IDENTIFICATION OF GRAM-NEGATIVE BACTERIA 557 Figure 2. Side view of modificd Lidwcll applicator. The arm on the left with the 26 loops can be lowered into the wells of thc nylon template and then rotated to the right to inoculate a plate of solid culture medium • • •. . -* . .•. • .•-..• . -.. ...•- i%•....... •'- .- ...• . ,... ,: •,.:.•::•.•.' .... Figure 3. A plate of solid diagnostic medium showing response of 26 isolates. Two isolates did not •ow. A color change (dye response to pH change) was noted for 4 isolates known gram-negative species for each solid medium test to determine similarity with test responses previously known for broth cultures of these same organisms. Since the solid medium allows the microbial isolates to grow under highly oxidized conditions, some fermentation reactions that require reduced O-R potentials can be altered. What is significant is that a typical pattern of response has been achieved for each o.• a wide spectrum of known gram-negative contaminants of topical products. It is hoped to eventually place all of the reactions for each microbial isolate characterized to date in a computer bank to facilitate easier comparisons of future isolates with response patterns previously developed.

558 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table HI--Diagnostic Reactions Reactions a Differential Media Carbohydrate Utilization Species Identifica- tion P. aeruginosa q- q- P. stutzeri -- q- P. multivorans q- q- -- Enterobacter aerogenes -- q- S. marcescens (--) q- E. coil -- q- (--) Klebsiella -- q- Alcaligenes f aecalis q- q- q- Adnetobacter anitratus -- q- (--) ( q- ) ( q- ) K/-- -- ( q- ) -- q- -- - - K/-- -- -- (-) -- - - K/- + + + (+)(-) + + -- -- A/A (+) + + q- -- q- q- q- + q- -- -- K/-- (+) + + -- + + + + (+) + -- -- -- A/A (q-) q- q- q- + q- q- 4-, (+) -- -- A/A (q-) + q- q- -- q- 4- q- q- q- (q-) - K/-- -- -- + + + (+) (+) + a q- = indicator change -- = no indicator change K = alkaline A =actd P----peptonization (+)= plated medium. e NOs reduction. al Optimum growth temperature. Since the introduction of the Lidwell applicator to our speciation ef- forts in 1968, the group has speciated 1550 gram-negative bacteria from raw ingredients, production facilities, and finished topical products. Table IV presents the identity of these isolates by 6 gram-negative group- ings. Members of the genus Pseudomonas represent 50% of the gram- negative isolations, while Klebsiella and members of the genera Escherichia-Enterobacter represent 11 and 10%, respectively, of_ the isolates. In addition, another 88 gram-positive cocci and rods and 10 yeast and mold cultures have been speciated but not included in Table IV. Serological and Pyocine Typing Procedures for the Identification of Pseudomonas Early in the work on the speciation of gram-negative bacteria from topicals, the authors' group encountered significant bacterial contamina- tion of a detergent-antiseptic product (17). Initial growth patterns and biochemical responses were similar to those associated with Flavobac- terium lutescens. However, the organism isolated by the Center for Disease Control (CDC) was characterized as a variant of. Pseudomonas multivorans known as EO-1. At the time of. our identification the iso- lated organism was nonmotile. A comparison of results with the CDC