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).

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In the fiscal year of year 1962, that is, during the period from April, 1962 to March, 1963, health guidance of three year old infants was made under the Juvenile Welfare Law at all of the health centers in Tokyo City. At that time health guidance charts were filled out, and among these charts, 61, 333 complete charts were selected as the object of study. When the annual average of the infants having dental diseases and the occuring conditions were observed, the following results were obtained.
1. As for the dental caries, the percentage of infants affected was 67.9%. In case the contents are classified as, A Type: Infants having dental caries in the upper jaw front teeth only or upper and lower jaw molar teeth only. B Type: Infants having dental caries in upper and lower jaw molar teeth and upper jaw front teeth. C Type: Infants having dental caries in both the upper and lower jaw front teeth and the upper and lower jaw molar teeth, there were 48.0% of Type A, 38.2% of Type B, and 12.8% of Type C. Furthermore, the average number of teeth affected by caries per infant was 3.3.
2. As for the malocclusion, the percentage of occurrence was 3.0%. If the contents are classified into a. Progenie b. upper protrusion c. open bite d. crowding e. diasthema, the respective percentage was as follows:
a. 66.5% b. 13.4% c. 9.4% d. 5.7% and e. 5.3%
3. As for the soft tissue disease of oral cavity, the percentage of infants affected with it was 1.4%. If the local ones are expressed as L type, and the system ones are expressed as the S type, the percentage of the L type was 86.5% and the S type 13.5%.
4. As for other extraordinary conditions, the occurrence percentage was 0.4%.
5. If each of the above figures are compared with the national average, although the occurrence rate of malocclusion and other extraordinary conditions were rather low, the soft tissue diseases of oral cavity were slightly high.
6. Of all the health centers in Tokyo City, 27.391 health guidance charts were obtained from the health centers which conducted examination on infants which reached three year of age in the month preceding the examination month. When similar observations were made on these data classified by the month of birth, the following results were obtained.
1) As for the number of infants affected with dental diseases and the annual average of occurrence, similar results to those of all health centers were obtained.
2) As for the number of infants affected with dental caries, since the number of births in April, May, September, and October were great, they showed high percentage, and for infants born in June and July, the percentage was very low.
3) As for the occurrence rate of malocclusion, the rate was high for the infants born in March and September, and low for those born in December and February. As for the contents, the majority was held by a. A large number of the infants affected by a, were born in August. Contrary to this, those born in January showed a low rate.
4) As for the infants affected by the soft tissue diseases of the oral cavity, those born in May showed a high percentage, and those born in September showed a low percentage.
5) As for the other extraordinary conditions, the results were similar to those affected by dental caries.
6) By the observations made on the infants classified by the month of birth as described above, the percentage of three year old infants affected by dental diseases and the rate of occurrence was presumed to be closely related with the characteristics of the Japanese climatical environment which is high temperature high humidity in summer, and low temperature low humidity in winter, causing the nutritional condition of the mother to become bad when the said three year old infant is still in an embryonic stage.

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