SYNERGY OF PRESERVATIVES 3 5 5 Table V Determination of Sterilization Times of Several Pseudomonas Species in MP, Na2EDTA, and MP + Na2EDTA Solutions 0.2% MP solutions: Test organism 0 hr 2 hr 4 hr 24 hr 48 hr ST MPST P, aeruginosa 9027 + + + + + P. aeruginosa 27853 + + + + + P, aeruginosa 9721 + + + + + P. aeruginosa 10145 + + + + + P. cepacia 13945 + + + + - P. cepacia 25416 + + + + + P. fluorescens 13525 + + + + + [2] P. putid• 12633 + + + + - P. stutzeri 17588 + + + + + Pseudomonas sp. 9230 + + + + + S. aureus 6538 + + + + + E. coli 8739 + + + + + 48 48 48 48 48 48 -- 48 48 __ 48 48 48 48 0.01% Na2EDTA solutions: Test organism 0 hr 2 hr 4 hr 24 hr 48 hr ST MPST P. aeruginosa 9027 + + + + + -- 48 P. aeruginosa 27853 + + + + + -- 48 P. aeruginosa 9721 + + + + + -- 48 P. aeruginosa 10145 + + + + + -- 48 P. cepacia 13945 + + + + + -- 48 P. cepacia 25416 + + + + + -- 48 P. fluorescens 13525 + + + [s] + + -- 48 P. putid• 12633 + + +[s] +[2] - 48 -- P. stutzeri 17588 + - +[1] - - 24 -- Pseudomonas sp. 9230 + + + + + -- 48 S. aureus 6538 + + + + + -- 48 E. coli 8739 + + + + + -- 48 0.2% MP + 0.01% Na2EDTA solution: Test organism 0 hr 2 hr 4 hr 24 hr 48 hr ST MPST P. aeruginosa 9027 + + [s] - - - 4 -- P. aeruginosa 27853 + +[s] +[2] - - 24 -- P. aeruginosa 9721 + + [s] - - - 4 -- P. aeruginosa 10145 + +[1] - - - 4 -- P. cepacia 13945 + + + + - 48 -- P. cepacia 25416 + .... 2 -- P. fluorescens 13525 + .... 2 -- P. putid• 12633 + .... 2 -- P. stutzeri 17588 ..... 0.1' -- Pseudomonas sp. 9230 + + + [ 1] - - 24 -- S. aureus 6538 + + + + + -- 48 E. coli 8739 + + + + + -- 48 Deionized water (control): Test organism 0 hr 2 hr 4 hr 24 hr 48 hr ST MPST P. aeruginosa 9027 + + + + + P. aeruginosa 27853 + + + + + P. aeruginosa 9721 + + + + + P. aeruginosa 10145 + + + + + 48 48 48 48 (continued)

356 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table V (Continued) Deionized water (control): Test organism 0 hr 2 hr 4 hr 24 hr 48 hr ST MPST P. cepacia 13945 + + + + + -- 48 P. cepacia 25416 + + + + + -- 48 P. fluorescens 13525 + + + + + -- 48 P. putida 12633 + + + + + -- 48 P. stutzeri 17588 + + + + + -- 48 Pseudomonas sp. 9230 + + + + + -- 48 S. aureus 6538 + + + + + -- 48 E. coli 8739 + + + + + -- 48 Explanation of symbols: +, growth on TSALT -, no growth on TSALT numbers in brackets (i.e., [1] and [2]) indicate the number of colonies growing on TSALT [s] is used to designate scant growth (i.e., 8-15 colonies growing on TSALT). ST, sterilization time in hr. MPST, minimum possible sterilization time in hr. * P. stutzeri was not recovered, so ST was set at 0.1 hr because this is the approximate time required for setting up the series of samples after inoculation. APC of the saline suspensions inocula: P. aeruginosa 9027 = 1.1 X 107/ml. P. aeruginosa 9721 = 1.8 X 107/ml. P. aeruginosa 27853 = 1.5 X 107/ml. P. aeruginosa 10145 = 2.0 X 107/ml. P. cepacia 13945 = 1.7 X 107/mi. P. cepacia 25416 = 9.9 X 106/ml. P. fluorescens 13525 = 1.6 X 107/ml. P. putida 12633 = 1.6 x 107/ml. P. stutzeri 17588 = 1.7 x 107/ml. Pseudomonas sp. 9230 = 6.3 x 106/ml. S. aureus 6538 = 1.4 x 107/ml. E. coli 8739 = 6.3 X 106/ml. MP. It was not possible to calculate STs, slopes, and D-values when the organisms were recovered at 48 hr (i.e., ST 48 hr), as for all strains of P. aeruginosa in MP or Na2EDTA. Nevertheless, the survivor curve slope method may be used to determine synergy when the experimentally determined rate of death (slope) is a larger negative number than the sum of the MPSlopes of each of the components. Use of MPSlopes in determining synergy is discussed in greater detail below. No synergy was observed for S. aureus or E. coli in these experiments. The results in Table VII show the growth response of P. aeruginosa 9027 on TSALT after exposure to various combinations of P and/or N in saline. As one reviews the growth responses of P. aeruginosa following exposure to various combinations of P and N, it is apparent that several of the combinations killed the population of P. aeruginosa faster than the same concentrations of either N or P used alone. The test organism was not recovered from test systems containing 0.1% N (except for one tube containing both 1% P and 0.1% N, in which one colony developed on TSALT at the 0 hr sam- pling). P. aeruginosa grew on TSALT streaked with 0.1% N consequently, preservative carryover was not responsible for the lack of growth on TSALT in systems containing the highest concentration of N. Synergy was observed when the slope of the survivor curve was a larger negative number than the sum of the slopes (or MPSlopes) for the components. The systems in which synergism was observed are marked with an asterisk (Table VIII).