Culture method of microaerobic bacteria and application of microaerobic bacteria in drug sensitivity test

Abstract

The invention provides a culture method of micro-aerobic bacteria and application of the micro-aerobic bacteria in a drug sensitivity test, the culture method comprises the following steps: step S1, selecting a porous sponge material with a proper size, sterilizing at a high temperature, and placing in a culture vessel; the porous sponge material is bamboo fiber, cotton fiber or polyurethane; s2, preparing a liquid culture medium, performing high-temperature sterilization, inoculating to-be-cultured micro-aerobic strains, and fully and uniformly mixing to obtain a suspension, so as to obtain the liquid culture medium containing the strains; s3, dropwise adding a liquid culture medium containing strains onto the porous sponge material in the culture vessel; and putting the culture vessel into a culture box and culturing for 12-72 hours. By adopting the technical scheme provided by the invention, the result is stable, the repeatability is high, the operation is simple, the batch experiment can be carried out, and the culture effect is better.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a method for culturing microaerobic bacteria and an application thereof in drug sensitivity testing. Background Art

[0002] Microaerophilic bacteria are a type of bacteria that grow better under conditions where the oxygen concentration is lower than the normal oxygen level in the atmosphere (about 21%). The oxygen concentration they require is usually between 2% and 10%, and too much oxygen is toxic to them. Such bacteria usually live in low-oxygen environments, such as deep soil, the bottom of water bodies, and animal intestines. Common microaerophilic bacteria include Campylobacter, Helicobacter, Pseudomonas, etc. Microaerophilic bacteria are closely related to human health. The human intestine belongs to a microaerobic environment, and a large number of intestinal probiotics and pathogens can only grow under microaerobic conditions. For example, Helicobacter pylori and Campylobacter jejuni are both microaerophilic bacteria. Research on such bacteria requires strict culture conditions, generally a microaerobic environment of 5% O2, 10% CO2, and 85% N2.

[0003] For microaerophilic bacteria, solid plate culture is generally used. After the bacteria are inoculated on the surface of the solid culture medium, they are placed in a low-oxygen environment for culture. This culture method has the following problems: First, the surface area of ​​the solid culture medium is limited; second, the diffusion effect of nutrients on the solid culture medium is poor, and the bacteria cannot continue to grow after consuming the surrounding nutrients; in addition, the operation of solid plate culture is cumbersome and time-consuming. Therefore, it is difficult to collect a large number of microaerophilic bacteria by solid plate culture. Some studies have also reported the use of liquid culture methods to culture microaerophilic bacteria, but in practical operations, liquid culture of microaerophilic bacteria has the problem of poor stability. The main reason is that the dissolved oxygen content in the liquid is affected by multiple factors such as oxygen concentration in the air, liquid stirring frequency and speed, and liquid viscosity. Researchers can only control the oxygen concentration in the air in the culture environment, and cannot accurately control the dissolved oxygen content in the liquid. Too low or too high dissolved oxygen will lead to failure of microaerophilic bacterial culture. Therefore, it is necessary to develop a more efficient microaerophilic bacterial culture method.

[0004] Antibiotics are the most important drugs for treating bacterial infections. In recent years, drug-resistant bacteria and even superbugs have continued to emerge. Clinical treatment urgently needs new antibiotics, which has also promoted the screening and drug research of new antibiotics. Conventional antibacterial activity tests (also known as drug sensitivity tests) generally use filter paper diffusion method or liquid culture method. The filter paper diffusion method is to drop the drug on the filter paper and stick it on the surface of the solid culture medium inoculated with bacteria. After a period of culture, the size of the transparent circle around the filter paper is measured (representing that there is no bacterial growth here) to confirm whether it has antibacterial activity. The disadvantage of the filter paper method is that it is difficult to control the actual concentration of the drug diffused from the paper into the culture medium, and it is impossible to give a clear dose-effect relationship. Liquid culture is to add different concentrations of drugs to the liquid, quantitatively measure the growth of bacteria, and then give a clear dose-effect relationship. This method is suitable for conventional aerobic bacteria and anaerobic bacteria, but because microaerobic bacteria are unstable in liquid culture medium, it is not suitable for antibacterial testing of microaerobic bacteria such as Helicobacter pylori. Therefore, it is necessary to develop new antibacterial testing methods for microaerobic bacteria. Summary of the invention

[0005] In view of the above technical problems, the present invention discloses a method for culturing microaerobic bacteria and its application in drug sensitivity testing, which has simple operation, low cost and good culturing effect.

[0006] To this end, the technical solution adopted by the present invention is:

[0007] A method for culturing microaerobic bacteria comprises the following steps:

[0008] Step S1, selecting a porous sponge material of appropriate size, sterilizing it at high temperature, and placing it in a culture vessel; the porous sponge material is bamboo fiber, cotton fiber or polyurethane;

[0009] Step S2, preparing a liquid culture medium, sterilizing it at high temperature, inoculating the microaerobic bacteria to be cultured, and fully mixing it into a suspension to obtain a liquid culture medium containing the bacteria;

[0010] Step S3, dripping the liquid culture medium containing the bacterial strain onto the porous sponge material in the culture vessel; placing the culture vessel in an incubator for culturing for 12-72 hours.

[0011] Using this technical solution, a porous sponge structure material is added to the liquid culture medium, which forms a scaffold in the liquid. The surface tension causes a liquid film to form on the surface of the porous sponge material, which is more conducive to the cultivation of bacteria, and the cultivation effect is significantly better than the simple liquid culture method.

[0012] As a further improvement of the present invention, the method for culturing microaerophilic bacteria further includes step S4, after the bacteria grow to a sufficient amount, squeezing the sponge, washing the sponge with a sterile culture medium or aqueous solution, and collecting the liquid to obtain the required microaerophilic bacteria.

[0013] As a further improvement of the present invention, the density of the porous sponge material is 18-55g / m 3 Furthermore, the density of the porous sponge material is 18-45 g / m 3 ; Further preferably, the density of the porous sponge material is 30-45g / m 3 .

[0014] As a further improvement of the present invention, the porous sponge material is a polyurethane sponge.

[0015] As a further improvement of the present invention, in step S3, the environment of the incubator is a three-gas environment of 5% O2, 10% CO2, and 85% N2.

[0016] As a further improvement of the present invention, in step S2, the liquid culture medium includes BHI broth culture medium.

[0017] The invention also discloses the application of the above-mentioned microaerobic bacteria culture method in drug sensitivity testing.

[0018] As a further improvement of the present invention, the application includes: after the culturing in step S3 is completed, adding MTT solution into the culture vessel, squeezing the porous sponge material to mix the liquid, and continuing to culture at 37° C. for 4 hours;

[0019] Then, add anhydrous ethanol into the culture vessel to dissolve the blue-purple crystals of formazan formed after the metabolism of MTT;

[0020] Take the solution from the culture vessel into a cuvette or ELISA plate, and measure the absorbance at 570 nm using a spectrophotometer or ELISA reader.

[0021] Furthermore, the volume of anhydrous ethanol added is 2-4 times the volume of the liquid in the culture vessel.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The technical solution of the present invention is adopted, and a suitable porous material is selected to be placed in a liquid culture medium to form a support structure. The liquid forms a thin film on the surface of the porous material through surface tension, thereby forming a stable microaerobic liquid environment. The environment of microaerobic bacteria in the organism is simulated in the liquid, and the growth rate of microaerobic bacteria is faster, and the total amount of harvested bacteria is more. Further, different concentrations of drugs can be added to the system as needed, and the bacterial activity can be determined by the MTT method, and then the total amount of bacteria can be measured, and the antibacterial activity test can be performed. The results obtained by this method are stable, highly repeatable, simple to operate, can be batched for experiments, have high sensitivity to drugs, and can provide a clear dose-effect relationship, and can be widely used in the cultivation and drug sensitivity testing of microaerobic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the comparison result of the effect of culturing Helicobacter pylori using different types of porous materials in Example 1 of the present invention.

[0025] Figure 2 This is the comparison result of the effect of culturing Helicobacter pylori using polyurethane sponges of different densities in Example 2 of the present invention.

[0026] Figure 3 The results of culturing Helicobacter pylori using polyurethane sponge and conventional liquid methods are compared in Example 3 of the present invention.

[0027] Figure 4 The results are compared between Example 3 of the present invention using a polyurethane sponge and a conventional liquid method to culture Campylobacter jejuni.

[0028] Figure 5 This is the result of using polyurethane sponge and conventional culture methods for antimicrobial drug screening in Example 4 of the present invention. DETAILED DESCRIPTION

[0029] The preferred embodiments of the present invention are described in further detail below.

[0030] Example 1

[0031] In this example, a variety of porous materials and liquid culture methods were used to culture Helicobacter pylori. 1.5×10 8 CFU / mL bacterial solution was cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2-3 days, and the bacterial activity was determined by MTT method. The specific steps include:

[0032] 1. Preparation of culture media and various porous materials: Columbia blood agar and BHI broth culture media were prepared, and porous materials of uniform shape were prepared using a hole puncher, and all were placed in a high-pressure steam sterilizer and sterilized at 121°C for 20 minutes.

[0033] 2. After dilution, Helicobacter pylori was spread on Columbia blood agar plates and cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2-3 days. The bacterial moss was scraped into BHI broth medium and the concentration of the bacterial solution was determined by McFarland turbidimetry. The solution was diluted to 0.5 McFarland turbidimetric value (1.5×10 8 CFU / mL) was used as the stock solution, which was further diluted 10 times with culture medium to serve as the test solution.

[0034] 3. Prepare a 24-well plate and add different types of sponge materials to each well. The experimental groups are as follows: the control group is liquid culture (no porous material), experimental group 1 is bamboo fiber sponge material, experimental group 2 is cotton fiber sponge material, and experimental group 3 is polyurethane sponge. The density of the three sponge materials is 30g / m 3 300 μL of culture medium (test solution) containing bacteria was added to each well, and the cells were cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days.

[0035] 4. Determination of bacterial activity of Helicobacter pylori using MTT method:

[0036] (1) Add 30 μL of MTT to each well of a 24-well plate, squeeze the sponge to mix the liquid, and incubate at 37°C for 4 h.

[0037] (2) Transfer the liquid in the wells of the 24-well plate to a 1.5 mL EP tube, then add 600 μL of anhydrous ethanol to the sponge in each well and squeeze repeatedly to fully dissolve and extract the blue-purple substance.

[0038] (3) Then transfer 600 μL of anhydrous ethanol in the 24-well plate to the 1.5 mL EP tube in step (2), mix well and centrifuge at 12,000 × g for 5 min, and transfer 100 μL of the supernatant to each 96-well ELISA plate.

[0039] (4) The absorbance value at 570 nm was measured by enzyme-linked detector. The specific test results are as follows: Figure 1 It can be seen that the effect of using different types of porous materials (bamboo fiber, cotton fiber, polyurethane sponge) to culture Helicobacter pylori is better than the traditional liquid culture method, among which polyurethane sponge has the best culture effect.

[0040] Example 2

[0041] In Example 1, sponge materials of different materials (bamboo fiber, cotton fiber, polyurethane) have different properties (such as water absorption), which affect the surface tension of the liquid and further affect the growth of Helicobacter pylori, but the culture effect is better than the liquid culture method of the prior art. Because the pore size of the sponge material may also affect the surface tension of the liquid, this example selects polyurethane sponge material on the basis of Example 1 to examine the effect of sponge materials with different pore sizes, that is, different densities, on the culture effect of Helicobacter pylori. Specifically, 18g / m 3 , 30g / m 3 , 45g / m 3 , 55g / m 3 Helicobacter pylori was cultured with polyurethane sponges of four densities, and the effects of polyurethane sponge materials with different densities on the growth of Helicobacter pylori were compared.

[0042] The specific implementation methods and steps are carried out according to Example 1, and the test results are as follows. Figure 2 As shown. It can be seen that the pores of sponges with different densities are different. The higher the density, the smaller the pores. In the polyurethane sponges with different densities for culturing Helicobacter pylori, the pores of sponges with different densities are different. The ... 3 Density range: As the density of polyurethane sponge increases, the culture effect of Helicobacter pylori is better. And when the density of polyurethane sponge reaches 55g / m 3 The culture effect of Helicobacter pylori decreased slightly.

[0043] Example 3

[0044] This example is a comparison of the effect of using polyurethane sponge and conventional liquid method to culture Helicobacter pylori and Campylobacter jejuni. 3 The polyurethane sponges were inoculated with 1.5×10 5 , 1.5×10 6 , 1.5×10 7 , 1.5×10 8 CFU / mL Helicobacter pylori or Campylobacter jejuni culture liquid was cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days, and the bacterial activity was determined by MTT method. The specific steps include:

[0045] 1. Preparation of culture medium and sponge materials: Columbia blood agar and BHI broth culture medium were prepared, and polyurethane sponge sheets of uniform shape were prepared using a puncher and sterilized in a steam sterilizer at 121°C for 20 min.

[0046] 2. After Helicobacter pylori and Campylobacter jejuni were diluted and smeared on Columbia blood plates, they were cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2-3 days. The bacterial mosses were scraped into BHI broth medium, and the concentration of the bacterial solution was determined by McFarland turbidimetry, and the solution was diluted to 0.5 McFarland turbidimetric value (1.5×10 8 CFU / mL).

[0047] 3. Prepare four 24-well plates, two for polyurethane sponge culture and two for BHI broth liquid culture:

[0048] 4. Inoculation of Helicobacter pylori in 2 24-well plates: one 24-well plate was covered with polyurethane sponge material, and the other 24-well plate was not covered with sponge material. The experimental groups in each 24-well plate were as follows: 1.5×10 5 , 1.5×10 6 , 1.5×10 7 and 1.5×10 8 CFU / mL of four concentrations of bacterial solution, 4 replicate wells in each group, 300 μL of liquid in each well, and cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days.

[0049] 5. Two 24-well plates were inoculated with Campylobacter jejuni: one 24-well plate was covered with polyurethane sponge material, and the other 24-well plate did not contain sponge material. The experimental groups of each 24-well plate were as follows: the blank group was BHI liquid culture medium, and the experimental groups were 1.5×10 5 , 1.5×10 6 , 1.5×10 7 and 1.5×10 8 CFU / mL of four concentrations of bacterial solution, 4 replicate wells in each group, 300 μL of liquid in each well, and cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days.

[0050] 6. The bacterial activities of Helicobacter pylori and Campylobacter jejuni were determined according to the MTT method in Example 1. 30 μL of MTT was added to each well, the liquid was mixed by squeezing the sponge, and then incubated at 37° C. for 4 h.

[0051] 7. Transfer the liquid in the 24-well plate to a 1.5 mL EP tube, add 600 μL of anhydrous ethanol to each well, squeeze repeatedly to fully dissolve and extract the blue-purple substance.

[0052] 8. Transfer 600 μL of anhydrous ethanol in the 24-well plate to the 1.5 mL EP tube in the previous step, mix well, centrifuge at 12000×g for 5 min, and transfer 100 μL of the supernatant to each 96-well ELISA plate.

[0053] 9. Use an ELISA reader to measure the absorbance at 570 nm. Compare the effects of the two methods in culturing Helicobacter pylori. Figure 3 As shown in the figure, the effect of the two methods on the cultivation of Campylobacter jejuni is compared. Figure 4 As shown in the figure, it can be seen that at four different inoculation concentrations, the proliferation and activity of Helicobacter pylori and Campylobacter jejuni in sponge culture are better than those in liquid culture.

[0054] Example 4

[0055] This example uses sponge material and liquid culture to compare the sensitivity of Helicobacter pylori to drugs. 3 The polyurethane sponge material with a density of 1.5×10 7 CFU / mL bacterial solution and adding different concentrations of anti-Helicobacter pylori positive drugs, co-cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days, and the bacterial activity was determined by MTT method. Specifically, the following steps are included:

[0056] 1. Preparation of culture medium and sponge materials: Columbia blood agar and BHI broth culture medium were prepared, and a number of sponge pieces with the same appearance were prepared using a hole puncher. The pieces were sterilized in a high pressure steam sterilizer at 121°C for 20 min.

[0057] 2. Preparation of anti-Helicobacter pylori quadruple drug stock solution, the stock solution contains ① 500 mg / mL amoxicillin, ② 500 mg / mL clarithromycin, ③ 20 mg / mL omeprazole and ④ 220 mg / mL bismuth potassium citrate. The stock solution is filtered and sterilized using a 0.22 μm filter membrane for later use.

[0058] 3. After Helicobacter pylori was diluted and smeared on Columbia blood agar plates, it was cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2-3 days. The bacterial moss was scraped into BHI broth medium and the concentration of the bacterial solution was determined by McFarland turbidimetry. The solution was diluted to 0.5 McFarland turbidimetric value (1.5×10 8 CFU / mL).

[0059] 4. Prepare two 24-well plates, one containing sponge material and the other without sponge material, for BHI broth liquid culture:

[0060] 5. Inoculation of Helicobacter pylori in 2 24-well plates: The experimental groups in each 24-well plate are as follows:

[0061] The control group was 1.5×10 7CFU / mL bacterial solution, experimental group 1 was a bacterial solution containing 0.625 mg / mL amoxicillin + 0.625 mg / mL clarithromycin + 0.025 mg / mL omeprazole + 0.275 mg / mL bismuth potassium citrate, experimental group 2 was a bacterial solution containing 1.25 mg / mL amoxicillin + 1.25 mg / mL clarithromycin + 0.05 mg / mL omeprazole + 0.55 mg / mL bismuth potassium citrate, experimental group 3 was a bacterial solution containing 2.5 mg / mL amoxicillin + 2.5 mg / mL clarithromycin + 0.1 mg / mL omeprazole + 1.1 mg / mL bismuth potassium citrate, and experimental group 4 was a bacterial solution containing 5 mg / mL amoxicillin + 5 mg / mL clarithromycin + 0.2 mg / mL omeprazole + 2.2 mg / mL bismuth potassium citrate, with 4 replicates in each group. Each well contains 300 μL of 1.5 × 10 7 CFU / mL of bacterial liquid was cultured in a three-gas incubator (5% O2, 10% CO2, 85% N2) at 37°C for 2 days.

[0062] 6. MTT method was used to determine the bacterial activity of Helicobacter pylori:

[0063] (1) Four 24-well plates for Helicobacter pylori were plated. 30 μL of MTT was added to each well. The liquid was mixed by squeezing the sponge and then incubated at 37°C for 4 h.

[0064] (2) Transfer the liquid in the wells of the 24-well plate to a 1.5 mL EP tube, add 600 μL of anhydrous ethanol to each well, and squeeze repeatedly to fully dissolve.

[0065] (3) Transfer 600 μL of anhydrous ethanol in the 24-well plate to the 1.5 mL EP tube in the previous step, mix well, and centrifuge at 12,000 × g for 5 min. Transfer 100 μL of the supernatant to each 96-well ELISA plate.

[0066] (4) Use an ELISA reader to measure the absorbance at 570 nm. The specific test results are as follows: Figure 5 As shown. It can be seen that in conventional liquid culture, antibiotics have a significant inhibitory effect on Helicobacter pylori, but there is no significant difference between treatment groups with different concentrations, and the difference in results between the treatment group with antibiotics and the control group without antibiotics is small. In contrast, after the addition of polyurethane sponge material, bacterial growth is better than that without sponge, the difference between the treatment group with drug addition and the control group without drug addition is greater, and the difference between treatment groups with different drug concentrations is also significant. This shows that when the method of adding polyurethane sponge is used for drug sensitivity testing, Helicobacter pylori is more sensitive than the ordinary liquid culture method. Therefore, this method is more effective for antimicrobial drug screening, while when using conventional liquid culture methods, drugs with potential development value may be missed due to low sensitivity.

[0067] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A method for culturing microaerobic bacteria, characterized in that: The steps include: Step S1, selecting a porous sponge material of appropriate size, sterilizing it at high temperature and placing it in a culture vessel; the porous sponge material is bamboo fiber, cotton fiber or polyurethane; Step S2, preparing a liquid culture medium, sterilizing it at high temperature, inoculating the microaerobic bacteria to be cultured, and fully mixing it into a suspension to obtain a liquid culture medium containing the bacteria; Step S3, dripping the liquid culture medium containing the bacterial strain onto the porous sponge material in the culture vessel; placing the culture vessel in an incubator for culturing for 12-72 hours.

2. The method for culturing microaerophilic bacteria according to claim 1, characterized in that: The method further includes step S4, wherein after the bacteria grow to a sufficient amount, the sponge is squeezed, the sponge is rinsed with a sterile culture medium or aqueous solution, and the liquid is collected to obtain the required microaerophilic bacteria.

3. The method for culturing microaerophilic bacteria according to claim 1, characterized in that: The density of the porous sponge material is 18-55 g / m 3 .

4. The method for culturing microaerobic bacteria according to claim 3, characterized in that: The density of the porous sponge material is 30-45 g / m 3 .

5. The method for culturing microaerophilic bacteria according to claim 4, characterized in that: The porous sponge material is polyurethane sponge.

6. The method for culturing microaerobic bacteria according to claim 1, characterized in that: In step S3, the environment of the incubator is a three-gas environment of 5% O2, 10% CO2, and 85% N2.

7. The method for culturing microaerophilic bacteria according to claim 1, characterized in that: In step S2, the liquid culture medium includes BHI broth medium.

8. Use of the microaerophilic bacteria culture method according to any one of claims 1 to 7 in drug sensitivity testing.

9. The use of the microaerophilic bacteria culture method according to claim 8 in drug sensitivity testing, characterized in that: After the incubation in step S3 is completed, add MTT solution into the culture vessel, squeeze the porous sponge material to mix the liquid, and continue to culture at 37° C. for 4 hours; Then, add anhydrous ethanol into the culture vessel to dissolve the blue-purple crystals of formazan formed after the metabolism of MTT; Take the solution from the culture vessel into a cuvette or ELISA plate, and measure the absorbance at 570 nm using a spectrophotometer or ELISA reader.

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