IDENTIFICATION OF GRAM-NEGATIVE BACTERIA 555 ,o,uc TAT BROTH I DILUTIONS IN TAT STERILITY * lO -t - lO -• l•f Positive, Do B,) •• SCO V-J POTAAGAR* TAT AGAR AGAR* DEX. INCUBAT'E DILUTIONS J, •..• BHI BROTH •AC CONKEY'S AGAR • COAGULASE TEST TSI AGAR BIOCHEIVilCALS *Gram Stain of TAT Broth Dictates Whether These Tests Are Performed Figure 1. Schematic diagram of sequence of microbiological determinations in current isola- tion procedure. Note: Aerobic sterility test in TAT broth is n•ot an official sterility test as defined in USP XVIII TAT broth, which have been incubated at 35-37øC for 24-28 hours, are used. From each tube or bottle showing growth a plate of MacConkey's agar is streaked and incubated for 24 hours at 35-37øC (13). At least one type from each group of colonies having distinct morphological charac- teristics is isolated to triple sugar-iron agar (TSI) slants for further iden- tification (12). From the TSI cultures a battery of media are inoculated to allow determination of biochemical reactions which characterize the various gram-negative species. Comparative Studies of Aerobic Plate Count Procedures A variation of the above procedure for determining aerobic plate count was evaluated along with 6 other plate count methods in a labora- tory study performed by the Microbial Content Subcommittee of the Cos- metic Toiletry Fragrances Association (14). Three separate oil/water lotions deliberately spiked with known bacteria or fungi were distributed to participating laboratories for determination of microbial content. Each laboratory examined the preparations by a plate count method de- vised by the Subcommittee and by "in-house" procedures. The "in- house" methods were either plate count or tube-dilution techniques and included a modified FDA procedure. All methods were found to be equally satisfactory despite variations in technique, diluents, and media.

556 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Repetitive studies by one laboratory demonstrated excellent correlation between all plate count methods. Use of Solid Media to Characterize Gram-Negative Isolates Current legal interpretation of the Food, Drug, and Cosmetic (FDgcC) Act as it applies to microbial contaminants in topical products impels the FDA to speciate all gram-negative isolates for health hazard evaluation (15). The classification of microorganisms to genera and species requires the determination of a series of morphological, biochemical, and fre- quently serological characteristics. In the past, basic tests such as carbo- hydrate utilization, IMViC patterns, gelating hydrolysis, protein de•a- dation, gTowth inhibitions, and optimum growth temperatures were usu- ally performed for each isolate in separate tubes of broth culture media. More recently, the interest in automation of microbial diagnostic pro- cedures has encouraged an evaluation of solid or semisolid media to al- low biochemical determinations for a group of isolates to be performed on a single plate of culture medium. Because of the large number of speciations required to support recall requests of contaminated products by District Laboratories, combined with severe limits on the technical manpower in many FDA laboratories, a solid media concept has been adapted to allow the rapid characterization of the many isolates obtained from topical products. A preliminary report on the use of a modified Lidwell phage appli- cator for the simultaneous application of 26 bacterial isolates to a plate of solid culture medium was made in 1969 (16). Figure 2 shows the modified phage applicator with its 26 platinum loops. After flame steril- ization, the loops are cooled and then simultaneously dipped into a tem- plate made of autoclavable nylon that contains 26 wells, each of which can hold up to 1 ml of a suspension of a test organism. When the ap- plicator has picked up a charge of test organisms, it is rotated and allowed to make contact with a 15 X 100 mm petri dish of solid culture media. Figure 3 is an example of a culture plate showing the reactions of 26 bac- terial isolates after 24 hours incubation at 32øC. Typical test responses for the most objectionable gram-negative contaminants in topical prod- ucts are presented in Table III along with a notation as to which of these tests are determined from solid media reactions. It will be noted in Table III that some reactions still have to be per- formed in broth media. Thus, tests for indole production, nitrate reduc- tion, and the methyl red-Voges Proskauer reactions utilize broth media. Furthermore, it has been necessary to catalog the reactions of many