A medium for inhibiting the growth of gram-negative bacteria and a method for preparing the same

Abstract

The present application relates to the field of biotechnology, especially to the field of A01G18 / 20, and more particularly to a culture medium for inhibiting growth of gram-negative bacteria and a preparation method thereof.The raw materials of the culture medium include a nitrogen source, inorganic salt, carbon source, coagulant, bacteriostatic drug, animal blood and water, wherein the bacteriostatic drug includes aztreonam and avibactam.The culture medium prepared by the present application can effectively inhibit the growth of gram-negative bacteria, and has no obvious inhibitory effect on other non-gram-negative bacteria strains, so that directional screening of specific mixed bacteria strains can be realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, particularly to the field of A01G18 / 20, and specifically to a culture medium for inhibiting the growth of Gram-negative bacteria and a method for preparing the same. Background Technology

[0002] Culture media typically contain essential nutrients that maintain microbial nutrition and promote microbial proliferation, while also providing a suitable environment for microbial growth and reproduction. Common culture media can be classified into various types, including natural media, synthetic media, semi-synthetic media, solid media, liquid media, semi-solid media, and screening media. Different types of culture media are used to meet the basic growth needs of different types of microorganisms.

[0003] Adding antimicrobial agents to culture media can inhibit the growth of unwanted microorganisms, ensuring a more suitable growth environment for target microorganisms. Antibiotics are among the most commonly used antimicrobial agents, and they are classified into several categories, such as β-lactams, aminoglycosides, and macrolides. These antibiotics work by inhibiting protein synthesis and other mechanisms, exhibiting different inhibitory effects against different types of bacteria.

[0004] Gram-negative bacilli are a class of bacteria, generally divided into two main categories: fermenting bacteria and non-fermenting bacteria. Fermenting bacteria are mainly Enterobacteriaceae, including Escherichia coli, Shigella, Salmonella, and Klebsiella pneumoniae, which can cause intestinal or extraintestinal infections and may cause symptoms such as abdominal pain and diarrhea. Non-fermenting bacteria include Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, and Haemophilus influenzae, which mainly cause infections in the respiratory tract, urinary tract, blood, skin, and soft tissues.

[0005] Clinically, when an infection is suspected to be caused by Gram-positive bacteria (such as Staphylococcus, Streptococcus, or Enterococcus), but the sample may contain a large number of interfering Gram-negative bacteria, selective culture media can be used to inhibit the growth of Gram-negative bacteria, thereby highlighting the isolation of the target Gram-positive bacteria. Additionally, when the sample source is susceptible to Gram-negative bacteria contamination, such as urine or wound secretions, it may contain intestinal Gram-negative bacteria (such as Escherichia coli). By inhibiting Gram-negative bacteria, false positive results can be avoided, and the pathogenic Gram-positive bacteria can be identified more reliably.

[0006] In clinical practice, compound antibiotics are often used to treat the symptoms of bacterial infections. For example, prior art CN202080060861.2 discloses an oral administration combination of a β-lactam antibiotic and an avibactam derivative for treating bacterial infections. However, there are few reports on using compound antibiotics to prepare culture media that inhibit Gram-negative bacteria and to accurately screen for target bacterial species. Summary of the Invention

[0007] To address the aforementioned technical problems, the first aspect of this invention provides a culture medium for inhibiting the growth of Gram-negative bacteria, the raw materials of which include: a nitrogen source, inorganic salts, a carbon source, a coagulant, an antibacterial drug, animal blood, and water.

[0008] nitrogen source Preferably, the nitrogen source includes an inorganic nitrogen source and / or an organic nitrogen source.

[0009] Preferably, the inorganic nitrogen source includes: ammonium salt (NH4) + ), nitrates (NO3) - One or more of the following: ) and ammonia (NH3·H2O).

[0010] Preferably, the organic nitrogen source includes one or more of the following: yeast powder, beef extract, peptone, amino acids, and protein hydrolysates.

[0011] More preferably, the nitrogen source includes peptone.

[0012] More preferably, the peptone includes trypsinone and special peptone.

[0013] Nitrogen sources in culture media provide the nitrogen element necessary for microbial growth and metabolism. This invention preferably uses a combination of tryptone and a specific peptone as the nitrogen source, both of which are rich in vitamins and growth factors, providing essential nitrogen and amino acids to maintain microbial growth and reproduction.

[0014] Inorganic salts Inorganic salts play a crucial role in culture media, providing the trace elements necessary for microbial growth and metabolism, maintaining cell osmotic pressure, and providing a stable external environment for cells. This invention does not further limit the inorganic salts; any salt that can provide essential elements for microorganisms is acceptable, including but not limited to sodium chloride, potassium chloride, magnesium chloride, potassium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, and calcium carbonate. Sodium chloride is the preferred inorganic salt in this invention.

[0015] carbon source Preferably, the carbon source includes: sugars, alcohols, and organic acids; in this invention, the carbon source is a sugar; sugars include one or more of glucose, sucrose, fructose, maltose, and starch.

[0016] More preferably, the starch includes one of the following: corn starch, sweet potato starch, potato starch, wheat starch, mung bean starch, tapioca starch, and pea starch.

[0017] Carbon sources in culture media play a crucial role in the growth and metabolic processes of microorganisms; they provide the energy and carbon required for microbial cell growth and have a decisive effect on the growth rate and physiological characteristics of microorganisms.

[0018] coagulant Preferably, the coagulant comprises agar powder and / or peptone.

[0019] More preferably, the coagulant is agar powder.

[0020] Agar powder enables the culture medium described in this invention to maintain high stability at high temperatures, preventing changes in the morphology of the culture medium due to temperature variations and ensuring the accuracy of microbial culture experiments. In addition, the porous nature of agar powder culture medium makes it suitable for measuring the motility and migration of microorganisms, and allows for more convenient observation of the inhibition of growth of Gram-negative bacteria.

[0021] Antibacterial drugs Preferably, the antibacterial drug includes aztreonam and / or avibactam.

[0022] More preferably, the antibacterial drugs include aztreonam and avibactam.

[0023] More preferably, the mass ratio of aztreonam to avibactam is (8-64):(1-8).

[0024] Aztreonam belongs to the monocyclic β-lactam antibiotic class. Its antibacterial effect is mainly achieved by inhibiting bacterial cell wall synthesis, thereby killing bacteria. Aztreonam has strong antibacterial activity against Gram-negative bacteria, such as *Pseudomonas aeruginosa* and *Escherichia coli*; however, its inhibitory effect on drug-resistant Gram-positive bacteria is relatively poor. However, the inventors have also discovered that some Gram-negative bacteria can secrete β-lactamases and also exhibit some drug resistance. Therefore, aztreonam's inhibitory effect on drug-resistant Gram-negative bacteria that secrete β-lactamases is relatively poor.

[0025] Avibactam is a β-lactamase inhibitor. Avibactam itself has no antibacterial effect against Gram-negative bacteria, but it can inhibit various drug-resistant enzymes produced by Gram-negative bacteria. It can inhibit those enzyme-producing Gram-negative drug-resistant bacteria, thus playing a further inhibitory role against Gram-negative bacteria.

[0026] During the actual research and development process, the inventors discovered that when aztreonam and avibactam are used in combination, especially when the mass ratio of aztreonam to avibactam in this culture medium is (8-64):(1-8), aztreonam can enhance the inhibitory effect of avibactam on the secretion of β-lactamase by Gram-negative bacteria, thereby enabling the culture medium to better inhibit Gram-negative bacteria. The combination of the two can ultimately inhibit most Gram-negative bacteria (common negative bacteria + drug-resistant negative bacteria) without affecting the growth of the target cultured bacteria (Gram-positive bacteria), thus enabling this culture medium to play a role in targeted screening of microorganisms.

[0027] animal blood Preferably, the animal blood includes sheep blood and / or horse blood.

[0028] Animal blood contains water, inorganic salts, vitamins, carbohydrates, fats, amino acids, and other nutrients, providing essential nutrition for microorganisms cultured in vitro. Although the amounts of these nutrients in animal blood are relatively small, they are crucial for the growth and metabolism of microorganisms.

[0029] Preferably, each 1L of the culture medium comprises: 15-20g of special peptone, 5-10g of tryptone, 5g of sodium chloride, 1-3g of corn starch, 10-15g of agar powder, 8-64mg of aztreonam, 1-8mg of avibactam, 50-70mL of animal blood, and the remainder being water.

[0030] A second aspect of the present invention provides a method for preparing a culture medium for inhibiting the growth of Gram-negative bacteria, comprising: S1. Add nitrogen source, inorganic salt, carbon source, coagulant, antibacterial agent and water to the flask in proportion; S2. Place the flask in an autoclave and sterilize it at high temperature; S3. Cool the culture medium, add animal blood and stir well; S4. Immediately dispense the well-stirred culture medium into petri dishes and let it cool and solidify.

[0031] Preferably, the high-temperature sterilization temperature in step S2 is 110-135℃, and the high-temperature sterilization time is 10-20 minutes.

[0032] Preferably, the cooling temperature in step S3 is 50-65°C.

[0033] Beneficial effects (i) The culture medium provided by the present invention can effectively inhibit the growth of Gram-negative bacteria and has a better antibacterial effect than other common culture media.

[0034] (ii) The culture medium provided by the present invention has no significant inhibitory effect on other non-Gram-negative bacterial species, and can achieve targeted screening of specific mixed bacterial species.

[0035] (III) The antibacterial drugs selected in this invention include aztreonam and avibactam. Aztreonam can enhance the inhibitory effect of avibactam on the secretion of β-lactamase by Gram-negative bacteria, thereby enabling the culture medium to better inhibit Gram-negative bacteria. The combination of the two can ultimately inhibit most of the Gram-negative bacteria (common negative bacteria + drug-resistant negative bacteria) without affecting the growth of the target culture bacteria (Gram-positive bacteria), thereby enabling this culture medium to play a role in targeted screening of microorganisms.

[0036] (iv) By selecting specific carbon sources, nitrogen sources, coagulants and inorganic salts in this invention, a suitable living environment can be ensured for the growth and reproduction of microorganisms.

[0037] (v) The raw materials for the culture medium in this invention are widely available and have low cost. The preparation method is also relatively simple, which is expected to achieve large-scale production and generate considerable market revenue. Attached Figure Description

[0038] Figure 1 This shows the growth of Escherichia coli in ordinary blood culture medium.

[0039] Figure 2 This shows the growth of Pseudomonas aeruginosa in ordinary blood culture medium.

[0040] Figure 3 This describes the growth of Salmonella typhimurium in ordinary blood culture medium.

[0041] Figure 4 This describes the growth of Klebsiella pneumoniae in ordinary blood culture medium.

[0042] Figure 5 This shows the growth of Escherichia coli in the culture medium of Example 1.

[0043] Figure 6 This shows the growth of Pseudomonas aeruginosa in the culture medium of Example 1.

[0044] Figure 7 This shows the growth of Salmonella typhimurium in the culture medium of Example 1.

[0045] Figure 8 This shows the growth of Klebsiella pneumoniae in the culture medium of Example 1.

[0046] Figure 9 Escherichia coli ATCC25922 and Streptococcus pneumoniae ATCC49619 were mixed and inoculated into ordinary blood culture medium.

[0047] Figure 10 Escherichia coli ATCC25922 and fecal enterovirus ATCC29212 were mixed and inoculated into ordinary blood culture medium.

[0048] Figure 11 Escherichia coli ATCC25922 and Streptococcus pneumoniae ATCC49619 were mixed and inoculated into the culture medium of Example 1.

[0049] Figure 12 Escherichia coli ATCC25922 and fecal ATCC29212 were mixed and inoculated into the culture medium of Example 1.

[0050] Figure 13 Klebsiella pneumoniae ATCC BAA 2814 and Streptococcus pneumoniae ATCC49619 were mixed and inoculated into ordinary blood culture medium.

[0051] Figure 14 Klebsiella pneumoniae ATCC BAA 2814 + fecal enterovirus ATCC 29212 were mixed and inoculated into ordinary blood culture medium.

[0052] Figure 15 Example 1 culture medium was used to inoculate Klebsiella pneumoniae ATCC BAA 2814 and Streptococcus pneumoniae ATCC49619 together.

[0053] Figure 16 Example 1 culture medium was prepared by inoculating a mixture of Klebsiella pneumoniae ATCC BAA 2814 and fecal ATCC29212.

[0054] Figure 17 The growth of clinical sample 1 (i.e., blood sample) in ordinary culture medium (mixed growth of large intestine, mutated and bird intestine).

[0055] Figure 18 The growth of clinical sample 1 in the culture medium of Example 1 (only bird intestines grew).

[0056] Figure 19 The growth of clinical sample 2 (i.e., drainage fluid sample) in ordinary culture medium (mixed growth of Serratia marcescens and Staphylococcus aureus).

[0057] Figure 20 The growth of clinical sample 2 in the culture medium of Example 1 (only Staphylococcus aureus grew). Detailed Implementation

[0058] Experimental Group 1 Example 1 The first aspect of this example provides a culture medium for inhibiting the growth of Gram-negative bacteria, each 1L comprising the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 30mg of aztreonam (antibacterial agent), 4mg of avibactam (antibacterial agent), 50mL of horse blood (animal blood); the remainder being deionized water.

[0059] All of the above raw materials are ordinary commercially available products.

[0060] The second aspect of this example provides a method for preparing a culture medium for inhibiting the growth of Gram-negative bacteria, comprising: S1. Add nitrogen source, inorganic salt, carbon source, coagulant, antibacterial agent and water to the flask in proportion; S2. Place the flask in an autoclave and sterilize at 121°C for 15 minutes. S3. Cool the culture medium to 55°C, add animal blood and stir well; S4. Immediately dispense the well-stirred culture medium into petri dishes and let it cool and solidify.

[0061] Example 2 The first aspect of this example provides a culture medium for inhibiting the growth of Gram-negative bacteria, each 1L comprising the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 10mg of aztreonam (antibacterial agent), 4mg of avibactam (antibacterial agent), 50mL of horse blood (animal blood); the remainder being deionized water.

[0062] All of the above raw materials are ordinary commercially available products.

[0063] The second aspect of this example provides a method for preparing a culture medium for inhibiting the growth of Gram-negative bacteria, comprising: S1. Add nitrogen source, inorganic salt, carbon source, coagulant, antibacterial agent and water to the flask in proportion; S2. Place the flask in an autoclave and sterilize at 121°C for 15 minutes. S3. Cool the culture medium to 55°C, add animal blood and stir well; S4. Immediately dispense the well-stirred culture medium into petri dishes and let it cool and solidify.

[0064] Experimental Group 2 Example 3 This example provides a culture medium for inhibiting the growth of Gram-negative bacteria. Each 1L contains the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 10mg of aztreonam, 50mL of horse blood (animal blood); and the remainder is deionized water.

[0065] Example 4 This example provides a culture medium for inhibiting the growth of Gram-negative bacteria. Each 1L contains the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 20mg of aztreonam (antibacterial drug), 50mL of horse blood (animal blood); and the remainder is deionized water.

[0066] Example 5 This example provides a culture medium for inhibiting the growth of Gram-negative bacteria. Each 1L contains the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 40mg of aztreonam (antibacterial drug), 50mL of horse blood (animal blood); and the remainder is deionized water.

[0067] Example 6 This example provides a culture medium for inhibiting the growth of Gram-negative bacteria. Each 1L contains the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 60mg of aztreonam (antibacterial drug), 50mL of horse blood (animal blood); and the remainder is deionized water.

[0068] Experimental Group 3 Example 7 This example provides a culture medium for inhibiting the growth of Gram-negative bacteria. Each 1L contains the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 40mg of aztreonam (antibacterial agent), 2mg of avibactam (antibacterial agent), 50mL of horse blood (animal blood); and the remainder is deionized water.

[0069] Example 8 This example provides a culture medium for inhibiting the growth of Gram-negative bacteria. Each 1L contains the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 40mg of aztreonam (antibacterial agent), 4mg of avibactam (antibacterial agent), 50mL of horse blood (animal blood); and the remainder is deionized water.

[0070] Example 9 This example provides a culture medium for inhibiting the growth of Gram-negative bacteria, each 1L comprising the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 40mg of aztreonam (antibacterial agent), 6mg of avibactam (antibacterial agent), 50mL of horse blood (animal blood); the remainder being deionized water.

[0071] Example 10 This example provides a culture medium for inhibiting the growth of Gram-negative bacteria. Each 1L contains the following ingredients: 18g of special peptone (nitrogen source), 5g of tryptone (nitrogen source), 5g of sodium chloride (inorganic salt), 2g of corn starch (carbon source), 10g of agar powder (coagulant), 40mg of aztreonam (antibacterial agent), 8mg of avibactam (antibacterial agent), 50mL of horse blood (animal blood); and the remainder is deionized water.

[0072] Performance testing Test Method 1 Gram-negative bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Klebsiella pneumoniae were prepared into 0.5M bacterial suspensions using sterile inoculation water. 20 μL of the 0.5M bacterial suspension was inoculated into Example 1 and ordinary blood agar medium, respectively, and the bacterial growth was observed. Each 1L of standard blood agar medium contains the following ingredients: 18g special peptone, 5g tryptone, 5g sodium chloride, 2g corn starch, 10g agar powder, 50mL horse blood; the remainder is deionized water. Experimental results Gram-negative bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Klebsiella pneumoniae showed normal growth in all three zones of ordinary blood agar medium after 24 hours of culture, with no inhibition.

[0073] Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Klebsiella pneumoniae were cultured in the culture medium of Example 1 for 24 hours, with only a small amount of bacteria growing in one zone or no growth at all, indicating that bacterial growth was significantly inhibited.

[0074] For detailed test results, please see Figures 1-8 .

[0075] Test Method 2: (1) Gram-negative bacteria Escherichia coli and Gram-positive bacteria Streptococcus pneumoniae and Enterococcus faecalis were mixed and inoculated on ordinary blood agar medium and the culture medium of Example 1 of the invention, and the bacterial growth was observed.

[0076] (2) The Gram-negative bacteria Klebsiella pneumoniae and the Gram-positive bacteria Streptococcus pneumoniae and Enterococcus faecalis were mixed and inoculated on ordinary blood agar medium and the culture medium of Example 1 of the invention, and the bacterial growth was observed.

[0077] Experimental results Gram-negative Escherichia coli (ATCC25922) and Gram-positive Streptococcus pneumoniae and Enterococcus faecalis were inoculated together on ordinary blood agar medium and the inventive medium (Example 1) and cultured for 24 hours. Results showed that on ordinary blood agar medium, Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, and Streptococcus pneumoniae all grew normally and were not inhibited. On the In Example 1 medium, Gram-negative Escherichia coli showed no growth or its growth was significantly inhibited, while Gram-positive Streptococcus pneumoniae and Enterococcus faecalis grew normally and were not inhibited. See detailed test results below. Figures 9-12 .

[0078] Gram-negative Klebsiella pneumoniae (ATCC BAA 2814) was mixed with Gram-positive Streptococcus pneumoniae and Enterococcus faecalis and inoculated into ordinary blood agar medium and the inventive medium of Example 1, respectively. After 24 hours of incubation, the results were observed as follows: In ordinary blood agar medium, all Gram-negative Klebsiella pneumoniae, Gram-positive Streptococcus pneumoniae, and Enterococcus faecalis grew normally without inhibition. In the In Example 1 medium, no growth of Gram-negative Klebsiella pneumoniae was observed or its growth was significantly inhibited, while the growth of Gram-positive Streptococcus pneumoniae and Enterococcus faecalis was normal and uninhibited. For detailed test results, please see Figures 13-16 .

[0079] Test Method 3: Clinical samples containing Gram-negative and Gram-positive bacteria were inoculated into Example 1 and ordinary blood agar medium, respectively, and bacterial growth was observed. Experimental results Clinical samples containing Gram-negative and Gram-positive bacteria were cultured in ordinary blood agar medium for 24 hours. Gram-negative and Gram-positive bacteria grew together, and some of the Gram-negative bacteria covered the mixed Gram-positive pathogens. Gram-negative bacteria could not be well distinguished from Gram-positive bacteria, which was not conducive to the isolation and purification of pathogens.

[0080] Clinical samples containing Gram-negative and Gram-positive bacteria were cultured in the culture medium of Example 1 of the invention for 24 hours. Gram-negative bacteria were significantly inhibited, while Gram-positive bacteria grew normally and were not inhibited.

[0081] For detailed test results, please see Figures 17-20 .

[0082] Test Method 4: The antibacterial effects of different concentrations of the antibacterial drugs avibactam and aztreonam in blood agar plates were tested. The test results are detailed in Table 2.

[0083] The blood agar plate consists of 39 g / L Columbia medium and 50 mL / L sheep blood (the sheep blood is added when the medium is autoclaved at 50-60°C); the blood agar plate needs to be sterilized at 121°C for 30 min.

[0084] The test results for the above tests are detailed in Table 1.

[0085] Table 1

Claims

1. A culture medium for inhibiting the growth of Gram-negative bacteria, characterized in that, The raw materials for preparation include: nitrogen source, inorganic salt, carbon source, coagulant, antibacterial drug, animal blood, and water.

2. The culture medium for inhibiting the growth of Gram-negative bacteria according to claim 1, characterized in that, The nitrogen source includes inorganic nitrogen sources and / or organic nitrogen sources.

3. The culture medium for inhibiting the growth of Gram-negative bacteria according to claim 2, characterized in that, The organic nitrogen source includes one or more of the following: yeast powder, beef extract, peptone, amino acids, and protein hydrolysates.

4. The culture medium for inhibiting the growth of Gram-negative bacteria according to claim 1, characterized in that, The coagulant includes agar powder and / or peptone.

5. The culture medium for inhibiting the growth of Gram-negative bacteria according to claim 1, characterized in that, The antibacterial drugs mentioned include aztreonam and / or avibactam.

6. The culture medium for inhibiting the growth of Gram-negative bacteria according to claim 5, characterized in that, The antibacterial drugs mentioned include aztreonam and avibactam.

7. The culture medium for inhibiting the growth of Gram-negative bacteria according to claim 6, characterized in that, The mass ratio of aztreonam to avibactam is (8-64):(1-8).

8. A method for preparing a culture medium for inhibiting the growth of Gram-negative bacteria according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Add nitrogen source, inorganic salt, carbon source, coagulant, antibacterial agent and water to the flask in proportion; S2. Place the flask in an autoclave and sterilize it at high temperature; S3. Cool the culture medium, add animal blood and stir well; S4. Immediately dispense the well-stirred culture medium into petri dishes and let it cool and solidify.

9. The method for preparing the culture medium for inhibiting the growth of Gram-negative bacteria according to claim 8, characterized in that, In step S2, the high-temperature sterilization temperature is 110-135℃, and the high-temperature sterilization time is 10-20 minutes.

10. The method for preparing the culture medium for inhibiting the growth of Gram-negative bacteria according to claim 8, characterized in that, The cooling temperature in step S3 is 50-65℃.

Images