Isolation of Pure Cultures

These instruments are typically calibrated using saturated is especially common when organisms are recovered at salt solutions at 25°, as listed in Table 3. atypically high rates or in numbers that exceed recommended levels for specific categories of products. Additionally, microbial identification is useful in aseptic processing Table 3. Standard Saturated Salt Solutions Used to Calibrate and is necessary where sterility test positives have occurred Water Activity Determination Instruments and in the assessment of contamination recovered from Saturated Salt Solutions ERH (%) aW failed aseptic process simulations, i.e., media fills. Potassium sulfate (K2SO4) 97.3 0.973 Microbiological identification systems are based on different analytical methodologies, and limitations may be inherBarium chloride (BaCl2) 90.2 0.902 ent to the method and/or arise from database limitations. Sodium chloride (NaCl) 75.3 0.753 Identification is accomplished by matching characteristics Magnesium nitrate [Mg(NO3)2] 52.9 0.529 (genotypic and/or phenotypic) to an established standard Magnesium chloride (MgCl2) 32.8 0.328 (reference) organism such as a type strain. If a microorganism is not included in the database it will not be identified, so manufacturers should review the breadth of the database of the identification system they plan to use and its applicability to their needs. Users should consider which microbiological identification system(s) is (are) most applicable to their requirements. Bearing in mind both these limitations and the level of identification required (genus, species, strain), users also must select the appropriate technology to Add the following: use in routine microbiological identification testing. The need for microbial identification is specifically cited in USP general test chapter Microbiological Examination of Non▲〈1113〉 MICROBIAL sterile Products: Tests for Specified Microorganisms 〈62〉. This chapter indicates a requirement for confirmatory identificaCHARACTERIZATION, tion tests for organisms that grow on selective or diagnostic media and demonstrate defined morphological characterisIDENTIFICATION, AND STRAIN tics. Also, USP general test chapter Sterility Tests 〈71〉 allows for invalidation of the test, if after identification of the miTYPING croorganisms isolated from the test, the growth of this (or these) species may be unequivocally ascribed to faults with respect to the material and/or the technique used in conducting the sterility test procedure. USP general information chapter Microbiological Evaluation of Clean Rooms and Other INTRODUCTION Controlled Environments 〈1116〉 recommends that microbial isolates be identified at a rate sufficient to support the enviMicroorganisms, if detected in drug substances, excipironmental monitoring program. ents, water for pharmaceutical use, the manufacturing environment, intermediates, and finished drug products, typically undergo characterization. This may include ISOLATION OF PURE CULTURES identification and strain typing, as appropriate. [NOTE—A Glossary of Terms is provided at the end of this chapter.] The first step in identification is to isolate a pure culture Routine characterization of microorganisms may include the for analysis. This is typically accomplished by successive determination of colony morphology, cellular morphology streaking of the colony of interest in a quadrant pattern on (rods, cocci, cell groupings, modes of sporulation, etc.), appropriate general microbiological solid media with the obGram reaction or other differential staining techniques, and jective of obtaining discreet colonies that usually yield pure certain key biochemical reactions (e.g., oxidase, catalase, cultures. This technique also allows phenotypic expression and coagulase activity) that can be diagnostic. Microbial and growth of sufficient inoculum for succeeding identificacharacterization to this level is sufficient for many risk-assesstion procedures. Analysts should recognize that expression ment purposes in nonsterile pharmaceutical manufacturing of the microbial phenotype (i.e., cell size and shape, sporuoperations and in some sterile product manufacturing lation, cellular composition, antigenicity, biochemical activenvironments. ity, and sensitivity to antimicrobial agents) may be affected In some cases a more definitive identification of the miby isolate origins, media selection, and growth conditions croorganisms yields genusand species-level identification. (see Table 1). Therefore, the preparatory media for identifiBeyond this, available methodologies can perform straincation and the number of subcultures may affect the results level identification, which can be useful in an investigation of phenotype identification methods. to determine the source of the microorganism. Identification