Chocolate agar culture medium formula and preparation method thereof

Abstract

The invention discloses a chocolate agar culture medium formula and a preparation method thereof, the chocolate agar culture medium formula comprises the following components in proportion: 3.0-5.0% of blood agar basal culture medium, 0.5-1.5% of freeze-dried hemoglobin particles, and the balance of purified water; the preparation method comprises the following steps: step 1, raw material pretreatment; step 2, weighing and dissolving; step 3, high-pressure sterilization; step 4, solution mixing; and step 5, subpackaging the flat plate. According to the formula of the chocolate agar culture medium and the preparation method of the chocolate agar culture medium, the purpose of rapid sterilization can be achieved, operation is easy, and the risk that the culture medium is polluted is avoided.

Description

Technical Field

[0001] The invention relates to the technical field of chocolate agar culture medium, in particular to a chocolate agar culture medium formula and a preparation method thereof. Background Art

[0002] Chocolate agar medium is a medium used for microbial culture, mainly used for the culture of Haemophilus and Neisseria, and is also suitable for the proliferation of Neisseria. This medium has important application value in microbiological research and can help researchers effectively culture and observe the growth of these microorganisms.

[0003] Patent CN106978363A describes a chocolate agar medium and a preparation method thereof, and patent 101550404A describes an improved chocolate agar medium and a preparation method thereof. Both adopt the traditional preparation method. After the medium base solution is sterilized by high pressure, it is cooled to 45-50°C, sterile defibrinated sheep blood is added, mixed evenly, heated in a 70-80°C water bath for 30 minutes, and then dispensed into plates. The water bath heating step in the traditional preparation method is complicated to operate, time-consuming and labor-intensive, and also increases the risk of medium contamination. The purpose of the present invention is to overcome the deficiencies in the above-mentioned prior art and provide a chocolate agar medium formula and a preparation method thereof. Summary of the invention

[0004] The object of the present invention is to provide a chocolate agar culture medium formula and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a chocolate agar culture medium, wherein the chocolate agar culture medium comprises, by proportion, 3.0-5.0% of blood agar basal culture medium, 0.5-1.5% of freeze-dried hemoglobin particles, and the remainder being purified water.

[0006] Preferably, the chocolate agar medium formula comprises, by proportion, 4.5% blood agar basal medium, 1.0% freeze-dried hemoglobin particles, and the remainder is purified water.

[0007] Preferably, the chocolate agar medium formula comprises, in proportion, 5.0% blood agar basal medium, 1.5% freeze-dried hemoglobin particles, and the remainder purified water.

[0008] A method for preparing a chocolate agar medium comprises the following steps:

[0009] Step 1: Pretreatment of raw materials: Grind freeze-dried hemoglobin particles with a grinder until they are in powder form;

[0010] Step 2, weighing and dissolving: Place the weighed freeze-dried hemoglobin powder in a clean and dry container, add purified water to the container, heat and stir to dissolve; Place the weighed blood agar basal medium in another clean and dry container, add purified water to the container, heat and stir to dissolve;

[0011] Step 3, high pressure sterilization: sterilize the above solution with high pressure steam, and cool to 45-50°C after sterilization;

[0012] Step 4, solution mixing: under sterile conditions, add the hemoglobin solution to the blood agar basal medium and mix thoroughly;

[0013] Step 5, filling plates: under sterile conditions, pouring sterile plates to prepare the culture medium.

[0014] Preferably, the high pressure steam sterilization condition in step three is 115-121° C. sterilization for 10-15 minutes.

[0015] Preferably, the high pressure steam sterilization condition in step three is preferably sterilization at 121° C. for 15 minutes.

[0016] Preferably, the cooling temperature after sterilization in the step 3 is preferably 45°C. If the solution temperature is lower than 40°C, in the step 4, the blood agar basal medium begins to solidify and cannot be fully mixed with the hemoglobin solution; if the solution temperature is higher than 70°C, in the step 4, floccules precipitate after the solution is mixed, affecting the quality of the chocolate agar medium.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The chocolate agar culture medium formula and preparation method described in the present invention can achieve the purpose of rapid sterilization, and the operation is simple, thereby avoiding the risk of culture medium contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a flow chart of the preparation method of the present invention;

[0020] Figure 2 It is the bacterial colony growth diagram of the present invention:

[0021] Specifically, Figures 2a to 2b are graphs showing the colony growth of the quality control strain after inoculation into a chocolate agar medium and cultured at 36±1°C for 18 to 24 hours. DETAILED DESCRIPTION

[0023] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] See attached Figure 1-2 The present invention provides a chocolate agar culture medium. The chocolate agar culture medium formula comprises 3.0-5.0% of blood agar basal culture medium, 0.5-1.5% of freeze-dried hemoglobin particles, and the rest is purified water.

[0025] A method for preparing a chocolate agar medium comprises the following steps:

[0026] Step 1: Pretreatment of raw materials: Grind freeze-dried hemoglobin particles with a grinder until they are in powder form;

[0027] Step 2, weighing and dissolving: Place the weighed freeze-dried hemoglobin powder in a clean and dry container, add purified water to the container, heat and stir to dissolve; Place the weighed blood agar basal medium in another clean and dry container, add purified water to the container, heat and stir to dissolve;

[0028] Step 3, high pressure sterilization: sterilize the above solution with high pressure steam, and cool it to 45-50°C after sterilization; the high pressure steam sterilization condition is 115-121°C sterilization for 10-15 minutes, the high pressure steam sterilization condition is preferably 121°C sterilization for 15 minutes, and the cooling temperature after sterilization is preferably 45°C.

[0029] Step 4, solution mixing: under sterile conditions, add the hemoglobin solution to the blood agar basal medium and mix thoroughly;

[0030] Step 5, filling plates: under sterile conditions, pouring sterile plates to prepare the culture medium.

[0031] Embodiment 1:

[0032] Each 1L of culture medium includes the following components: 3.0 g of blood agar basal medium, 0.5 g of freeze-dried hemoglobin particles, and 1000 ml of purified water.

[0033] As attached Figure 1 Chocolate agar medium was prepared as shown.

[0034] Step 1: Pretreatment of raw materials: Grind freeze-dried hemoglobin particles with a grinder until they are in powder form;

[0035] Step 2, weighing and dissolving: Place the weighed freeze-dried hemoglobin powder in a clean and dry container, add purified water to the container, heat and stir to dissolve; Place the weighed blood agar basal medium in another clean and dry container, add purified water to the container, heat and stir to dissolve;

[0036] Step 3, high pressure sterilization: sterilize the above solution with high pressure steam at 115°C for 10 minutes, and cool to 45°C after sterilization;

[0037] Step 4: Solution mixing: under sterile conditions, add the hemoglobin solution to the blood agar basal medium and mix thoroughly;

[0038] Step 5, filling plates: under sterile conditions, pouring sterile plates to prepare the culture medium.

[0039] The high pressure steam sterilization conditions of Example 1 at 115° C. for 10 minutes resulted in a decrease in the sterilization effect of the solution, increasing the risk of contamination of the culture medium.

[0040] Embodiment 2:

[0041] Each 1L of culture medium includes the following components: 3.0 g of blood agar basal medium, 0.5 g of freeze-dried hemoglobin particles, and 1000 ml of purified water.

[0042] As attached Figure 1 Chocolate agar medium was prepared as shown.

[0043] Step 1: Pretreatment of raw materials: Grind freeze-dried hemoglobin particles with a grinder until they are in powder form;

[0044] Step 2, weighing and dissolving: Place the weighed freeze-dried hemoglobin powder in a clean and dry container, add purified water to the container, heat and stir to dissolve; Place the weighed blood agar basal medium in another clean and dry container, add purified water to the container, heat and stir to dissolve;

[0045] Step 3, high pressure sterilization: sterilize the above solution with high pressure steam at 121°C for 15 minutes, and cool to 45°C after sterilization;

[0046] Step 4: Solution mixing: under sterile conditions, add the hemoglobin solution to the blood agar basal medium and mix thoroughly;

[0047] Step 5, filling plates: under sterile conditions, pouring sterile plates to prepare the culture medium.

[0048] Embodiment 3:

[0049] Each 1L of culture medium includes the following components: 4.0 g of blood agar basal medium, 1.0 g of freeze-dried hemoglobin particles, and 1000 ml of purified water.

[0050] The chocolate agar medium was prepared using the same steps as in Example 2.

[0051] Embodiment 4:

[0052] Each 1L of culture medium includes the following components: 5.0 g of blood agar basal medium, 1.5 g of freeze-dried hemoglobin particles, and 1000 ml of purified water.

[0053] The chocolate agar medium was prepared using the same steps as in Example 2.

[0054] Embodiment 5:

[0055] Each 1L of culture medium includes the following components: 5.0 g of blood agar basal medium, 0.5 g of freeze-dried hemoglobin particles, and 1000 ml of purified water.

[0056] The chocolate agar medium was prepared using the same steps as in Example 2.

[0057] Embodiment 6:

[0058] Each 1L of culture medium includes the following components: 4.5g of blood agar basal medium, 1.0g of freeze-dried hemoglobin particles, and 1000ml of purified water.

[0059] The chocolate agar medium was prepared using the same steps as in Example 2.

[0060] Effect experiment of cultivating quality control strains.

[0061] Haemophilus influenzae ATCC 10211 and Neisseria meningitidis ATCC 13090 were prepared into a bacterial suspension of 1.0×108 cfu / mL, diluted at 1:100, and 10 μL of the diluted bacterial suspension (about 104 cfu / mL) was taken on the chocolate agar medium prepared in Examples 2 to 6, inoculated using the four-zone streak method, and cultured in a constant temperature incubator at 36±1°C for 18 to 24 hours, as shown in Table 1.

[0062] Table 1: Colony growth of quality control strains inoculated into chocolate agar medium

[0063]

[0064]

[0065] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A chocolate agar medium, characterized in that The formula of the chocolate agar culture medium comprises, by proportion, 3.0-5.0% of a blood agar basic culture medium, 0.5-1.5% of freeze-dried hemoglobin particles, and the remainder being purified water.

2. A chocolate agar medium according to claim 1, characterized in that: The formula of chocolate agar medium includes 4.5% blood agar basal medium, 1.0% freeze-dried hemoglobin particles, and the rest is purified water.

3. A chocolate agar medium according to claim 1, characterized in that: The formula of the chocolate agar culture medium comprises, in proportion, 5.0% of blood agar basic culture medium, 1.5% of freeze-dried hemoglobin particles, and the remainder is purified water.

4. The method for preparing a chocolate agar medium according to any one of claims 1 to 3, characterized in that: The steps include: Step 1: Pretreatment of raw materials: Grind freeze-dried hemoglobin particles with a grinder until they are in powder form; Step 2, weighing and dissolving: Place the weighed freeze-dried hemoglobin powder in a clean and dry container, add purified water to the container, heat and stir to dissolve; Place the weighed blood agar basal medium in another clean and dry container, add purified water to the container, heat and stir to dissolve; Step 3, high pressure sterilization: sterilize the above solution with high pressure steam, and cool to 45-50°C after sterilization; Step 4, solution mixing: under sterile conditions, add the hemoglobin solution to the blood agar basal medium and mix thoroughly; Step 5, filling plates: under sterile conditions, pouring sterile plates to prepare the culture medium.

5. The method for preparing a chocolate agar medium formula according to claim 4, characterized in that: The high pressure steam sterilization condition in step 3 is 115-121° C. for 10-15 minutes.

6. The method for preparing a chocolate agar medium formula according to claim 5, characterized in that: The high pressure steam sterilization condition in step 3 is preferably sterilized at 121° C. for 15 minutes.

7. The method for preparing a chocolate agar medium formula according to claim 4, characterized in that: The cooling temperature after sterilization in step 3 is preferably 45°C.

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