JP2535488B2 - Candida simple identification medium - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The discrimination medium of the present invention is Candida albicans, which is a causative bacterium of vaginal candidiasis.
bicans, hereinafter C.I. Albicans or CA) and Candida glabrata (hereinafter C.
(Abbreviated as glabrata or CG) can be easily identified by the color tone of the colonies on the medium. By using this, the causative agent of vaginal candidiasis can be easily and quickly determined, and treatment measures can be established early. can do.

[0002]

BACKGROUND OF THE INVENTION Candidiasis is caused by C. Albicans is the major causative agent, but recently in C. vaginalis, especially in vaginal candidiasis.
The number of those causing glabrata as the causative bacteria is increasing, and most of the present cases are C. Albicans and C.I. Caused by any single or mixed infection of glabrata. Especially C.I. Infections of glabrata are often intractable and pose a problem in the field of gynecology.

Therefore, in order to identify the pathogenic bacterium and establish a therapeutic policy at an early stage, C.I. Albicans and C.I. It is necessary to identify the glabrata, and several types of identification media are already on the market. All of these media are for distinguishing CA and CG by the color tone of the colony, and are as follows depending on the mechanism of color development.

(1) Those that utilize the difference in copper ion uptake ability (for example, Nissui Grant Candida medium manufactured by Nissui Pharmaceutical, CA-TG medium manufactured by Serotech). CA, which has a weak copper ion uptake ability, is C. Grabrata exhibits dark brown colonies. (2) Those that utilize the difference in the uptake ability of copper ions and 2,3,5-triphenyltetrazolium chloride (hereinafter referred to as TTC) (for example, Kamedew manufactured by Kyokuto Pharmaceutical Co., Ltd.). CA shows green brown to light brown, and CG shows pink colonies.

[0005]

However, in the medium utilizing the difference in copper ion uptake ability as described in (1) above, the color tone of colonies varies depending on the clinical isolates, and the difference in color tone is small. , CA and CG are difficult to distinguish. Further, in the medium described in (2) above, there is a difference in color tone between CA and CG, but there is a problem that the color tone varies depending on the clinical isolate, and the degree of variation increases particularly when the culture time is long.

On the other hand, "Pagano-Levin medium" is used as a supplement to the above-mentioned drawbacks of the conventional medium. According to "Yeast classification and identification method" (University of Tokyo Press), the composition of the medium is 100 μg of TTC which is a redox indicator.
/ Ml, yeast extract 0.1g / 100ml, peptone 1
%, Glucose 4.5%, agar 1.5%, neomycin 500 μg / 100 ml, and pH 6.0-6.2. However, in this Pagano-Levin medium, C. Albicans and C.I. The growth of glabrata is slow and it takes time to distinguish, and C.I. Albicans and C.I. There is a problem that it is difficult to identify because the difference in coloration between the glabrata colonies is small and the color changes as the culture time increases.

The present invention has been made by paying attention to the above-mentioned problems of the prior art, and the purpose thereof is CA and CG.
It is intended to provide a discrimination medium capable of quickly and simply and clearly discriminating.

[0008]

Means for Solving the Problems The medium for simple identification of Candida of the present invention which was able to solve the above problems is a Sabouraud medium having a pigment, which is obtained by adding a tetrazolium compound and a yeast extract. The following general formula that has never been used as a compound

[0009]

Embedded image

[Wherein R ₁ , R ₂ and R ₃ are the same or different from each other and may be an aryl group which may have either a lower alkyl group or a halogen as a substituent, and X is an acid residue]. (However, 2,3,5-triphenyltetrazolium chloride is excluded)].

[0011]

[Functions and Examples] Tetrazolium compounds are almost colorless, but are easily reduced, have a unique maximum absorption wavelength, and are water-insoluble colored formazan (Fo).
rmazan) is generated. This tetrazolium compound utilizes its reducibility to produce redox enzymes, corticoids,
It has been used for quantification of reducing sugars.

The inventors of the present invention have conducted various studies focusing on tetrazolium compounds other than TTC in order to solve the above-mentioned problems of the prior art. As a result, CA and CG were obtained by adding a tetrazolium salt having a certain structure to the medium. It has been found that the present invention is excellent in differentiating property, and has completed the present invention.
The details will be described below.

<Test Example 1> As a tetrazolium salt, a group represented by R or X in the chemical structural formula shown in Chemical Formula 2 above satisfies the definition of the present invention (Sample Nos. 1 to 13: Examples), And those in which the groups represented by R and X do not satisfy the definition of the present invention (Sample Nos. 14 to 29: Comparative Examples), and the distinguishability when using them was examined (Tables 1 to 4).
reference).

Yeast extract 10 g, magnesium sulfate 2.1 g, potassium dihydrogen phosphate 2.0 g, in 1 L of Sabouraud agar medium containing 10 mg of patent blue as a pigment,
Thiamine hydrochloride 50 mg was added, and further various tetrazolium compounds 20 μg / ml shown in Tables 1 to 4 were added,
An agar medium was prepared according to a conventional method. C. in each test medium Albicans and C.I. The color tone of the colonies was examined after inoculating glabrata and culturing at 37 ° C. for 2 days. The results are shown in Tables 1 to 4.

[0015]

[Table 1]

[0016]

[Table 2]

[0017]

[Table 3]

[0018]

[Table 4]

As is clear from Tables 1 to 4, it can be seen that in the medium using the compound of the present invention, the difference in color tone between CA and CG is clear and the distinguishability is excellent.

<Test Example 2> Sample No. 10 and Sample No. 3 showing particularly excellent color difference in the above test example, that is,
2,3-diphenyl-5- (p-chlorophenyl) tetrazolium chloride (hereinafter referred to as TZ-1), 2,5
-Diphenyl-3- (o-tolyl) tetrazolium chloride (hereinafter referred to as TZ-2) was used as an indicator to obtain the medium of the present invention having the same composition as the above. As a comparison medium, T
A medium was prepared using TTC instead of Z-1 or TZ-2. 42 clinical isolates of CA and 2 of CG
Four strains were smeared on each of the above-mentioned medium, cultured at 37 ° C. for 2 days, and the color tone of colonies was compared and examined. The results are shown in Table 5.

[0021]

[Table 5]

As is clear from Table 5, TZ-1 or TZ
In the medium of the present invention using -2, CA shows all strains red to red-purple, CG shows all strains white, and it can be seen that the color tone difference is larger than TTC.

TEST EXAMPLE 3 Next, the optimum concentration of yeast extract was examined for TZ-1, TZ-2 and 2,3,5-triphenyltetrazolium bromide (Sample No. 13) (hereinafter referred to as TTB). . As in Test Example 1, TZ-1 was added to Sabouraud's agar medium containing Patent Blue.
20 μg / ml of TZ-2 or TTB was added, and yeast extracts of various concentrations shown in Table 6, Table 7, and Table 8 below were added to prepare agar medium according to a conventional method, and each test medium was added. After inoculating CA and CG and culturing at 37 ° C. for 2 days, the color tone of the colonies was examined. The results are shown in Tables 6, 7 and 8.

[0024]

[Table 6]

[0025]

[Table 7]

[0026]

[Table 8]

As can be seen from Table 6 above, in the case of TZ-1, the optimum concentration of the yeast extract was 0.05 to 1 g / 100 ml, particularly 0.1 to 0.3 g / 100 ml. As can be seen from Table 7 above, in the case of TZ-2, the optimum concentration of the yeast extract was 0.1 to 1 g / 100 ml, particularly 0.1 to 0.3 g / 100 ml. Further, as can be seen from Table 8 above, in the case of TTB, good distinguishability was exhibited at all of the tested yeast extract concentrations of 0 to 5.0 g / 100 ml, and particularly good at 0 to 0.5 g / 100 ml.

<Test Example 4> Next, TZ-1 and TZ-2
The optimum concentration of was investigated. An agar medium was prepared in the same manner as in Test Example 1 above with various concentrations of TZ-1 and TZ-2 shown in Tables 9 and 10 below, and the color tone of the colonies of CA and CG was examined. The results are shown in Tables 9 and 10.

[0029]

[Table 9]

[0030]

[Table 10]

As can be seen from Tables 9 and 10, TZ-1,
The optimum concentration of TZ-2 was 5 to 30 μg / ml. Due to the above, yeast extract 0.05 to 1 g / 100 m
1, the tetrazolium compound of the present invention in the range of 5 to 30 μ / ml, C.I. Albicans and C.I. It can be seen that the degree of distinction based on the color difference of the glabrata colonies is particularly excellent.

<Test Example 5> The color discrimination of colonies on the medium is affected by the pH of the medium. Next, the optimum pH of the medium using TZ-1 was examined. TZ-1 concentration 20μ
After the culture medium was prepared and CA and CG were cultured in the same manner as in Test Example 1 except that the pH was changed to g / ml and various pH values shown in Table 11 below, the color tone of the colonies was examined. The results are shown in Table 11. It should be noted that CA and CG grow well under weak acidity or neutrality.

[0033]

[Table 11]

As can be seen from Table 11, the optimum p of TZ-1
H was 5.0 to 6.5. <Test Example 6> Next, the color stability of the colonies when examined with time was examined. TTC, TZ-1, TZ-2
For the above, a medium was prepared in the same manner as in Test Example 1, and CA, CG
Were cultured, and the color tone after each elapsed day was observed. Table 12 shows the results.

[0035]

[Table 12]

As can be seen from Table 12, within 2 days of culturing, TZ-1 and TZ-2 are superior to TTC in distinguishability due to the difference in color between CA and CG colonies.

<Examples> Next, clinical material samples were examined using the optimum Candida simple discrimination medium obtained from the results of the above-mentioned test examples. First, a method for producing a medium will be described. Glucose 40 g, polypeptone 10 g, yeast extract 2 g and chloramphenicol 200 mg, patent blue 10 mg, magnesium sulfate 2.1 g, potassium dihydrogen phosphate 2.0 g, and thiamine hydrochloride 50 mg are dissolved in 1 liter of water to prepare pH 6.2. After that, agar 15g
Was sterilized in an autoclave for 10 minutes. This is 50
TZ-1 (20 mg) that had been kept warm at ℃ and sterilized by filtration was added, and after stirring, it was poured into a vial and solidified.

41 samples of clinical material collected from the vaginal mucosa with a cotton swab were smeared on the above medium and cultured at 37 ° C. for 3 days. At the same time, the case of using a conventional medium containing TTC was also examined. Of the 41 samples of clinical material, there are 10 positive cases, and the judgment results for them are shown in Table 13.

[0039]

[Table 13]

As shown in Table 13, in the case of TZ-1, all CA-positive cases showed a bright red color, which was completely in agreement with Candida check performed using the Candida identification kit. In addition, there was one case of CG, and white colonies were exhibited as in the case of TTC medium.

[0041]

As described above, in the medium for simple identification of Candida of the present invention, C. Is there Albicans?
C. Excellent discrimination when judging the color tone of colonies on the medium to determine whether glabrata or both are present, and there is no variation in color tone among clinical isolates, and can accurately cause vaginal candidiasis. It has the effect of being able to judge the bacteria.

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Claims (1)

(57) [Claims] 1. A Sabouraud medium containing a dye is represented by the general formula: [Wherein R ₁ , R ₂ and R ₃ are the same or different from each other and may be an aryl group which may have either a lower alkyl group or a halogen as a substituent, and X is an acid residue (provided that 2,3,5-triphenyltetrazolium chloride)]], and a tetrazolium compound and a yeast extract are added to the medium for simple identification of Candida.