CULTURE MEDIUM COMPOSITION

Abstract: A culture medium composition for examining the presence or absence of dental caries pathogen in the mouth of a subject (adult, child, infant or baby) as one means for preventing dental caries, which comprises in combination of (a) a basal culture medium having a good storage stability, which is used for culturing collected saliva (0.1 ml) of the subject and determining the presence or absence of the microorganism and/or the number of cells and (b) a dry support containing various test reagents (in the dry state) to be added to the basal culture medium, optionally with (c) a vessel containing the basal culture medium (a) and the support (b) therein, which functions also as a vessel for culturing the microorganism.

Description: Background of the Invention The human tooth decay is a so-called multi-factorial disease which is caused by many factors which are connected with one another in a complicated manner. One of the main factors is that dental plaque is formed from saliva and sucrose taken by streptococci always present in the mouth and tooth decay is readily caused by this dental plaque. As the streptococci synthesizing this dental plaque, there are known Streptococcus sanguis, Streptococcus mutans and Streptococcus salivarius. Among these streptococci, it is Streptococcus mutans that synthesizes water-insoluble polysaccharides and forms dental plaque inducing the dental caries. Accordingly, in order to prevent. the dental caries, it is important to know the environmental condition in the mouth, especially the amount of Strepyococcus mutans present in the mouth. For this purpose, there was previously developed and proposed a method of measuring the dental caries activity ( see Japanese Patent Application Laid-Open Specification No. 55260/1980 ). The MSB broth that is used in this conventional method, however, has several defects as described below. More specifically, it has been found that the composition of the MSB broth is changed with the lapse of time after preparation of the MSB broth and propagation of Streptococcus mutans ( hereinafter referred to as " dental caries pathogen " ) and inhibition of growth of other bacteria become uncertain. It has been considered that this defect is based on the essence of the composition of the MSB broth and is therefore unavoidable. The reason is that it is impossible to store an antibiotic substance such as Bacitracin at normal temperatures in the culture medium for a long time without reduction of the antibiotic activity thereof. Accordingly, a fresh MSB broth is prepared when the test is carried out. Therefore, in the clinical examination, it is practically impossible for a dental practitioner to apply the above-mentioned method using this MSB broth to diagnosis. Description of the Invention The present invention relates to a culture medium for testing the dental caries activity, in which two or three of ingredients of a liquid medium capable of selectively propagating bacteria which causes dental caries, Streptococcus mutans, are contained. These ingredients are not added to said culture medium in the initial stage but are present outside in the dry state and just before use of said culture medium, said ingredients are included in said culture medium to obtain a desirable composition of the culture medium. We made researches with a view to provide an improved simple MSB broth having the same capacity as that of the original MSB broth, which would be conveniently used by a practising dentist in an ordinary clinical examination and would be manufactured at a low cost. The compositions of the MSB medium and the culture medium of the present invention are shown in Table 1. Table 1 Compositions (g/1000 ml) of MSB Broth and Culture Medium of Present Invention Culture Medium of Present Ingredients MSB Broth Invention EMI3.1 <tb> tryptosel) <SEP> 10 <SEP> 10 <tb> peptone2) <SEP> 10 <SEP> 10 <tb> glucose <SEP> 1 <SEP> 1 <SEP> # <SEP> liquid <SEP> basal <SEP> medium <tb> sucrose <SEP> 200 <SEP> 200 <tb> dipotassium <SEP> phosphate <SEP> 4 <SEP> 4 <tb> Bacitracin <SEP> 200 <SEP> u <SEP> 200 <SEP> u <tb> potassium <SEP> tellurite <SEP> 0.01 <SEP> 0.01 <SEP> additive <SEP> reagents <tb> <SEP> # <SEP> (kept <SEP> <SEP> in <SEP> dry <SEP> state <tb> <SEP> outside <SEP> until <SEP> just <tb> Crystal <SEP> Violet3) <SEP> 0.0008 <SEP> 0.0008 <SEP> <SEP> before <SEP> use) <tb> Trypan <SEP> Blue <SEP> 0.075 <SEP> 0.075 <tb> Note 1) Bacto Tryptose ( Difco 2) Proteose Peptone No. 3 ( Difco 3) Crystal Violet may be included in the liquid basal medium We carefully examined mutual reactions among the respective ingredients of the MSB broth and as the result, we have created a combination of culture medium composition as described below. A liquid basal medium having the composition from which Bacitracin ( hereinafter referred to as "BA" ), potassium tellurite ( hereinafter referred to as "TE" ), and Trypan Blue ( hereinafter referred to as "TB" ) and/or Crystal Violet, have been removed is prepared, and this liquid basal medium is sterilized and is stored aseptically. Separately, BA, TE and TB are made independently present as additive reagents in the dry state on an appropriate support, and the additive reagent-containing support is sterilized and is stored aseptically. When the test is actually carried out, a dentist dissolves the additive reagents in the liquid basal medium sterilely to form an instant MSB broth simply. In this case, TB, a coloring material which is contained in the support is dissolved in the liquid basal medium to color the medium. By this coloration, completion of dissolution of other ingredients can be confirmed. From the results of the experiments made by us, it was found that the above-mentioned combination of culture medium composition is comparable to the conventional MSB broth as a clinical medium for selectively culturing the dental caries pathogen in saliva ( see Table 2 ) and that even if the combination of culture medium composition is subjected to 6 months' shelf life test, the capacity is not degraded ( see Table 3 ). We have now completed the present invention based on the foregoing findings. Table 2 Results of Determination of Caries Actiyity by MSB Broth and Culture Medium of Present Invention Sample MSB Present Invention 1 + + 2 Sample MSB Present Invention 3 - - 4 +++ 5 +++ 6 +++ +++ 7 + + 8- - 9 ++ ++ 10 + + Note Mark "-" indicates no propagation of the dental caries pathogen, and each of marks "+" through "+++" indicates the degree of propagation of the dental caries pathogen, which has a relation to the number of cells of the bacteria in saliva. Test Method: 0.1 ml each of saliva collected from 10 healthy adults was added to 2 ml of the ordinary MSB broth and to one sample of the culture medium described in Example 2 given hereinafter, respectively, and cultured at 37 C for 24 hours according to routine procedures. The degree of propagation was determined by the naked eye examination according to the standard customarily adopted in this field. Table 3 Capacity of Culture Medium of Present Invention after Shelf Test Times of Repetition of Test Shelf Test Period (months) O(control) 2 4 6 1 +++ +++ +++ +++ Times of Repetition of Test Shelf Test Period (months) 0 (control) 2 4 6 2 +++ +++ +++ +++ 3 +++ +++ +++ +++ Test Method: Culture Medium: Trial product Lot No. 5402 prepared in Example 1 given hereinafter. Shelf Life Test: Trial product Lot No. 5402 was stored in a incubator maintained at 40 C. A part of the products was taken out at an interval of 2 months, and immediately stored in a freezer maintained below - 5 0C. Control: Trial product Lot No. 5402 was stored in a freezer maintained below - 5 0C directly without the shelf life test. Sample (fresh saliva): Saliva of a 23-years-old healthy man in whom high dental caries activity was found by a preliminary test. Operation: 0.1 ml of the sample was added to the culture medium ( control culture medium or culture medium subjected to the shelf life test ), and cultured at 37Qfor 24 hours according to rountine procedures. In carrying out the present invention, the vessel in which the liquid basal medium is contained functions also as a culturing device, and therefore, a colorless transparent glass vessel having an inner diameter of about 70 mm, for example, an ampoule, a vial, a tubing bottle or a test tube, can advantageously be used. The additive reagents, that is, BA, TE and TB, are independently freeze-dried or spread on a paper such as a filter paper. In each case, solvent is eliminated as much as possible to obtain a product of the present invention. The present invention will now be described in detail with reference to the following Examples. Incidentally, the liquid basal medium shown in Table 1 was commonly used in these Examples. Example 1 (A) A colorless transparent glass vial having an inner diameter of 12 mm and a diameter of about 8 mm in the vicinity of the opening was charged with 2 ml of the liquid basal medium and the glass vial was sealed with a rubber stopper. Sterilization was performed at 1150C for 15 minutes. (B) A circular filter paper having a thickness of 0.8 mm and a diameter of 8 mm was used as the support for the additive reagents. The additive reagents, BA, TE and TB were independently dissolved and included in the support in manners as described below, and the support was dried under reduced pressure and sealed in a vessel. 1) BA: A solution of 0.4 U of Bacitracin in about 0.002 ml of water was poured onto the support by using a micropipette. 2) TE: A solution of 0.4 mg of potassium tellurite in about 0.002 ml of water was poured onto a different support by using a micropipette. 3) TB: A solution of 0.15 mg of Trypan Blue in about 0.002 ml of water was poured onto a different support by using a micropipette. then the culture medium composition was actually used, the rubber stopper was taken out from the vessel containing the basal medium, which was obtained in (A) above, and the reagent-containing supports obtained in (3-1), (B-2) and (B-3) above were thrown into the basal medium described in (A) above. After 30 seconds, the supports were taken out and 0.1 ml of saliva of the patient was added to the culture medium. Then, the vessel was sealed again with the rubber stopper and cultivation was carried out at 370C for 24 hours. Example 2 (A) A 3 ml-capacity ampoule having an inner diameter of about 9.5 mm and an opening diameter of about 4 mm ( the diameter of the broken face on the tubular body top when the ampoule was broken ) was charged with 2 ml of the liquid basal medium, and the top end of the ampoule was melt-sealed and sterilization was carried out at 1150C for 15 minutes. (B) A square filter paper having a thickness of 0.5 mm and one side of 3.5 mm was used as the support. Solutions of the additive reagents BA, TE and TB were independently poured onto supports in the same manner as described in (B) of Example 1 to obtain a product. When the culture medium composition was actually used, the ampoule (A) was broken, and the reagent-containing paper supports (B-l), (B-2) and (B-3) were added to the basal medium (A) in the same manner as described in Example 1. Then, 0.1 ml of saliva of the patient was added to the medium and the opening was covered with a sterilized aluminum cap. Cultivation was carried out at 370C for 24 hours. Example 3 (A) A basal medium was prepared in the same manner as in (A) of Example t except that Crystal Violet was not added. Then, 2 ml of the basal medium was charged in a small glass tube having an inner diameter of about 10 mm and a length of 60 mm, and the opening was covered with a closely fitting polypropylene stopper. Sterilization was carried out at 1150C for 15 minutes. (Bì Additive reagent-containing three square filter paper supports having a thickness of 0.5 mm and one side of 3.5 mm were prepared in the same manner as described in Example 2, and they were bonded in a line to a transparent plastic sheet having a width of 3.5 mm, a length of 50 mm and a thickness of 0.3 mm by using an appropriate adhesive. In this case, the three supports were not separated from one another but combined together, and therefore, they could be handled very easily. The BA and TE supports were prepared in the same manner as described in Example 2, but the TB support was prepared in a manner as described in 3) below. 3) TB: A mixed coloring material solution of 0.15 mg of Trypan Blue and 0.0016 mg of Crystal Violet in about 0.002 ml of water was poured onto the support ( filter paper ) by using a micropipette.

Claims: 1. What Is Claimed Is: 1. A culture medium composition comprising a liquid culture medium and an additive reagent-containing support, which culture medium composition comprises a liquid basal medium sealed in a transparent vessel, said liquid basal medium comprising main nutritional sources of ingredients of an MSB broth which is a Streptococcus mutans-growing liquid medium containing sucrose, and predetermined amounts of additive reagents to be added to said broth, which are independently spread on or incorporated in a paper or plastic support, said support being attached to said vessel containing the culture medium therein to form one package, wherein when a sample is cultured, the additive reagent-containing support is inserted in the liquid basal.medium contained in said vessel or tÅakenout from said vessel to dissolve out the additive reagents into the culture medium from the support, whereby the culture medium composition is used as a simple MSB broth. 2. A culture medium composition as set forth in claim 1, wherein the additive reagents are Bacitracin ( antibiotic substance ), potassium tellurite (K2TeO3) and at least one coloring material. 3. A culture medium composition as set forth in claim 1, where the additive reagent-containing support is a plate-like, filmy, rod-like, spherical or cubic support composed of paper, pulp, synthetic fiber, glass fiber, fabric or water-absorbing plastic material, predetermined amounts of the additive reagents being spread on or incorporated in said support. 4. A culture medium composition as set forth in claim 1, wherein the additive reagent-containing support is formed by continuously bonding a non-water-absorbing belt-like film integrally to the additive reagent-containing support set forth in claim 3. 5. A culture medium composition as set forth in claim 1, wherein the additive reagent-containing support is formed by homogeneously dissolving or suspending the additive reagents independently in water or other appropriate solvent, absorbing or incorporating or spreading the solutions or suspensions in or on a support and drying the support under a reduced pressure or at a low temperature to hold predetermined amounts of the additive reagents on the support while removing the solvent. 6. A culture medium composition as set forth in claim 1, wherein the transparent vessel can be utilized for culturing the microorganisms to be tested.

Inventors: ASAMI, KUNIAKI, MATSUKUBO, TAKASHI, MATSUMOTO, KIYOYUKI, OHTA, KOSEI, SAITO, HITOSHI, TAKAZOE, ICHIRO, TAKEUCHI, MITSUHARU, TANIMURA, MASAYUKI
Applicants: MATSUKUBO, TAKASHI, OHTA, KOSEI, SHOWA YAKUHIN KAKO CO., LTD., TAKAZOE, ICHIRO, TAKEUCHI, MITSUHARU
Application year: 1982
Main classification: C12Q1
IPC codes: C12R1, C12Q1