ESTIMATION OF THE SALIVARY ANTIBACTERIAL ACTIVITY IN TRYPTICASE SOY AGAR BY CUP PLATE METHOD BY Hajime TAKAHATA, Toshio MORIOKA and Toshiharu MATSUMURA The peroxidase antibacterial action in human saliva against species of lactobacilli has been described by Klebanoff and Luebke1), Iwamoto et al.1), Inoue3), Saji, Morioka and Matsumura4) have reported that the antibacterial activity is distributed in certain tissues of rat including parotid, submaxillary, lacrimal, and mammary gland. In each study, however, this antibacterial activity was detected in SL broth5) against lactobacilli. Inoue et al.6) have also reported that the salivary antibacterial (S.A. Factor) activity was obtained in the selected media including Trypticase soy broth by treating with the-sh group reagent, CuSO4. It is conceivable that the salivary peroxidase antibacterial system plays an important role as a natural defensive function in the oral cavity. In order to clarify the defensive function of the antibacterial system, non-selected culture medium such as Trypticase soy other than SL broth should be used obtaining the wide variety of antibacterial spectrum against oral microorganisms. As the growth rate of the oral microorganisms in culture media, in general, is low, an estimation of the growth inhibition by the antibacterial system on the microorganisms is hard to quantify, and the viable cell count of the certain microorganisms such as cariogenic streptococci is also hard to carry out. It is suggested that in these cases the antibacterial activity would be adequately estimated by the cup plate method. In the present work, experiments were carried out to determine whether the antibacterial activity could demonstrate quantitatively in the non-selective medium such as Trypticase soy. The salivary peroxidase antibacterial activity was successfully demonstrated in the cup plate method with Trypticase soy agar containing 8.3 ~ 10-5M CuSO4. Growth of Lactobacillus plantarum ATCC 8014 was apparently inhibited by the antibacterial system, and the growth inhibition is completely depressed by catalase. The cup plate method is strongly suggested to be useful for the quantitative estimation of the salivary peroxidase antibacaterial activity against various species of oral microorganisms. * Department of Preventive Dentistry, Osaka University Dental School, 32 Joancho, Kitaku, Osaka (Prof. T. Matsumura)

Table 2 Formation of growth inhiditory zone in trypticase soy agar dy salivary peroxidase antidacterial spstem Table 1 Effect of CuSO4 on the salivary peroxidase antibacterial activity

Table 3 Relationship between the amount salivary peroxidase and the zone of growth inhibition Conditions were as described in Tab. 1, except that the various amount of salivary peroxidase as indicated in the table were used. Conditions were as described in Tab. 3. Fig.2 Correlation between the amount of salivary peroxidase and the zone of growth inhibition

salivary antibacterial factor (S.A. Factor). Archs. oral Biol. 12:1009-1012, 1967. 4) Saji, S., Morioka, T. and Matsumura, T.: Detection of peroxidase mediated antibacterial activity in the tissue extract of salivary glands and other certain tissues of rat. Abstract of the 16th Annual Session, Japanese Division of International Association for Den- Research. Nov. 16-17, 1968 P.23. tal 5) Rogosa, M., Mitchel, J.A. and Weiseman, R.F.: A selective medium for the isolation and enumeration of oral lactobacilli. J. dent. Res. 30:682-689, 1951. 6) Inoue, M., Saji, S., Hamada, K., Iwamoto, Y., Morioka, T. and Matsumura, T.: Regeneration of the activity of the salivary antibacterial factor (S.A. Factor) in selected media. Archs. oral Biol. 12:167-169, 1967. 7) Iwamoto, Y. and Matsumura, T.: Purification and characterization of the salivary antibacterial factor (S.A. Factor). Archs. oral Biol 11:667-676, 1966. 8) Matsumura, T., Morioka, T., Onishi, T.and Iwamto, Y.: Studies on anti-lactobacillary factor in human saliva. III. Relationship between active saliva and concentration of bacterial suspension. J. Osaka Univ. Dent. School., 1:31-37, 1961. 9) Matsumura, T., Morioka, T., Onishi, T. and Go, S.: Studies on antilactobacillary factor in human saliva. Dent. Bull. Osaka Univ. 1:3-11, 1960. 10) Aldridge, W.N.: Cynogen bromide-pyridine benzidine method for thiocyanate determination. Analyst. 69:262, 1944. 1) Klebanoff, S.J. and Lueke, R.G.: The antilactobacillus system of saliva. Role of salivary peroxidase. Proc. Soc. expl. Biol. Med. 118:483-486, 1965. 2) Iwamoto, Y., Inoue, M., Tsunemitsu, A. and Matsumura, T.: Some properties of the ) Oram, J.D. and Reiter, 11B.: The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. Biochem. J. 100: 373-381, 1966.

Conditions were as described in Table 1 and 2. Fig. 1. Inhibition zone in trypticase soy agar of lactobacillus growth developed by the salivary peroxidase antibacterial system