A Candida chromogenic culture medium and its preparation method and application

Abstract

The present invention belongs to the field of microbial culture and identification technology, and provides a Candida chromogenic culture medium and its preparation method and application. The present invention utilizes a specific concentration of chlorinated nitro blue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, and 5-bromo-6-chloro-3-indole-β-D-galactoside as the main color-developing component in the Candida chromogenic culture medium, and further utilizes the Candida chromogenic culture medium of the present invention to cultivate different types of Candida for a period of time, and can display different colors according to different types of Candida, wherein Candida albicans can display obvious red, which is different from the colors of other types of Candida, and the red colony is not only conspicuous, but also easier to identify than light green or green colonies, and can be well distinguished from the colors of other colonies, so that identification personnel can directly observe and identify Candida albicans with the naked eye.

Description

Technical Field

[0001] The invention relates to the technical field of microbial culture and identification, and more specifically to a Candida chromogenic culture medium and a preparation method and application thereof. Background Art

[0002] Candida albicans has the highest average proportion among all forms of human candidiasis isolates, reaching more than 80% (RA Calderone, Candida and Candidiasis, ASM Press, Washington DC, 2002, pp. 15-25). Therefore, the identification of Candida albicans is particularly important for the performance of Candida colorimetric culture medium. Chinese patent (publication number CN101948902A) discloses a Candida colorimetric culture medium, which can be used to identify the type of Candida by the color displayed by the culture medium after the bacteria are inoculated into the culture medium. For example, if light green or green colonies appear on the plate, it means that Candida albicans is present in the sample; if dark blue or cyan colonies appear, it means that tropical Candida is present in the sample; if light purple or purple-red flat colonies with white edges appear, it means that Candida krusei is present in the sample; if light pink or pink smooth colonies appear, it means that Candida glabrata or other yeasts are present in the sample. However, for Candida albicans, this Candida chromogenic culture medium shows light green or green colonies, which are difficult for identification personnel to identify by direct observation with the naked eye, and are also difficult to distinguish from other types of colonies (for example, tropical Candida can be cyan, which is close to the color of light green or green Candida albicans colonies).

[0003] Therefore, it is urgent to develop a Candida albicans chromogenic culture medium that can easily identify Candida albicans by direct observation with the naked eye and has a high color distinction from other types of Candida colonies. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a Candida color development medium and a preparation method and application thereof. After different types of Candida are cultured in the Candida display medium of the present invention for a period of time, Candida albicans will be obviously red, and other colonies will be green-like (such as tropical Candida is cyan colonies, Candida krusei is green colonies, and Candida glabrata is bright cyan colonies). The red colonies are not only conspicuous, but also easier to identify than light green or green colonies, and can be well distinguished from the colors of other colonies, so that identification personnel can directly observe and identify Candida albicans with the naked eye.

[0005] A first aspect of the present invention provides a Candida chromogenic culture medium.

[0006] Specifically, a Candida chromogenic culture medium comprises a chromogenic composition, wherein the chromogenic composition comprises the following components:

[0007] Nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, and 5-bromo-6-chloro-3-indole-β-D-galactoside;

[0008] In the Candida chromogenic culture medium, the concentration of the nitro blue tetrazolium chloride is 0.01-0.02 mg / mL, the concentration of the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt is 0.02-0.04 mg / mL, and the concentration of the 5-bromo-6-chloro-3-indole-β-D-galactoside is 0.25-0.5 mg / mL.

[0009] The invention utilizes a combination of nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indole-β-D-galactoside as the main color-developing components in a Candida chromogenic culture medium, so that after culturing different types of Candida for a period of time, the Candida chromogenic culture medium can display different colors according to different types of Candida, wherein Candida albicans can display a conspicuous red color, which is different from the colors of other types of Candida (for example, Candida albicans presents red colonies, Candida tropicalis presents cyan colonies, Candida krusei presents green colonies, and Candida glabrata presents bright cyan colonies), and has high differentiation, thereby well distinguishing Candida albicans from other types of Candida.

[0010] Preferably, the Candida chromogenic culture medium uses Sabouraud medium as the basic culture medium.

[0011] Preferably, the Sabouraud medium contains 5-15 g / L of peptone, 10-30 g / L of agar, and 30-50 g / L of glucose.

[0012] Further preferably, the Sabouraud medium contains 10-15 g / L of peptone, 14-20 g / L of agar, and 40-50 g / L of glucose.

[0013] Preferably, the Sabouraud medium also includes chloramphenicol.

[0014] More preferably, the Sabouraud culture medium also includes 0.1-0.2 mg / L of chloramphenicol.

[0015] A second aspect of the present invention provides a method for preparing a Candida chromogenic culture medium.

[0016] A method for preparing a Candida chromogenic culture medium comprises the following steps:

[0017] (1) taking a basal culture medium, sterilizing it, and obtaining a sterilized culture medium;

[0018] (2) Mixing the color-developing composition and the solvent to obtain a color-developing application liquid, then adding the color-developing application liquid to the sterilized culture medium, adjusting the pH value to 6±0.5, sealing, heating at 90-110° C. for 5-15 min, and then cooling to 50-70° C. to obtain a prepared culture medium, and then transferring the prepared culture medium to a culture dish to obtain the Candida color-developing culture medium.

[0019] In the process of preparing the Candida chromogenic medium of the present invention, after adjusting the pH value, a heating treatment is also performed, which is beneficial to the uniform dispersion of the chromogenic reagent in the medium and the control of contaminating bacteria in the medium. However, it should be noted that the reheating should not be too long, otherwise the chromogenic reagent will be decomposed, affecting the color development effect of the medium.

[0020] Preferably, in step (1), the sterilization is high-pressure sterilization, the pressure of the high-pressure sterilization is 100-120 kPa, and / or the temperature of the high-pressure sterilization is 110-130° C., and / or the time of the high-pressure sterilization is 10-20 min.

[0021] Further preferably, in step (1), the pressure of the high pressure sterilization is 100-115 kPa.

[0022] Preferably, in step (2), the solvent is acetone and / or ethanol.

[0023] Preferably, in step (2), the color development application liquid is filtered before being added to the sterilized culture medium for mixing.

[0024] Preferably, the filtration uses a sterile organic microporous filter membrane, and the pore size of the filter membrane is 0.20-0.25 μm.

[0025] Further preferably, the filtration uses a sterile organic microporous filter membrane, and the pore size of the filter membrane is 0.22-0.24 μm.

[0026] Preferably, the pH value is adjusted by using hydrochloric acid solution and / or sodium hydroxide solution.

[0027] Preferably, the heating treatment is water bath heating.

[0028] Preferably, when the diameter of the culture dish is 70 mm, the injection volume of the culture medium is 8-12 mL.

[0029] Preferably, after the prepared culture medium is transferred to a culture dish, vacuum dehumidification is performed and sealed and packaged to obtain the Candida chromogenic culture medium.

[0030] The third aspect of the present invention provides a use of a Candida chromogenic culture medium.

[0031] The invention discloses an application of a Candida chromogenic culture medium in the identification and cultivation of Candida.

[0032] Preferably, the Candida species include at least two of Candida albicans, Candida tropicalis, Candida krusei and Candida glabrata.

[0033] A fourth aspect of the present invention provides a method for identifying Candida species.

[0034] A method for identifying Candida albicans comprises the following steps:

[0035] The strains to be tested are inoculated into the Candida colorimetric culture medium respectively, and cultured at 30-40° C. for 24-40 hours; the strains to be tested include Candida albicans.

[0036] Preferably, the culture is carried out at 37-40°C for 36-40 hours.

[0037] Preferably, the bacterial species to be tested further includes at least one of Candida tropicalis, Candida krusei and Candida glabrata.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] The present invention utilizes a combination of nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, and 5-bromo-6-chloro-3-indole-β-D-galactoside of a specific concentration as the main color-developing component in the Candida color-developing culture medium. After different types of Candida are cultured using the Candida color-developing culture medium of the present invention for a period of time, different colors can be displayed according to different types of Candida, wherein Candida albicans can display an obvious red color, which is different from the colors of other types of Candida (for example, Candida albicans presents red colonies, Candida tropicalis presents cyan colonies, Candida krusei presents green colonies, and Candida glabrata presents bright cyan colonies). The red colonies are not only conspicuous, but also easier to identify than light green or green colonies, and can be well distinguished from the colors of other colonies, so that identification personnel can directly observe and identify Candida albicans with the naked eye. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a photo of a Candida albicans colony cultured with the Candida chromogenic medium of Example 1 of the present invention;

[0041] Figure 2 This is a photo of a Candida tropicalis colony cultured using the Candida chromogenic medium of Example 1 of the present invention;

[0042] Figure 3This is a photograph of a Candida krusei colony cultured using the Candida chromogenic medium of Example 1 of the present invention;

[0043] Figure 4 This is a photograph of a Candida glabrata colony cultured with the Candida chromogenic medium of Example 1 of the present invention;

[0044] Figure 5 This is a photo of the Candida albicans colony after being cultured using the Candida chromogenic medium of Comparative Example 1;

[0045] Figure 6 A photograph of a Candida tropicalis colony cultured with the Candida chromogenic medium of Comparative Example 1;

[0046] Figure 7 This is a photo of the Candida krusei colony after being cultured using the Candida chromogenic medium of Comparative Example 1;

[0047] Figure 8 This is a photo of the Candida glabrata colony cultured with the Candida chromogenic medium of Comparative Example 1;

[0048] Fig. 9 This is a photo of the Candida albicans colony after being cultured using the Candida chromogenic medium of Comparative Example 2;

[0049] Fig.10 A photograph of a Candida tropicalis colony cultured with the Candida chromogenic medium of Comparative Example 2;

[0050] Fig.11 This is a photo of the Candida krusei colony after being cultured using the Candida chromogenic medium of Comparative Example 2;

[0051] Fig.12 This is a photo of the Candida glabrata colony after being cultured using the Candida chromogenic medium of Comparative Example 2;

[0052] Fig.13 This is a photo of the Candida albicans colony after being cultured using the Candida chromogenic medium of Comparative Example 3;

[0053] Fig.14 This is a photo of a Candida tropicalis colony cultured with the Candida chromogenic medium of Comparative Example 3;

[0054] Fig.15 This is a photograph of the Candida krusei colony after being cultured using the Candida chromogenic medium of Comparative Example 3;

[0055] Fig.16 This is a photo of the Candida glabrata colony after being cultured using the Candida chromogenic medium of Comparative Example 3;

[0056] Fig.17This is a photo of the Candida albicans colony after being cultured using the Candida chromogenic medium of Comparative Example 4;

[0057] Fig.18 A photograph of a Candida tropicalis colony cultured with the Candida chromogenic medium of Comparative Example 4;

[0058] Fig.19 This is a photograph of the Candida krusei colony after being cultured using the Candida chromogenic medium of Comparative Example 4;

[0059] Fig. 20 This is a photo of the Candida glabrata colony after being cultured using the Candida chromogenic medium of Comparative Example 4;

[0060] Fig.21 This is a photo of the Candida albicans colony after being cultured using the Candida chromogenic medium of Comparative Example 5;

[0061] Fig. 22 A photograph of a Candida tropicalis colony cultured with the Candida chromogenic medium of Comparative Example 5;

[0062] Fig.23 This is a photograph of the Candida krusei colony after being cultured using the Candida chromogenic medium of Comparative Example 5;

[0063] Fig.24 This is a photograph of the Candida glabrata colony after being cultured using the Candida chromogenic medium of Comparative Example 5. DETAILED DESCRIPTION

[0064] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0065] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0066] Example 1

[0067] The invention discloses a Candida chromogenic culture medium, which adopts Sabouraud culture medium as a basic culture medium, contains 10 g / L of peptone, 15 g / L of agar, 40 g / L of glucose, 0.1 mg / L of chloramphenicol and a color development composition, wherein the color development composition has the following components: nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indolyl-β-D-galactoside, wherein in the Candida chromogenic culture medium, the concentrations of nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indolyl-β-D-galactoside are 0.01 mg / mL, 0.03 mg / mL and 0.4 mg / mL respectively.

[0068] The preparation method of the Candida chromogenic culture medium comprises the following steps:

[0069] (1) Prepare Sabouraud medium (containing 40 g glucose, 10 g maltose, 10 g peptone, 15 g agar, and 10 g yeast powder per 1000 mL) as the basal medium, add 0.1 mg / L chloramphenicol, sterilize at 121° C., 115 kPa for 15 min, and then take out to obtain the sterilized medium for later use;

[0070] (2) Dissolve nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, and 5-bromo-6-chloro-3-indole-β-D-galactoside in acetone to prepare a color development application solution. After filtering the color development application solution through a sterile organic microporous filter membrane (pore size 0.22 μm), add it to the sterilized culture medium and mix it evenly. Adjust the pH of the culture medium to 6.2±0.1 with hydrochloric acid solution and reseal it; heat the culture medium in a water bath at 100°C for 2 minutes and then take it out; cool the prepared culture medium to 60°C and transfer it to a culture dish. Each culture dish with a diameter of 70 mm is injected with 10 mL of culture medium; vacuum dehumidify the culture dish loaded with the culture medium, seal it and package it to prepare a Candida color development culture medium.

[0071] Example 2

[0072] A Candida chromogenic medium, which is different from Example 1 in that, in the Candida chromogenic medium, the concentrations of nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indole-β-D-galactoside are 0.01 mg / mL, 0.02 mg / mL and 0.5 mg / mL respectively. The preparation method is the same as that of Example 1.

[0073] Example 3

[0074] A Candida chromogenic medium, which is different from Example 1 in that, in the Candida chromogenic medium, the concentrations of nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indole-β-D-galactoside are 0.02 mg / mL, 0.04 mg / mL and 0.25 mg / mL respectively. The preparation method is the same as that of Example 1.

[0075] Comparative Example 1

[0076] A Candida chromogenic medium, which is different from that in Example 1 in that the Candida chromogenic medium does not contain nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indole-β-D-galactoside. The preparation method is the same as that in Example 1.

[0077] Comparative Example 2

[0078] A Candida chromogenic medium, which is different from Example 1 in that, in the Candida chromogenic medium, the concentrations of nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indole-β-D-galactoside are 0.03 mg / mL, 0.03 mg / mL and 0.3 mg / mL respectively. The preparation method is the same as that of Example 1.

[0079] Comparative Example 3

[0080] A Candida chromogenic medium, which is different from Example 1 in that, in the Candida chromogenic medium, the concentrations of nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indole-β-D-galactoside are 0.025 mg / mL, 0.01 mg / mL and 0.1 mg / mL respectively. The preparation method is the same as that of Example 1.

[0081] Comparative Example 4

[0082] A Candida chromogenic medium, which is different from Example 1 in that the concentrations of nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indole-β-D-galactoside in the Candida chromogenic medium are 0.015 mg / mL, 0.04 mg / mL and 0.1 mg / mL respectively. The preparation method is the same as that in Example 1.

[0083] Comparative Example 5

[0084] A Candida chromogenic medium, which is different from Example 1 in that, in the Candida chromogenic medium, the concentrations of nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indole-β-D-galactoside are 0.02 mg / mL, 0.08 mg / mL and 0.25 mg / mL respectively. The preparation method is the same as that of Example 1.

[0085] Product effect testing

[0086] In order to study the color development of each of the above culture media, the following tests were performed:

[0087] Take 4 identical candida colorimetric culture media of Example 1, and then inoculate four candida of equal mass and concentration, namely, Candida albicans (strain number: ATCC 90028), Candida tropicalis (strain number: ATCC 750), Candida krusei (strain number: ATCC 6258), and Candida glabrata (strain number: ATCC 2001) in the candida colorimetric culture media of Example 1, and then grow them in an incubator at 37° C. for 36 hours, and then observe and record the color change of the culture media. The culture media of other embodiments and comparative examples are tested using the same method as above.

[0088] The bacterial colonies after culture in the culture medium of Example 1 are as follows Figure 1-4 As shown, Candida albicans appears as red colonies ( Figure 1 ), Candida tropicalis appears as cyan colonies ( Figure 2 ), Candida krusei appears as green colonies ( Figure 3 ), Candida glabrata presents bright cyan colonies ( Figure 4 ), the colors of various fungi are significantly different. Among them, because Candida albicans has red colonies, it forms a sharp "red-green contrast" with other cyan, green or bright cyan colors, which improves the recognizability of Candida albicans and makes it easy to identify Candida albicans with the naked eye.

[0089] The growth of colonies after culture in the culture medium of Example 2 and Example 3 is the same as that of Example 1, with Candida albicans showing red colonies, Candida tropicalis showing cyan colonies, Candida krusei showing green colonies, and Candida glabrata showing bright cyan colonies, and Candida albicans can be easily distinguished by naked eyes.

[0090] The bacterial colonies after culture in the culture medium of Comparative Example 1 are as follows Figure 5-8 As shown, since there is no color development reagent, Candida albicans ( Figure 5 ), Candida tropicalis ( Figure 6 ), Candida krusei ( Figure 7 ), Candida glabrata ( Figure 8 ) are all white transparent colonies.

[0091] The bacterial colonies after culture in the culture medium of Comparative Example 2 are as follows Figure 9-12 As shown in the figure, due to the increase in the concentration of nitro blue tetrazolium chloride, Candida albicans showed light red colonies and its growth was partially inhibited ( Fig. 9 ); Candida tropicalis showed pale cyan colonies, and its growth was partially inhibited ( Fig.10 ); Candida krusei showed light green colonies, and its growth was partially inhibited ( Fig.11 ); Candida glabrata showed bright cyan colonies, and its growth was partially inhibited ( Fig.12 ).

[0092] The bacterial colonies after culture in the culture medium of Comparative Example 3 are as follows Figure 13-16 As shown in the figure, due to the increase in the concentration of nitro blue tetrazolium chloride and the decrease in the concentrations of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and 5-bromo-6-chloro-3-indole-β-D-galactoside, Candida albicans showed gray colonies and its growth was partially inhibited ( Fig.13 ); Candida tropicalis showed gray colonies, and its growth was partially inhibited ( Fig.14 ); Candida krusei appeared as gray colonies, and its growth was partially inhibited ( Fig.15 ), Candida glabrata showed gray colonies, and its growth was partially inhibited ( Fig.16 ).

[0093] The bacterial colonies after culture in the culture medium of Comparative Example 4 are as follows Figure 17-20 As shown in the figure, due to the reduction of the concentration of 5-bromo-6-chloro-3-indole-β-D-galactoside, Candida albicans showed light cyan colonies ( Fig.17 ), Candida tropicalis appears as cyan colonies ( Fig.18 ), Candida krusei appears as green colonies ( Fig.19 ), Candida glabrata presents bright cyan colonies ( Fig. 20 ), among which Candida albicans is close to Candida tropicalis and Candida glabrata in color, and it is not easy to distinguish Candida albicans with the naked eye.

[0094] The bacterial colonies after culture in the culture medium of Comparative Example 5 are as follows Figure 21-24 As shown, due to the increase in the concentration of 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, Candida albicans showed cyan colonies ( Fig.21 ), Candida tropicalis appears as green colonies ( Fig. 22 ), Candida krusei appears as green colonies ( Fig.23 ), Candida glabrata appears as bright green colonies ( Fig.24 ), wherein Candida albicans is cyan, Candida tropicalis, Candida krusei and Candida glabrata are green, and compared with the red colonies of Candida albicans in Example 1, it is not easy to distinguish Candida albicans by naked eyes.

Claims

1. A Candida chromogenic culture medium, characterized in that The invention comprises a color developing composition, wherein the color developing composition comprises the following components: Nitro blue tetrazolium chloride, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, and 5-bromo-6-chloro-3-indole-β-D-galactoside; In the Candida chromogenic culture medium, the concentration of the nitro blue tetrazolium chloride is 0.01-0.02 mg / mL, the concentration of the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt is 0.02-0.04 mg / mL, and the concentration of the 5-bromo-6-chloro-3-indole-β-D-galactoside is 0.25-0.5 mg / mL.

2. The Candida chromogenic culture medium according to claim 1, characterized in that The Candida chromogenic culture medium uses Sabouraud medium as the basic culture medium.

3. The Candida chromogenic culture medium according to claim 2, characterized in that The Sabouraud culture medium contains 5-15 g / L of peptone, 10-30 g / L of agar, and 30-50 g / L of glucose.

4. The method for preparing the Candida chromogenic culture medium according to any one of claims 1 to 3, characterized in that: The steps include: (1) taking a basal culture medium, sterilizing it, and obtaining a sterilized culture medium; (2) Mixing the color-developing composition and the solvent to obtain a color-developing application liquid, then adding the color-developing application liquid to the sterilized culture medium, adjusting the pH value to 6±0.5, sealing, heating at 90-110° C. for 5-15 min, and then cooling to 50-70° C. to obtain a prepared culture medium, and then transferring the prepared culture medium to a culture dish to obtain the Candida color-developing culture medium.

5. The preparation method according to claim 4, characterized in that: In step (1), the sterilization is high-pressure sterilization, the pressure of the high-pressure sterilization is 100-120 kPa, and / or the temperature of the high-pressure sterilization is 110-130° C., and / or the time of the high-pressure sterilization is 10-20 min.

6. The preparation method according to claim 4, characterized in that: In step (2), the solvent is acetone and / or ethanol.

7. The preparation method according to claim 4, characterized in that: In step (2), the heating treatment is water bath heating.

8. The preparation method according to claim 4, characterized in that: In step (2), when the diameter of the culture dish is 70 mm, the injection volume of the culture medium is 8-12 mL.

9. Use of the Candida chromogenic culture medium according to any one of claims 1 to 3 in the identification and cultivation of Candida.

10. A method for identifying Candida albicans, characterized in that: The method comprises the following steps: inoculating the strains to be tested into the Candida chromogenic culture medium according to any one of claims 1 to 3, respectively, and culturing at 30-40° C. for 24-40 hours; the strains to be tested include Candida albicans.

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