554 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table II Detection of Microbial Contamination during Preliminary Testing of TAT Broth with Commercial Products • No. of Units Contaminated/ No. Tested 2% Aerobic Plate TAT tryptone Count Organisms ø Speciated Item broth broth (cells/ml) from TAT Broth Liquid bath cleanser Lot 1 48/48 48/48 9 X 104-2 X 10 • P. aeruginosa A. faecalis S. marcgscens Lot 2 48/48 48/48 1.3-5.8 X 10 • Same as lot 1 Lot 3 45/48 45/48 6 X 10a-8 X 10 • P. aeruginosa A. faecalis P. fluorescens S. marcescens Lot 4 0/72 0/72 Not done None Antacids Brand 1 2/2 2,/2 2.1-2.9 X 10• Flavobacterium sp. coilforms Brand 2 2/2 2/2 3.2-4.0 X 106 P. aeruginosa Aerosol shave cream 0/30 0/30 Not done None Baby conditioning oil 0/30 0/30 Not done None Skin lotion 2/2 2/2 1-2 X 104 P. putida Pseudomonas sp. Baby lotion Lot 1 12/12 12/12 1-10 X 10,• A. faecalis Lot 2 5/12 5/12 1-10 X 102 Micrococcus sp. Lot 3 0/12 0/12 Not done None Eye cream 2/2 2/2 100 •*licrococcus sp. • Levels or types of preservatives not known. b Complete names of organisms: Pseudomonas aeruginosa P•eudomonas fluorescens Alcaligenes f aecalis Serratia marcescens Pseudomonas putida. 10 • to 10 -4 dilutions are prepared with potato dextrose agar (13) 2 plates are incubated at room temperature and 2 plates at 37øC for 7 days. For the isolation of S. aureus, 0.5 ml of each dilution is transferred and spread over the surface of each of duplicate plates of Vogel-Johnson (V-J) agar (13). These plates are inverted and incubated for 48 hours at 35-37øC. Colonies are picked to 0.3 ml of sterile Brain Heart Infusion Broth, in- cubated at least 18 hours at 35-37øC and tested for coagulase reaction by adding 0.5 mI of reconstituted coagulase (rabbit) plasma containing 0.1% EDTA (13). All cultures giving a positive reaction in 6 hours are con- sidered as S. aureus. For the isolation and enumeration of gram-negative bacteria, the TAT broth used in the aerobic sterility test plus the product dilutions in

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.