JP6847628B2 - Coloring medium for Candida differentiation - Google Patents

Description

The present invention relates to a color-developing medium for Candida differentiation, which is capable of rapidly differentiating Candida bacteria, which is the causative bacterium of Candidiasis, and in which the color development of colonies is stable without discoloration even after long-term culture.

In diagnosing the infection status, treatment status, recurrence status, etc. in candidiasis, it is clinically important to confirm by isolation / identification of the bacteria as well as the habitat form, tissue lesions, macroscopic inflammatory findings of the candida bacteria.

For confirmation by the number of such isolated and identified bacteria, a culture method is widely used, and various media such as Mizuno-Takada medium and Sabouraud medium are already on the market. All of these media contain carbon sources, nitrogen sources, and substances that suppress the growth of general bacteria, and are intended to detect only Candida (Non-Patent Documents 1 and 2).

In using these media, the current state of candidiasis is diagnosed by culturing the sample collected from the affected area in the medium for about 2 to 3 days and then observing the number and morphology of the colonies with the naked eye. However, with the conventional medium, it takes two or three days to culture the bacteria, so that it is not possible to make a prompt diagnosis, and there is a problem that the treatment cannot be started promptly.

Therefore, in an aqueous solution containing at least amino acids, peptides, sugars, and a bacterial growth inhibitor, a pH indicator that discolors in an acidic region that suppresses the growth of germs is adjusted to a pH that also suppresses germs. A medium for diagnosing candidiasis has been proposed, which can easily diagnose candidiasis in a short time without requiring skill by using a medium blended with the above-mentioned medium (Patent Document 1).

On the other hand, Candida albicans is known to be the main causative agent of candidiasis, but in addition to this, those caused by Candida glabrata and those caused by Candida tropicalis are increasing. ..

Of these Candida bacteria, Candida albicans and Candida glabrata can be easily identified by the color tone of the colonies on the medium supplemented with the tetrazolium compound, and by using this, the causative bacteria of candidiasis can be easily and quickly investigated. However, a medium for simple Candida differentiation has been proposed that can establish therapeutic measures at an early stage (Patent Documents 2 and 3).

However, since Candida albicans and Candida tropicalis not only have similar morphological properties but also similar biochemical properties, there is a problem that they cannot be distinguished at all.

Therefore, two types of chromogens, which are substrates of enzymes produced by Candida albicans and Candida tropicalis, are added to the medium, and after hydrolysis of the chromogens, a color different from the basic color of the chromophore. A method for detecting Candida bacteria that distinguishes between the two has been proposed (Patent Documents 4 and 5).

The present inventor also contains 5-blom-6-chloro-3-indrill-β-D-galactopyranoside and 5-blom-6-chloro-3-indrill-β-D-glucopyranoside as color-developing substrates. By doing so, we have proposed a color-developing medium for Candida differentiation that can easily distinguish Candida albicans and Candida tropicalis in a short time (Patent Document 6).

On the other hand, Candida albicans, Candida glabrata, and Candida tropicalis are currently used in the Candida identification coloring medium (trade name: Candida gar Candida, publisher: Kanto Chemical Co., Ltd., hereinafter referred to as Chromocandida medium). , Candida albicans, and Candida parapsillosis are reported to be presumably identifiable (Non-Patent Document 3).

However, although five types of Candida can be identified in this Chromocandida medium, according to the study of the present inventor, it takes 40 hours or more to culture all five types of Candida. It has been confirmed that there is.

The present inventor cultivates five types of Candida bacteria, Candida albicans, Candida grabulata, Candida tropicalis, Candida crusei, and Candida parapsillosis, for about 20 hours by containing a color-developing enzyme substrate and an oxidation-reduction reagent. We have already proposed a color-developing medium for Candida differentiation that can be differentiated at the same time (Patent Document 7).

Special Fair 1-4615 Japanese Unexamined Patent Publication No. 6-78793 Japanese Unexamined Patent Publication No. 6-277094 Special Table 9-500790 Special Table No. 9-500791 Japanese Unexamined Patent Publication No. 2003-310298 Japanese Unexamined Patent Publication No. 2005-085744

Medical Technology, 6, 109; 1978 Laboratory tests, 26, 1498; 1982 Clinical and Microbiology, 22, 735-739; 1995

In the color-developing medium for Candida differentiation of Patent Document 7, which contains a color-developing enzyme substrate and an oxidation-reduction reagent, five types of Candida bacteria can be identified by culturing for about 20 hours, but the culture time becomes long (40). The color of the colony will change after about 20 hours, and it will not be possible to distinguish between Candida albicans, Candida glabrata and Candida parapsillosis. Therefore, if for some reason the culture cannot be determined for about 20 hours, the culture should be restarted. There was a problem that it had to be diagnosed quickly.

In addition, since Candida glabrata is not colored, it is easy to mistake it for a germ, and since the difference in color tone between Candida albicans and Candida parapsillosis is small, it may be difficult to distinguish by colony color alone.

Therefore, the present invention has been made by paying attention to such a conventional problem, and the five types of Candida bacteria are highly discriminating and can be discriminated by culturing for about 20 hours, and the color development of the colony is cultured. An object of the present invention is to provide a color-developing medium for Candida differentiation that is stable for a long time without being affected by time.

As a result of diligent research to develop a medium in which many types of Candida can be easily differentiated in a short time and the color of colonies is stable for a long time, the present inventor differentiates Candida containing an enzyme substrate and an oxidation-reduction reagent. The present invention has been completed by finding that the distinctiveness of five types of Candida bacteria is improved and the color development is stabilized for about 50 hours by further adding tellurate to the color-developing medium for use.

The present invention has the following configuration.
(1) A color-developing medium for Candida differentiation, which comprises an enzyme substrate, a redox reagent, and a tellurate salt.
(2) The color-developing medium for Candida differentiation according to (1), wherein the terluate is potassium telurate and / or sodium tellurate.
(3) The color-developing medium for Candida differentiation according to (1) or (2), wherein the content of tellurate is in the range of 0.0005 to 0.015 g / L.
(4) The enzyme substrate is 5-bromo-4-chloro-3-indrill phosphate. 2Na (hereinafter referred to as x-phos. 2Na) and / or 5-bromo-4-chloro-3-indolyl-N-acetyl-β-D-galactosaminide (hereinafter referred to as x-galactosamide) (1) to ( The color-developing medium for Candida differentiation according to any one of 3).
(5) The color-developing medium for Candida differentiation according to any one of (1) to (4), wherein the content of the enzyme substrate is in the range of 0.01 to 0.1 g / L.
(6) The color-developing medium for Candida differentiation according to any one of (1) to (5), wherein the redox reagent is a tetrazolium salt.
(7) The color-developing medium for Candida differentiation according to (6), wherein the tetrazolium salt is tetrazolium violet (hereinafter referred to as TTV) and / or tetrazolium blue (hereinafter referred to as TTB).
(8) The color-developing medium for Candida discrimination according to any one of (1) to (7), wherein the content of the redox reagent is in the range of 0.001 to 0.01 g / L.
(9) Per 1 L of medium, 10 to 30 g of peptone, 1 to 10 g of yeast extract, 1 to 20 g of white sugar, 1 to 10 g of glucose, 0.001 to 0.01 g of TTV, x-phos. The color-developing medium for Candida differentiation according to any one of (1) to (8), which contains 0.01 to 0.1 g of 2Na, 0.0005 to 0.015 g of potassium sterlate and 3 to 20 g of agar.

The present invention provides a color-developing medium for Candida differentiation, which contains an enzyme substrate, a redox reagent, and a tellurate salt, so that the color is strong and easy to distinguish, and the color of the colony does not change even after long-term culture. Further, according to the color-developing medium for Candida differentiation of the present invention, five types of Candida bacteria can be differentiated at the same time by culturing for about 20 hours, and the colony color does not change even after culturing for 48 hours and the differentiation property does not decrease. The significance of this is also considered to be great.

Hereinafter, preferred embodiments of the present invention will be described.
The feature of the color-developing medium for Candida differentiation of the present invention is that the medium contains an enzyme substrate, a redox reagent, and a tellurate salt.

In the present invention, by containing tellurate in the medium in addition to the enzyme substrate and the redox reagent, the color development of the colonies becomes clearer, and the discoloration of the color-developed colonies is suppressed even after long-term culture. .. That is, the enzyme substrate, the redox reagent, and the tellurate salt together produce a synergistic effect, and the discrimination ability can be further improved as compared with the case where the enzyme substrate and the redox reagent are used, and the colony. It is possible to suppress the discoloration of the color for a long time.

The enzyme substrate, redox reagent, and tellurate form a synergistic effect, and the five types of Candida bacteria form colonies with their own unique colors in a short time, so they can be easily distinguished. Each colony can be distinguished by the color of the colony even for about 50 hours. That is, at 18 to 24 hours of culture, Candida albicans is pink to dark pink, C. glabrata is central gray-brown to central gray or central black, and C. tropicalis. ) Is blue, Candida albicans is light blue, Candida albicans is pale pink to pink, and the color of these colored colonies is 40 to 48 hours after cultivation. The color of C. albicans and C. parapsilos may become darker, but other than these, there is no change, and 5 types of Candida can be easily differentiated even after 40 to 48 hours of culture. Can be done.

The tellurate can be appropriately selected from known ones. Specific examples thereof include potassium sterlate and sodium terlate.

The content of this tellurate is preferably in the range of 0.0005 to 0.015 g / L, more preferably in the range of 0.001 to 0.0125 g / L, and even more preferably in the range of 0.001 to 0.01 g / L. Is. If the content of tellurate is less than 0.0005 g / L, the discriminating power is lowered and stable color development cannot be maintained for a long time. On the contrary, when it exceeds 0.015 g / L, the colony is discolored by long-term culture and the discrimination ability is lowered.

The enzyme substrate can be appropriately selected from known substrates as long as it is a substrate for phosphatase and / or N-acetyl-β-D-galactosaminidase produced by Candida. Specific examples thereof include, for example, x-phos. Examples thereof include 2Na and x-galactosamine.

The content of this enzyme substrate is preferably in the range of 0.01 to 0.1 g / L, more preferably in the range of 0.03 to 0.08 g / L. When the content of the enzyme substrate is less than 0.01 g / L, it takes time to develop the color of the colony and the detection sensitivity is lowered. On the contrary, if it exceeds 0.1 g / L, the growth of the colony may be inhibited.

The redox reagent is preferably a tetrazolium salt, and can be appropriately selected from known tetrazolium salts. Specific examples of the tetrazolium salt include TTV and TTB.

The content of this redox reagent is preferably in the range of 0.001 to 0.01 g / L, more preferably in the range of 0.003 to 0.008 g / L. When the content of the redox reagent is less than 0.001 g / L, it takes a long time to develop the color of the colonies. On the contrary, if it exceeds 0.01 g / L, the growth of the colony may be inhibited.

The Candida differential color-developing medium of the present invention contains at least 1 L of medium as a component of x-phos. 2Na 0.01-0.1g, TTV 0.001-0.01g, Potassium Tellurate 0.0005-0.015g, Peptone 10-30g, Yeast Extract 1-10g, White Sugar 1-20g, Glucose 1-10g and Agar It preferably contains 2 to 20 g. In addition to these components, as components that promote the growth of Candida, nitrogen sources such as ammonium sulfate, sugars such as maltose and sucrose, and optionally minerals and vitamins can be included.

Antibacterial agents for suppressing the growth of contaminants include aminoglycoxide antibiotics such as gentamicin, streptomycin, kanamycin and neomycin, broad antibiotics such as chloramphenicol and tetracycline, and penicillin having a broad antibacterial spectrum. Cephalosporins can be mentioned, with chloramphenicol being preferred.

The medium of the present invention may take any form of liquid, semi-fluid, or solid, but from the viewpoint of ease of detection and the like, a solid medium is preferable, and a flat plate solid medium is more preferable. Examples of the solidifying agent for the solid medium include commonly used agents such as agar, carrageenan, and locust bean gum.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

Example 1 Examination of Color Development by Addition Concentration of Potassium Tellurate 51.3 g of the medium component shown in Table 1 was weighed and dissolved in 1000 mL of purified water. After dissolution, the pH was adjusted to 6.0 ± 0.2 and autoclaved at 121 ° C. After cooling, x-phos. After 2Na, TTV and potassium sterate were added by filtration sterilization, 20 mL each was dispensed into a petri dish and solidified. The one to which potassium terrelate was not added was used as a comparative example.

In the medium prepared above, 10 strains (A) to (J) shown in Tables 3 and 4 below were subcultured in advance with a commercially available potato dextrose agar medium, and the obtained strains were smeared with a loop loop. Then, the cells were cultured at 35 ° C. for 48 hours. The results of culturing for 18 to 24 hours are shown in Table 3, and the results of culturing for 40 to 48 hours are shown in Table 4.

As is clear from Table 3, in the color-developing medium for Candida differentiation of the present invention containing an enzyme substrate, a redox reagent, and a tellurate, five types of Candida colonies are easily colored in 18 to 24 hours of culture. It was possible to distinguish between them. On the other hand, in the color-developing medium for Candida differentiation (comparative example) that does not contain terluate, the difference in color tone between Candida albicans and Candida parapsillosis is small. It was difficult, and Candida glabrata did not develop color, so it was not possible to distinguish it from contaminants by the color of the colony.

In addition, as is clear from Table 4, in the Candida differential color-developing medium of the present invention containing an enzyme substrate, an oxidation-reduction reagent, and a terluate, Candida albicans and Candida parapsillosis were observed in 40 to 48 hours of culture. Although the color development may be deep, no discoloration of the colonies of other fungi was observed, and five types of Candida colonies could be easily distinguished. On the other hand, in the color-developing medium for Candida differentiation (comparative example) containing no tellurate, the color of the colonies of Candida albicans, Candida glabrata and Candida parapsillosis changed, and these could not be differentiated. ..

According to the present invention, by containing an enzyme substrate, an oxidation-reduction reagent and a tellurate, a strong color of Candida colonies can be developed in a short time, and five types of Candida bacteria can be differentiated at the same time. Since the color of the colony does not change even after long-term culture, five types of Candida can be differentiated at the same time even after long-term culture.

Claims (9)

Enzyme substrate with a content of 0.01-0.1 g / L, redox reagent with a content of 0.001-0.01 g / L and subs with a content of 0.0005-0.015 g / L a Candida differential for coloring medium you containing tellurium salt,
The enzyme substrate is 5-bromo-4-chloro-3-indrill phosphate. 2Na and / or 5-bromo-4-chloro-3-indrill-N-acetyl-β-D-galactosaminide and
A medium characterized in that the redox reagent is a tetrazolium salt . The redox reagent is tetrazolium violet and / or tetrazolium blue, and
The medium according to claim 1, wherein the terluate is potassium tellurate and / or sodium tellurate. The enzyme substrate is 5-bromo-4-chloro-3-indrill phosphate. The medium according to claim 1 or 2, wherein the medium is 2Na, the redox reagent is tetrazolium violet, and the tellurate salt is potassium tellurate. 10 to 30 g of peptone, 1 to 10 g of yeast extract, 1 to 20 g of white sugar, 1 to 10 g of glucose, 0.001 to 0.01 g of tetrazolium violet, 5-bromo-4-chloro-3-indolyl phosphate per 1 L of medium. The medium according to any one of claims 1 to 3, which contains 0.01 to 0.1 g of 2Na, 0.0005 to 0.015 g of potassium sterate and 3 to 20 g of agar. A method for differentiating Candida bacteria, which comprises inoculating a sample into the medium according to any one of claims 1 to 4 and culturing the sample, and then confirming the colony color of the bacterium. In the color-developing medium for Candida differentiation to confirm the colony color development of Candida after culturing,
Enzyme substrate with a content of 0.01-0.1 g / L, redox reagent with a content of 0.001-0.01 g / L and subs with a content of 0.0005-0.015 g / L It is a method for stabilizing colony color development of Candida bacteria containing tellurate.
The enzyme substrate is 5-bromo-4-chloro-3-indrill phosphate. 2Na and / or 5-bromo-4-chloro-3-indrill-N-acetyl-β-D-galactosaminide,
A method characterized in that the redox reagent is a tetrazolium salt. The redox reagent is tetrazolium violet and / or tetrazolium blue, and
The method according to claim 6, wherein the terluate is potassium tellurate and / or sodium tellurate. The enzyme substrate is 5-bromo-4-chloro-3-indrill phosphate. The method according to claim 6 or 7, wherein the redox reagent is 2Na, tetrazolium violet, and the tellurate salt is potassium tellurate. The medium is 10 to 30 g of peptone, 1 to 10 g of yeast extract, 1 to 20 g of white sugar, 1 to 10 g of glucose, 0.001 to 0.01 g of tetrazolium violet, 5-bromo-4-chloro-3-indrill per liter. Yeast. The method according to any one of claims 6 to 8, which contains 0.01 to 0.1 g of 2Na, 0.0005 to 0.015 g of potassium sterlate and 3 to 20 g of agar .