MICROBIAL CONTAMINATION OF COSMETICS AND TOILETRIES 567 bacteria were made at 25øC for 5 days instead of at 30øC for 3 days. Counts of yeasts and moulds were made on Sabouraud Dextrose Agar (SDA Oxoid) incubated for 5 days at 25øC. Counts of anaerobic sporeforming bacteria were made on Reinforced Clostrial Agar (RCA Oxoid) after pasteurization (30 min at 75øC) of dilutions made up in freshly steamed Reinforced Clostridial Medium (Oxoid). The plates were incubated for 3 days at 37øC in an atmosphere of 95• hydrogen plus 5}/0 CO2. Clostridia were identified by the methods described by Willis (13). Dilutions of lipstick and toothpaste samples were tested for the presence of presumptive coliforms by inoculation into MacConkey Bile Salt Broth (Oxoid) which was incubated at 37øC and examined after 1 and 2 days. After incubation the number of colonies was recorded for each plate. Arithmetic mean counts were derived for each item from those plates having from 30 to 300 colonies. In the case of samples with low counts the number of colonies recorded on the first dilution tested was used to derive the count. In some instances, the presence of an antimicrobial agent in the product was shown by a carryover effect where the count at high dilution was sometimes greater than the count at low dilution. Repeat analyses were always undertaken in such cases. MouM growth on compressed eye make-up The remainder of each sample after surface scraping was aseptically dissected into three portions, each of which was placed on a moistened sterile filter paper in a petri dish. The surface of the make-up was moistened with sterile water and the samples were incubated in a moist atmosphere for several weeks at 25øC sterile water was added as necessary to the samples. Statistical analysis When replicate samples were tested, the significance of the difference in mean count for samples from different sources (e.g. surface scrape or mixed sample) or for replicate plates incubated at different temperatures was tested using two methods. For those results where large numbers of 'sterile' items (i.e. 10 cfu g4) were obtained, Wilcoxson's Signed Ranks test was used (14). Where mainly definitive results were obtained, the mean difference of the log•0 counts from zero was tested using Student's t. Counts of 10 g-• were in all cases assigned the numerical value of 10 in calculations of the t value.

568 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS RESULTS A total of 190 analyses were carried out on the 172 items purchased during the survey. The additional 18 tests were conducted to compare the counts on top and bottom samples from all tubed products, e.g. toothpaste, hair dressing, etc. A further 60 analyses were made on items of selected products purchased locally. Aerobic bacteria The distributions of aerobic bacterial colony counts at 30 ø and 37øC are summarized in Tables H and III. Over 80•o of the items tested contained fewer than 300 cfu g-•. Viable bacteria were not recovered from over 50•o of the items examined. No difference was seen in the bacterial colony counts on samples from the top and bottom ends of 16 tubed products. Most samples yielded no viable bacteria from either end of the tube but in one case colony counts of 2.6 x 104 and 3.0 x 104 cfu g-• were recorded for the top (nozzle end) sample at 30 ø and 37øC respectively whereas counts from the crimped end sample were 1.32 x 105 and 1.34 x 105 cfu g-X respec- tively. In this instance general contamination of the product prior to filling may have occurred. In contrast, significantly different counts were noted in two items. The counts at 37øC on these items were 1.65 x 104 and 2.5 x 10 a cfu g-• for the top samples and 10 and 35 cfu g-• respectively for the bottom samples. In these two instances it is probable that the nozzle end of the tubes was contaminated prior to filling with the product. Coliform bacteria were not detected in 0.1 g of any toothpaste sample nor on any lipstick sample examined. The predominant microorganisms isolated from high count products were Gram negative non-sporing rods, but no attempt was made to identify the organisms. Anaerobic bacteria Samples of each item of talcum powder, face powder and 'complete make-up' were examined for spores of mesophilic anaerobic bacteria. The distribution of counts is summarized in Table IV. Statistically significant counts (300 cfu g-•) were not obtained from any product examined in the survey, but such counts were later observed in the repeat analyses (see below). Selected colonies of anaerobes were subcultured and examined both microscopically and culturally. The organisms were typically mesophilic