat the solution again without boiling, and filterwhile hot through moistened filter paper. Add the resazurinsodium solution, mix, and place the medium in suitable ves-sels that provide a ratio of surface to depth of medium suchthat not more than the upper half of the medium has un- 71 STERILITYTESTS dergone a color change indicative of oxygen uptake at theend of the incubation period.

7 Sterilize using a validated pro-cess. If the medium is stored, store at a temperature be-FPortions of this general chapter have been harmonizedtween 2 and 25 in a sterile, airtight container. If morewith the corresponding texts of the European Pharmacopeiathan the upper one-third of the medium has acquired aand/or the Japanese Pharmacopeia. Those portions that arepink color, the medium may be restored once by heatingnot harmonized are marked with symbols (FF) to specify thisthe containers in a water-bath or in free-flowing steam pink color disappears and by cooling quickly, takingThese Pharmacopeial procedures are not by themselvescare to prevent the introduction of nonsterile air into thedesigned to ensure that a batch of product is sterile or hascontainer.

8 Do not use the medium for a longer storage pe-been sterilized. This is accomplished primarily by validationriod than has been the sterilization process or of the aseptic from May 1, 2012 Copyright (c) 2011 The United States Pharmacopeial Convention. All rights from by nEwp0rt1 on Thu Dec 01 21:55:30 EST 201170 71 STERILITY TESTS / Microbiological TestsUSP 35 Fluid thioglycollate Medium is to be incubated at 30 35 .Table 1. Strains of the Test Microorganisms Suitable for Use inFor products containing a mercurial preservative that cannotthe Growth Promotion Test and the Method Suitabilitybe tested by the membrane filtration method, Fluid Thiog-Test (Continued)lycollate Medium incubated at 20 25 may be used insteadBacillus subtilisATCC 6633, CIP ,of Soybean Casein Digest Medium provided that it has beenNCIMB 8054, NBRC validated as described in Growth Promotion Test of Aerobes,3134 Anaerobes, and Fungi.

9 Where prescribed or justified and au-Pseudomonas aeruginosaF1 FATCC 9027, NCIMB thorized, the following alternative thioglycollate medium8626, CIP , NBRC might be used. Prepare a mixture having the same composi-13275tion as that of the Fluid thioglycollate Medium, but omittingAnaerobic bacteriumthe agar and the resazurin sodium solution. Sterilize as di-rected above. The pH after sterilization is Heat inClostridium sporogenesF2 FATCC 19404, CIP ,a water bath prior to use and incubate at 30 35 underNCTC 532 or ATCC anaerobic , NBRC 14293 FungiSoybean Casein Digest MediumCandida albicansATCC 10231, IP ,NCPF 3179, NBRC 1594 Pancreatic Digest of gAspergillus brasiliensisATCC 16404, IP ,Papaic Digest of Soybean g(Aspergillus Niger)IMI 149007, NBRC 9455 Sodium gF1An alternative microorganism is Kocuria rhizophila (MicrococcusDibasic Potassium gluteus)

10 ATCC gF2An alternative to Clostridium sporogenes, when a nonspore-formingPurified Water1000 mLmicroorganism is desired, is Bacteroides vulgatus (ATCC 8482).FThe media used comply with the following TESTS , carriedpH after sterilization: before, or in parallel, with the test on the product to beDissolve the solids in the Purified Water, heating effect a solution. Cool the solution to room temperature,and adjust the pH with 1N sodium hydroxide so that, aftersterilization, it will have a pH of Filter, if necessarySterilityto clarify, dispense into suitable containers, and sterilize us-ing a validated procedure. Store at a temperature betweenIncubate portions of the media for 14 days.