140 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS mixtures of them must be investigated. In fact, most cosmetics today re- quire a mixture of preservatives in order to broaden the antibacterial spec- trum. A product, shown to be adequately preserved, should not be neglected but should be examined periodically for microt3rganisms. I recall one cosmetic manufacturer who had been making an emulsion for quite a few years which appeared satisfactory in every way. For some unknown reason, he subjected it to a bacteriological examination. Imagine his surprise when over 2,000,000 bacteria per gram were found! Consider his con- sternation when control samples a year old, from various plants, also showed this condition! Certainly, he had a stable, marketable product no one knew it was "loaded" with bacteria--not even the manufacturer-- but, what would happen if some consumer got a skin rash and stated that it was caused by the use of this product ? Even if the rash were caused by some other means, the fact that the cosmetic was a "carrier" of bacteria would most likely be sufficient to warrant any judgement in favor of the consumer. The adverse publicity that goes along with such cases should not be con- sidered lightly. Therefore, in order to be certain that your product is ade- quately maintaining its preserved state, examine it periodically for micro- organisms. In order to test the preservation properties of a cosmetic, it is recom- mended that one has available about eight resistant cultures. It is pref- erable that these micro/3rganisms had been isolated from a deteriorated product. It is further recommended that the cultures consist of at least two different strains of Pseudomonas such as aeruginosa and fluorescens derobacter aerogenes the Coliform type a yeast a Penicillium and an d3pergillus. Let us first consider preservation against bacteria: To be sure that the cosmetic product is not highly contaminated with bacteria before one starts the test, it is necessary to make a bacteria count of the product. In making bacteria counts of products that are entirely water-soluble, the regular serial-dilution technique can be followed. However, in examin- ing emulsions such as lotions, creams, pastes, suspensions, powders, cake make-up, etc., it is recommended that from three to six grams of sample be placed in 300 ml. of sterile water contained in a sterile Waring Blendor. The speed of the Blendor can be controlled by a rheostat which is often necessary when handling products that may produce considerable foam. After mixing or dispersing the sample, this dilution can be used for plating and making further serial dilutions. One will also find this technique most helpful in the examination of water-in-oil emulsions.

THAT UNWANTED COSMETIC INGREDIENT--BACTERIA 141 As for determining the preservation properties of the cosmetic against bacteria, it is suggested that one place 10 grams of sample in a sterile 1 ounce wide-mouthed jar. A twenty-four-hour test culture is standardized by serial dilutions and plated on the appropriate media, usually Difco's Count Plate Agar or Tryprose Phosphate. Simultaneously, one inoculates the 10 grams of sample with 0.1 ml. of the same culture. The mixture is thoroughly stirred with a sterile spatula for approximately three minutes, capped and stored at room temperature. After any reasonable time period, usually two to four days, a 1-gram ali- quot is removed and the number of surviving bacteria determined. The residual inoculated sample is kept at room temperature for further testing should it become necessary. Standardizing the test culture at the time of inoculating the sample shows the number of live bacteria per gram actually placed into the cos- metic sample. Determining the number of surviving bacteria, after two to four days contact, informs one whether the population of the test organism is being reduced gradually, drastically or even increased. Of course, the most desirable result is to have no bacterial growth at the end of two to three days contact. If necessary, the original inoculated cos- metic can be tested again at the end of six days, and so on, depending upon how rapidly the population is decreasing. Usually, the inoculum amounts to about 6 million organisms per gram and, if the count is reduced to a few thousand per gram within a period of six days, the product may be considered to be adequately preserved against this test organism. However, at this point care must be taken not to accept these results as final because the few surviving bacteria in the inoculated sample have been known to acclimate themselves to the environment reproducing the count increasing sometimes gradually and other times rapidly. Thus, if live bacteria are found after four. or six days contact, it becomes necessary to keep the inoculated sample under test until one has evidence that the number of surviving bacteria do not increase. This may require periodic testing over a period of two to three weeks. As mentioned above, the ideal condition is for the sample to kill all the inoculum within two to four days contact. The same technique should be followed with all the various cultures with which the product is to be tested. The testing of a cosmetic product for its preservation action against yeast and mold growth is not quite so "clear-cut" as that against bacteria. Luckily, modern preservatives are apparently most effective toward arrest- ing the growth of these microiSrganisms because they are seldom encoun- tered in commercial cosmetic items. However, they are encountered, al- though infrequently, so it is necessary to determine the ability of the cos- metic to inhibit or kill them.