COSMETIC PRESERVATION 205 likelihood of selecting organisms without the resistant factor is high when using tra- ditional "streak for isolation" pure culture concepts. Instead, one should rely on assess- ing the purity of the population by its homogenous appearance on a lawned agar plate. Preferably this should be done on a medium that has the preservative in an active state (not neutralized) incorporated into the agar. Once the population is grown up as a lawn, the entire lawn should be harvested for freezing or lyophilization. An area needing more research is the effect of growing the inoculum in broth or on solid media. Greater resistance to preserved product has been described for broth-grown cultures compared to cultures grown on solid medium. However, this result may have been due to the carryover of broth into the product acting as a neutralizing agent of the preservative in the product rather than to any intrinsic resistance gained by the bacteria by growing in broth (63,64). Another area needing further research is the investigation of the importance of the growth phase of the challenge inoculum. The growth phase affects the physiological state of the organisms used as the inoculum. For example, Holm-Hansen found that ATP per cell is decreased as cells reach stationary phase (65). This physiological change and potentially other changes may affect an organism's resistance to preservatives. Inocu/um.' Concentration and recha//enge. In the CTFA's PET, the recommended inoculum level for bacteria is 1 x 108 colony-forming units per milliliter (CFU/ml). If 20 grams of product are inoculated with 0.2 ml (a 100:1 ratio), then the final CTFA recom- mended concentration of 1 x 106 colony-forming units per gram (CFU/g) of product is obtained. Other PETs may use different inoculum levels. The key issue is to keep the dilution of product by the inoculum to a minimum a good rule is to not dilute the product over 1% with the inoculum. In like manner, fungi and yeast are introduced into the product. However, their concentration is only 1 X 10 4 CFU/g of product in the CTFA method. The assumption that these counts represent fungal spores may not be valid since hyphae can also give rise to fungal colonies. How to standardize the concentration of the inoculum is left up to the microbiologist in the CTFA method. A transmittance of 30-40% at 425 nm of bacteria suspended in buffer will usually yield 1.0 x 108 CFU/ml. However, any reference to standard microbiological methods will provide the specifics for determining microbial concen- trations (66). Rechallenge is the addition of fresh inoculum to a product that has already killed off the first challenge after an appropriate time. CTFA provides for a rechallenge if desired but does not require it. Some studies suggest this practice does not provide any more information than single challenges (67). The manufacturer, however, may be able to make a case for multiple challenges. For example, mascaras are commonly subjected to repeat insults by the consumer. In this case, the microbiologist should select a challenge level that is reasonable and likely from consumer use (1 x 102 CFU/gm) rather than the high levels recommended in the compendial methods. These levels could be determined by allowing people to use unpreserved products and analyzing the level of organisms introduced into the product after that use. Another area of concern regarding the inoculum is whether or not to use pure or mixed challenges. This question refers to the use of several pure cultures that are mixed together after they were grown up and harvested. Use of this mixed inoculum may be more representative of actual conditions of contamination since microorganisms do not

206 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS exist as pure cultures in nature but as interacting populations within communities of microorganisms. If one assumes that co-metabolism or synergism occurs within a com- munity biology dynamic, mixed cultures may provide greater stress to the preservative system than pure challenges (68). In fact, cometabolism and vitamin and cofactor synthesis help stimulate mixed interactions within communities of microorganisms (69,70). The idea that such interactions occur during a PET is supported by the observations of Henriette et al. (71), who described a mixed-bacterial community that developed in disinfectants and antibiotics. None of the individual species were resistant to the antimicrobials. Only the community showed resistance. In contrast to the above, however, it is the idea that mixed populations are more robust that forms one of the objections to their use. The claim is that it introduces the variable of microbial population dynamics into the challenge test. Alternatively, some feel that the mixed cultures may be less stringent than pure challenges because one organism may produce metabolic factors that are antagonistic against other microorganisms in the challenge (72) or that the organisms will compete with each other for limiting substrates and growth factors such as iron (73). Resolution of the issue will take more research. Plate counts and other assumptions. There are two assumptions that microbiologists make that are false regarding plate counts . . . and yet we still rely on them: 1) one organism gives rise to one colony, and 2) organisms are evenly distributed as single cells and do not exist as clumps. A new paradigm of organisms existing as nonuniformly distributed clumps that later break up into individual cells may help to explain the anomalous results one occasionally gets in preservative efficacy testing. It must be emphasized that the following is only a model as it applies to preservative testing. It is, however, a valid model since it is based on a historically well known fact that organisms do exist predominantly in clumps rather than as single individuals, even in shake flask cultures (74,75). It is also based on reports about the clumping nature of bacteria due to hydrophobicity (76) and on the newer reports about biofilm and aggregate formation, particularly when exposed to biocides (77). The following enigmatic scenario is sometimes seen during a PET. An initial kill occurs at 7 days (seen as a decrease in CFU) but is followed by an increase in CFU at 14 days, followed by another decrease at 21 days. Usually this is passed off as experimental error such as use of the wrong culture conditions or recovery system or incorrect dilution/ pipetting techniques. Occasionally one gets these results despite controlling all these factors. When this happens, the experimenter may pass off the result as an anomaly of biological systems. However, all these explanations assume that a CFU comes from single organisms that are evenly dispersed throughout the sample. Let's explore the new paradigm that provides at least a hypothetical model that may help explain these results better. Most people working with bacteria exposed to disinfectants and antibiotics are very well aware that bacteria do not exist as uniformly distributed individuals but as biofilms and as Poisson-distributed or negative binomial-distributed clumps or aggregates (66,78-80). If one uses the paradigm of microbes existing in aggregates (or clumps), the enigma may be explained without having to claim "exper- imental error" (Figure 1). The initial kill at 7 days may have been due to killing of the cells in smaller clumps, where the entire clump of cells is killed but the larger clumps have a few cells within them that remain alive because they were protected. Our model is that CFU are really derived from clumps rather than individual cells. The surviving