404 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS when endeavoring to duplicate zone sizes or find end-points of bacterio- stasis by the various dilution pro- cedures. It is recognized that this simpli- fied procedure does not determine directly the exact concentration of "M" which is the final limiting bacteriostatic concentration, al- though this figure can be obtained by indirect methods if it is ever deemed necessary to have it. The amount of medication in the solu- tion into which the filter paper is dipped is not necessarily the same as that present in the seeded agar in the area where bacteriostasis is going on. Variations in the pick-up value of the paper, .its retentive power for the chemical, the diffusion power of the material when placed on the agar, the lag phase of the organism, etc., all enter in to affect the end result. If one so wishes, however, these factors can be de- termined, with a good degree of ac- curacy, by weighing the filter paper before and after dipping into the solution, for the pick-up value, then by analyzing the paper for its chem- ical content before and after it has been on the agar for the incubation period, followed by calculating the volume of the agar in which the re- duction of count occurs. From such calculations one can obtain very close to what is the actual bacterio- static concentration of the chemical in question. However, experi- mental evidence, as shown in the table of data to follow, shows quite conclusively that the differences be- tween the concentration as present in the solution into which the paper is dipped before placing on the agar and that producing the bacterio- static effect in the agar are not sufFi- ciently great to make a material difference for the purpose at hand. This assumption is based upon the fact that these dilutions so found parallel so closely those as f•)und by the commonly used dilution method, and the closeness with which one can duplicate a result is approxi- mately the same for either method. Furthermore, as mentioned above, the lag-phase phenomena of bac- terial growth plays an important role in determining the actual zone size one obtains when using the standard zone-of-inhibition test. The extent of this lag phase is in- fluenced by, and thus varies with, factors that are not related to the bacteriostatic phenomena. Thus, the zone size is not a direct criterion or a relative variable of the bacterio- static power of the substance being tested by the zone method. However, in our Zone-Reduction test method this variable is elim- inated. The end point read is' that which represents the concen- tration of the chemical (i.e., mole- cules, M,) that just neutralizes a definite number of molecules of bacteria (B). The lag-phase phe- nomenon does not interfere with this reaction, as it is immaterial as to how long it takes for the bacteria to overcome local resistance effects and get started growing, or how far the chemical may diffuse due to solubility influences in the vehicle (including both the agar at•d the

DETERMINING BACTERIOSTATIC POTENCY OF CHEMICALS Chart $.--Bacteriostatic Dilutions 405 "Dilution" Chemical "Zone-reduction" Method Method Phenylmercuric benzoate 1-5,000,000 to 1-10,000,000 1-10,000,000 Gentian Violet 1-2,000,000 to 1-5,000,000 1-5,000,000 Mercuric Chloride 1-500,000 to 1-1,000,000 1-1,000,000 Quaternary ammonium cpd. 1-500,000 to 1-1,000,000 1-1,000,000 Hexachlorophene 1-500,000 to 1-1,000,000 1-1,000,000 Acriflavine 1-300,000 to 1-600,000 1-500,000 8-Hydroxyquinolin sulfate 1-50,000 to 1-100,000 1-100,000 Hexylresorcinol 1-20,000 to 1-50,000 1-20,000 Chlorothymol 1-5,000 to 1-10,000 1-10,000 Phenol 1-20 to 1-50 1-200 cream, or whatever you are testing). ii?:i: The regulating factor, which de- the end-point (i.e., the bac- teriostatic dilution) is the relative •:• concentrations of the two com- ponent parts of the chemical teac- :!? tion involved (M-+-B) and not the point in a special artificial medium (agar) where the "M" and the "B" i:11::,.(chemical and bacteria) meet to react. SUMMARY AND CONCLUSIONS . : '!!7111 It has been the endeavor in prepar- ii:11:11' ing this paper to present first a brief over-all description of the phenom- ena of bacteriostasis, how it acts, how it may be made use of in the field of cosmetics, and how its rela- tive efficiencies and specific values can be determined. Finally we have presented a new method, simple to perform, usable for determining the correlative, as well as actual, bac- teriostatic potencies of chemicals. BIBLIOGRAPHY' (1) Cade, A. R., Soap and Sanitary Chem- icals, 111 (February, 1944) Ibid., 138 (April, 1947) y. oeact., 53,504 (1947). (2) Klarmann, E.G., and Wright, E. S., Soap and Sanitary Chemicals, 21, No. 1, 113, (1945) 22, No. 1, 125, (1946) and 22, No. 8, 139, (1946). (3) Stuart, L. S. Ibid., 135 (September, 1947) y. Assoc. Off. Agr. Chem., 401, (1949). (4) Cade, A. 12., Soap and Sanitary Chemi- cals (April, 1947). (5) Cade, A. R., 2•roc. Sci. Section Toilet- Goods Assoc., 16 (May, 1947) Ibid., 31 (May, 1948).