RESIDUAL ANTIMICROBIAL ACTIVITY ON HUMAN SKIN 413 5. After the 4-hour period the plates are removed, incubated at the opti- mum temperature for the organism being studied, and then counted. 6. To determine the length of time activity remains on the treated site, new seeded plates are applied to the identical site at various time periods. 7. The counts obtained from test sites are then compared with suitable controls to determine per cent reduction of growth due to exposure to treated areas. Evaluation of AntiraicrobiaZ Skin Cleansers and Soap In the studies reported here the organism used for seeding the plates was Staphylococcus "S. epidermis" (ATCC 155).* This organism was chosen be- cause S. epidermidis together with Staphylococcus albus is considered to be a maior inhabitant of practically all regions of the skin with S. epidermidis tending to be predominant (7). A typical experiment consists of the follow- ing steps, A. Preparation of Plates The organism, S. epidermidis ATCC 155 is grown in brain heart in- fusion broth (from Difeo). The culture is standardized and diluted to contain approximately g00 to 300 organisms per milliliter using normal saline as the diluent. The plates used are the covers of tissue culture dish #3001 made by Faleon©t Plastics and are 38 mm in diameter and 5 mm in depth. The area of the surface is approximately 11.3 em = (Fig. 1). Seeded agar is prepared as follows. To every 5 ml Mannitol salt agar (from Difeo) is added 1 ml of the diluted culture described previously. This mixture is prepared in bulk and kept at 45øC. Five ml of seeded agar is added to each plate using a Cornwall piperror. This amount fills the plate almost to the top. These seeded plates are in turn placed into larger sterile petri dishes to prevent contamina- tion prior to use (Fig. •). It is permissible to prepare all the plates needed for the entire test on the same day and store them in a refrig- erator. Mannitol salt agar was used because most organisms, other than Staphyloeoeei, are inhibited by this medium. ]3. Test Procedure 1. For at least 3 days before the test, subjects wash with a nonmedicated soap to remove all traces of antimicrobial agents from the skin. *American Type Culture Collection, 12301 Parklawn Dr., Bockeville, Md. 20852. '•Falcon Division of Becton Dickinson & Co., 1950 Williams Dr., Oxnard, Calif. 93030.

414 JOURNAL OF THE SOCIETY OF COSMETIC CItEMISTS Figure I. Tissue culture dish used in test .•] -•.. '"' .•.• .' •.. -•....!• ½,•.:. &? ?: .... .: ..: :: • :' •'"• :.:..""" Figure 2. Test plates placed in sterile petri dish