2.6. BIOLOGICAL TESTS - uspbpep.com

1 EUROPEAN PHARMACOPOEIA BIOLOGICAL TESTS01/2008 STERILITYThe test is applied to substances, preparations or articleswhich, according to the Pharmacopoeia, are required to besterile. However, a satisfactory result only indicates thatno contaminating micro-organism has been found in thesample examined in the conditions of the test. Guidancefor using the test for sterility is given at the end of this AGAINST MICROBIAL CONTAMINATIONThe test for sterility is carried out under aseptic order to achieve such conditions, the test environmenthas to be adapted to the way in which the sterility test isperformed.

2 The precautions taken to avoid contaminationare such that they do not affect any micro-organisms whichare to be revealed in the test. The working conditions inwhich the TESTS are performed are monitored regularly byappropriate sampling of the working area and by carryingout appropriate controls (such as those indicated in theappropriate European Community Directives and associatedguidance documents on GMP).CULTURE MEDIA AND INCUBATION TEMPERATURESM edia for the test may be prepared as described below, orequivalent commercial media may be used provided thatthey comply with the growth promotion the test for sterility.

3 Fluid thioglycollate medium isprimarily intended for the culture of anaerobic bacteria;however, it will also detect aerobic bacteria. Soya-beancasein digest medium is suitable for the culture of both fungiand aerobic media may be used provided that they pass the growthpromotion and the validation thioglycollate gAgar, granulated (moisture content not inexcess of 15 per cent) gSodium gGlucose gYeast extract (water-soluble) gPancreatic digest of gSodium thioglycollate gThioglycollic mlResazurin sodium solution (1 g/l ofresazurin sodium), freshly mlWater R1000 mlpH of the medium after sterilisation theL-cystine, agar, sodium chloride, glucose,water-soluble yeast extract and pancreatic digest of caseinwith thewater Rand heat until solution is effected.

4 Dissolvethe sodium thioglycollate or thioglycollic acid in the solutionand, if necessary, add1 M sodium hydroxideso that, aftersterilisation, the solution will have a pH of Iffiltration is necessary, heat the solution again without boilingand filter while hot through moistened filter paper. Add theresazurin sodium solution, mix and place the medium insuitable vessels which provide a ratio of surface to depthof medium such that not more than the upper half of themedium has undergone a colour change indicative of oxygenuptake at the end of the incubation period. Sterilise using avalidated process.

5 If the medium is stored, store at 2-25 Cin a sterile, airtight container. If more than the upper thirdof the medium has acquired a pink colour, the medium maybe restored once by heating the containers in a water-bathor in free-flowing steam until the pink colour disappears andcooling quickly, taking care to prevent the introduction ofnon-sterile air into the container. Do not use the medium fora longer storage period than has been thioglycollate medium is to be incubated at 30-35 casein digest mediumPancreatic digest of gPapaic digest of soya-bean gSodium gDipotassium hydrogen gGlucose gWater R1000 mlpH of the medium after sterilisation the solids inwater R,warmingslightlytoeffectsolution.

6 Cool the solution to room temperature. Add1 Msodium hydroxide, if necessary, so that after sterilisationthe medium will have a pH of Filter, if necessary,to clarify, distribute into suitable vessels and sterilise usinga validated process. Store at 2-25 C in a sterile well-closedcontainer, unless it is intended for immediate use. Do notuse the medium for a longer storage period than has casein digest medium is to be incubated at20-25 media used comply with the following TESTS , carriedout before or in parallel with the test on the product to Incubate portions of the media for 14 days.

7 Nogrowth of micro-organisms promotion test of aerobes, anaerobes and each batch of ready-prepared medium and each batchof medium prepared either from dehydrated medium orfrom the ingredients. Suitable strains of micro-organismsare indicated in Table portions of fluid thioglycollate medium with asmall number (not more than 100 CFU) of the followingmicro-organisms, using a separate portion of medium foreach of the following species of micro-organism:Clostridiumsporogenes,Pse udomonas aeruginosa,Staphylococcusaureus. Inoculate portions of soya-bean casein digestmedium with a small number (not more than 100 CFU) ofthe following micro-organisms, using a separate portion ofmedium for each of the following species of micro-organism.

8 Aspergillus niger,Bacillus subtilis,Candida for not more than 3 days in the case of bacteria lot culture maintenance techniques (seed-lot systems)are used so that the viable micro-organisms used forinoculation are not more than 5 passages removed from theoriginal master (1) SterilityEUROPEAN PHARMACOPOEIA TESTC arry out a test as described below under Test for sterility ofthe product to be examined using exactly the same methodsexcept for the following filtration. After transferring the contents of thecontainerorcontainerstobetestedtothem embraneaddaninoculum of a small number of viable micro-organisms (notmore than 100 CFU) to the final portion of sterile diluentused to rinse the inoculation.

9 After transferring the contents of thecontainer or containers to be tested (for catgut and othersurgical sutures for veterinary use: strands) to the culturemedium add an inoculum of a small number of viablemicro-organisms (not more than 100 CFU) to the both cases use the same micro-organisms as thosedescribed above under Growth promotion test of aerobes,anaerobes and fungi. Perform a growth promotion test asa positive control. Incubate all the containers containingmedium for not more than 5 clearly visible growth of micro-organisms is obtainedafter the incubation, visually comparable to that in thecontrol vessel without product, either the product possessesno antimicrobial activity under the conditions of the testor such activity has been satisfactorily eliminated.

10 Thetest for sterility may then be carried out without clearly visible growth is not obtained in the presence ofthe product to be tested, visually comparable to that inthe control vessels without product, the product possessesantimicrobial activity that has not been satisfactorilyeliminated under the conditions of the test. Modify theconditions in order to eliminate the antimicrobial activityand repeat the validation validation is performed:a) when the test for sterility has to be carried out on a newproduct,b) whenever there is a change in the experimental conditionsof the validation may be performed simultaneously with thetest for sterility of the product to be FOR STERILITY OF THE PRODUCT TO BEEXAMINEDThe test may be carried out using the technique of membranefiltration or by direct inoculation of the culture media withthe product to be examined.