<62> Microbiological Examination Of Nonsterile Products ...

1 USP 31 Microbiological Tests / 62 Microbiological Examination1 Staphylococcus aureussuch as ATCC 6538, NCIMB 62 MICROBIOLOGICAL9518, CIP , or NBRC13276 Examination OF NONSTERILEP seudomonas aeruginosasuch as ATCC 9027, NCIMB8626, CIP , or NBRCPRODUCTS:TESTS FOR13275 Escherichia colisuch as ATCC 8739, NCIMBSPECIFIED MICROORGANISMS8545, CIP , or NBRC3972 Salmonella enterica ssp. enter-such as ATCC 14028ica serotype typhimuriumor, as an alternative,INTRODUCTIONS almonella enterica ssp. enter-such as NBRC 100797, NCTCThe tests described hereafter will allow determination of the ab-ica serotype abony6017, or CIP of, or limited occurrence of, specified microorganisms thatCandida albicanssuch as ATCC 10231, NCPFmay be detected under the conditions , IP , or NBRCThe tests are designed primarily to determine whether a sub-1594stance or preparation complies with an established specification formicrobiological quality.

2 When used for such purposes, follow theUse Buffered Sodium Chloride Peptone Solution pH orinstructions given below, including the number of samples to bePhosphate Buffer Solution pH to make test suspensions. Use thetaken, and interpret the results as stated within 2 hours or within 24 hours if stored at 2 to 8 .Alternative Microbiological procedures, including automatedmethods, may be used, provided that their equivalence to thePharmacopeial method has been Clostridium sporogenes such as ATCC 11437 (NBRCGENERAL PROCEDURES14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532or CIP ).

3 Grow the clostridial test strain under anaerobic condi-The preparation of samples is carried out as described in Micro-tions in Reinforced Medium for Clostridia at 30 to 35 for 24 to 48biological Examination of Nonsterile Products : Microbial Enumer-hours. As an alternative to preparing and then diluting down a freshation Tests 61 .suspension of vegetative cells of Cl. sporogenes, a stable spore sus-If the product to be examined has antimicrobial activity, this ispension is used for test inoculation. The stable spore suspensioninsofar as possible removed or neutralized as described in Microbi-may be maintained at 2 to 8 for a validated Examination of Nonsterile Products : Microbial Enumera-tion Tests 61.

4 If surface-active substances are used for sample preparation, theirNegative Controlabsence of toxicity for microorganisms and their compatibility withany inactivators used must be demonstrated as described in Micro-To verify testing conditions, a negative control is performed us-biological Examination of Nonsterile Products : Microbial Enumer-ing the chosen diluent in place of the test preparation. There mustation Tests 61 .be no growth of AND INHIBITORYG rowth Promotion and Inhibitory PropertiesPROPERTIES OF THE MEDIA ANDof the MediaSUITABILITY OF THE TESTTest each batch of ready-prepared medium and each batch of me-dium prepared either from dehydrated medium or from ability of the test to detect microorganisms in the presence ofVerify suitable properties of relevant media as described in Table product to be tested must be established.

5 Suitability must beconfirmed if a change in testing performance or a change in theTest for Growth-Promoting Properties, Liquid Media product that may affect the outcome of the test is a portion of the appropriate medium with a small number(not more than 100cfu) of the appropriate microorganism. Incubateat the specified temperature for not more than the shortest period ofPreparation of Test Strainstime specified in the test. Clearly visible growth of the microorga-nism comparable to that previously obtained with a previouslyUse standardized stable suspensions of test strains as stated be-tested and approved batch of medium Seed-lot culture maintenance techniques (seed-lot systems) are Test for Growth-Promoting Properties, Solid Media Per-used so that the viable microorganisms used for inoculation are notform Surface-Spread Method (see Plate-Count Methods under Mi-more than five passages removed from the original master Examination of Nonsterile Products .)

6 Microbial Enu-meration Tests 61 ), inoculating each plate with a small number(not more than 100cfu) of the appropriate microorganism. IncubateAEROBIC MICROORGANISMSat the specified temperature for not more than the shortest period oftime specified in the test. Growth of the microorganism comparableGrow each of the bacterial test strains separately in containersto that previously obtained with a previously tested and approvedcontaining Soybean Casein Digest Broth or on Soybean Caseinbatch of medium Agar at 30 to 35 for 18 to 24 hours. Grow the test strainfor Candida albicans separately on Sabouraud Dextrose Agar or inTest for Inhibitory Properties, Liquid or Solid Media Inoc-Sabouraud Dextrose Broth at 20 to 25 for 2 to 3 the appropriate medium with at least 100cfu of the appropri-ate microorganism.

7 Incubate at the specified temperature for notless than the longest period of time specified in the test. No growthof the test microorganism for Indicative Properties Perform Surface-SpreadMethod (see Plate-Count Methods under Microbiological Examina-tion of Nonsterile Products : Microbial Enumeration Tests 61 ), in-oculating each plate with a small number (not more than 100cfu) ofDate:11-AUG-2008 Time:16:36\\managewise\share\SHARE\USPNF \PRINTQ\pager\xmlIn\ 62 Microbiological Examination / Microbiological TestsUSP 31the appropriate microorganism. Incubate at the specified tempera-ized, then it is to be assumed that the inhibited microorganism willture for a period of time within the range specified in the test.

8 Colo-not be present in the are comparable in appearance and indication reactions to thosepreviously obtained with a previously tested and approved batch ofTESTING OF of the Test MethodBile-Tolerant Gram-Negative BacteriaFor each new product to be tested perform sample preparation asdescribed in the relevant paragraph under Testing of Products . AtSample Preparation and Pre-Incubation Prepare a samplethe time of mixing, add each test strain in the prescribed growthusing a 1 in 10 dilution of not less than 1g of the product to bemedium. Inoculate the test strains individually. Use a number of mi-examined as described in Microbiological Examination of Nonster-croorganisms equivalent to not more than 100cfu in the inoculatedile Products .

9 Microbial Enumeration Tests 61 , but usingtest Casein Digest Broth as the chosen diluent, mix, and incu-Perform the test as described in the relevant paragraph underbate at 20 to 25 for a time sufficient to resuscitate the bacteria butTesting of Products using the shortest incubation period sufficient to encourage multiplication of the organisms (usuallyThe specified microorganisms must be detected with the indica-2 hours but not more than 5 hours).tion reactions as described under Testing of for Absence Unless otherwise prescribed, use the volumeAny antimicrobial activity of the product necessitates a modifica-corresponding to 1g of the product, as prepared in Sample Prepara-tion of the test procedure (see Neutralization/Removal of Antimicro-tion and Pre-Incubation, to inoculate Enterobacteria Enrichmentbial Activity under Microbiological Examination of NonsterileBroth Mossel.)

10 Incubate at 30 to 35 for 24 to 48 hours. SubcultureProducts: Microbial Enumeration Tests 61 ).on plates of Violet Red Bile Glucose Agar. Incubate at 30 to 35 For a given product, if the antimicrobial activity with respect to afor 18 to 24 for which testing is prescribed cannot be neutral-The product complies with the test if there is no growth Test Selection and Subculture Inoculate suitable quantities of Enter-obacteria Enrichment Broth Mossel with the preparation as directedunder Sample Preparation and Pre-Incubation and/or dilutions of itTable 1. Growth Promoting, Inhibitory, and Indicative Properties of MediaTest/MediumPropertyTest StrainsTest for bile-tolerant Gram-negative bacteria Enterobacteria Enrichment Broth MosselGrowth promotingE.