MICROBIAL CONTAMINATION OF PRODUCTS 437 retail outlets in Cairo. There were no manufacture or expiration dates on the containers however, a batch number was recorded on each one. All products were manufactured in Egypt. Their contents, name of the manufacturer, etc., are listed in the Index of Special- ities (10). MEDIA The following dehydrated media (Oxoid, Ltd., England) were used during the course of the study: trypticase soya agar (TSA), Sabouraud's dextrose agar (SDA), MacConkey agar and broth (MA and MB), eosin methylene blue agar (EMB), mannitol salt agar (MSA), cooked meat medium (CMM), and Staphylococcus medium No. 110 (SM). In addition, cetrimide broth (CB) was prepared as follows: pancreatic digest of gelatin (20 g), MgCI 2 (1.4 g) K 2 SO 4 (10 g) cetrimide (0.3 g) and glycerol (10 ml). The pH was adjusted to 7.2 -+ 0.2. Cetrimide agar (CA) was prepared by addition of 1.5% agar to cetrimide broth. All media were sterilized by autoclaving at 121øC for 20 min. QUANTITATIVE DETERMINATION OF VIABLE MICROORGANISMS Viable counts for total bacterial, coliform, and fungal counts were determined by the conventional pour plate method as described by Collins and Lyne (11), using normal saline as diluent. In the case of total bacterial count, TSA plates were incubated at 37øC and examined daily. For coliform count, MA plates were used and incubated at 37øC and examined after 24 hrs. Fungal counts were carried out by plating on SDA medium, and the plates were incubated at 25øC and examined daily for seven days. The presence of either yeast or mold colonies was then noted. DETECTION AND IDENTIFICATION OF POTENTIALLY HAZARDOUS BACTERIA Two ml or 2 g of the preparation were aseptically mixed in a flask with 20 ml of one of the enrichment media and incubated (Table II). Several loopfuls were then transfered onto the surface of the selective media. Developed colonies were subjected to different identification schemes for different organisms as follows: Staphylococcus species. Colonies which developed on MSA and SM were identified ac- cording to the scheme described by Adegoke (12). Solares which proved to be gram- positive cocci, catalase (+), coagulase ©, mannitol fermenter, and which produced acid from glucose aerobically and anaerobically were considered to be S. aureus. Table II Media and Incubation Conditions for Isolation of Certain Bacteria Microorganisms Enrichment conditions Selective conditions Staphylococcus species CMM containing 10% NaC1 at 37øC MSA and SM at 37øC for 48 hr for 72 hr CB at 37øC for 48 hr MB at 37øC for 24 hr P seudomonas species Gram-negative rods CA at 37øC for 48 hr MA and EMB at 37øC for 48 hr

438 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Pseudomonas species. Colonies which developed on CA were subjected to gram stain and biochemical tests. Isolates which proved to be gram-negative rods, and which were oxidase-positive, non-lactose fermenters, motile, and utilized sugar by oxidation rather than fermentation were considered to be Pseudomonas species. The species was determined according to established methods (7). Identification of other gram-negative rods. Developed colonies on MA and EMB were gram- stained and tested for oxidase. The gram-negative, oxidase-negative rods were further identified by inoculation into the Enterotube I! (Roche) scheme for identification of gram-negative rods. The species was determined by applying the instructions of the Enterotube system (12). RESULTS A total of 144 samples representing eight toothpastes and four mouthwashes were ex- amined for their total aerobic bacterial, coliform, and fungal counts. Tests for detection of some potentially hazardous bacteria in the same sample were also carried out. The distribution of the total aerobic bacterial and coliform counts are summarized in Table I. A significant difference was noted between the total bacterial count of tooth- pastes and mouthwashes. More than 60% of the tested mouthwashes contained fewer than 100 CFU/ml, compared to only 25% of the toothpastes which contained the same count of bacteria/g. Viable bacteria were not recovered from 39% of the mouthwashes and 12% of the toothpastes. Similarly, while 6% of the toothpastes were heavily con- taminated (contained more than 10,000 CFU/g), only 2% of the mouthwashes were heavily contaminated to the same level. No coliform bacteria were recovered from any of the mouthwashes, while 7% of the Table III Distribution of Hazardous Bacteria in Tested Preparations No. and % of items containing items Other Other Preparation tested S. aureus Staph. sp., P. aeruginosa Pseudomonas sp. E. coli A. Toothpaste I 12 1(8) 0(0) 0 1(8) 0 2 12 0(0) 0(0) o o 1(8) 3 12 1(8) 1(8) 3(25) 0 0 4 12 0(0) 1(8) 0 0 0 5 12 1(8) 0(0) 0 2(17) 0 6 12 0(0) 1(8) 0 0 0 7 12 0(0) 1(8) 0 0 1(8) 8 12 0(0) 1(8) 0 0 0 Total 96 3(3) 5(5) 3(3) 3(3) 2(2) B. Mouthwash 1 12 0(0) 2(17) 0 0 0 2 12 0(0) 1(8) 0 0 0 3 12 0(0) 1(8) 0 0 0 4 12 0(0) 1(8) 0 0 0 Total 48 0(0) 5(11) 0(0) 0(0) 0(0)