Dermatophyte culture medium, preparation method and method for culturing dermatophytes

Abstract

The present invention discloses a culture medium for dermatophytes, a preparation method and a method for culturing dermatophytes, and belongs to the field of biotechnology. The culture medium for dermatophytes comprises components including starch, peptone, chloramphenicol, agar, a soluble magnesium salt, a soluble cesium salt, cycloheximide and gentamicin. The preparation method of the culture medium for dermatophytes comprises: stirring and dissolving starch, peptone, agar, a soluble magnesium salt and a soluble cesium salt according to a concentration ratio, sterilizing the mixture, and then adding cycloheximide, gentamicin and chloramphenicol to obtain the culture medium. The present invention also proposes a culture medium for dermatophytes or a method for culturing dermatophytes using the culture medium for dermatophytes, comprising: inoculating dermatophytes into a culture medium and culturing at 28-30°C for more than 3 days; further, the dermatophytes are preferably Microsporum canis. The culture medium can accelerate the colony growth rate of dermatophytes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture medium for dermatophytes, a preparation method thereof, and a method for culturing dermatophytes. Background Art

[0002] Dermatophytes are keratinophilic filamentous fungi, primarily including Trichophyton, Microsporum, and Epidermophyton. Zoophilic dermatophytes include Microsporum canis, Trichophyton mentagrophytes, and Microsporum gypseum. Dermatophytes primarily invade the stratum corneum, disrupting the skin's ecology and causing various pathologies such as hair loss, itching, and erythema, impacting both human and animal health.

[0003] Dermatophyte Test Medium (DTM Medium) is a selective culture medium. Its principle is to use the characteristics of dermatophytes to decompose the skin stratum corneum to produce ammonia and alkali. On the basis of the original SDA medium, the carbon-nitrogen ratio is changed, and phenol red is used as an indicator.

[0004] After culturing dermatophytes on this medium for about a week, they will grow colonies and the medium around the colonies will turn from yellow to red. However, most yeasts and molds will remain yellow even when colonies grow, allowing dermatophytes to be distinguished from non-dermatophytes. Although the identification of fungi cannot be accurate to the species, it greatly facilitates clinical medication and treatment. How to accelerate the growth rate of colonies and shorten the color change time to make the results easier to interpret, thereby facilitating the rapid diagnosis of fungal diseases and targeted and rational medication, is a difficult problem in the existing technology. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above technical deficiencies, provide a culture medium for dermatophytes, a preparation method and a method for culturing dermatophytes, and solve the technical problem of how to accelerate the growth rate of colonies in the prior art.

[0006] To achieve the above technical objectives, the technical solution of the present invention provides a culture medium for dermatophytes, the components of which include: starch, peptone, chloramphenicol, agar, soluble magnesium salt, soluble cesium salt, cycloheximide, and gentamicin.

[0007] Furthermore, an indicator is included.

[0008] Furthermore, the indicator is phenol red.

[0009] Furthermore, the soluble magnesium salt is magnesium chloride; and / or the soluble cesium salt is cesium chloride.

[0010] Furthermore, the concentrations of the components are as follows: starch concentration is 12-15 g / L, peptone concentration is 9-11 g / L, chloramphenicol concentration is 0.05-0.15 g / L, agar concentration is 18-22 g / L, soluble magnesium salt concentration is 280-320 nmol / L, soluble cesium salt concentration is 280-320 nmol / L, cycloheximide concentration is 450-550 mg / L, and gentamicin concentration is 90-110 mg / L.

[0011] Furthermore, the concentrations of the components are as follows: starch concentration is 12-15 g / L, peptone concentration is 6-10 g / L, chloramphenicol concentration is 0.1-0.3 g / L, agar concentration is 18-22 g / L, soluble magnesium salt concentration is 280-320 nmol / L, soluble cesium salt concentration is 280-320 nmol / L, cycloheximide concentration is 450-550 mg / L, gentamicin concentration is 90-110 mg / L, and phenol red concentration is 0.1-0.3 g / L.

[0012] In addition, the present invention also provides a method for preparing the above-mentioned dermatophyte culture medium, comprising the following steps:

[0013] Starch, peptone, agar, soluble magnesium salt and soluble cesium salt are stirred and dissolved according to the concentration ratio, and then sterilized, and then cycloheximide, gentamicin and chloramphenicol are added to obtain the culture medium.

[0014] Furthermore, the sterilization is high-pressure sterilization, and the sterilization temperature is 120-125°C.

[0015] In addition, the present invention also provides a method for culturing dermatophytes using the above-mentioned culture medium for dermatophytes or the culture medium prepared by the above-mentioned culture medium preparation method, comprising the following steps: inoculating dermatophytes into the culture medium and culturing at 28-30°C for more than 3 days.

[0016] Furthermore, the dermatophyte is Microsporum canis.

[0017] Compared with the prior art, the beneficial effects of the present invention include: the culture medium for dermatophytes proposed by the present invention includes: starch, peptone, chloramphenicol, agar, soluble magnesium salt, soluble cesium salt, cycloheximide, and gentamicin. The culture medium can accelerate the growth rate of dermatophytes, and obvious dermatophytes can be observed after 3 days of culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the OD value of Microsporum canis cultured in a culture medium containing different concentrations of ZnCl2, CsCl, MgCl2 and keratin;

[0019] Figure 2 is a Pareto chart of Example 1 of the present invention;

[0020] Figure 3 This is a graph showing the results of a Microsporum canis climbing experiment in Example 1 of the present invention;

[0021] Figure 4 OD value results of Example 2, Comparative Example 1 and Comparative Example 2 of the present invention;

[0022] Figure 5 OD value results of Example 1, Comparative Example 3 and Comparative Example 4 of the present invention;

[0023] Figure 6 It is a comparison chart of culturing Microsporum canis using the culture medium of Example 1 of the present invention and the DTM culture medium. DETAILED DESCRIPTION

[0024] This specific embodiment provides a culture medium for dermatophytes, the components of which include: starch, peptone, chloramphenicol, agar, a soluble magnesium salt, a soluble cesium salt, cycloheximide, and gentamicin; the soluble magnesium salt is magnesium chloride; and / or the soluble cesium salt is cesium chloride.

[0025] In certain embodiments, in order to facilitate direct observation of colony formation, an indicator is further included, and further, the indicator is phenol red.

[0026] Furthermore, the concentrations of the components are as follows: starch concentration is 12-15 g / L, peptone concentration is 9-11 g / L, chloramphenicol concentration is 0.1-0.3 g / L, agar concentration is 18-22 g / L, soluble magnesium salt concentration is 280-320 nmol / L, soluble cesium salt concentration is 280-320 nmol / L, cycloheximide concentration is 450-550 mg / L, and gentamicin concentration is 90-110 mg / L.

[0027] In the embodiment including phenol red, the concentrations of the components are as follows: starch concentration is 12-15 g / L, peptone concentration is 6-10 g / L, chloramphenicol concentration is 0.1-0.3 g / L, agar concentration is 18-22 g / L, soluble magnesium salt concentration is 280-320 nmol / L, soluble cesium salt concentration is 280-320 nmol / L, cycloheximide concentration is 450-550 mg / L, gentamicin concentration is 90-110 mg / L, and phenol red concentration is 0.1-0.3 g / L.

[0028] In addition, this specific embodiment also proposes a method for preparing the above-mentioned dermatophyte culture medium, comprising the following steps:

[0029] Starch, peptone, agar, soluble magnesium salt, and soluble cesium salt are stirred and dissolved according to a concentration ratio, and then sterilized, and then cycloheximide, gentamicin, and chloramphenicol are added to obtain the culture medium; further, the sterilization is high-pressure sterilization, and the sterilization temperature is 120-125°C.

[0030] Furthermore, this specific embodiment also proposes a culture medium for the above-mentioned dermatophytes or a method for culturing dermatophytes using the culture medium for the above-mentioned dermatophytes, comprising the following steps: inoculating dermatophytes into the culture medium and culturing at 28-30°C for more than 3 days; further, the dermatophytes are preferably Microsporum canis.

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] Exploration test

[0033] The effects of culture media with different concentrations of ZnCl2, CsCl, MgCl2 and keratin on the OD value of Microsporum canis were investigated. The relevant concentrations are shown in Table 1.

[0034] Table 1 Concentrations of ZnCl2, CsCl, MgCl2 and keratin in culture medium

[0035] factor a b c <![CDATA[Zn 2+ ]]> 1000nM / L 500nM / L 200nM / L <![CDATA[Cs + ]]> 1000nM / L 500nM / L 200nM / L <![CDATA[Mg 2+ ]]> 1000nM / L 500nM / L 200nM / L Keratin 25ml / L 10ml / L 5ml / L

[0036] The concentrations of other components in the culture medium were as follows: 15 g / L starch, 10 g / L peptone, 0.1 g / L chloramphenicol, 20 g / L agar, 500 mg / L cycloheximide, 100 mg / L gentamicin, and 0.2 g / L phenol red.

[0037] The results are as follows Figure 1 As shown, from Figure 1 It can be seen that the OD values of the Mgb and Csa groups reached 2.309 at 7 days, and the difference between the Mgc group and the blank group was extremely significant, with the OD value reaching 6.033 at 7 days. However, the experimental groups of Zn salt and keratin inhibited the growth rate of Microsporum canis. Therefore, Mg 2+ and Cs 2+ It is the key factor to improve the culture medium.

[0038] Based on this, the following embodiments are further proposed:

[0039] Example 1

[0040] This example provides a culture medium for dermatophytes, in which the concentrations of the components are as follows: 15 g / L starch, 10 g / L peptone, 0.1 g / L chloramphenicol, 20 g / L agar, 300 nmol / L MgCl2, 300 nmol / L CsCl, 500 mg / L cycloheximide, 100 mg / L gentamicin, and 0.2 g / L phenol red.

[0041] This embodiment also provides a method for preparing a culture medium for dermatophytes, comprising the following steps:

[0042] Starch, peptone, agar, MgCl2, and CsCl are stirred and dissolved according to a concentration ratio, and then sterilized by high pressure at a sterilization temperature of 121° C. Then, cycloheximide, gentamicin, and chloramphenicol are added to obtain the culture medium.

[0043] In order to further explore the most influential factors in this culture medium system, this example conducted a Pb experimental design:

[0044] Seven real factors and four fictitious variables were set to estimate the error. Each factor had two levels, with the low level being the original culture condition and the high level being 1.5 times the low level.

[0045] There's often a difference in response values between high and low levels of the same factor. This difference is larger for key factors, while smaller for non-key factors. Therefore, using a Pareto chart to show the difference in response values for different factors can help us intuitively identify key factors with large differences in response values. The chart plots the absolute value of the effect as a reference dashed line; factor effects exceeding this line are likely significant.

[0046] SAS software was used for Plackett-Burman (N=12) experimental design, and Minitab software was used for experimental analysis. The main effect of each factor, that is, the influence of the factor on the response value when other factors remained unchanged, was subjected to significance analysis and regression analysis. The significant influencing factors affecting the discoloration rate of Microsporum canis were screened out from the seven culture medium components.

[0047] Table 2 Design of PB experiment

[0048]

[0049] Table 2 shows the design of the PB experiment. The horizontal axis represents the influencing factors, and the vertical axis represents the experimental groups. +1 represents that the factor in that group is 1.5 times the standard value, and -1 represents that the factor in that group is the standard value. This design was used to screen for significant factors.

[0050] Depend on Figure 2 It can be seen that after 7 days of culture, there is a significant influencing factor among the 7 factors in the culture medium that may affect the discoloration rate of Microsporum canis: peptone (P<0.05). Among these 7 factors, the regression equation coefficient shows that factors A, F, and G are positive effect factors, and high concentrations of these factors are selected in subsequent climbing experiments. Factors B, C, D, and E are negative effect factors, and low concentrations are selected in subsequent climbing experiments. Among them, A: starch; B: peptone; C: chloramphenicol; D: cycloheximide; E: gentamicin; F: MgCl2; G: CsCl.

[0051] The regression equation expressed in uncoded units:

[0052] -Y^-0.5=-0.5897+0.0102A-0.0789B-0.0720C-0.0057D-0.0691E+0.0592F+0.0287G

[0053] The steepest climbing experiment was designed based on the fitting equation obtained from the PB experiment. The experimental design table is shown in Table 3. The experimental results are shown in Figure 3 As shown by Figure 3 As can be seen, the change in peptone content had little effect on the absorbance on the 7th day, but starting from the 3rd day, the absorbance of group A (10g / L) was higher than that of the other groups. Therefore, the optimal peptone concentration is 10g / L.

[0054] Table 3 Concentration of peptone in the steepest climbing experiment

[0055] factor A B C D E Peptone 10g / L 9g / L 8g / L 7g / L 6g / L

[0056] Example 2

[0057] This example provides a culture medium for dermatophytes, in which the concentrations of the components are as follows: 15 g / L starch, 8 g / L peptone, 0.2 g / L chloramphenicol, 18 g / L agar, 280 nmol / L MgCl2, 320 nmol / L CsCl, 550 mg / L cycloheximide, 90 mg / L gentamicin, and 0.1 g / L phenol red.

[0058] This embodiment also provides a method for preparing a culture medium for dermatophytes, comprising the following steps:

[0059] Starch, peptone, agar, MgCl2, and soluble CsCl are stirred and dissolved according to a concentration ratio, and then sterilized by high pressure at a sterilization temperature of 120°C. Then, cycloheximide, gentamicin, and chloramphenicol are added to obtain the culture medium.

[0060] This embodiment also provides a method for culturing dermatophytes in the culture medium of the embodiment, comprising the following steps: inoculating dermatophytes in the culture medium, culturing at 28° C. for 7 days, and observing and recording the growth of the colonies.

[0061] Example 3

[0062] This example provides a culture medium for dermatophytes, wherein the concentrations of the components are as follows: 13 g / L starch, 6 g / L peptone, 0.3 g / L chloramphenicol, 22 g / L agar, 320 nmol / L MgCl2, 280 nmol / L CsCl, 450 mg / L cycloheximide, 110 mg / L gentamicin, and 0.3 g / L phenol red.

[0063] This embodiment also provides a method for preparing a culture medium for dermatophytes, comprising the following steps:

[0064] Starch, peptone, agar, MgCl2, and soluble CsCl are stirred and dissolved according to a concentration ratio, and then sterilized by high pressure at a sterilization temperature of 125° C., and then cycloheximide, gentamicin, and chloramphenicol are added to obtain the culture medium.

[0065] This embodiment also provides a method for culturing dermatophytes in the culture medium of the embodiment, comprising the following steps: inoculating dermatophytes in the culture medium, culturing at 28° C. for 7 days, and observing and recording the growth of the colonies.

[0066] Comparative Example 1

[0067] The only difference between this comparative example and Example 1 is that glucose is used instead of starch, and the other raw materials, amounts and preparation methods are the same as those in Example 1.

[0068] Comparative Example 2

[0069] The only difference between this comparative example and Example 1 is that sucrose is used instead of starch, and the other raw materials, amounts and preparation methods are the same as those in Example 1.

[0070] Comparative Example 3

[0071] The only difference between this comparative example and Example 1 is that beef powder is used instead of peptone, and the other raw materials, amounts and preparation methods are the same as those in Example 1.

[0072] Comparative Example 4

[0073] The only difference between this comparative example and Example 1 is that yeast powder is used instead of peptone, and the other raw materials, amounts and preparation methods are the same as those in Example 1.

[0074] Related trials:

[0075] Method for culturing dermatophytes in culture medium

[0076] The culture medium of Example 1, Example 2, and Comparative Examples 1-4 was used to culture Microsporum canis, and DTM culture medium was used as a control group for culture, including the following steps: Microsporum canis was inoculated into SDA culture medium for activation and culture for 7 days, and then washed with sterile water to prepare a bacterial suspension. 200 μl of the bacterial suspension was inoculated into DTM culture medium and homemade culture medium respectively and evenly spread, and cultured at 28°C. The growth was recorded daily. The results are shown in FIG. Figure 4-5 shown; from Figure 4 It can be seen that the absorbance change rate of Microsporum canis in Example 2 at a wavelength of 560nm is the best, and there is a gap between the other two groups on the fifth day of the experiment, and the absorbance reaches 2.104 on the seventh day. Figure 5 It can be seen that Example 1 has the best absorbance change rate at a wavelength of 560 nm, which differs from the other two groups on the fourth day of the experiment, and reaches an absorbance of 2.691 on the seventh day.

[0077] It should be noted that the components of DTM culture medium are: peptone 10 g / L, glucose 10 g / L, phenol red 0.2 g / L, chloramphenicol 0.1 g / L, agar 20 g / L; gentamicin 100 mg / L, cycloheximide 500 mg / L.

[0078] It should also be noted that the reason why the OD values of Example 1 in each figure are different is that the inoculation amount of Microsporum canis was different when each comparative test was performed (the results of each figure were obtained at different times), while the inoculation amount of Microsporum canis in the tests of the same group, i.e., the tests in the same sub-figure, was the same.

[0079] from Figure 6 It can be seen that at 3 days, colonies began to grow in the homemade culture medium of Example 1 and the culture medium turned light red near the colonies, proving that while Microsporum canis grew, it decomposed the nutrients in the culture medium and produced alkali, causing the indicator phenol red to change color, but no colonies grew and no color change occurred in the DTM culture medium. At 5 days, large-scale colony growth appeared in the homemade culture medium, and the culture medium turned dark red; small colonies grew in the DTM culture medium and there was a light red color change around the colonies. At 7 days, the homemade culture medium was almost completely covered with colonies, and the color of the culture medium was still dark red; small-scale colonies grew in the DTM culture medium and the culture medium around the colonies turned red. It can be seen that the culture medium of this embodiment is significantly better than the DTM culture medium in culturing Microsporum canis.

[0080] Because dermatophytes cause identification culture media to change color based on a similar principle, the present invention uses Microsporum canis as an example and studies the effects of several key factors on the culture media's color change rate based on the original formula, thereby further accelerating the colony growth rate and advancing its color change time, making the results easier to interpret, thereby facilitating the rapid diagnosis of fungal diseases and targeted and rational use of drugs.

[0081] Other beneficial effects:

[0082] 1) Starch as the carbon source in the culture medium has a faster color change rate than glucose as the only carbon source.

[0083] 2) Adding trace elements magnesium ions and cesium ions to the culture medium can increase the growth rate of fungi and accelerate the color change of the culture medium.

[0084] 3) Because dermatophytes produce alkali as they grow, causing the indicator to change color, their growth is synchronized with the color change of the culture medium. Non-dermatophytes, on the other hand, produce acid first and then alkali during their growth, and therefore generally exhibit growth followed by color change. Therefore, the criteria for identification are: if the culture medium changes color at the same time as colony formation, it is a dermatophyte; otherwise, it is a non-dermatophyte.

[0085] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A culture medium for dermatophytes, characterized in that The invention comprises the following components: starch, peptone, chloramphenicol, agar, soluble magnesium salt, soluble cesium salt, cycloheximide, and gentamicin; the concentrations of the components are as follows: starch concentration is 12-15 g / L, peptone concentration is 9-11 g / L, chloramphenicol concentration is 0.1-0.3 g / L, agar concentration is 18-22 g / L, soluble magnesium salt concentration is 280-320 nmol / L, soluble cesium salt concentration is 280-320 nmol / L, cycloheximide concentration is 450-550 mg / L, and gentamicin concentration is 90-110 mg / L; and the invention also comprises an indicator, which is phenol red.

2. The culture medium for dermatophytes according to claim 1, characterized in that The soluble magnesium salt is magnesium chloride; and / or the soluble cesium salt is cesium chloride.

3. The culture medium for dermatophytes according to claim 1, characterized in that The concentrations of the components are as follows: starch concentration is 12-15 g / L, peptone concentration is 6-10 g / L, chloramphenicol concentration is 0.1-0.3 g / L, agar concentration is 18-22 g / L, soluble magnesium salt concentration is 280-320 nmol / L, soluble cesium salt concentration is 280-320 nmol / L, cycloheximide concentration is 450-550 mg / L, gentamicin concentration is 90-110 mg / L, and phenol red concentration is 0.1-0.3 g / L.

4. A method for culturing dermatophytes using the dermatophyte culture medium according to any one of claims 1 to 3, characterized in that: The following steps are involved: The dermatophytes are inoculated into a culture medium and cultured at 28-30° C. for more than 3 days; the dermatophytes are Microsporum canis.

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