MEDIUM FOR CITRATE UTILIZATION TEST

Abstract: A medium for citrate utilization test, comprising: 0.5 to 5 g of citrate, 0.1 to 1 g of dialkali metal monohydrogen phosphate, 0.2 to 2 g of monoalkali metal dihydrogen phos­ phate, 0.3 to 6 g of magnesium salt, 1 to 10 g of sodium chloride, and 0.01 to 0.2 g of a pH indicator, wherein the component ratio of dialkali metal monohydrogen phosphate to monoalkali metal dihydrogen phosphate by weight is in the range of 1 : 1.5 to 1 : 5, which culture medium exhibits a pH value in the range of 5.5 to 7.0 when dissolved in 1 liter of water.

Description: MEDIUM FOR CITRATE 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 citrate utilization test on microorganisms such as enteric bacteria by the method of biochemical indentification. 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 citrate utilization test. The medium of this invention is advantageously used for the citrate utilization test. Description of the Prior Art: The citrate utilization test is intended to determine whether or not citric acid can be utilized as a carbon source in the culture of a given microorganism. At present, it is one of the most important tests performed on microorganisms for biochemical behavior. This test is performed by culturing the given microorganism in a medium containing citrate and a pH indicator as main components. Recently, as a medium for use in this test, a composition comprising yeast extract, sodium citrate, glucose, glutathione (reduction type), nicotine adenine dinucleotide, sodium chloride, disodium monohydrogen phosphate, 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 following 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 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 citrate utilization test. Another object of this invention is to provide a medium for the citrate utilization test which does not specifically limit culture time and enables the identifica tion 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 citrate 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 described above are attained by a medium for the citrate utilization test, which comprises 0.5 to 5 g of a citrate, 0.1 to 1 g of dialkali metal monohydrogen phosphate, 0.2 to 2 g of monoalkali metal dihydrogen phosphate, 0.3 to 6 g of magnesium salt, 1 to 10 g of sodium chloride and 0.01 to 0.2 g of a pH indicator, wherein the component ratio of dialkali metal monohydrogen phosphate to monoalkali metal dihydrogen phosphate by weight is in the range of 1 : 1.5 to 1 : 5, which medium assumes a pH value in the range of 5.5 to 7.0 when dissolved in 1 liter of water. DESCRIPTION OF THE PREFERRED EMBODIMENT The medium of this invention comprises citrate, dialkali metal monohydrogen phosphate, monoalkali metal dihydrogen phosphate, magnesium salt, sodium chloride and pH indicator. The composition of this medium comprises 0.5 to 5 g, preferably 1 to 4 g of citrate, 0.1 to 1 g, preferably 0.2 to 0.6 g of dialkali metal monohydrogen phosphate, 0.2 to 2 g, preferably 0.3 to 1.2 g of monoalkali metal dihydrogen phosphate, 0.3 to 6 g, preferably 0.5 to 3 g of magnesium salt, 1 to 10 g, preferably 2 to 8 g of sodium chloride and 0.01 to 0.2 g, preferably 0.06 to 0.14 g of a pH indicator in 1 liter of water, wherein the component ratio of dialkali metal monohydrogen phosphate to monoalkali metal dihydrogen phosphate by weight is in the range of 1 1.5 to 1 : 5, preferably 1 : 2 to 1 : 3. The medium in accordance with the present inven tion has a feature that dialkali metal monohydrogen phosphate and monoalkali metal dihydrogen phosphate as pH adjusters control buffering ability of the medium. If the buffering ability is suppressed, it is liable to be pseudopositive when the determination is carried out next day, because it is designed that the reaction occurs in a short time cultivation. If the buffering activity is hastened in order to avoid such defect, it becomes difficult to occur the reaction in the short time. Thus it is necessry to use specified amounts of dialkali metal monohydrogen phosphate and monoalkali metal dihydrogen phosphate, namely the amount in which the component ratio of dialkali metal monohydrogen phosphate to monoalkali metal dihydrogen phsophate by weight is in the range of 1 : 1.5 to 1 : 5, in order to make possible either on the same day and the following day. As the alkali metal salts thereof, potassium or sodium salts are preferable. As for magnesium salt, a mineral acid salt, such as magnesium sulfate, magnesium chloride, magnesium nitrate or magnesium phosphate, is preferably usable. As for citrate, a alkali metal, such as sodium, potassium or lithum, or anrmonium salt of citric acid is usable. 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, bromophenol red, chlorophenol red and the like. Bromothymol blue is best choice. The amount of the pH indicator is to be varied to some extent with its kind. For example, as bromothymol blue is used, the amount thereof is desired to be in the range of 0.06 to 0.14 g per a liter of water. The proportions of the components in the medium of this invention are critical. In particular, the proportion of the monoalkali metal dihydrogen phosphate and the dialkali metal monohydrogen phosphate is fixed so that the pH value of the medium will fall in the range of 5.5 to 7.0, preferably 6.0 to 6.5, and the color of the color reaction will appear distinctly. Also the proportion of the citrate is selected so as to permit reduction in the culture time. Further, for the medium of the present invention, the proportion of the pH indicater and the proportion of the magnesium salt are selected in order that 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 citrate utilization test is effected. The medium of the present invention is used in much the same way as the conventional medium for citrate 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. Now, one working exmaple of the actual use of the medium of this invention in the citrate utilization test will be described below. Examples I-IV and Controls I-III Example Control composition of medium I II III IV V VI I II III Yeast extract (g)* - - - - - - 1.0 - Glutathione, reduced form (g) - - - - - - 3.0 - NAD (g)** - - - - - - 0.05 - Disodium monohydrogen phosphate (g) - - - - - - 0.55 - Dipotassium monohydrogen phosphate (g) 0.35 0.35 0.1 0.6 0.2 0.2 - 0.35 1.5 Monopotassium dihydrogen phosphate (g) 0.7 0.7 0.4 1.2 0.3 0.6 - 0.7 1.5 Magnesium sulfate (g) 1.0 1.0 1.0 3.0 0.5 0.5 - 0.2 1.0 Sodium chloride (g) 5.0 5.0 5.0 8.0 2.0 2.5 4.0 5.0 5.0 Trisodium citrate (g) 2.0 2.0 2.0 4.0 1.0 1.0 6.0 2.0 2.0 Bromothymol blue (g) 0.12 0.08 0.12 0.14 0.06 0.1 0.14 0.12 0.12 Purified water (liter) 1 1 1 1 1 1 1 1 1 pH 6.4 6.4 6.2 6.0 6.5 6.4 6.0 6.4 6.4 * Yeast extract produced by Oxoid Corp. ** NAD: nicotinamide adenine dinucleotide Culture conditions The medium 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 35 to 37 0C 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 370C for a prescribed length of time. By the change in color of the resultant solutions, presence or absence of the citric acid decomposition reaction was evaluated. The results are shown in Table 1. Table 1 EMI9.1 <SEP> Medium <SEP> of <SEP> Control <SEP> Control <SEP> Control <tb> <SEP> Examples <SEP> I-IV <SEP> medium <SEP> I <SEP> Medium <SEP> II <SEP> medium <SEP> III <tb> 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> Klebsiella <tb> <SEP> oxytoca <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <tb> Escherichia <tb> <SEP> coli <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> Morganella <tb> <SEP> morganii <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> - <SEP> <tb> Proteus <tb> <SEP> mirabilis <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> + <tb> Enterobacter <tb> <SEP> sakazakii <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> + <tb> Serratia <tb> <SEP> marcescens <SEP> + <SEP> + <SEP> - <SEP> + <SEP> # <SEP> - <SEP> - <SEP> + <SEP> - <SEP> # <SEP> - <SEP> + <tb> Serratis <tb> <SEP> marinorubra <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> + <SEP> # <SEP> - <SEP> + <SEP> + <tb> Serratia <tb> <SEP> liquefaciens <SEP> + <SEP> + <SEP> # <SEP> - <SEP> + <SEP> + <SEP> # <SEP> - <SEP> + <SEP> + <SEP> # <SEP> - <SEP> - <SEP> # <SEP> - <SEP> + <tb> + : Color of positive test + - + : 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 that the use of the medium of this invention in the citrate utilization test permits 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 hours' 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 citrate utilization test, comprising: 0.5 to 5 g of citrate, 0.1 to 1 g of dialkali metal monohydrogen phosphate, 0.2 to 2 g of monoalkali metal dihydrogen phosphate, 0.3 to 6 g of magnesium salt, 1 to 10 g of sodium chloride, ad 0.01 to 0.2 g of a pH indicator, wherein the component ratio of dialkali metal monohydrogen phosphate to monoalkali metal dihydrogen phosphate by weight is in the range of 1 : 1.5 to 1 : 5, which culture medium exhibits a pH value in the range of 5.5 to 7.0 when dissovled in 1 liter of water. 2. A medium according to Claim 1, which comprises: 1 to 4 g of citrate, 0.2 to 0.6g of dialkali metal monohydrogen phosphate, 0.3 to 1.2 g of monoalkali metal dihydrogen phosphate, 0.5 to 3 g of magnesium salt, 2 to 8 g of sodium chloride, 0.06 to 0.14 g of a pH indicator, wherein the component ratio of dialkali metal monohydrogen phosphate to monoalkali metal dihydrogen phosphate by weight is in the range of 1 : 2 - 1 : 3. 3. A medium according to Claim 2, wherein the pH value of the medium is in the range of 6.0 to 6.5. 4. A medium according to Claim 1, wherein said citrate is an alkali metal or ammonium salt. 5. A medium according to Claim 4, wherein said alkali metal citrate is potassium or sodium citrate. 6. A medium according to Claim 1, wherein said dialkali metal monohydrogen phosphate and monoalkali metal dihydrogen phosphate are potassium or sodium salts. 7. A medium according to Claim 1, wherein said magnesium salt is a mineral acid salt. 8. A medium according to Claim 7, wherein said mineral acid is one compound selected from the group consisting of chloride, nitrate, sulfate and phosphate. 9. A medium according to Claim I, wherein said pH indicator is one compound selected from the group consisting of phenol red, bromothymol blue, bromocresol purple, cresol red and bromophenol red. 10. A medium according to Claim 9, 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