138 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS which finally resulted in a well preserved active ingredient and shampoo-- so it was thought! In fact, the final product could be classified as being sterile. These well preserved shampoos became cloudy within a few days and again separated into two layers. Further bacteriological tests showed that they were free from bacteria--what, then, was going on ? The only logical answer was that there must be an enzyme causing hydrolysis. To prove this, a culture was made of the isolated Pseudomonas aeru- ginosa, treated with formaldehyde to kill the organism, aseptically filtered and stored in the refrigerator. The enzyme formed by the Pseudomonas aeruginosa, called pyocyanase, was in this sterile filtered solution. Simul- taneously, samples of freshly prepared shampoo were sterilized in the auto- clave. Upon cooling, aliquots were then inoculated with the sterile enzyme solution. Those stored at incubation temperature hydrolized overnight while those at room temperature required several days to produce hy- drolysis. With this information, it was evident that the appropriate preservative must be incorporated into the concentrated active ingredients at an early stage during manufacture. In this manner, the bacteria are retarded or killed--preferably the latter--and, since the enzyme was not given a chance to be formed, the active ingredient did not hydro]ize. SufFicient preservative was added to the concentrated active ingredient at this stage so that the finished formulated shampoo contained enough preservative to retard any bacterial growth. It required a year of steady work to solve this problem to the point where a stable, marketable shampoo was produced. One can readily see how expensive a problem of this sort can be, not only from the standpoint of solving the problem but, also, from the sales lost during this period. However, this company can consider itself fortunate in that the trouble was encountered before marketing rather than afterwards. C^sE No. 5 A marketed cream shampoo exhibited numerous little black specks on the surface. Every one visually examining the product considered these specks to be incipient mold growth. However, extensive bacteriological work showed that they were caused by the organism zflcaligenes viscosus. Eventually, the source of the contamination was traced to the sodium lauryl sulfate and was isolated only after frantic efforts. Extreme difficulty was encountered in growing the organism until it reached the point where the discolored spots were streaked on every type of agar media in the laboratory--preparing two sets of plates--incubating one at room temperature and the other at 37øC. This technique is known as "the last resort."

THAT UNWANTED COSMETIC INGREDIENT--BACTERIA 139 The bacteria grew plentifully on calcium carbonate-yeast extract agar at room temperature. It did not grow on any of the other media at either temperature. ?11caligenes viscosus is a gram-negative, nonspore forming organism that is found in water and around dairy barns. Once the organism was isolated and its culturing habits known, it was then possible to run through the gamut of preservatives and solve the preservation problem. The manufacturer of the sodium lauryl sulfate was approached and he eventually found this organism in the large storage tank. He believed it got there by taking returned drums of sodium lauryl sulfate and adding them to the storage tank, mixing thoroughly, in order to maintain a uniform product In this way, they believe their entire storage tank became con- taminated. It should be mentioned that this case took place during the latter stages of World War II when sodium lauryl sulfate was rather difficult to obtain and was thus the reason for the manufacturer accepting returned goods. Bacteriological problems in the cosmetic field have become more nu- merous with the extensive use of modern emulsifiers. It does not appear to make too much difference whether they are anionic, cationic or nonionic. The type of bacteria most commonly encountered are: Pseudornonas aeru- ginosa, Pseudomonas fluorescens, .4erobacter aerogenes and the coliform type. I mentioned that I would outline to you the procedure found to be quite efficient in determining whether or not a product is adequately pre- served. These tests require from ten to fourteen days. It should be pointed out, however, that no test has ever been devised that is infallible in respect to showing whether a product is absolutely free from invasion of microbrganisms. The best one can hope for is that his product is adequatel y preserved against most of the common micro Srganisms likely to be encountered. Each cosmetic formulation should be examined separately for its preserva- tion properties. Just because a preservative gives excellent results in one product does not mean that similar results will be obtained in a different formulation. One can liken preservation work to formulating emulsions. If a certain emulsifier, or mixtures of emulsifiers, produces an excellent, stable product, this is no reason why one can expect the same to hold true in other formula- tions. In such instances, various emulsifiers and their combinations are investigated in order to obtain the one best suited for producing the prod- uct desired. The same holds true when it comes to preserving cosmetic products. Bactericidal and bacteriostatic potencies of the preservative must be con- sidered in combination with color, odor, irritation, solubility and compati- bility. If a single preservative does not produce satisfactory results, then