Abstract
In the preliminary examinations it was proved that epithelial cells stained positively by gram staining coincided with the cells rich in sulfhydryl group, detectable by means of Chèvremond and Fréderic technique (Fig. 1 In Japanese original). And maj ority of gram negative cells were deficient in sulfhydryl group (thus less keratinized) and more keratinized gram negative cells (Weinmann's F cell) appeared in exceptional number on the smear slides from the palate (Table 1 and Fig. 2). Based on these findings, a quantitative method to enable the estimation of the keratinization were developed from gram staining of the oral specimen. This method was adopted on some oral problems in which were often interested by several investigators in the past because of that the keratinization of oral mucosa is one of the most important sign of the general and local state of the body as well as the skin is so. The method did not provide only the quantitative estimation of oral keratinization, but also reduced the error orignating from different examiners.
The procedure was practiced in following manner.
1. Make smear by scraping taken from the mucosa on full of quarter area of a slide.
2. After drying, it was stained by standardized gram staining, without any previous fixation.
a. Hucker's christal violet, kept at 30°C, was applied for three mins. Dye was washed away in water bath for one min. Blotting dry.
b. Put the slide in the bath of Lugol's solution, kept at 30°C, for three mins. Wash and blotting dry.
c. Decolorize in ethanol bath, kept at 30°C, for two mins. Then ethanol was washed away in the water bath. Blotting dry.
d. Stain with one percent bismark brown, prepared by phosphate buffer pH 5.85, kept at 30°C, for three mins. Wash and then blotting dry.
e. Decolorize with aceton, dropping on the slide.
f. Read concentrations of christal violet and bismark brown at the wave length 575 mμ and 430 mμ respectively.
Degree of keratinization of the specimen was expressed by the antilog of the quotient of both extinctions on 575mμ and 430mμ, because the ratios of several mixtures of gram positive Candida species culture and gram negative Eschericia coli culture are proportional to the antilog quotients of extinctions of these mixtures (Fig. 3).
Forty one young female with healthy gum, aged from 19 to 21 years old, were examined on the several respects of keratinization of their oral mucosa. Degree of keratinization, estimated by the new method, are not the same at the different area of the mouth and its order are hard palate, mandibular anterior attached gingiva, maxillar anterior attached gingiva, tongue and cheek, and their average degrees and mean errors were 22.9±1.15, 13.54±1.11, 9.76± 1.13, 9.76±1.12, and 3.39±1.09 respectively (Fig. 4 and Table 2). It was also found large deviation among individuals.
Rise of keratinization at the hard palate was very obvious at the middle phase (ovulation) of sexual cycle (Table 3 and Fig. 5) and it proceeded parallel with bulk of desquamated epithelium recovered in water gargled the mouth (Table 4 and Fig. 7).
Similar sexual change of keratinization on the cheek did not observed (Table 3). However, it was considered that inaccuracy of the present method could not detect slighter change on the cheek because of another fact that keratinization of the cheek related to that of palatal epithelium (Fig. 6).
Brushing with a tooth brush on the cheek did not cause any change in its keratinization (Table 5).
