When the human whole saliva is fractionated by centrifugation into supernatant and sediment, the latter contains oral bacteria in addition to other insoluble materials. The respiration and lactic acid fermentation of the sediment are enhanced by the addition of the supernatant, and the substance responsible for this stimulatory effect was called the metabolic factor by Hartles and others. Although studies on the chemical nature of the factor have revealed that it may not be a single chemical compound but is probably active as a multicomponent system consisting of proteins, peptides and amino acids, the reaction mechanism and detailed chemical nature of the components remain unclear. On the other hand, the assay of the metabolic factor has been carried out manometrically by means of Warburg thechnique using the salivary sediment as the source of bacteria to determine the oxygen consumption directly and the lactate production indirectly as CO₂ evolved from a CO₂-bicarbonate buffer. This method, however, is not suitable for assaying a large number of samples for a short period because of its rather complicated and time-consuming procedures. In addition, the use of salivary sediment as the source of bacteria does not always give reproducible results.
In the present study, methods were explored which can be used in a mass examination. The substances in the salivary supernatant were fractionated on a column of Sephadex gel, and each fraction was assayed by the method described above for the stimulatory activity in organic acid production. The fractions with a high activity were combined and used as the fraction of the metabolic factor. The active fraction was found to promote the initial growth of several strains of bacteria tested, particularly that of Streptococcus lactis. Thus, this strain was selected and used as the test material instead of the salivary sediment. The activity of the metabolic factor was then assayed by a turbidometry, a titration method and by a pulpdisk method on agarplate. The experimental conditions were studied for a pulpdisk method.
All the three methods examined were found to be satisfactory. Among these pulpdisk method was fonud to be better than the other methods. It was concluded that the pulp-disk method established may be used routinely for the purpose of examination in a large scale.

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on of its distribution in individuals, it is prerequisite to clean the surface for biopsy removing extrinsic deposit. However, damage or alteration of the enamel surface due to abrasion should be avoided.
As series of the determination of the acid resistance of the surface enamel, this study aimed at examining the influence of cleaning the tooth surface with abrasives and at refinements of the method without difficult procedures in order to evaluate the acid resistance of the surface enamel in vivo.
For abrasion of the tooth surface, several abrasives were employed in this study, i. e. cleaning the tooth surface with a polishing rush. singly or by polishing with pumice, zirconium silicate, and tooth paste based on calcium carbonate or dicalcium phosphate.
Observations were made on the degree and presence of the abraded surface of the enamel using a Vickers microhardness tester. The effects of cleaning by using the abrasives mentioned above in relation to calcium and phosphorus dissolved from the labial surface of upper central humann incisor were examined following successive application of agar plate (pH 4.0) for each 3 minutes.
Some observations were made on the morphological changes of the surface of the enamel using a electron microscope.
As results of these experiments, it was found that quantitative alteration of the surface abraded could be recognized. The depths abrased from the surface were 3-5 μ in the case of zirconium silicate abrasive and 2-3 μ in the case of pumice. Cleaning with these abrasives produced the alteration of the tooth surface increasing the dissolution of phosphorus and calcium. The results showed that tooth paste of calcium carbonate base was to be recommended for the agar plate method. Furthermore, from the results of a clinical study with the agar plate method, a suitable method can be developed to evaluate the weakness and strength of the acid resistance of the individual and to employ the determination of the enamel solubility at a lower pH than that of the agar plate method with pH 4.0.
Alternative procedures using non-woven paper plates were developed by the author. The non-woven paper plates consists of two-ply plate which punched of 6mm in diameter. The plate was moistened with 0.05ml of 0.1M lactate buffer solution of pH 2.5 and applied to the labial surface of upper central incisor for 3 minutes. The determinations were done on the phosphorus dissolved from the surface enamel of 30 subjects of 6th grade children.
The results showed that the distribution pattern of phosphorus extracted by means of the nonwoven paper plate method revealed a nearly normal curve.
It is concluded that the non-woven paper plate method is promising for application as a suitable procedure in vivo to evaluate the acid resistance of the surface enamel with high accuracy and without difficulty.

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It has been reported in our laboratory that Streptococcus mutans strain BHT formed bacterial deposits on human enamel after cultivation at 37°C for 4 days in 5% sucrose broth and that by the treatment of the artificial plaque with the bacteriolytic enzyme derived from Streptomyces glovisporus strain 1829, strain BHT cells in the plaque were lyzed into the string form and the membranous bands which intersected with each other and with the fibrils protruduing from the cell peripheries, and constructed filamentous networks. The purposes of the present manuscript were to demonstrate that the residual networks could be removed by the treatment with various kinds of enzymes such as dextranase and lysozyme, and to obtain a clue for clarifying the nature of the lyzate which persisted on the enamel surface.
The artificial plaque on enamel piece was incubated with the bacteriolytic enzyme in vitro under the same conditions described elsewhere. After the residual networks were further treated for 1 hour with dextranse from Spicaria violaceae, a coarse networks intersected with membranous bands with a clear outline appeared and remained on the enamel surfaces, whereas the fine network formed by the fibrils and the string virtually disappeared. Intact cells were also observed to be reduced in number. On the contrary, when the artificial bacterial deposition was incubated for 1hour with the lytic enzyme and egg-white lysozyme simultaneously, the membranous bands and intact cells disappeared and a monolayer of relatively fine network structure formed by the fibrils and the string still remained. Furthermore the artificial plaque was almost completely removed from the tooth surface after the treatment with those three enzymes. Dextranase P, trypsin and nucleases had no effect on either the nontreated control or the lytic enzyme-treated plaque.
Based on the results obtained, the mechanisms of the plaque-eliminating ability of lysozyme and dextranase as the synergist with the bacteriolytic enzyme were discussed in relatiot to the natures of the filamentous and membranous components which constructed the residual networks after the lytic enzyme treatment of the artificial plaque.

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It has been shown that a bacterolytic enzyme derived from Streptomyces globisoporus strain 1829 was able to lyze rapidly glucose-grown cells of many odontopathic plaque microorganism and that the enzyme inhibited the formation of human dental plaque in vivo. The present report described the effects of the bacteriolytic enzyme on the plaque-like deposits produced on human enamel pieces by cariogenic plaque bacteria cultivated in Brain Heart Infusion broth supplemented with 5% sucrose.
The susceptibilities to the bacteriolytic enzyme of the sucrose-grown cells were estimated by following the reduction in optical density at 550_nm, of the cell suspensions. The extents of lysis of the bacterial strains tested were in the following order from high to low: Streptococcus mutans BHT>Lactobacillus casei ATCC 4646>Streptococcus sanguis 10557>Actinomyces viscosus 15987 (T₉) >>S. mutans AHT≈IB≈P4≈B14≈S. salivarius HHT>>>S. mutans K1R. The sucrose-grown cells of each strain were less sensitive than their glucose-grown cells.
All streptococcal strains except S. sanguis formed plaque-like deposits on the enamel pieces after cultivation in the 5% sucrose broth at 37°C for 4 days. The artificial plaque forming ability of strain K1R was show to be the most marked and that of strain BHT the least, and those of the other five strains were intermediate between the two strains. S. sanguis, L. casei and A. viscosus failed to produce “atrificial plaque”, and only a few bacterial cells were scattered on the enamels surface.
The microbial deposits were treated at 37°C for 60 min. with the bacteriolytic enzyme, freeze-dried and examined with a scanning electron microscope. The bacterial cells in the plaque were lyzed on the enamel surface in situ and losing their original forms became amorphous masses, membranous strings of fibrils which intersected each other and formed a network or honey-comb and sponge-like caves. The intensity of the morphological changes of a plaque was roughly consistent with the susceptibility to the enzyme of the strain which formed the plaque.

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Three different agents were added to dentifrice used daily for the prevention and treatment of periodontal diseases and the effects of the three dentifrices were evaluated.
Healthy adult women were selected as subjects. Twenty were assigned to a group using a dentifrice containing Tranexamic acid, 19 to a group using a dentifrice containing stearylglycyrrhetinate, and the remaining 20 to a group using a dentifrice containing α-amylase. They were instructed to apply each dentifrice with a toothbrush for one month continually. All subjects received 5 examinations of the periodontal tissue at intervals of 7 days and changes of subjective and objective conditions were observed at each time.
The following results were obtained: the dentifrice containing Tranexamic acid was effective in the reduction of gingival inflammation, in the regression of gingival bleeding and swelling and tooth mobility; the dentifrice containing Stearylglycyrrhetinate was effective in the removal of bad breath and oral ill feeling; the dentifrice containing α-amylase, however, was not significantly effective, compared to the other two dentifrices.
Therefore, Tranexamic acid was encouraging when added to the dentifrice as an agent for preventing gingival diseases, and so with Stearylglycyrrhetinate for removing bad breath.

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It has been reported in our laboratory that a bacteriolytic enzyme fromStreptomyces globisporusstrain 1829 possessed marked bacteriolytic activity against various strains of cariogenic plaque bacteria.
The purpose of the present investigation was to determine the effects of the enzyme on plaque accumulation, caries development and alveolar bone resorption in hamsters infected withStreptococcus mutans.
An NIH (U.S.A.) strain of golden hamsters weighing around 50gr. was infected orally with the Brain Heart Infusion broth cultures of streptomycin-resistant strains ofS. mutansAHT-R or K1-R and fed cariogenic Diet it 2000 with or without addition at 0.025 or 0.1% (w/w) of the bacteriolytic enzyme. After rearing on the diets for 34 days (AHT-R) or 30 days (K1-R), the animals wete sacrificed and the extents of plaque, caries and alveolar bone loss of the molar dentition were estimated.
When the hamsters inoculated with strain AHT-R or K1-R were provided with Diet it # 2000 without the enzyme, a large amount of microbial plaques were deposited around the molars, and rampant dental caries and alveolar bone resorption were extensively developed. The extent of the carious lesions of the molars were in the following order: M₁<M₂<M₃. Neither plaque formation not the pathologic changes of tooth and alveolar bone could be observed in the non-infected control hamsters.
By addition of the bacteriolytic enzyme to the diet at 0.1%, the plaque formation was reduced, and the caries development and the bone resorption were inhibited with statistical significance, but this suppression was not observed in the case of supplement with 0.025% enzyme.

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The bacterial population in the dentogingival area under a calculus splint was examined.
Streptococcus salivarius with urease activity decreased and granular organisms (A. viscosus) increased significantly.
The dry weight of the deposition decreased significantly.
There was no viable count of cariogenic bacteria, Streptococcus mutans either before or after wearing the calculus splint.
Therefore, although the glicolitic bacteria increased when the calculus splint was worn and the local PHdecreased, there is no fear of suffering from dental caries.
The ratio of Streptococcus mutans to total Streptococcus was measured at various times after a 10% glucose rinse.
Streptococcus mutans could be counted only rarely on the labio-lingual surface of the anterior teeth, but in the pits and fissures of the molars, the maximum ratio was measured between 5 and 15 minutes after rinsing after 30 minutes there was a sharp decrease and after 60 minutes the ratio was gradually returning to the initial ratio.

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In an attempt to evaluate the acidresistance of surface enamel by the biopsy method, the effects of pre-test food uptake and the use of SnF₂dentifrice were investigated.
The biopsy method used was as follows: after the labial surface of the upper central incisor was polished with common dentifrice prepared with calcium carbonate base, an agar plate, impregnated with 0.1M lactate buffer solution of pH 4.0, of 2mm in thickness and 6.5 mm in diameter, was placed on the central part of it for 3 minutes for conditioning. A second agar plate was placed on it for 3 minutes and then the phosphorus dissolved in the second agar plate was spectrophotometrically determined by the molybdate-safranin method.
In 8 adults, significant increases in phosphorus dissolution were observed after taking corn sirup and caramel (1% level of significance), and mandarin orange (5% level of significance), before the biopsy test as compared with control values. The results suggested that the biopsy test of the acid-resistance of surface enamel should be done before a meal or between meals, but not after a meal.
In a second series of in vivo experiments, the effects of the use of freshly prepared SnF₂dentifrice (1, 000 ppm as F) before the biopsy test were examined in 6 (A group) and 34 children (B group) of an average of 10 years of age. The amount of phosphorus dissolution after the use of SnF₂dentifrice for 3 minutes (A group) was significantly lower than in controls at the 5% level.
When the amount of phosphorus dissolution was determined at the 4th day after a 3 minbrushing with SnF₂dentifrice once a day for 3 days (B group), it was significantly lower at the 1% level. However, since the activity of fluorine ions in dentifrice decreases with time, the data on the use of dentifrice containing fluoride on the biopsy test should be cautiously interpreted.

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As part of the experiments on enamel destruction planned by our study group, microscopical observation was made on the changes of the destructive process in enamel by EDTA in the acidic range, employing the sandwich method devised by Ishii and Yoshida, as outlined below. Sections approximately 30μ thick of undecalcified teeth were cemented between the slide and cover glass using polyvinylmethylether as the adhesive, with exposure of the surface area of enamel alone. The decalcifying solution was then allowed to act on the enamel surface to study the main changes continuously under the microscope. As the decalcifying solution, 0.01 M EDTA 2 Na solution (pH 4.8) was used with a flow rate of about 1 mm/5 min. The process of change occurred was studied under the phase contrast microscope and recorded by 16 m/m automatic microspeed photographic apparatus. In order to compare the pattern of destruction in the inner enamel layer with that in decalcification with organic acid, 0.1 N acetic acid buffer solution, pH 5.0 was used for decalcification by the same method described above.
In both experiments, microradiograms of decalcified specimens were obtained by the contact method.
The following results were obtained.
1) As to the attack entry on the enamel surface, micro-channels of 1-2 pm in diameter, ends of striae of Retzius and some small defects can be cited. These are identical with those already reported in the study on acid decalcification.
2) The spread of destruction was considerably more rapid than that in acid decalcification.
3) The degree of destruction was also much more pronounced than in acid decalcification. Due to this reaction, the prismless layer and striae of Retzius have almost no influence the pattern of destruction. Considerable injury appears to be inflicted on the mechanism of recalcification of enamel.

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