36 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Many types of cosmetic and topical preparations contain microorganisms, but their presence is difficult to detect because the cells are entrapped in oil-water emulsions or ointment based matrices. Physical and chemical procedures are employed to free entrapped cells so that they can grow in culture media. Many products contain preservatives which kill or inhibit the growth of microorganisms. In testing such products, it is desirable to neutralize the preservative so that any viable cells present can grow in the test culture medium. It may not be obvious why we want to look for microorganisms that cannot grow in a preserved cosmetic or topical drug. The problem is that although initially the preservative may work well in the product, upon storage its effectivensss can be reduced by chemical changes. Furthermore, upon application of a product to the body, the effectiveness of a preservative can be reduced due to dilution with moisture and organic matter or even by evaporation of the preservative. Previously inhibited microorganisms are now capable of growth and can be potentially pathogenic if they gain entrance into the body. The present methods employed by FDA for the microbiological analysis of cosmetics and topical drugs are outlined in the 5th edition of the Bacteriological Analytical Manual (BAM) (1). The ultimate objective of the examination is to provide a microbiological profile of the product which is as complete and comprehensive as possible. A considerable improvement in our ability to detect contaminating microor- ganisms has been achieved through the use of modified letheen broth as both an enrichment medium and diluent. Modified letheen, a highly enriched medium containing lecithin, polysorbate $0, and sodium bisulfite, supports the growth of most microorganisms. Both the lecithin and polysorbate 80 serve as surface-active agents and emulsifiers that promote microbial contact with the aqueous phase of the broth medium. These two ingredients also serve to inactivate or neutralize several kinds of preservatives, including quarternary ammonium compounds, parabens, and phenolics. Viable cells that are present but inhibited by these preservatives in products are therefore able to grow in culture media. Sodium bisulfite, another ingredient in modified letheen, neutralizes some of the aidehyde-type preservatives, e.g., 2% glutaraldehyde (2). In practice, we have found that modified letheen broth is a very effective and versatile medium. However, it seems possible that future research will lead to development of media that are even more effective in neutralizing preservatives than modified letheen. For example, Bruch and Kohn reported that polysorbate 20 is more effective than polysorbate $0 in neutralizing parabens (3). As in all microbiological testing, the sample should be analyzed as soon as possible after collection. The containers are examined visually for any defects or irregularities, such as cracks or leaks. Once established that a container is sound, the external surface is disinfected with a suitable antimicrobial agent and air dried under a laminar flow hood. The analytical methodology can be divided into four distinct stages: screening, plating, enrichment, and if required, identification of isolates. An initial screening test is performed to determine if the product is contaminated. If growth is detected in the screening media, then a variety of methods are used to determine plate counts of specific groups of microorganisms including aerobes, anaerobes, Staphylococcus aureus, yeasts, and molds. Every sample containing gram negative bacilli is subjected to an enrichment procedure for the enumeration and further identification of these organisms.

FDA METHODOLOGY FOR MICROBIOLOGICAL ANALYSIS 37 SCREENING TEST The screening test is in effect a rapid and simple test for the presence of microorga- nisms. Broth media are inoculated with either 1 g or 1 ml of sample. The media employed depend on the type of product being tested. Liquids, creams, ointments and most other products are inoculated into both modified letheen broth, to recover bacteria, and Sabouraud dextrose broth for yeasts and molds. For talcum and other powders, fluid thioglycollate medium is used to detect aerobic and anaerobic sporeformers. The fluid thioglycollate is incubated at 35øC, and the other media at 30øC, for at least seven days. Media are examined for growth periodically by checking for turbidity. Many products themselves, such as powders, creams, and lotions, cause turbidity in broth media and growth must be confirmed by plating on modified letheen agar, malt extract agar containing 40 ppm chlortetracycline, or acidified potato dextrose agar. If no growth is detected at the screening stage, the sample is considered negative and no further testing is necessary. If growth is detected, serial dilutions of the original sample are prepared in enrichment medium. These dilutions are used for both plate counts and microbial enrichment. PLATE COUNTS For aerobic plate counts, the sample is serially diluted from 10 -• to 10 -6 in modified letbeen broth. Each dilution is mixed thoroughly, and 0.5 ml is surface plated in duplicate on modified letheen agar. Surface plating, rather than pour plating, is employed to avoid the possibility of killing stressed organisms with melted agar. Plates are incubated for 48 hours at 30øC prior to enumeration of colonies for calculation of the number of organisms per g or ml of sample. These same dilutions are tested for the presence and number of $taphylococcus aureus by plating on Vogel-Johnson (v-J) agar. Typical staphylococci produce black colonies, caused by the reduction of potassium tellurite to elemental tellerium, surrounded by yellow zones indicating the fermentation of mannitol. Baird-Parker (BP) agar, an extremely effective medium for recovering stressed staphylococci, can be substituted for V-J agar (4). Sodium pyruvate, an ingredient of BP agar, destroys peroxides formed during the oxidative degradation of lipids and carbohydrates present in many products, and allows for better microbial recovery (4). Although many microorganisms have defense mechanisms (e.g., catalase and superoxide dismutase) against peroxides and free radicals, antimicrobial effects can still occur when high concentrations of toxic materials are produced or defense systems have been damaged. On BP agar, staphylococcal colonies appear black and are surrounded by either a clear zone, an opaque zone, or both, caused by the proteolysis or lipolysis of egg yolk in the medium. The only criticism against the use of BP agar is that polysorbates, from product or modified letheen broth, can clear the opaque medium and diminish its diagnostic value. However, this is an insignificant drawback in comparison to the overall advantages of using BP agar. Characteristic colonies are picked and streaked on tryptic soy agar slants and subsequent bacterial growth is used to test for coagulase activity. All strains yielding a positive coagulase reaction are considered to be $taphylococcus aureus, and no further testing is required. All product dilutions are also tested for numbers of yeasts and molds by plating them on either malt extract agar supplemented with 40 ppm chlortetracycline or potato