Culture medium for preventing diffuse growth of proteus and method for preparing the same

Abstract

The present application relates to the technical field of biology, in particular to the field of A01G18 / 20, more particularly to a culture medium for preventing Proteus diffuse growth and a preparation method thereof.The preparation raw materials of the culture medium include: special proteose peptone, tryptone, inorganic salt, starch, agar powder, animal blood, bacteriostatic agent, water.The culture medium provided by the present application has a good inhibitory effect on the diffuse growth of Proteus, has a better bacteriostatic effect compared with other common culture media; and has no obvious inhibitory effect on other non-Proteus bacterial species, so that directional screening of specific mixed bacterial species can be realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, particularly to the field of A01G18 / 20, and more specifically to a culture medium for preventing the diffuse growth of Proteus mirabilis and a method for preparing the same. Background Technology

[0002] Proteus is a group of Gram-negative bacilli widely found in nature. They exhibit various morphologies, including spherical, rod-shaped, and filamentous forms, hence the name "Proteus." Proteus is commonly found in soil, water, decaying organic matter, and the intestines of humans and animals. While generally non-pathogenic, it can cause food poisoning under specific conditions, such as high temperatures or contaminated and incompletely cooked food. Clinically, Proteus mainly includes *Proteus vulgaris* and *Proteus mirabilis*. Additionally, *Proteus moggans*, *Proteus reticularis*, and *Proteus agnosticus*, although also belonging to the *Proteus* genus, are relatively rare clinically.

[0003] The migration and growth of *Proteus vulgaris* and *Proteus mirabilis* on agar plates is one of their biological characteristics. In clinical testing, the diffuse growth of *Proteus vulgaris* makes the isolation of other bacterial species more difficult. How to effectively and precisely inhibit the migration of *Proteus vulgaris* without significantly affecting the growth of other bacterial species is an urgent industry-wide technical challenge. Literature: A culture method for inhibiting the migration and growth of *Proteus vulgaris* discloses an antibacterial culture medium containing peptone, agar powder, sodium chloride, and water; however, it does not show specificity for inhibiting migrating bacterial species. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a culture medium for preventing the diffuse growth of Proteus mirabilis and a method for preparing the same.

[0005] The first aspect of the present invention provides a culture medium for preventing the diffuse growth of Proteus, the raw materials of which include: special peptone, tryptone, inorganic salts, starch, agar powder, animal blood, antibacterial agent, and water.

[0006] Preferably, the concentration of the special peptone in the culture medium is 15-20 g / L.

[0007] Preferably, the mass concentration of tryptone in the culture medium is 5-10 g / L.

[0008] Both tryptone and special peptones are rich in vitamins and growth factors, providing the nitrogen source and nutrients needed for microbial growth and promoting their growth and metabolism. Furthermore, tryptone and special peptones can also act as solidifying agents, providing a solid culture medium.

[0009] Preferably, the inorganic salt includes one of NaCl, KCl, MgCl2, KH2PO4, MgSO4, and FeSO4.

[0010] More preferably, the inorganic salt is NaCl.

[0011] More preferably, the NaCl concentration in the culture medium is 5-10 g / L.

[0012] An appropriate amount of sodium chloride is necessary to maintain the normal growth and function of microbial cells because sodium ions are related to maintaining cell osmotic pressure. Therefore, a small amount of sodium chloride is often added to the culture medium. In addition, the inventors have found that an appropriate amount of sodium chloride can also specifically inhibit the diffuse growth of Proteus in this culture medium.

[0013] 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.

[0014] More preferably, the starch is corn starch.

[0015] Corn starch is a carbon source in culture media, providing the necessary energy for the growth and metabolism of microorganisms. Corn starch can be enzymatically hydrolyzed into monosaccharides by microorganisms, and then releases energy through processes such as glycolysis, citric acid cycle and oxidative phosphorylation, supporting the growth and reproduction of microorganisms.

[0016] Preferably, the agar powder has a mass concentration of 10-40 g / L in the culture medium.

[0017] More preferably, the agar powder has a mass concentration of 15-40 g / L in the culture medium.

[0018] More preferably, the agar powder has a mass concentration of 30-40 g / L in the culture medium.

[0019] In their practical research, the inventors discovered that the agar content significantly affects the migration of Proteus miraculous bacteria. When the agar content is too low, the culture medium may become semi-liquid, affecting its stability. Increased fluidity of the medium may prevent Proteus from forming stable colonies. Furthermore, Proteus, with its active motility and pleomorphism, is more prone to diffusion and growth on media with low agar content, promoting its migration and growth. Additionally, agar, as part of the culture medium, not only provides physical support but also participates in nutrient exchange. Low agar content may increase nutrient fluidity, potentially leading to uneven nutrient distribution and affecting the absorption and utilization of nutrients by Proteus. Conversely, high agar content increases the hardness of the culture medium, affecting colony morphology and growth characteristics. Proteus may not form normal colonies on overly hard media, inhibiting colony growth. However, it may also have a significant inhibitory effect on other non-Proteus species, affecting the specific inhibitory properties of the culture medium. Therefore, the optimal agar concentration in this invention is 30-40 g / L.

[0020] Preferably, the animal blood includes sheep blood and / or horse blood.

[0021] 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.

[0022] Preferably, the antibacterial agent includes: organic alcohols, inorganic compounds, plant extracts, and biological enzymes.

[0023] More preferably, the organic alcohol includes one of pentanediol, hexanediol, ethanol, octanediol, and decanediol.

[0024] More preferably, the organic alcohol is ethanol.

[0025] Ethanol, as an organic solvent, can penetrate bacterial cell membranes, affecting their integrity and permeability, thereby interfering with normal bacterial physiological functions. For Proteus vulgaris, ethanol can inhibit its motility and thus prevent its migration and growth by disrupting its cell membrane structure. Furthermore, ethanol can affect the absorption and utilization of nutrients by Proteus vulgaris, interfering with its metabolic processes. When bacteria are in unfavorable environments, such as in culture media with high ethanol concentrations, their growth and reproduction are inhibited, and migration and growth phenomena are correspondingly weakened or eliminated. However, because ethanol is volatile, the volume content of ethanol in the culture medium in this invention is 3-5%.

[0026] Preferably, the inorganic compound includes: Hg 2+Salt, Cd salt, Cu 2+ Salt, Zn 2+ A type of salt, specifically an azide salt.

[0027] More preferably, the inorganic compound is an azide salt, and as an example, the azide salt is NaN3.

[0028] Sodium azide can generate cytotoxic azidoic acid (HN3) under certain conditions. This substance can cross the cell membrane and damage intracellular biomolecules. Because Proteus has the characteristic of migratory growth, that is, it can form migratory colonies on certain culture media, the addition of sodium azide may interfere with key links in its migration process, such as signal transduction, cell movement or energy metabolism, thereby inhibiting its migratory growth. However, sodium azide also has a certain inhibitory effect on non-Proteus species, and its specific inhibitory effect is relatively poor.

[0029] Preferably, the plant extract includes one of the following: Portulaca oleracea extract, Origanum vulgare extract, and Soapnut Saponin.

[0030] More preferably, the plant extract is Sapindus mukorossi saponin.

[0031] Sapindus saponins have a certain inhibitory effect on Proteus. Sapindus saponins can destroy the bacterial cell wall and cell membrane, and interfere with the normal metabolic process of bacteria. Furthermore, Sapindus saponins can penetrate into the bacterial cell and bind to receptors on the Proteus cell wall, interfering with the synthesis and stability of the cell wall, thereby inhibiting its migration ability.

[0032] Preferably, the biological enzyme includes one or more of lysozyme, glucose oxidase, cellulase, and antimicrobial peptides.

[0033] More preferably, the bioenzyme is lysozyme.

[0034] Although Proteus, a Gram-negative bacterium, has a cell wall that differs from that of Gram-positive bacteria, it still contains key components such as peptidoglycan. Lysozyme can effectively hydrolyze these components, especially the β-1,4 glycosidic bonds in peptidoglycan, which are the main structural support of the bacterial cell wall. When lysozyme acts on Proteus, it specifically recognizes and hydrolyzes these key chemical bonds on the cell wall, leading to the destruction of the cell wall structure. Due to the concentration difference between the inside and outside of the cell, a large amount of water rushes into the cell, causing the bacteria to swell and eventually rupture, thus exerting an antibacterial effect.

[0035] More preferably, the antibacterial agent includes ethanol, Sapindus mukorossi saponins, and lysozyme; wherein the mass concentration of Sapindus mukorossi saponins in the culture medium is 1-5 g / L; and the mass concentration of lysozyme in the culture medium is 1-3 g / L.

[0036] A second aspect of the present invention provides a method for preparing a culture medium for preventing the diffuse growth of Proteus vulgaris, comprising: S1. Add the special peptone, tryptone, inorganic salts, starch, agar powder, animal blood, antibacterial agent, and water to the flask; 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.

[0037] Preferably, the high-temperature sterilization temperature in step S2 is 110-140℃, and the high-temperature sterilization time is 10-30 minutes.

[0038] Preferably, the cooling temperature in step S3 is 50-75°C.

[0039] Beneficial effects (i) The culture medium provided by the present invention has a good inhibitory effect on the diffuse growth of Proteus, and has a better antibacterial effect than other common culture media.

[0040] (ii) The culture medium provided by the present invention has no significant inhibitory effect on other non-Proteus species, and can achieve targeted screening of specific mixed species.

[0041] (III) The preferred concentration of agar powder in this invention is 10-40 g / L. If the agar powder content is too low, Proteus may not be able to form a stable colony morphology. Furthermore, Proteus exhibits active motility and pleomorphism, making it easier for it to spread and grow on media with low agar powder content, thus promoting bacterial migration and growth. Conversely, if the agar powder content is too high, it will increase the hardness of the culture medium, which will affect the colony morphology and growth characteristics. Proteus may not be able to form a normal colony morphology on an overly hard culture medium, or its colony growth may be inhibited. However, it will also have a significant inhibitory effect on other non-Proteus species, thus affecting the specific inhibitory properties of the culture medium.

[0042] (iv) The present invention preferably uses specific antibacterial agents, including ethanol, soapberry saponins, and lysozyme, which can effectively inhibit the migration of Proteus.

[0043] (v) The raw materials for the culture medium in this invention are widely available and have low cost. The preparation method is relatively simple. The antibacterial effect of Proteus is excellent, and it is expected to achieve large-scale mass production, which is expected to generate considerable market revenue. Attached Figure Description

[0044] Figure 1 A schematic diagram of inoculating Proteus on ordinary blood agar medium.

[0045] Figure 2 A schematic diagram of the culture medium used for inoculating Proteus mirabilis in Example 1.

[0046] Figure 3 A schematic diagram of inoculating Escherichia coli with ordinary blood agar powder culture medium.

[0047] Figure 4 A schematic diagram of the culture medium used for inoculating Escherichia coli in Example 1.

[0048] Figure 5 A schematic diagram of inoculating Staphylococcus aureus onto a standard blood agar medium.

[0049] Figure 6 A schematic diagram of the culture medium used for inoculating Staphylococcus aureus in Example 1.

[0050] Figure 7 A schematic diagram of inoculating Pseudomonas aeruginosa onto ordinary blood agar medium.

[0051] Figure 8 Schematic diagram of the culture medium for inoculating Pseudomonas aeruginosa in Example 1.

[0052] Figure 9 A schematic diagram of inoculating Proteus + Escherichia coli onto ordinary blood agar medium.

[0053] Figure 10 A schematic diagram of the culture medium used for inoculating Proteus + Escherichia coli in Example 1.

[0054] Figure 11 A schematic diagram of inoculating Proteus + Staphylococcus aureus onto ordinary blood agar medium.

[0055] Figure 12 A schematic diagram of the culture medium used for inoculating Proteus + Staphylococcus aureus in Example 1.

[0056] Figure 13 A schematic diagram of inoculating Proteus + Pseudomonas aeruginosa onto ordinary blood agar medium.

[0057] Figure 14 Schematic diagram of the culture medium for inoculating Proteus + Pseudomonas aeruginosa in Example 1.

[0058] Figure 15A schematic diagram of inoculating clinical wound samples with ordinary blood agar powder culture medium.

[0059] Figure 16 A schematic diagram of the culture medium used for inoculating clinical wound samples in Example 1.

[0060] Figure 17 A schematic diagram of inoculating clinical urine samples with ordinary blood agar powder culture medium.

[0061] Figure 18 A schematic diagram of the culture medium used for inoculating clinical urine samples in Example 1.

[0062] Figure 19 A schematic diagram of inoculating clinical sputum samples with ordinary blood agar powder culture medium.

[0063] Figure 20 This is a schematic diagram of the culture medium for clinical sputum samples in Example 1. Detailed Implementation

[0064] Example 1 The first aspect of this example provides a culture medium for preventing the diffuse growth of Proteus mirabilis, each 1L comprising the following ingredients: 18g of special peptone (brand: OXOID), 5g of tryptone, 5g of sodium chloride (inorganic salt), 2g of corn starch, 30g of agar powder, 40mL of ethanol (antibacterial agent), 1g of lysozyme LYM (antibacterial agent), 3g of Sapindus mukorossi saponin (antibacterial agent), 50mL of sheep blood (animal blood), and the remainder of deionized water.

[0065] Unless otherwise specified, all of the above raw materials are ordinary commercially available products.

[0066] 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 the special peptone, tryptone, inorganic salts, starch, agar powder, animal blood, antibacterial agent, and water to the flask in the specified proportions; 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.

[0067] The diagrams illustrating the inoculation of different bacterial species and clinical samples onto standard blood agar medium and the medium used in this case are shown below. Figures 1-20 As shown.

[0068] During the test, 20 μL of a 0.5 M bacterial suspension was inoculated into the culture medium of Example 1 and ordinary blood agar powder culture medium, respectively.

[0069] Example 2 The first aspect of this example provides a culture medium for preventing the diffuse growth of Proteus mirabilis, each 1L comprising the following ingredients: 18g of special peptone, 5g of tryptone, 5g of sodium chloride (inorganic salt), 2g of corn starch, 40g of agar powder, 40mL of ethanol (antibacterial agent), 1g of lysozyme LYM (antibacterial agent), 3g of Sapindus mukorossi saponin (antibacterial agent), 50mL of sheep blood (animal blood), and the remainder of deionized water.

[0070] 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 the special peptone, tryptone, inorganic salts, starch, agar powder, antibacterial agent, and water to the flask in the specified proportions; S2. Place the flask in an autoclave and sterilize at 121°C for 15 minutes. S3. Cool the culture medium to 55°C, add sterile animal blood and stir well; S4. Immediately dispense the well-stirred culture medium into petri dishes and let it cool and solidify.

[0071] Performance Evaluation Test Method 1 Proteus was prepared into a 0.5M suspension using sterile inoculation water. 20 μL of the 0.5M Proteus suspension was inoculated into the culture medium of Example 1 and ordinary blood agar powder, respectively. The time for the appearance of single colonies and the time for fusion were observed. The test results are detailed in Table 1.

[0072] Each 1L of ordinary blood agar powder culture medium includes the following ingredients: 18g special peptone, 5g tryptone, 5g sodium chloride, 2g corn starch, 10g agar powder, 50mL horse blood, and the remainder deionized water.

[0073] Single colony appearance time: The time when a moderately sized, smooth, round, white single colony appears.

[0074] Merging time: The time it takes for colonies to migrate and merge into a sheet.

[0075] Table 1

[0076] Test Method 2 (1) Prepare a 0.5M Proteus suspension by using sterile inoculation water to prepare Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Take 20 μL of the 0.5M Proteus suspension and inoculate it into ordinary blood agar powder medium of Example 1 respectively, and observe the growth of the pathogens.

[0077] Experimental results: Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa showed normal growth after 24 hours of incubation in the culture medium of Example 1, which was not significantly different from the phenomenon observed after 24 hours of incubation in ordinary blood agar powder culture medium. The culture medium of Example 1 had no significant antibacterial effect on the target isolated pathogens.

[0078] Test Method 3 After mixing 0.5M Proteus bacterial suspension with 0.5M Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae bacterial suspensions, 20 μL of the mixed bacterial suspension was inoculated onto the culture medium of Example 1 and ordinary blood agar powder culture medium, and the growth of Proteus and pathogens was observed.

[0079] Experimental results: When Proteus and pathogens were cultured in the culture medium of Example 1 for 24 hours, Proteus and the mixed bacteria could be well distinguished. There was no phenomenon of Proteus covering other pathogens due to diffuse growth, which is beneficial for the isolation and purification of pathogens.

[0080] When Proteus and mixed bacteria are cultured in ordinary blood agar powder medium for 24 hours, Proteus grows diffusely and covers other pathogens. Proteus cannot be easily distinguished from pathogens, which is not conducive to the isolation and purification of pathogens.

[0081] (2) Clinical samples containing Proteus were inoculated onto the culture medium of Example 1-2 and ordinary blood agar powder culture medium, respectively, and the growth of Proteus and pathogens was observed.

[0082] Clinical samples containing Proteus were cultured in the culture medium of Examples 1-2 for 24 hours. Proteus and pathogens could be well distinguished, and there was no phenomenon of Proteus covering the pathogens due to diffuse growth, which is conducive to the isolation and purification of pathogens.

[0083] When clinical samples containing Proteus were cultured in ordinary blood agar powder for 24 hours, the Proteus bacteria diffused and covered other pathogens. The Proteus bacteria could not be well distinguished from the pathogens, which was not conducive to the isolation and purification of the pathogens.

[0084] Test 4: Impact of antibacterial agent performance test The test results are detailed in Table 2 (except for agar powder and antibacterial agent, the contents of the other components are the same as in Examples 1-2).

[0085] Table 2

[0086] The culture results of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa are detailed in Table 3.

[0087] Table 3

Claims

1. A culture medium for preventing the diffuse growth of Proteus vulnerabilities, characterized in that, Raw materials include: Special peptone, tryptone, inorganic salts, starch, agar powder, animal blood, antibacterial agent, water.

2. The culture medium for preventing diffuse growth of Proteus vulnerabilities according to claim 1, characterized in that, The inorganic salts mentioned include one of NaCl, KCl, MgCl2, KH2PO4, MgSO4, and FeSO4.

3. The culture medium for preventing diffuse growth of Proteus vulnerabilities according to claim 1, characterized in that, The starch mentioned includes one of the following: corn starch, sweet potato starch, potato starch, wheat starch, mung bean starch, tapioca starch, and pea starch.

4. The culture medium for preventing diffuse growth of Proteus according to claim 1, characterized in that, The agar powder has a mass concentration of 10-40 g / L in the culture medium.

5. The culture medium for preventing diffuse growth of Proteus vulnerabilities according to claim 1, characterized in that, The antibacterial agents include one or more of the following: organic alcohols, inorganic compounds, plant extracts, and biological enzymes.

6. The culture medium for preventing diffuse growth of Proteus according to claim 5, characterized in that, The organic alcohols mentioned include one of the following: pentanediol, hexanediol, ethanol, octanediol, and decanediol.

7. The culture medium for preventing diffuse growth of Proteus according to claim 5, characterized in that, The inorganic compounds mentioned include: Hg 2+ Salt, Cd 3+ Salt, Cu 2+ Salt, Zn 2+ A type of salt, specifically an azide salt.

8. The culture medium for preventing diffuse growth of Proteus according to claim 5, characterized in that, The plant extracts mentioned include one of the following: purslane extract, oregano extract, and soapberry saponins.

9. The culture medium for preventing diffuse growth of Proteus according to claim 5, characterized in that, The biological enzymes mentioned include one or more of the following: lysozyme, glucose oxidase, cellulase, and antimicrobial peptides.

10. A method for preparing a culture medium for preventing diffuse growth of Proteus according to any one of claims 1-9, characterized in that, include: S1. Add the special peptone, tryptone, inorganic salts, starch, agar powder, animal blood, antibacterial agent, and water to the flask; 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.

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