LACTOBACILLUS TEST IN CONTROL OF DENTAL CARIES 81 bacteria in human metabolism, it appears that there are no oral organisms essential for human metabolism. C•A•P. MA•: Are there any other com- ments ? MP.. LEw•s: May I also answer the ques- tion on the "t" correlation of the statistics of this paper. I was privileged to do a sta- tistical study on this particular report and found that the "t" figure between the test and control group was 5.62, a level of signifi- cance of about 1 chance in 100,000 that the results could be similar. There is no statisti- cal question that the difference between the test and control groups is real and significant. (Unfortunately, further discussion was not legible on the wire recm&ng. As a result additional remarks by Drs. Hill and Kesel, as well as the summaries by all three con- tributors to the symposium cannot be printed.--Editor.) THE ROLE OF THE LACTOBACILLUS TEST IN THE CONTROL OF DENTAL CARIES* By PHILIP JAY Professor of Dentistry, School of Dentistry, University of Michigan, /inn/lrbor, Mich. ONE OF THE MOST trouble- some aspects of dental caries re- search is the difficulty of evaluating dental caries activity. Because of its chronic nature, it is necessary to conduct consecutive clinical exam- inations several months apart in order to determine the progress of this disease. It is not possible to recognize caries activity in a single clinical examination. With the greatest scrutiny it is merely pos- sible to determine the extent of tooth destruction which has already, taken place. It is for this reason that.a laboratory test is extremely important. The Lactobacillus counting method has been useful since it has been shown by various investigators that gram positive acid producing rods may be demonstrated on tooth * Presented at the December 5, 1950, Meeting, New Ycrk City. surfaces and in the saliva from six to eighteen months before new carious lesions can be demonstrated clini- cally. As far back as 1907 Goadby, and in 1915 Kligler, associated Lactobacilli with carious lesions. Since that time Mcintosh, Rodri- guez, Bunting, Hadley, Jay, En- right, Arnold, McClure, Becks, and others have shown a diagnostic rela- tionship between the presence of Lactobacilli in the saliva and the de- velopment ofcarious lesions. At the present time the test developed at the University of Michigan is in wide use. Hadley (1) found that a modifica- tion of Kulp's tomato agar produces Lactobacillus colonies which can be differentiated from yeast and vari- ous coccal forms by their colonial characteristics. Most workers have found this method satisfactory, but it must be reproduced in every de- tail.

82 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Two types of media are required: 1 per cent dextrose meat infusion, broth, pH 5.0, and tomato agar, pH 5.0. Preparation of the Broth (1 Liter): 1. Infuse 1 pound of ground lean beef in 1 liter of distilled water in the refrigerator overnight. 2. Skim off the fat. 3. Strain through a cheese cloth. 4. Add 10 gin. of peptone and 5 gin. of sodium chloride. 5. Boil vigorously for fifteen minutes. 6. Strain through a wire gauze strainer. 7. Adjust to pH 5.0 with hydrochloric acid. 8. Boil vigorously for thirty minutes. 9. Add 10 gin. ofdextrose. 10. Cool to room temperature. 11. Filter through paper. 12. Pour into flask and place in autoclave at 120øC., 15 pounds, for twenty minutes. Preparation of Tomato Agar (5 Liters): 1. Filter a large can of tomatoes (6 pounds 6 ounces) through a thin layer of cotton. If filtra- tion is allowed to continue overnight, this amount should yield 2000 cc. of clear straw- colored juice. 2. Add 50 gin. of peptonized milk and 50 gin. of peptone. 3. Heat gently to dissolve do not boil. 4. At this point the tomato , o juice is generally approxi- mately pH 5.0, but since, in order to obtain optimum growth for Lactobaci//i, the media must contain lactic acid, alkalize the solution with approximately 15 cc. of normal sodium hydroxide and then adjust to pH 5.0 with normal lactic acid. This usu- ally requires about 20 cc. of the acid. Add 3000 cc. of distilled water and stir. Measure out 25 gm. of pow- dered agar in each of five flasks, or pyrex bottles if the media are to be shipped. Add 1000 cc. of tomato juice solution to each flask. Autoclave at 120 øC., 15 pounds, for twelve minutes. Prolonged heating will induce hydrolysis resulting in a mushy agar. This should be borne in mind when the media are melted for pouring plates. Inoculation of Media. When large numbers of saliva specimens are examined for the purpose of estimat- ing caries incidence rates, it is pref- ,erable to inoculate the cultures on the day the specimens are collected. Since the process of eating results in a temporary drop in the oral bac- terial count, it is advisable to obtain the saliva at least two hours after the last meal. When the test is used as a check on dietary treatment for individual patients, the patients themselves ordinarily obtain the specimens immediately upon arising