• DEGERMING EFFICIENCY OF HEXACHLOROPHENE SOAPS 283 Procedure 1. Use at least three individuals as subjects per test. Keep clearly in mind the fact that two distinct types of soap are being used in this test. One is the hexachlorophene or medicated soap employed daily for the two weeks' test period to bring about the desired degerming effects. The other is plain soap used where the wash water is collected in the various basins for determining the counts of the bacteria removed. These latter applications of plain soap should not be made until at least two hours has elapsed since the prior use of the medicated soap. 2. Each series of tests should be started on a Monday morning, which is considered as the first day of the washing series. This first washing series is made with plain soap, and only after this has been done does the subject start to use the medicated soap for a period of two weeks, at least three times daily. Bacterial count tests are to be made on four days during the two weeks' period, as described in detail below, washing with blank soap only for this purpose. 3. In the bacterial count or washing test, three basins are used. These are equivalent to the first, fourth, and fifth basins of a "Price series" of hand-washing tests. Enamel or aluminum pans of about 4000 cc. capacity are preferred. Tap water may be used (2000 cc. per basin) provided tests have been made to insure that the water after being placed in the basins (properly cleansed) will contain less than 10 bacteria per cc. before it is used for any washing purposes. Sterile water and sterile basins, of course, would be the ideal but to save time and cost the above is satisfactory for this test, provided counts are made on the water before using and these subtracted from the counts as found after washing, to get the actual counts of the bacteria removed from the hands by the washing process in the basins. 4. The detailed procedure for the washing test follows: (a) Roll sleeves up to elbow. (b) Add 2000 cc. of lukewarm water from the tap into the first basin. Remove 2-1 cc. samples by means of a sterile pipette and plate same in agar for getting the blank bacterial count of the water. (c) Moisten the hands, only up to wristline, then apply blank soap (containing no germicidal material) for fifteen seconds, followed by a thorough lathering and washing for sixty seconds, collecting all of the water in the basin. Rinse off both hands as thoroughly and completely as possible into the basin, allowing fifteen seconds for this process. (d) The hands are then rinsed off lightly in running lukewarm tap water. The latter is discarded as far as collecting for counting purposes is concerned. (e) Two more washings are then carried out successively under the tap, but the water therefrom is not collected or tested in order to save

284 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS time and cost. However, to col- lect these respective washings and to determine the counts thereof, would make the test that much more accurate, since it would natu- rally produce five points for deter- mining the curve instead of three. Also, for these two washings, the area covered should extend three- fourths the way up the arm to the elbow. This is done so as to remove all transient bacteria as well as a part of the resident flora from the area just above the wristline, which line serves as the limiting edge of the normal washing area. By so doing, we simplify the process through eliminating the necessity for wearing rubber or plastic sleeves, strips of adhesive tape, or other marking means as guides to limit the wash area. This modification also makes the washings to follow more accurate by eliminating the danger of adding extraneous bac- teria from the skin at the tape or sleeve edge, which bacteria may have been washed from the skin at that point by the softening action of the detergent solution collected there during the normal washing procedure. It is thus apparent that the bac- terial counts from these two wash- ing procedures, if collected, would constitute data for basins 2 and 3, respectively, of a Price hand-wash- ing test series. (f) Subiect should then. proceed to Basin 4 centaining 2000 cc. of lukewarm 'water and carry out a washing process exactly as was done in the first basin above (para- graphs b, c, and d). (g) Subject then washes in Basin 5, using the same complete proce- dure as was carried out in the pre- ceding basin test. 5. Immediately after subject has finished washing and rinsing in each respective basin, two 1 cc. and two 0.1 cc. samples of the 2000 cc. of wash-water in the basin are removed by the technician and each placed into a separate sterile Petri dish, to which is then added immediately 15-18 cc. of 42-45øC. warm nutrient agar, containing 1 per cent normal horse-serum as an antidote to neu- tralize any possible bacteriostatic effects, mixing thoroughly by rotat- ing, allowing to cool and harden, and incubating same at 37øC. for a period of forty-eight hours. At the end of this time the number of colonies present, indicating the num- ber of bacteria per aliquot removed, are determined by means of a Quebec colony counter. The counts thus obtained are multiplied by 2000 for the 1 cc. samples, and 20,000 for the 0.1 cc. ones to get the total num- bers removed. Before removing the aliquot samples, the 2000 cc. of wash-water should be mixed thoroughly by stir- ring with the sterile pipette to be used for withdrawing first the 0.1 cc. and then the 1.0 cc. samples. Re- member to use a sterile pipette for each basin. 6. Tests as outlined in para- graphs 3 and 4 above should be car- ried out on the first Monday, when the test starts and before the test