ß PRESE'RVATION OF TOILET PREPARATIONS CONTAINING NONIONICS 223 TABLE VIII Each Nonionic at 2% With Benzalkonium Chloride at 0'1% Active Inactive. Gram _ve Gram +re Gram _re Gram -I -ve Strength of culture. I 2 3 4 56 r 2 3 4 5 6 i 2 3 4 5 6 I 2 3 4 56 Benzalkonium chloride alone, Glycerine Sorbitol Propylene (11ycol Tallow P. E.G. 2OO•4OO & 600 R E,G. 2pOO &10,OOO RE.G. 400 Laurate. _ _ Nonex 64 & 99 _ Cremophor. E.L. Glyceryl monostearate. __ Tween. 20 Tween. 40 Tween, 80 -- Span. 20 Arlacel 83 I_ubrol W Tcxofor D I Sucrose esters ,

224 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS (f) Benzalkonium chloride is more active against Gram-positive organ- isms than Gram-negatives when inactivation occurs, the Gram- negatives are allowed to grow. , At the ratios of nonionic to preservative used in this experiment, none of the nonionics affected the activity of benzalkonium chloride against Gram-positive organisms. Effect of Nonionics on Resistance of Bacteria to Preservatives During the course of this work it has often been observed that when bacteria have grown in media containing nonionics their staining character- istics change. This change is sometimes reversible. Gram-positive organisms have, on occasion, become Gram-negative after a period of contact with non- ionics and, on subculture, have reverted to Gram-positive. A change has also been observed with Gram-negative organisms which become Gram- positive for a period and, when transferred to media without nonionic, once again become Gram-negative. This phenomenon has also been observed by N. and A. Delmotte. • It was thought that these observations indicated some change at the cell wall. The change in staining characteristics might indicate some protective effect at the cell wall whereby effective contact between the germicide and the cell is hindered. This phenomenon has been studied with a view to determining whether it influences the resistance of bacteria to preservatives. Two types of experiment have been carried out: one in which the bacteria were grown in nonionic media before transfer to a bacteriostatic agar medium, and the other where the organisms were suspended in a saline/nonionic mixture prior to exposure to the preservative. (a) Organisms Grown in Media containing Nonionics Four test organisms were grown for a period of 72 hours in broths con- taining various nonionics at 2 per cent. The organisms were also grown in plain nutrient broth as a control. After the incubation period, the organisms were spun down and transferred to nutrient agar containing 0.1 per cent Nipa 82121. These plates were incubated for four days at 37 ø C. Results are shown in Table IX. KEY TO TABLES IX and X -- no growth after 4 days. + = growth after 3 days. + + -• growth after 48 hours. + + = growth after 24 hours.