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.

142 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Such tests are performed by using the usual Agar Cup-Plate Method to- gether with inoculating the sample itself with mold spores. The yeast and fungi cultures are grown on slants of Orange Serum Agar, No. 77, for ten days at room temperature. This media is manufactured by Sunkist Growers, Ontario, California, and supports copious yeast and mold growth. It contains concentrated orange serum, yeast hydrolysate, peptone, buffer salts, cysteine and agar. Yeast and mold counts are gen- erally greater using this medium when compared to the use of Sabouraud's Dextrose Agar. The mold is transferred from the slant into a small, sterile, Pyrex bottle containing glass beads. Sterile physiological salt solution is added the bottle stoppered and placed in a shaking machine for ten minutes. The resultant suspension is then filtered through sterile glass wool producing a suspension free from clumps which is used to inoculate the agar plates. Inoculation is carried out by means of a sterile "Q-Tip," swabbing the sur- face of the solidified agar medium. A cup 11/2 cm. in diameter is made by means of a sterile cork borer. The cup is filled with sample and the plate incubated in a humidified chamber and observed on the third, sixth and tenth days of incubation. If there are any fungistatic properties present, the three-day period usually shows a sharp clear halo that may measure from 3 to 10 m.m. However, by the sixth day, the sharp clear halo may be covered with mycelial growth and, by the tenth day, mold growth could be growing up to the edge of the sample. Of course, the ideal situation would be to have a sharp clear 3 to $ mm. halo present after the ten-day incuba- tion period. However, when the mold grows up to and adjacent to the sample area, but not over it, it is considered to be adequately preserved against this mold. Careful observation should be made under five power to be sure that the mycelia have not become imbedded in the surface of the sample. In other words, it is not considered necessary to produce a halo around the sample area to prove it will not support mold or yeast growth. If a halo is produced, so much the better. One must take into consideration the fact that the sample has been stored in the presence of countless mold spores for ten days, under conditions favorable to their growth. So, the absence of growth on the sample is indicative that it is adequately preserved against the test organism. In conjunction with the above test, it is recommended that the cosmetic product under investigation be inoculated on the surface with dry mold spores. The inoculated sample is placed in a humidified chamber and stored for thirty days and examined under five power periodically during this period. It has been found that mold or yeast growth, if and when it takes place, will usually do so within ten to fourteen days incubation under these conditions. The results of this test are then correlated with those of