MEDIUM FOR MALONATE UTILIZATION TEST

Abstract: 1. A medium for malonate utilization test, comprising: 0.2 to 4 g of yeast extract, 1 to 20 g of peptone, 0.3 to 3 g of glucose, 0.1 to 2 g of sodium chloride, 0.1 to 2 g of ammonium sulfate, 0.2 to 8 g of monoalkali metal dihyd­ rogen phosphate, 4 to 40 g of a malonate, and 0.01 to 0.2 g of a pH indicator, which culture medium exhibits a pH value in the range of 6.0 to 6.5 when dissolved in 1 liter of water.

Description: MEDIUM FOR MALONATE UTILIZATION TEST BACKGROUND OF THE INVENTION Field of the Invention: This invention relates to a medium for testing microorganisms for biochemical behavior. More particularly, this invention relates to an improved medium to be used in performing malonate utilization test on microorganisms such as enteric bacteria by the method of biochemical identification. For administration of a proper medication to a patient of an infectious disease, it is essential to identify a pathogenic microorganism responsible for the disease, subjecting the patient to a sensitivity test, and selecting an effective medicine. In the identification of such a pathogenic microorganism, many biochemical tests are performed. One of them is a malonate utilization test. The medium of this invention is advantageously used for the malonate utilization test. Description of the Prior Art: The malonate utilization test is intended to determine whether or not malonic acid can be utilized as a carbon source in the culture of a given microorganism. This test is performed by culturing the given microorganism in a culture medium containing malonate. Recently, as a culture medium for use in this test, a composition comprising proteose peptone, glucose, sodium malonate, L-tryptophane, monosodium dihydrogen phosphate, sodium chloride, bromothymol blue and purified water has been proposed (Japanese Patent Publication No. SHO 58(1983)-20263). This medium permits the time required for culture to be notably decreased as compared with the conventional countertype. Specifically, the culture time has been one to two days in the conventional medium, whereas it is four to five hours in the medium under discussion, making it possible to conduct the test and find the result of the test on one same day. Depending on the kind of the microorganism under test, however, the culture performed for four to five hours in this medium does not permit the reaction to proceed sufficiently for required evaluation. In this case, the culture time must be elongated and the evaluation of the test result made on the following day. In the circumstance, the desirability of developing a medium in which any microorganism can be cultured sufficiently in a matter of four to five hours to permit clear distinction between positive and negative test of the reaction has found growing approval. For early medication, the identification of a pathogenic microorganism is desired to be carried out as rapidly as permissible. There are, however, times when the evaluation of the test result is compelted to be performed on the fcllowing day because of the convenience of the work involved in the test. In this case, the medium is desired to be such that the culture time is not rigidly specified and, therefore, the culture time may be elongated to suit the convenience of the work and, despite the elongation of the culture time, the reaction is not suffered to proceed excessively and the evaluation of the test result can be conducted accurately. For the efficiency of work involved, many biochemical tests are frequently performed all at once by the use of a multi-well culture plate. Thus, it becomes necessary for culture times of different test items to be equalized to one another. Again in this case, the culture medium is desired not to involve any rigid limitation of culture time. An object of this invention, therefore is to provide a novel medium for the malonate, utilization test. Another object of this invention is to provide a medium for the malonate utilization test which does not specifically limit culture time and enables the identification of the pathogenic microorganism to be performed on the same day as the culture is carried out or on the day following the culture. A further object of this invention is to provide a medium for malonate utilization test which permits a color reaction to produce a distinct color and enables the evaluation of test result to be effected with ease. SUMMARY OF THE INVENTION The objects descirbed above are attained by a medium for the malonate utilization test, which comprises 0.2 to 4 g of yeast extract, 1 to 20 g of peptone, 0.3 to 3 g of glucose, 0.1 to 2 g of sodium chloride, 0.1 to 2 g of ammonium sulfate, 0.2 to 8 g of monoalkali metal dihydrogen phosphate, 4 to 40 g of a malonate and 0.01 to 0.2 g of a pH indicator, which composition assumes a pH value in the range of 6.0 to 6.5 when dissolved in 1 liter of water. DESCRIPTION OF THE PREFERRED EMBODIMENT The medium of this invention comprises yeast extract, peptone, glucose, sodium chloride, ammonium sulfate, monoalkali metal dihydrogen phosphate, a malonate and a pH indicator. The composition of this medium comprises 0.2 to 4 g, preferably 0.3 to 3 g,of yeast extract, 1 to 20 g, preferably 1.5 to 15 g, of peptone, 0.3 to 3 g, preferably 0.4 to 2 g, of glucose, 0.1 to 2 g, preferably 0.2 to 1.9 g, of sodium chloride, 0.1 to 2 g, preferably 0.2 to 1.9 g, of ammonium sulfate, 0.2 to 8 g, preferably 0.3 to 7 g, of monoalkali metal dihydrogen phosphate, 4 to 40 g, preferably 5 to 30 g, of a malonate, and 0.01 to 0.2 g, preferably 0.06 to 0.14 g, of a pH indicator in 1 liter of water. For the composition of the present invention, an alkali metal salt (preferably potassium or sodium salt) of malonic acid is advantageously used as the malonate. In this invention, the medium enjoys improved reactivity because it incorporates glucose. This yeast extract is intended to promote the enzymatic reaction. If glucose is incorporated in an excessive amount, the excess glucose decomposes itself to lower the pH value of the medium and the malonate utilization reaction consequently curbs the alkalization of the medium by positive microorganism. The monoalkali metal dihydrogen phosphate serves as a pH adjuster. The alkali metal salt is desired to be potassium or sodium. Although this invention does not discriminate the pH indicator by its kind, the pH indicator is desired to be phenol red, bromothymol blue, bromocresol purple, cresol red, bromophenot red, chlorophenol red and the like. The proportions of the components in the medium of this invention are critical. In particular, the proportion of the monoalkali metal dihydrogen phosphate is fixed so that the pH value of the medium will fall in the range of 6.0 to 6.5, preferably 6.2 to 6.4, and the color of the color reaction will appear distinctly. Also the proportion of the malonate is selected so as to permit reduction in the culture time. If the ammonium sulfate content of the medium is not sufficient, the malonate decomposition is liable to be pseudo-positive. For the medium of the present invention, bromothymol blue proves to be the most suitable pE indicator. When this pH indicator is used, the color of the color reaction is distinct enough to permit easy discrimination between positive and negative test even when the amount of the sample size is small. The medium of this invention is prepared for use by dissolving the components in their respective proportions in water. In recent years, it is normal for numerous tests to be performed all at once by the use of a multi-well culture plate. For the medium of this invention to be used in this manner, it is poured into the wells of the culture plate and dried to be used as dry medium. In the test, a suspension of a microorganism is dispensed in a prescribed volume to the individual wells and left standing therein to effect required culture for a prescribed length of time. By the change in color of the culture solution, the discrimination between positive test and negative test of the malonate utilization test is effected. The medium of the present invention is used in much the same way as the conventional medium for malonate utilization test. With the medium of this invention, although the culture time can be reduced to the order of three to five hours, it is not specifically limited to that range. The culture may be performed for a longer period. Thus, the culture medium permits the evaluation of the test result either on the same day as the sample is taken or on the following day. The color produced in color test is so distinct as to permit easy discrimination between positive test and negative test of the reaction. When the culture medium of this invention additionally incorporates L-tryptophane, it can be used for the tryptophane deaminase reaction test as well as for the malonate utilization test. The tryptophane deaminase reaction test is intended to detect a microorganism which deaminates tryptophane and forms indole pyruvic acid. Now, one working exmaple of the actual use of the medium of this invention in the malonate utilization test and referential examples of the use of the medium of this invention plus L-tryptophane in the malonate utilization test and the tryptophane deaminase test will be described below. Examples I-V and Controls I-III Example Control composition of medium I II III IV V I II III Yeast extract (g)* 0.5 0.5 1.0 0.3 3.0 - 0.5 0.5 Peptone (g)** 2.5 2.5 5.0 1.5 15.0 3.0 2.5 2.5 Glucose (g) 0.75 0.75 1.0 0.4 2.0 0.2 0.2 0.75 Sodium chloride (g) 1.0 1.0 0.5 0.2 1.9 1.0 1.0 1.0 Ammonium sulfate (g) 1.5 1.5 1.0 0.2 1.9 - 1.5 0 Monosodium dihydrogen phosphate (g) 2.0 2.0 3.0 0.3 7.0 5.5 2.0 2.0 Sodium malonate (g) 10.0 10.0 7.0 5.0 30.0 10.0 10.0 10.0 Bromothymol blue (g) 0.12 0.1 0.08 0.06 0.14 1.16 0.12 0.12 Purified water (liter) 1 1 1 1 1 1 1 1 pH 6.3 6.3 6.0 6.5 6.3 6.0 6.0 6.0 * Yeast extract produced by Oxoid Corp. ** Meat extract produced by Oxoid Corp. Culture conditions The media of this invention and control media having the compositions as shown above were dispensed in a unit volume of 50 p1 to the wells of a culture plate and dried at 40 OC. Separately various microorganisms were cultured on agar culture plates at 350 to 37 OC for 18 to 24 hours and suspended in 1.0 ml sterilized distilled water in a concentration of about 6 to 9 x 108 cells/ml. The suspensions were inoculated in a unit volume of 50 ul to the dry medium and, under a cover, cultured at 350 to 37 OC for a prescribed length of time. By the change in color of the resultant solutions, presence or absence of the malonate decomposition reaction was evaluated. The results are shown in Table 1. Table 1 EMI8.1 <SEP> Media <SEP> of <SEP> Control <SEP> Control <SEP> Control <tb> <SEP> Examples <SEP> I-V <SEP> medium <SEP> I <SEP> medium <SEP> II <SEP> medium <SEP> III <tb> <SEP> Microorganism <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <SEP> 4 <SEP> to <SEP> 5 <SEP> 6 <SEP> to <SEP> 20 <tb> <SEP> hours' <SEP> hours' <SEP> hours <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <tb> <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <tb> Enterobacter <tb> <SEP> cloacae <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Enterobacter <tb> <SEP> gergoviae <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Klebsiella <tb> <SEP> pneumoniae <SEP> + <SEP> + <SEP> + <SEP> + <SEP> # <SEP> - <SEP> + <SEP> + <SEP> + <SEP> # <SEP> - <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Hafnia <tb> <SEP> alvei <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Citrobacter <tb> <SEP> diversus <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Salmonella <tb> <SEP> arizonae <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Morganella <tb> <SEP> morganii <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> Providencia <tb> alcalifacins <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> Proyidencia <tb> <SEP> rettgeri <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> Providencia <tb> <SEP> stuartii <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> + <SEP> - <SEP> - <SEP> + : Color of positive test (number of +'s indicating the intensity of color) + @ + : Intermediate color closer to color of positive test + - - : Intermediate color closer to color of negative test - : Color of negative test Referential Example The malonate utilization test and the tryptophane deaminate reaction test were carried out by following the procedure of Example 1 using the medium and the control medium each additionally incorporating tryptophane. The results are shown in Tables 2 and 3. Medium of Control Control Control Composition of referential medium medium medium medium example IV V VI Yeast extract (g)* 0.5 - 0.5 0.5 Peptone (g)** 2.5 3.0 2.5 2.5 Glucose (g) 0.75 0.2 0.2 0.75 Sodium chloride (g) 1.0 1.0 1.0 1.0 Ammonium sulfate (g) 1.5 - 1.5 0 Monosodium dihydrogen phosphate (g) 2.0 5.5 2.0 2.0 Sodium malonate (g) 10.0 10.0 10.0 10.0 L-tryptophane (g) 1.5 1.5 1.5 1.5 Bromothymol blue (g) 0.12 0.16 0.12 0.12 Purified water (liter) 1 1 1 1 pH 6.3 pH 6.0 pH 6.0 pH 6.0 * and ** Same as in Example. Table 2 EMI10.1 <SEP> Culture <SEP> medium <SEP> Control <SEP> Control <SEP> Control <tb> <SEP> of <SEP> the <SEP> reference <SEP> medium <SEP> IV <SEP> medium <SEP> V <SEP> medium <SEP> VI <tb> <SEP> Microorganism <SEP> 4 <SEP> to <SEP> 16 <SEP> to <SEP> 20 <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <tb> <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <tb> <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <tb> Einterobacter <tb> <SEP> cloacae <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Einterobacter <tb> <SEP> gergoviae <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Klebsiella <tb> <SEP> pneumoniae <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> - <SEP> + <SEP> + <SEP> + <SEP> # <SEP> - <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Hafnia <tb> <SEP> alvei <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Citrobacter <tb> <SEP> diversus <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Salmonella <tb> <SEP> arizonae <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Morganella <tb> <SEP> morganii <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> # <SEP> - <SEP> - <SEP> Providencia <tb> alcalifaciens <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> # <SEP> - <SEP> - <SEP> Providencia <tb> <SEP> rettgeri <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> # <SEP> - <SEP> - <SEP> providencia <tb> <SEP> stuartii <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> # <SEP> - <SEP> - <SEP> + : Color of positive test (number of +'s indicating the intensity of color) + - + : Intermediate color closer to color of positive test # - - : Intermediate color closer to color of negative test - : Color of negative test Table 3 EMI11.1 <SEP> Medium <SEP> of <SEP> Control <SEP> Control <SEP> Control <tb> <SEP> the <SEP> reference <SEP> medium <SEP> IV <SEP> medium <SEP> V <SEP> medium <SEP> VI <tb> <SEP> Microorganism <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <SEP> 4 <SEP> to <SEP> 5 <SEP> 16 <SEP> to <SEP> 20 <tb> <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <SEP> hours' <tb> <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <SEP> culture <tb> Enterobacter <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <SEP> cloacae <tb> Enterobacter <tb> <SEP> gergoviae <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> Klebsiella <tb> <SEP> pneumoniae <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> Hafnia <tb> <SEP> alvei <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> Citrobacter <tb> <SEP> diversus <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> Salmonella <tb> <SEP> arizonae <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> Morganella <tb> <SEP> morganii <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Providencia <tb> alcalifaciens <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Providencia <tb> <SEP> rettgeri <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Providencia <tb> <SEP> stuartii <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> + : Color of positive test (number of +'s indicating the intensity of color) + - + : Intermediate color closer to color of positive test # - - : Intermediate color closer to color of negative test - : Color of negative test It is noted from the results of Table 1 and Table 2 that the use of the media of this invention in the malonate utilization test permit accurate identification of microorganisms without reference to length of culture time. Thus, with the medium of this invention, the evaluation of test result can be carried out either on the same day as the sample is taken or on the following day, depending on the convenience of the work involved in the test. In contrast, in the use of control media, the color produced after 4 to 5 nours' culture is not amply distinct to permit the evaluation of test result on the same day as the sample is taken. Moreover, the fact that the medium of this invention is not rigidly restricted by the length of culture time proves highly convenient where numerous biochemical tests are carried out all at once.

Claims: WHAT IS CLAIMED IS: 1. A medium for malonate utilization test, comprising: 0.2 to 4 g of yeast extract, 1 to 20 g of peptone, 0.3 to 3 g of glucose, 0.1 to 2 g of sodium chloride, 0.1 to 2 g of ammonium sulfate, 0.2 to 8 g of monoalkali metal dihydrogen phosphate, 4 to 40 g of a malonate, and 0.01 to 0.2 g of a pH indicator, which culture medium exhibits a pH value in the range of 6.9 to 6.5 when dissolved in 1 liter of water. 2. A medium according to Claim 1, which comprises: 0.3 to 3 g of yeast extract, 1.5 to 15 g of peptone, 0.4 to 2 g of glucose, 0.2 to 1.9 g of sodium chloride, 0.2 to 1.9 g of ammonium sulfate, 0.3 to 7 g of monoalkali metal dihydrogen phosphate, 5 to 30 g a malonate, and 0.06 to 0.14 g of a pH indicator, 3. A medium according to Claim 2, wherein the pH value of the medium is in the range of 6.2 to 6.4. 4. A medium according to Claim 1, wherein said monoalkali metal dihydrogen phosphate is potassium salt or sodium salt. 5. A medium according to Claim 1, wherein said malonate is an alkali metal salt. 6. A medium according to Claim 5, wherein said alkali metal salt is potassium or sodium salt. 7. A medium according to Claim 1, wherein said pH indicator is one compound selected from the group consisting of phenol red, bromothymol blue, bromocresol purple, cresol red, chlorophenol red and bromophenol red. 8. A medium according to Claim 7, wherein said pH indicator is bromothymol blue.

Inventors: ENDO, TOSHIHIKO, IGARASHI, TAKESHI, KOSHI, ISEI
Applicants: TERUMO KABUSHIKI KAISHA TRADING AS TERUMO CORPORATION
Application year: 1985
Main classification: C12Q1
IPC codes: C12Q1