STRUCTURE OF HUMAN SCALP HAIR−II 21 female and male, respectively. Although it is not mentioned herein, various other persons also donated their scalp hairs to the present study. All of the fi bers were straight, neither being stained with dyes nor subjected to any permanent setting processes. The fi bers were succes- sively washed with a 1.5% (w/w) aqueous solution of sodium dodecyl sulfate (SDS), deionized water, and 70% (v/v) ethanol and then stored at 4°C in a sealed plastic container. The reagents including an anthocyanin dye (from purple sweet potato a mixture of cyanidin acylglycoside and peonidin acylglycoside Kiriyachemi, Osaka, Japan) and purifi ed papain powder from Carica papaya (Wako Pure Chemical Industries, Osaka, Japan) were commercially available. MICROSCOPE OBSERVATION The biological microscope, Olympus, Tokyo, Japan model Vanox (AH), was used mainly for bright fi eld, phase contrast, and polarized light observations. The inverted microscope and the stereomicroscope, Olympus model CK30 and X-Tr, respectively, were modifi ed so as to place the observation chambers and the electrode cells (vide infra) on the micro- scopes. The microscopes were equipped with a digital camera, which was automatically controlled by a desktop computer to optimize for lighting, ISO levels, and focusing mea- sures. The images in JPEG and RAW formats were developed by means of Lightroom ver. 3 and Photoshop Elements ver. 9 software (Adobe Systems Inc., San Jose, CA). Image en- hancement included color level correction, noise reduction, and contrast and brightness adjustments the purpose of the enhancement was to make the image nearly identical to that seen actually by the observers. In the case of thick specimens, the images taken at various depths of fi eld were merged into a single deep focus picture by the use of the stacking software, Combine ZP (A. Hadley, Sheffi eld, United Kingdom) (17). SPECIMEN PREPARATION FOR STRUCTURAL ANALYSIS OF MEDULLA Enzymatic thinning of hair fi bers using the papain (a representative procedure). Several strands of the hair (jf8, about 15 mm in length) were warmed in pH 7/0.067 M phosphate buffer solution (PBS) at ambient temperature for 24 h. After washing briefl y with water and drying at ambient temperature, the fi bers, usually 1–3 strands, were placed parallel on a glass plate (22 × 22 × 0.17 mm) and both ends of each fi ber were glued to the plate with a rapid type–epoxy resin see Figure 2(A). Next, the glass plate having the fi bers was fi xed by the use of the epoxy resin on the window frame (20 × 20 mm) which had been made in the bottom of a plastic culture dish (diameter, 33 mm depth, 10 mm). After washing with PBS overnight at ambient temperature, the papain solution (20 units in 3 mL of PBS) containing its activator of SDS (12 mg) and 2-mercaptoethanol (ME) (65 μL) was put into the dish, and the digestion of the fi bers was allowed to proceed at 30°C. The inverted microscope was used to observe the morphological change of the fi bers. Random mechanical cutting of enzymatically thinned hair fi bers (a representative procedure). Sev- eral strands of the white fi bers (jf65 about 15 mm in length) were treated by dipping in PBS at ambient temperature for 24 h, and then warmed in the PBS solution (3 mL) of papain (20 units), SDS (12 mg), and ME (65 μL) at 30°C with occasional gentle shaking. After about 3 h, a new crop (10 units) of the enzyme was added to the reaction mixture. During incubation, one strand of the fi bers was periodically sucked into water (1 mL)

JOURNAL OF COSMETIC SCIENCE 22 using a pipette, then randomly cut with rapidly rotating poly(ethylene) blades in a manner similar to that mentioned previously (1). The resulting fragments were collected by means of a centrifuge, stained with a Giemsa’s solution (Wako Pure Chemical Industries, Osaka, Japan) (18), washed briefl y with a 50% (w/w) aqueous solution of glycerol, and then mounted to a microscope slide glass. A weight (30 g/cm2) was placed on the cover glass (grade: No. 1 Matsunami Glass, Osaka, Japan) while sealing the edges with Canada balsam. Mechanical slicing of a hair fi ber. Several strands of hair (cf16) were dried and embedded in an epoxy resin (Poly/Bed 812) according to the protocol of PolySciences (Warrington, PA). The solidifi ed resin block was sectioned to about 8 μm thickness by means of the microtome (American Optical, Buffalo, NY model 820) and stained with a 0.5% (w/v) aqueous solution of gentian violet. The microscope specimen was prepared in a man- ner similar to that described previously. MATERIAL FLOW PROPERTY OF MEDULLA H+ ion-fl ow through the medulla: the use of Congo red as an indicator. The hair fi bers (jf8, about 25 mm in length) were kept in an aqueous solution (5 mL) of 7 M urea, 2% (w/v) SDS, and 15% (v/v) ME at about 55°C for 20 min.1 The resulting fi bers were washed briefl y with water and dipped in a 0.025% (w/v) aqueous solution of Congo red (5 mL) for 1 d at ambient temperature. Three strands of the wholly stained hair were then placed, each in a U-letter shape, on the glass plate (22 × 22 × 0.17 mm) which had been glued with the epoxy resin to the window hole made in the bottom of a plastic Petrie chamber Figure 2. The o bservation chambers for the material fl ow study of the M. The type (A) was used for the in- verted microscope, whereas the type (B) was mainly employed for the upright microscope. 1 Treatments of hair and wool fi bers with the medium containing ME for long time at higher temperature was previously reported (19, 20). In “H+ ion-fl ow through the M: The use of Congo red as an indicator” process, the hair fi bers were softened by soaking in the presence of ME for a short time of period to wholly stain the fi ber body with Cong red. During the staining period, the swollen shafts were nearly brought back to the dimensions of the starting unheated wet fi bers. Although keratins, the main proteins of hair, might be structurally disturbed to some degree in the dyeing process, H+ ions was found to fl ow preferentially through the medulla. The “OH--fl ow through the M: the use of phenolphthalein as an indicator” process, on the other hand, did not use ME in the fl ow study of OH- ions.