METHOD FOR DANDRUFF SCALING 181 SCALE COLLECT ION BY SHAMPOOING •/• / II •• U / .' /.I ,,, ,,, ••Sink / B ,'','', ' .... ß .'. ',,. • , •'.'. X. ,' ' ,',' • Suspension of Scales X' ,' ,',',/ X' ' ' ' ß / Figure 1. Sink method for collecting scales. leading to a stainless steel screen whose pores were approximately 200 ix in diameter, trapping the larger scales. The residual fluid was similarly funneled through a nylon screen with a pore size of 100 ix, trapping small scales. A 100-ml aliquot of the residual fluid which had passed both screens was then filtered under pressure through an 8-.ix Millipore filter (Millipore system-PS 1), thereby collecting the single corneocytes. GRAVIMETRIC MEASUREMENT OF SCALES The first two screens were rinsed with water and dried overnight at 50øC. The scales on each screen were then scraped onto papers and weighed. CORNEOCYTE COUNTS The 8ix-Millipore filter was removed and put into a tube containing 10 ml of 0.1% Triton-X 100 phosphate buffer, pH 7.9, which was then vortexed for one minute. 5 ixl of Multiple Stain (Polysciences, Inc., PA) was added to a 50-ixl aliquot, and single corneocytes were counted in a Fuchs-Rosenthal hemocytometer. Dense suspensions may have to be diluted further to facilitate counting. The total single cell count per scalp was then calculated.

182 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS FILTRATION PROCEDURE Used waters (4 to 9 liters) (200 p x 200 F) I II !l ! I II I! ß LARGE SCALES (lOOp x 100 p) [ Aliquots (100 ml) SMALL SCALES ß (8 i • Millipore) ,---" • SINGLE CELLS Figure 2. Separation of large scales, small scales, and single cells by successive filtration. THE PARAKERATOTIC INDEX Estimating the percentage of nucleated cells required disaggregation of large and small scales by means of an ultrasonicator (Bransonic 2000), which produces a suspension of single corneocytes (Figures 3 and 4). About 5 mg of dried scales were placed into tubes containing 2 ml of the above Triton buffer. The tube was then sonicated for 20 minutes and centrifuged at 2500 rpm for five minutes, discarding the supernatant. 50 !xl of Triton buffer and 50 !xl of Giemsa stain (Fisher Scientific) were added to the sediment, with mixing. A smear of the suspension was made on a glass slide and the proportion of nucleated cells determined microscopically, counting a minimum of 500 cells. Cells collected on the 8-Ix Millipore filter were similarly treated in order to count nucleated cells. RESULTS AND DISCUSSION THOROUGHNESS OF SCALE REMOVAL When large and small pooled scales were collected in six successive shampooings, it was evident that practically all the scales were removed in the first two washings (Figure 5). In non-dandruff subjects (grades l and 2), scale mass was really trivial, amounting to a few milligrams in contrast to dandruff(grades 3, 4, and 5) in which the average amount was about 25 mg.