Enrichment culture medium, and preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of rapid detection of drug sensitivity for bacteria. Disclosed are an enrichment culture medium, and a preparation method therefor and the use thereof. The enrichment culture medium comprises the following raw materials in parts by mass: 15.0-20.0 parts of tryptone, 3.5-5.5 parts of peptone, 1.5-3.5 parts of glucose, 5.0-6.5 parts of yeast extract, 2.0-2.5 parts of dipotassium phosphate, 2.0-3.5 parts of disodium hydrogen phosphate, 1.0-1.5 parts of monopotassium phosphate, 2.5-7.5 parts of sodium chloride, 12.0-18.0 parts of proteose peptone, 15.0-22.0 parts of beef heart infusion powder, 8.0-12.0 parts of soya peptone, 1.0-5.0 parts of 0.1% nicotinamide adenine dinucleotide (NAD), 30-100 parts of newborn calf serum inactivated at 56°C for 30 min, 2.0-4.0 parts of mannitol, 3.0-5.0 parts of sodium pyruvate, 0.23-0.45 parts of cycloheximide, 0.8-2.0 parts of 0.05% bromothymol blue, 2.5-4.5 parts of 0.1% methylene blue, and 1000 parts of distilled water, with the pH value of 6.8-7.5. The enrichment culture medium is used to screen effective therapeutic drugs for bacterial diseases in poultry, and is convenient to use and has a single enrichment effect superior to that of respective selective enrichment broths of target bacteria. Therefore, the culture medium can realize rapid proliferation of common pathogenic bacteria in poultry.

Description

A bacterial enrichment culture medium and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of rapid bacterial drug sensitivity testing, in particular to a bacterial enrichment culture medium and a preparation method and application thereof. Background Art

[0002] In recent years, with the rapid growth in the scale and number of poultry farms, bacterial diseases have become increasingly harmful, with increasingly complex disease manifestations. Furthermore, bacterial resistance is becoming increasingly prevalent, with multidrug resistance becoming commonplace. The widespread spread of resistant strains presents significant challenges for clinical treatment. To address these challenges, the development of novel rapid bacterial susceptibility testing technologies that are simple, fast, low-cost, and easily interpretable is imperative. Currently, screening for effective therapeutics for poultry bacterial diseases primarily relies on susceptibility testing performed in specialized laboratories. However, common poultry bacterial pathogens, particularly Pasteurella multocida and Riemerella anatipestifer, have stringent nutritional requirements and are difficult to isolate and culture clinically. Existing commercial culture media, when used alone, can miss some bacterial pathogens. Consequently, laboratory susceptibility testing often requires the preparation of two or three culture media to process the sample. While this approach can provide specific and accurate guidance for clinical drug use, it also results in lengthy testing times and high costs. On the other hand, the drugs identified in laboratory susceptibility tests may not be available on farms, nor can they be purchased promptly. This makes it difficult to send samples for testing every time a disease breaks out. Farmers continue to blindly use drugs, which compromises treatment effectiveness and leads to increasingly serious mixed infections on farms. The difficulty in selecting effective drugs results in low cure rates and loss of drug costs. Summary of the Invention

[0003] The present invention aims to provide a bacterial enrichment culture medium and its preparation method and application, and to develop a simple, fast, low-cost, visual drug sensitivity test for realizing on-site rapid and accurate screening of effective therapeutic drugs for poultry bacterial pathogens.

[0004] The present invention provides a bacterial enrichment culture medium, which comprises, by weight, 15.0-20.0 parts of tryptone, 3.5-5.5 parts of peptone, 1.5-3.5 parts of glucose, 5.0-6.5 parts of yeast extract, 2.0-2.5 parts of dipotassium hydrogen phosphate, 2.0-3.5 parts of disodium hydrogen phosphate, 1.0-1.5 parts of potassium dihydrogen phosphate, 2.5-7.5 parts of sodium chloride, 12.0-18.0 parts of peptone, 15.0-22.0 parts of beef heart extract powder, 8.0-12.0 parts of soy peptone, 1.0-5.0 parts of 0.1% NAD nicotinamide adenine dinucleotide, 30-100 parts of newborn calf serum inactivated at 56°C for 30 minutes, 2.0-4.0 parts of mannitol, 3.0-5.0 parts of sodium pyruvate, 0.23-0.45 parts of cycloheximide, 0.8-2.0 parts of 0.05% bromothymol blue, 2.5-4.5 parts of 0.1% methylene blue, 1000 parts of distilled water, pH value is 6.8-7.5.

[0005] Preferably, the raw materials include, by mass: 18.0 parts of tryptone, 4.3 parts of peptone, 2.0 parts of glucose, 5.5 parts of yeast extract, 2.2 parts of dipotassium hydrogen phosphate, 2.5 parts of disodium hydrogen phosphate, 1.2 parts of potassium dihydrogen phosphate, 5.0 parts of sodium chloride, 15.0 parts of peptone, 17.5 parts of beef heart extract powder, 10.0 parts of soy peptone, 2.0 parts of 0.1% NAD (nicotinamide adenine dinucleotide), 50 parts of newborn calf serum inactivated at 56°C for 30 minutes, 2.6 parts of mannitol, 4.4 parts of sodium pyruvate, 0.25 parts of cycloheximide, 0.8-2.0 parts of 0.05% bromothymol blue, 2.5-4.5 parts of 0.1% methylene blue, 1000 parts of distilled water, and a pH value of 7.2.

[0006] The present invention provides a method for preparing a bacterial enrichment culture medium, comprising the following steps:

[0007] S1. Add tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, sodium pyruvate, and cycloheximide to distilled water in specific proportions, heat and boil until completely dissolved, cool to room temperature, and adjust the pH to 6.8-7.5 to obtain a prepared solution; sterilize the prepared solution under high pressure and cool to room temperature to obtain a culture medium liquid;

[0008] S2. Weigh nicotinamide adenine dinucleotide, completely dissolve it in sterile purified water, and filter it through a 0.22 μm filter membrane; add the filtrate to the culture medium obtained in step S1 and mix thoroughly;

[0009] S3. Inactivate the newborn calf serum and filter it through a 0.22 μm filter membrane; add the filtered serum to the culture medium obtained in step S2 and mix thoroughly;

[0010] S4. Weigh bromothymol blue, dissolve it in 0.05 M sodium hydroxide, and dilute to volume with sterile purified water to prepare a 0.05% solution; filter with a 0.22-μm filter membrane; weigh methylene blue, dissolve it in sterile purified water, and dilute to volume with sterile purified water to prepare a 0.1% solution; filter with a 0.22-μm filter membrane; separately draw the bromothymol blue filtrate and the methylene blue filtrate and add them to the culture medium liquid obtained in step S3, mix well, and store at 4°C.

[0011] The present invention provides an application of a bacterial enrichment culture medium in screening effective therapeutic drugs for poultry bacterial diseases.

[0012] The use of enrichment culture medium in screening effective therapeutic drugs for poultry bacterial diseases includes the following steps:

[0013] S1. Sampling: Collect samples of diseased material according to clinical symptoms;

[0014] S2. Add drugs. Select several therapeutic drugs to be screened and label them separately. After treatment, add them to the corresponding drug screening tubes.

[0015] S3. Add samples. Various samples are added to the screening tube in an appropriate manner.

[0016] S4. Culture test: Place the screening tube in a 37°C constant temperature incubator and culture for 16 hours, then observe the results. The negative control tube without sample and drug and the sample control tube with only sample but no drug are cultured simultaneously.

[0017] Preferably, in step S1, anal swabs or feces are collected for digestive tract symptoms; nasopharyngeal swabs are collected for respiratory tract symptoms; and tissues or organs with obvious lesions are collected from dead poultry.

[0018] Preferably, in step S2, the powdered drug is first diluted with purified water to 10 times the therapeutic dose of the drug, and then 1.0 mL is drawn and added to the corresponding drug screening tube; 1.0 mL of the liquid drug is directly drawn and added to the corresponding drug screening tube.

[0019] Preferably, in step S3, 0.1-0.2 mL of tissue exudate sample is drawn and added to the screening tube; soybean-sized amounts of feces, organs and tissues are drawn and added to the screening tube.

[0020] Preferably, the negative control tube does not fade or change color; the sample control tube fades and becomes turbid, indicating that pathogens are present in the sample; the color of the effective drug screening tube is consistent with that of the negative control tube, without fading or changing color.

[0021] Therefore, the present invention adopts the above-mentioned enrichment culture medium and its preparation method and application. This culture medium is easy to use and is not limited to the use environment. It can be used in breeding sites, veterinary medicine, feed and vaccine distributor stores, laboratories and other environments. Its single enrichment effect is better than the selective enrichment liquid of each target bacteria. The culture medium can achieve the rapid proliferation of common pathogenic bacteria in poultry such as Escherichia coli, Salmonella, Proteus, Klebsiella, Staphylococcus aureus, Pasteurella multocida, Streptococcus, Riemerella anatipestifer and Clostridium perfringens. When coexisting with non-target bacteria, the concentration of each bacteria increases from 10 CFU / mL to 10 after 3-16 hours of culture. 5 CFU / mL, the bacterial concentration increased by 4 orders of magnitude, and the bacterial enrichment effect was good.

[0022] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0024] Reference numerals

[0025] Figure 1 is a result diagram of Comparative Example 1.2;

[0026] Figure 2 is a result diagram of Comparative Example 2.2;

[0027] Figure 3 is a graph showing the results of Comparative Example 3.2;

[0028] Figure 4 is a graph showing the results of Comparative Example 4.2;

[0029] Figure 5 is a graph showing the results of Comparative Example 5.2, wherein a is a graph showing the target bacteria culture results in a culture medium with a color developer added, and b is a graph showing the target bacteria culture results in a culture medium without a color developer added;

[0030] Figure 6 is a graph showing the results of Comparative Example 6.2;

[0031] Figure 7 is a graph showing the results of Comparative Example 7.2;

[0032] FIG8 is a graph showing the color changes of the culture medium after culturing Escherichia coli, Salmonella, Proteus, Klebsiella, Staphylococcus aureus, Pasteurella multocida, Streptococcus, Riemerella anatipestifer, and Clostridium perfringens;

[0033] FIG9 is a diagram showing an operation method for screening effective therapeutic drugs for poultry bacterial diseases;

[0034] Figure 10 is the autopsy pathological condition of Example 5;

[0035] Figure 11 is the drug sensitivity observation results of Example 5;

[0036] FIG12 is the eosin-methylene blue culture results of Example 5;

[0037] FIG13 is the laboratory drug sensitivity test result of colistin sulfate in Example 5. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0039] The present invention provides a bacterial enrichment culture medium, which comprises, by weight (parts / 1000 parts), 15.0-20.0 parts of tryptone, 3.5-5.5 parts of peptone, 1.5-3.5 parts of glucose, 5.0-6.5 parts of yeast extract, 2.0-2.5 parts of dipotassium hydrogen phosphate, 2.0-3.5 parts of disodium hydrogen phosphate, 1.0-1.5 parts of potassium dihydrogen phosphate, 2.5-7.5 parts of sodium chloride, 12.0-18.0 parts of peptone, 15.0-22.0 parts of beef heart extract powder, and 10.0-25.0 parts of glutathione. .0 parts, soy peptone 8.0-12.0 parts, 0.1% NAD nicotinamide adenine dinucleotide 1.0-5.0 parts, newborn calf serum inactivated at 56°C for 30 minutes 30-100 parts, mannitol 2.0-4.0 parts, sodium pyruvate 3.0-5.0 parts, cycloheximide 0.23-0.45 parts, 0.05% bromothymol blue 0.8-2.0 parts, 0.1% methylene blue 2.5-4.5 parts, distilled water 1000 parts, pH value is 6.8-7.5.

[0040] Preferably, 18.0 parts of tryptone, 4.3 parts of peptone, 2.0 parts of glucose, 5.5 parts of yeast extract, 2.2 parts of dipotassium hydrogen phosphate, 2.5 parts of disodium hydrogen phosphate, 1.2 parts of potassium dihydrogen phosphate, 5.0 parts of sodium chloride, 15.0 parts of peptone, 17.5 parts of ox heart extract powder, 10.0 parts of soy peptone, 2.0 parts of 0.1% NAD (nicotinamide adenine dinucleotide), 50 parts of newborn calf serum inactivated at 56°C for 30 minutes, 2.6 parts of mannitol, 4.4 parts of sodium pyruvate, 0.25 parts of cycloheximide, 0.8-2.0 parts of 0.05% bromothymol blue, 2.5-4.5 parts of 0.1% methylene blue, 1000 parts of distilled water, and a pH value of 7.2.

[0041] The present invention provides a method for preparing a bacterial enrichment culture medium, comprising the following steps:

[0042] S1. Add tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, sodium pyruvate, and cycloheximide to distilled water in specific proportions, heat and boil until completely dissolved, cool to room temperature, and adjust the pH to 6.8-7.5 to obtain a prepared solution; sterilize the prepared solution under high pressure and cool to room temperature to obtain a culture medium liquid;

[0043] S2. Weigh nicotinamide adenine dinucleotide, completely dissolve it in sterile purified water, and filter it through a 0.22 μm filter membrane; then add the filtrate to the culture medium obtained in step S1;

[0044] S3. Inactivate the newborn calf serum and filter it through a 0.22 μm filter membrane; add the filtered serum to the culture medium obtained in step S2 and mix thoroughly;

[0045] S4. Weigh bromothymol blue, dissolve it in 0.05 M sodium hydroxide, and dilute to volume with sterile purified water to prepare a 0.05% solution; filter with a 0.22-μm filter membrane; weigh methylene blue, dissolve it in sterile purified water, and dilute to volume with sterile purified water to prepare a 0.1% solution; filter with a 0.22-μm filter membrane; separately draw the bromothymol blue filtrate and the methylene blue filtrate and add them to the culture medium liquid obtained in step S3, mix well, and store at 4°C.

[0046] The present invention provides an application of an enrichment culture medium in screening effective therapeutic drugs for poultry bacterial diseases, comprising the following steps:

[0047] S1. Sampling: Collect disease samples based on clinical symptoms; collect anal swabs or feces for digestive tract symptoms; collect nasopharyngeal swabs for respiratory symptoms; collect tissues or organs with obvious lesions from dead poultry;

[0048] S2. Add drugs. Select several therapeutic drugs to be screened and label them separately. After treatment, add them to the corresponding drug screening tubes. Powdered drugs are first diluted with purified water to 10 times the therapeutic dose of the drug, and then 1.0 mL is added to the corresponding drug screening tubes. Liquid drugs are directly added to the corresponding drug screening tubes with 1.0 mL.

[0049] S3. Add samples. Various samples are added to the screening tube in an appropriate manner. For samples such as tissue exudate, 0.1-0.2 mL is added to the screening tube. For feces, organs, and tissues, a soybean-sized amount is added to the screening tube.

[0050] S4. Culture test: Place the screening tube in a 37°C constant temperature incubator and culture for 16 hours, then observe the results. When bacteria grow in the screening tube, the color of the liquid in the tube will turn from blue to lighter or fade. When antibiotics are added to the tube, if the bacteria cannot grow, the liquid in the tube will not change color, indicating that the antibiotic is effective against the bacteria. If the bacteria can grow, the color of the liquid in the tube will become lighter or fade, indicating that the antibiotic is ineffective against the bacteria.

[0051] In the universal enrichment culture medium of the present invention, tryptone, peptone, yeast extract, peptone, beef heart extract powder, and soy peptone provide essential nutrients such as nitrogen source and carbon source for the growth and reproduction of bacteria; dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, and sodium chloride serve as buffers to prevent acid production during bacterial growth and reproduction, thereby inhibiting their growth; newborn calf serum and NAD nicotinamide adenine dinucleotide provide nutrition for bacteria with strict nutritional requirements such as Riemerella anatipestifer, Pasteurella multocida, and Streptococcus, ensuring their smooth growth; mannitol and sodium pyruvate serve as promoters to promote the growth and reproduction of target bacteria; cycloheximide can inhibit the growth of microorganisms such as yeast, mold, and protozoa; bromothymol blue and methylene blue can be used as inhibitors to inhibit the growth and reproduction of non-target bacteria such as yeast and mold, and can also be used as color indicators. When antibiotics are added to the culture medium, the effectiveness of the antibiotics can be visually identified by the change in color of the liquid in the tube.

[0052] Example 1.1

[0053] Prepare 1000 mL of culture medium by parts by mass (parts / 1000): 18.0 g tryptone, 4.3 g peptone, 2.0 g glucose, 5.5 g yeast extract, 2.2 g dipotassium hydrogen phosphate, 2.5 g disodium hydrogen phosphate, 1.2 g potassium dihydrogen phosphate, 5.0 g sodium chloride, 15.0 g peptone, 17.5 g ox heart extract powder, 10.0 g soy peptone, 2.6 g mannitol, 4.4 g sodium pyruvate, 0.25 g cycloheximide, 1000 mL distilled water, 2.0 mL 0.1% NAD (nicotinamide adenine dinucleotide), 50 mL newborn calf serum inactivated at 56°C for 30 min, 1.0 mL 0.05% bromothymol blue, and 3.0 mL 0.1% methylene blue. The pH value was 7.2.

[0054] Example 1.2

[0055] S1. Add tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, sodium pyruvate, and cycloheximide to distilled water according to the mass fractions in Example 1.1, heat and boil until completely dissolved, cool to room temperature, and adjust the pH to 7.2 to obtain a prepared solution; autoclave the prepared solution at 115°C for 30 min, and cool to room temperature to obtain a culture medium liquid;

[0056] S2. Weigh 1.0 g of NAD nicotinamide adenine dinucleotide, completely dissolve it in 1000.0 mL of sterile purified water, and filter it through a 0.22 μm filter membrane; draw 2.0 mL of the filtrate and add it to the culture medium obtained in step S1, and mix thoroughly.

[0057] S3: Newborn calf serum was inactivated at 56°C for 30 min and filtered through a 0.22 μm filter membrane; 50.0 mL of the filtered serum was added to the culture medium obtained in S2 and mixed.

[0058] S4. Weigh 0.05 g of bromothymol blue, dissolve it in 1.6 mL of 0.05 M sodium hydroxide, and dilute the volume to 100 mL with sterile purified water to prepare a 0.05% solution; filter with a 0.22-micron filter membrane; weigh 0.1 g of methylene blue, dissolve it in sterile purified water, and dilute the volume to 100 mL with sterile purified water to prepare a 0.1% solution; filter with a 0.22-micron filter membrane; respectively, draw 1.0 mL of bromothymol blue filtrate and 3.0 mL of methylene blue filtrate and add them to the culture medium liquid obtained in step S3, mix well, and store at 4°C.

[0059] Example 2.1

[0060] Prepare 1000 mL of culture medium by parts by mass (parts / 1000): 15.0 g tryptone, 3.5 g peptone, 1.5 g glucose, 5.0 g yeast extract, 2.0 g dipotassium hydrogen phosphate, 2.0 g disodium hydrogen phosphate, 1.0 g potassium dihydrogen phosphate, 2.5 g sodium chloride, 12.0 g peptone, 22.0 g ox heart extract powder, 8.0 g soy peptone, 2.0 g mannitol, 3.0 g sodium pyruvate, 0.23 g cycloheximide, 1000 mL of distilled water, 1.0 mL of 0.1% NAD (nicotinamide adenine dinucleotide), 30 mL of newborn calf serum inactivated at 56°C for 30 min, 0.8 mL of 0.05% bromothymol blue, and 2.5 mL of 0.1% methylene blue. The pH value is 6.8.

[0061] Example 2.2

[0062] Tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, sodium pyruvate, and cycloheximide were added to distilled water in the amounts by mass described in Example 2.1, heated and boiled until completely dissolved, cooled to room temperature, and the pH was adjusted to 6.8 to obtain a prepared solution; the prepared solution was autoclaved at 115° C. for 30 min, and cooled to room temperature to obtain a culture medium liquid;

[0063] S2. Weigh 1 g of NAD nicotinamide adenine dinucleotide, completely dissolve it in 1000.0 mL of sterile purified water, and filter it through a 0.22 μm filter membrane; draw 1.0 mL of the filtrate and add it to the culture medium obtained in step S1, and mix thoroughly.

[0064] S3: Newborn calf serum was inactivated at 56°C for 30 min and filtered through a 0.22 μm filter membrane; 30.0 mL of the filtered serum was added to the culture medium obtained in S2 and mixed.

[0065] S4. Weigh 0.05 g of bromothymol blue, dissolve it in 1.6 mL of 0.05 M sodium hydroxide, and dilute the volume to 100 mL with sterile purified water to prepare a 0.05% solution; filter with a 0.22-micron filter membrane; weigh 0.1 g of methylene blue, dissolve it in sterile purified water, and dilute the volume to 100 mL with sterile purified water to prepare a 0.1% solution; filter with a 0.22-micron filter membrane; respectively, take 0.8 mL of bromothymol blue filtrate and 2.5 mL of methylene blue filtrate and add them to the culture medium liquid obtained in step S3, mix well, and store at 4°C.

[0066] Example 3.1

[0067] Measured by mass (parts / 1000), 1000 mL of culture medium was prepared with the following formula: 20.0 g tryptone, 5.5 g peptone, 3.5 g glucose, 6.5 g yeast extract, 2.5 g dipotassium hydrogen phosphate, 3.5 g disodium hydrogen phosphate, 1.5 g potassium dihydrogen phosphate, 7.5 g sodium chloride, 18.0 g peptone, 22.0 g ox heart extract powder, 12.0 g soy peptone, 4.0 parts mannitol, 5.0 parts sodium pyruvate, 0.45 parts cycloheximide, 1000 parts distilled water, 5.0 mL 0.1% NAD nicotinamide adenine dinucleotide, 100 mL newborn calf serum inactivated at 56°C for 30 min, 2.0 mL 0.05% bromothymol blue, and 4.5 mL 0.1% methylene blue. The pH value was 7.5.

[0068] Example 3.2

[0069] Tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, sodium pyruvate, and cycloheximide were added to distilled water in the proportions by mass as described in Example 3.1, and the mixture was heated and boiled until completely dissolved. After cooling to room temperature, the pH was adjusted to 7.5 to obtain a prepared solution. The prepared solution was autoclaved at 115° C. for 30 min, and cooled to room temperature to obtain a culture medium liquid.

[0070] S2. Weigh 1.0 g of NAD nicotinamide adenine dinucleotide, completely dissolve it in 1000.0 mL of sterile purified water, and filter it through a 0.22 μm filter membrane; draw 5.0 mL of the filtrate and add it to the culture medium obtained in step S1, and mix thoroughly.

[0071] S3: Newborn calf serum was inactivated at 56°C for 30 min and filtered through a 0.22 μm filter membrane; 100.0 mL of the filtered serum was added to the culture medium obtained in S2 and mixed.

[0072] S4. Weigh 0.05 g of bromothymol blue, dissolve it in 1.6 mL of 0.05 M sodium hydroxide, and dilute the volume to 100 mL with sterile purified water to prepare a 0.05% solution; filter with a 0.22-micron filter membrane; weigh 0.1 g of methylene blue, dissolve it in sterile purified water, and dilute the volume to 100 mL with sterile purified water to prepare a 0.1% solution; filter with a 0.22-micron filter membrane; respectively, draw 2.0 mL of bromothymol blue filtrate and 4.5 mL of methylene blue filtrate and add them to the culture medium liquid obtained in step S3, mix well, and store at 4°C.

[0073] Comparative Example 1.1: In parts by mass (parts / 1000), 1000 mL of culture medium was prepared, the formula of which included: 18.0 g of tryptone, 4.3 g of peptone, 2.0 g of glucose, 5.5 g of yeast extract, 2.2 g of dipotassium hydrogen phosphate, 2.5 g of disodium hydrogen phosphate, 1.2 g of potassium dihydrogen phosphate, 5.0 g of sodium chloride, 15.0 g of peptone, 17.5 g of ox heart extract powder, 10.0 g of soy peptone, 2.6 g of mannitol, 4.4 g of sodium pyruvate, 0.25 g of cycloheximide, 1000 mL of distilled water, 2.0 mL of 0.1% NAD (nicotinamide adenine dinucleotide), 1.0 mL of 0.05% bromothymol blue, 3.0 mL of 0.1% methylene blue, and a pH value of 7.2.

[0074] Comparative Example 1.2

[0075] S1. Add tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, sodium pyruvate, and cycloheximide to distilled water according to the mass parts in Comparative Example 1.1, heat and boil until completely dissolved, cool to room temperature, and adjust the pH value to 7.2 to obtain a prepared solution; autoclave the prepared solution at 115° C. for 30 min, and cool to room temperature to obtain a culture medium liquid;

[0076] S2. Weigh 1.0 g of NAD nicotinamide adenine dinucleotide, completely dissolve it in 1000.0 mL of sterile purified water, and filter it through a 0.22 μm filter membrane; draw 2.0 mL of the filtrate and add it to the culture medium obtained in step S1, and mix thoroughly.

[0077] S3. Weigh 0.05 g of bromothymol blue, dissolve it in 1.6 mL of 0.05 M sodium hydroxide, and dilute the volume to 100 mL with sterile purified water to prepare a 0.05% solution; filter with a 0.22 μm filter membrane; weigh 0.1 g of methylene blue, dissolve it in sterile purified water, and dilute the volume to 100 mL with sterile purified water to prepare a 0.1% solution; filter with a 0.22 μm filter membrane; respectively, take 1.0 mL of bromothymol blue filtrate and 3.0 mL of methylene blue filtrate and add them to the culture medium liquid obtained in step S2, mix well, and store at 4°C.

[0078] Comparative Example 2.1: In parts by mass (parts / 1000), 1000 mL of culture medium was prepared, which included: 18.0 g of tryptone, 4.3 g of peptone, 2.0 g of glucose, 5.5 g of yeast extract, 2.2 g of dipotassium hydrogen phosphate, 2.5 g of disodium hydrogen phosphate, 1.2 g of potassium dihydrogen phosphate, 5.0 g of sodium chloride, 15.0 g of peptone, 17.5 g of beef heart extract powder, 10.0 g of soy peptone, 2.6 g of mannitol, 4.4 g of sodium pyruvate, 0.25 g of cycloheximide, 1000 mL of distilled water, 50 mL of newborn calf serum inactivated at 56°C for 30 min, 1.0 mL of 0.05% bromothymol blue, and 3.0 mL of 0.1% methylene blue, with a pH of 7.2. The results are shown in FIG1 . In the culture medium without the addition of newborn calf serum, Streptococcus, Pasteurella multocida, and Riemerella anatipestifer could not grow.

[0079] Comparative Example 2.2

[0080] S1. Add tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, sodium pyruvate, and cycloheximide to distilled water according to the mass parts in Comparative Example 2.1, heat and boil until completely dissolved, cool to room temperature, and adjust the pH value to 7.2 to obtain a prepared solution; autoclave the prepared solution at 115° C. for 30 min, and cool to room temperature to obtain a culture medium liquid;

[0081] S2: Newborn calf serum was inactivated at 56°C for 30 min and filtered through a 0.22 μm filter membrane; 50.0 mL of the filtered serum was added to the culture medium obtained in S1 and mixed.

[0082] S3. Weigh 0.05 g of bromothymol blue, dissolve it in 1.6 mL of 0.05 M sodium hydroxide, and dilute the volume to 100 mL with sterile purified water to prepare a 0.05% solution; filter with a 0.22 micron filter membrane; weigh 0.1 g of methylene blue, dissolve it in sterile purified water, and dilute the volume to 100 mL with sterile purified water to prepare a 0.1% solution; filter with a 0.22 micron filter membrane; respectively, draw 1.0 mL of bromothymol blue filtrate and 3.0 mL of methylene blue filtrate and add them to the culture medium liquid obtained in step S2, mix well, and store at 4°C. The results are shown in Figure 2. In the culture medium without NAD addition, Riemerella anatipestifer cannot grow.

[0083] Comparative Example 3.1: In parts by mass (parts / 1000), 1000 mL of culture medium was prepared, which included: 18.0 g of tryptone, 4.3 g of peptone, 2.0 g of glucose, 5.5 g of yeast extract, 2.2 g of dipotassium hydrogen phosphate, 2.5 g of disodium hydrogen phosphate, 1.2 g of potassium dihydrogen phosphate, 5.0 g of sodium chloride, 15.0 g of peptone, 17.5 g of ox heart extract powder, 10.0 g of soy peptone, 0.25 g of cycloheximide, 1000 mL of distilled water, 2.0 mL of 0.1% NAD (nicotinamide adenine dinucleotide), 50 mL of newborn calf serum inactivated at 56°C for 30 min, 1.0 mL of 0.05% bromothymol blue, and 3.0 mL of 0.1% methylene blue, with a pH of 7.2.

[0084] Comparative Example 3.2

[0085] S1. Add tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, and cycloheximide to distilled water according to the mass parts in Comparative Example 3.1, heat and boil until completely dissolved, cool to room temperature, and adjust the pH value to 7.2 to obtain a prepared solution; autoclave the prepared solution at 115° C. for 30 min, and cool to room temperature to obtain a culture medium liquid;

[0086] S2. Weigh 1.0 g of NAD nicotinamide adenine dinucleotide, completely dissolve it in 1000.0 mL of sterile purified water, and filter it through a 0.22 μm filter membrane; draw 2.0 mL of the filtrate and add it to the culture medium obtained in step S1, and mix thoroughly.

[0087] S3: Newborn calf serum was inactivated at 56°C for 30 min and filtered through a 0.22 μm filter membrane; 50.0 mL of the filtered serum was added to the culture medium obtained in S2 and mixed.

[0088] S4, weigh 0.05g bromothymol blue, add 1.6mL of 0.05M sodium hydroxide to dissolve, and then dilute to 100mL with sterile purified water to prepare a 0.05% solution; filter with a 0.22 micron filter membrane; weigh 0.1g methylene blue, dissolve in sterile purified water, and dilute to 100mL with sterile purified water to prepare a 0.1% solution; filter with a 0.22 micron filter membrane; draw 1.0mL of bromothymol blue filtrate and 3.0mL of methylene blue filtrate respectively and add them to the culture medium liquid obtained in step S3, mix well, and store at 4°C. The results are shown in Figure 3. The 9 tubes on the left are culture medium with mannitol and sodium pyruvate added, the 10th tube from the left is the negative control tube, and the 9 tubes on the right are culture medium without the addition of mannitol and sodium pyruvate. After 12h of culture, the culture medium without the addition of mannitol and sodium pyruvate, Streptococcus, Pasteurella multocida, and Riemerella anatipestifer, etc., grow slowly.

[0089] Comparative Example 4.1

[0090] Prepare 1000 mL of culture medium by parts by mass (parts / 1000): 18.0 g tryptone, 4.3 g peptone, 2.0 g glucose, 5.5 g yeast extract, 2.2 g dipotassium hydrogen phosphate, 2.5 g disodium hydrogen phosphate, 1.2 g potassium dihydrogen phosphate, 5.0 g sodium chloride, 15.0 g peptone, 17.5 g ox heart extract powder, 10.0 g soy peptone, 2.6 g mannitol, 4.4 g sodium pyruvate, 1000 mL distilled water, 2.0 mL 0.1% NAD (nicotinamide adenine dinucleotide), 50 mL newborn calf serum inactivated at 56°C for 30 min, 1.0 mL 0.05% bromothymol blue, and 3.0 mL 0.1% methylene blue. The pH value was 7.2.

[0091] Comparative Example 4.2

[0092] S1. Add tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, and sodium pyruvate to distilled water in proportion to the mass fraction in Comparative Example 4.1, heat and boil until completely dissolved, cool to room temperature, and adjust the pH to 7.2 to obtain a prepared solution; sterilize the prepared solution by autoclaving at 115° C. for 30 min, and cool to room temperature to obtain a culture medium liquid;

[0093] S2. Weigh 1.0 g of NAD nicotinamide adenine dinucleotide, completely dissolve it in 1000.0 mL of sterile purified water, and filter it through a 0.22 μm filter membrane; draw 2.0 mL of the filtrate and add it to the culture medium obtained in step S1, and mix thoroughly.

[0094] S3: Newborn calf serum was inactivated at 56°C for 30 min and filtered through a 0.22 μm filter membrane; 50.0 mL of the filtered serum was added to the culture medium obtained in S2 and mixed.

[0095] S4, weigh 0.05g bromothymol blue, add 1.6mL of 0.05M sodium hydroxide to dissolve, and dilute to 100mL with sterile purified water to prepare a 0.05% solution; filter with a 0.22 micron filter membrane; weigh 0.1g methylene blue, dissolve in sterile purified water, and dilute to 100mL with sterile purified water to prepare a 0.1% solution; filter with a 0.22 micron filter membrane; draw 1.0mL of bromothymol blue filtrate and 3.0mL of methylene blue filtrate respectively and add them to the culture medium liquid obtained in step S3, mix well, and store at 4°C. The results are shown in Figure 4. The culture medium without actinomycin is contaminated with mold after being placed at room temperature for 30 days, and the culture medium shows discoloration, turbidity, flocculent precipitation or floating matter.

[0096] Comparative Example 5.1

[0097] Measured in parts by mass (parts / 1000), 1000 mL of culture medium was prepared with the following formula: 18.0 g tryptone, 4.3 g peptone, 2.0 g glucose, 5.5 g yeast extract, 2.2 g dipotassium hydrogen phosphate, 2.5 g disodium hydrogen phosphate, 1.2 g potassium dihydrogen phosphate, 5.0 g sodium chloride, 15.0 g peptone, 17.5 g ox heart extract powder, 10.0 g soy peptone, 2.6 g mannitol, 4.4 g sodium pyruvate, 0.25 g cycloheximide, 1000 mL of distilled water, 2.0 mL of 0.1% NAD (nicotinamide adenine dinucleotide), 50 mL of newborn calf serum inactivated at 56°C for 30 min, and a pH of 7.2.

[0098] Comparative Example 5.2

[0099] S1. Add tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, sodium pyruvate, and cycloheximide to distilled water according to the mass fractions in Example 1.1, heat and boil until completely dissolved, cool to room temperature, and adjust the pH to 7.2 to obtain a prepared solution; autoclave the prepared solution at 115°C for 30 min, and cool to room temperature to obtain a culture medium liquid;

[0100] S2. Weigh 1.0 g of NAD nicotinamide adenine dinucleotide, completely dissolve it in 1000.0 mL of sterile purified water, and filter it through a 0.22 μm filter membrane; draw 2.0 mL of the filtrate and add it to the culture medium obtained in step S1, and mix thoroughly.

[0101] S3: Newborn calf serum was inactivated at 56°C for 30 min and filtered through a 0.22 μm filter membrane; 50.0 mL of the filtered serum was added to the culture medium obtained in S2, mixed, and stored at 4°C.

[0102] The culture medium without the addition of a chromogenic agent and the culture medium with the addition of a chromogenic agent were each dispensed into 9 mL tubes. Equal amounts of target bacteria were inoculated in each tube and incubated at 37°C for 12 hours. The results were observed. As shown in Figure 5, compared with the negative control tube, the target bacteria in the culture medium with the addition of a chromogenic agent all grew and proliferated, causing the culture medium to change color. Even with a very low inoculum, a noticeable color change occurred, making it easy to visually observe and judge the results. For the culture medium without the addition of a chromogenic agent, for target bacteria with strict nutritional requirements and slow growth, such as Streptococcus, Pasteurella multocida, and Riemerella anatipestifer, if the inoculum amount or the number of bacteria in the sample is very small, these target bacteria proliferate slowly and the number of bacteria is small, which does not cause particularly obvious turbidity in the culture medium, making it difficult to observe and distinguish the presence of the target bacteria with the naked eye. In the culture medium without the addition of a chromogenic agent, after 12 hours of incubation, no difference was visible to the naked eye between the culture tubes containing Streptococcus, Pasteurella multocida, and Riemerella anatipestifer and the negative control tube, making it impossible to judge whether the target bacteria had proliferated.

[0103] Therefore, chromogenic culture medium is necessary and essential for the culture of target bacteria and the judgment of subsequent drug sensitivity test results.

[0104] Comparative Example 6.1

[0105] Prepare 1000 mL of culture medium in parts by mass (parts / 1000): 2.0 g beef extract powder, 1.5 g soluble starch, 17.5 g acid-hydrolyzed casein, 2.6 g mannitol, 4.4 g sodium pyruvate, 0.25 g cycloheximide, 1000 mL distilled water, 2.0 mL 0.1% NAD (nicotinamide adenine dinucleotide), 50 mL newborn calf serum inactivated at 56°C for 30 min, 1.0 mL 0.05% bromothymol blue, and 3.0 mL 0.1% methylene blue. The pH value is 7.2.

[0106] Comparative Example 6.2

[0107] S1. Add beef extract powder, soluble starch, acid hydrolyzed casein, mannitol, sodium pyruvate, and cycloheximide to distilled water in the amounts by mass as in Comparative Example 6.1, heat and boil until completely dissolved, cool to room temperature, and adjust the pH to 7.2 to obtain a prepared solution; sterilize the prepared solution by autoclaving at 121° C. for 15 min, and cool to room temperature to obtain a culture medium liquid;

[0108] S2. Weigh 1.0 g of NAD nicotinamide adenine dinucleotide, completely dissolve it in 1000.0 mL of sterile purified water, and filter it through a 0.22 μm filter membrane; draw 2.0 mL of the filtrate and add it to the culture medium obtained in step S1, and mix thoroughly.

[0109] S3: Newborn calf serum was inactivated at 56°C for 30 min and filtered through a 0.22 μm filter membrane; 50.0 mL of the filtered serum was added to the culture medium obtained in S2 and mixed.

[0110] S4, weigh 0.05g bromothymol blue, add 1.6mL of 0.05M sodium hydroxide to dissolve, and dilute to 100mL with sterile purified water to prepare a 0.05% solution; filter with a 0.22-micron filter membrane; weigh 0.1g methylene blue, dissolve in sterile purified water, and dilute to 100mL with sterile purified water to prepare a 0.1% solution; filter with a 0.22-micron filter membrane; draw 1.0mL of bromothymol blue filtrate and 3.0mL of methylene blue filtrate respectively, add to the culture medium liquid obtained in step S3, mix well, and store at 4°C to obtain MH medium. The results are shown in Figure 6. After culturing for 12h in MH medium, Streptococcus, Pasteurella multocida, and Riemerella anatipestifer cannot grow.

[0111] Comparative Example 7.1

[0112] Prepare 1000 mL of culture medium by parts by mass (parts / 1000): 10.0 g tryptone, 17.5 g beef heart extract powder, 5.0 g sodium chloride, 2.0 g glucose, 2.5 g disodium hydrogen phosphate, 2.6 g mannitol, 4.4 g sodium pyruvate, 0.25 g cycloheximide, 1000 mL distilled water, 2.0 mL 0.1% NAD (nicotinamide adenine dinucleotide), 50 mL newborn calf serum inactivated at 56°C for 30 min, 1.0 mL 0.05% bromothymol blue, and 3.0 mL 0.1% methylene blue. The pH value is 7.2.

[0113] Comparative Example 7.2

[0114] S1. Add tryptone, beef heart extract powder, sodium chloride, glucose, disodium hydrogen phosphate, mannitol, sodium pyruvate, and cycloheximide to distilled water in the amounts by mass described in Comparative Example 7.1, heat and boil until completely dissolved, cool to room temperature, and adjust the pH to 7.2 to obtain a prepared solution; sterilize the prepared solution by autoclaving at 121° C. for 15 min, and cool to room temperature to obtain a culture medium liquid;

[0115] S2. Weigh 1.0 g of NAD nicotinamide adenine dinucleotide, completely dissolve it in 1000.0 mL of sterile purified water, and filter it through a 0.22 μm filter membrane; draw 2.0 mL of the filtrate and add it to the culture medium obtained in step S1, and mix thoroughly.

[0116] S3: Newborn calf serum was inactivated at 56°C for 30 min and filtered through a 0.22 μm filter membrane; 50.0 mL of the filtered serum was added to the culture medium obtained in S2 and mixed.

[0117] S4. Weigh 0.05 g of bromothymol blue, dissolve it in 1.6 mL of 0.05 M sodium hydroxide, and dilute the volume to 100 mL with sterile purified water to prepare a 0.05% solution; filter it with a 0.22 μm filter membrane; weigh 0.1 g of methylene blue, dissolve it in sterile purified water, and dilute the volume to 100 mL with sterile purified water to prepare a 0.1% solution; filter it with a 0.22 μm filter membrane; respectively, take 1.0 mL of the bromothymol blue filtrate and 3.0 mL of the methylene blue filtrate and add them to the culture medium liquid obtained in step S3, mix well, and store at 4°C to obtain BHI culture medium.

[0118] The results after 12 h of culture in BHI medium are shown in Figure 7 , and Pasteurella multocida could not grow.

[0119] Currently, MH medium and BHI medium have been commercialized, but they both have their own shortcomings in culturing nine common poultry pathogens, such as Escherichia coli, Salmonella, Proteus, Klebsiella, Staphylococcus aureus, Pasteurella multocida, Streptococcus, Riemerella anatipestifer, and Clostridium welchii. Neither medium can guarantee the cultivation and rapid proliferation of all pathogens. If a mixed infection with multiple pathogens occurs clinically, using these two mediums for drug sensitivity testing will result in missed detection of pathogens due to the defects of the medium itself, and the pathogens cannot be cultured. Naturally, it is impossible to accurately screen for the most effective antimicrobial drugs, which may result in the clinical application of effective drugs screened by laboratory drug sensitivity tests being ineffective, delaying the effectiveness of treatment. The universal enrichment medium disclosed in the present invention can ensure the rapid growth and proliferation of all target bacteria and provide timely and accurate guidance for clinical drug use.

[0120] Example 4

[0121] According to the ratio of Example 1.1 and the preparation method of Example 1.2, 10 culture tubes were prepared, one of which was used as a negative control, and the corresponding bacterial solution was inoculated into each of the other 9 tubes. The color development was observed after constant temperature incubation at 37°C for 3 to 16 hours. The color change is shown in Figure 8.

[0122] Example 5

[0123] The drug sensitivity test product obtained in Example 1.2 (taking the pre-filled product with a filling volume of 9 ml / tube as an example) was used to screen for effective therapeutic drugs for poultry Escherichia coli.

[0124] On November 12, 2023, a broiler farm in Weihai, Shandong, raising 200,000 Kebao broilers, suddenly fell ill in one of the 18-day-old chickens in one shed due to weather changes and improper husbandry. Sixty birds died that same day. On November 13, the farmer, based on experience, selected kanamycin sulfate for treatment. Two days of treatment failed to control the disease, and the mortality rate increased to over 400 birds. Upon arrival at the scene at 9:30 AM on November 15, the affected birds were listless, with their eyes closed and necks retracted, their wings drooping or lying prone. Their perianal feathers were soiled with feces, and there was white, loose feces on the ground. Some birds were breathing with their mouths open, and respiratory rales were evident. The autopsy findings, detailed in Figure 10, showed increased tracheal secretions, fibrinous pericarditis, fibrinous perihepatitis, mesenteric bleeding, intestinal bloating, enlarged kidneys, and urate deposits. During the operation, the diseased intestine and liver were first collected; then amoxicillin, colistin sulfate, and doxycycline were selected as the therapeutic drugs to be screened, and they were marked and added to the drug screening tubes respectively; then the samples were added to the screening tubes; the screening tubes were placed in a constant temperature incubator at 37°C and cultured for 16 hours, and the results were observed. The results are shown in Figure 11.

[0125] S4, culture detection,

[0126] At 14:00 on November 15, based on the drug screening results, colistin sulfate was selected for treatment.

[0127] Colistin sulfate: Mix with drinking water at 100 g / ton of water;

[0128] Baking soda: Mix with 3 kg / ton of water;

[0129] Antiviral Chinese medicine: 2 kg / ton of mixed material;

[0130] The above drugs were used continuously for 3 days.

[0131] The mental state, feed intake, feces and number of dead chickens of the flock were observed and counted every day.

[0132] On the evening of November 15, his mental state improved and the shape of his stool changed.

[0133] On November 16, the number of deaths decreased and feeding resumed. At 9:00 AM on November 16, laboratory drug sensitivity test results were observed and the pathogen was determined to be Escherichia coli. The culture results are shown in Figure 12. The colonies were dark purple with a metallic luster.

[0134] On November 17, only two chickens died and the flock had basically recovered.

[0135] The laboratory drug sensitivity test results at 9:00 on November 18 were observed. The laboratory drug sensitivity test results of colistin sulfate are shown in Figure 13.

[0136] The results of effective therapeutic drugs screened using universal enrichment media were consistent with those of laboratory susceptibility test kits. In this case, the farmer, relying solely on experience, mistakenly used ineffective antibiotics, missing the optimal treatment window and causing considerable financial losses. Using universal enrichment media for susceptibility screening helped the farmer identify effective drugs with minimal steps and time, thus controlling the disease.

[0137] The present invention has the following advantages:

[0138] 1. Easy to operate: No professional background is required, the operation is simple and easy to learn, and novices can quickly master it.

[0139] 2. Easy to Identify: Innovative color indicator technology is used to improve universal culture media. Once bacterial growth occurs, the color of the liquid in the culture tube will change from blue-green to lighter or faded. If the liquid color remains unchanged after adding antibiotics, it indicates that the antibiotics have an inhibitory effect on the bacteria; conversely, if the color becomes lighter or fades, it indicates that the antibiotics are ineffective.

[0140] 3. Wide adaptability: Supports a variety of sample types, including but not limited to nasopharyngeal swabs, anal swabs, environmental swabs, milk, tissue exudate, water samples, and tissue or organ samples of living or dead poultry.

[0141] 4. Multi-scenario application: Suitable for laboratories, feed mills, veterinary pharmacies, vaccine sales stores, breeding sites and other occasions.

[0142] 5. Reduce costs: One culture medium can simultaneously culture 9 pathogens including Escherichia coli, significantly reducing treatment costs.

[0143] 6. Shorten response time: It only takes 3 to 16 hours to complete the culture and obtain the results, so as to timely screen the effectiveness of antibiotics.

[0144] 7. Flexible and convenient: Adapt to local conditions and test existing drugs immediately, saving time and improving efficiency.

[0145] 8. Long-term storage: Pre-packaged products can be stably stored at 2 to 8°C for up to 12 months and can be used as needed.

[0146] 9. Precision veterinary decision support: Help clinical veterinarians shift from experience-based to empirical testing, and improve the accuracy of medication.

[0147] 10. Comprehensive evaluation tool: For farms and distributors, it can effectively evaluate the brand quality and efficacy of various antibiotic drugs. It can also be used to determine the actual disinfection effect of disinfectants on the farm, becoming an indispensable helper for management and evaluation.

[0148] Therefore, the present invention adopts the above-mentioned enrichment culture medium and its preparation method and application. The enrichment culture medium can simultaneously cultivate common poultry pathogens such as Escherichia coli, Salmonella, Proteus, Klebsiella, Staphylococcus aureus, Pasteurella multocida, Streptococcus, Riemerella anatipestifer, and Clostridium perfringens. A homemade bacterial-specific color indicator is added to the improved universal culture medium, which does not affect microbial growth and the efficacy of antibacterial drugs, and can easily identify color changes caused by microbial growth. When bacteria grow in the culture tube, the color of the liquid in the tube will fade from blue-green. When antibiotics are added to the tube, if the bacteria cannot grow, the liquid in the tube will not change color, indicating that the antibiotic is effective against the bacteria. Conversely, if the bacteria can grow, the color of the liquid in the tube will fade or become lighter, indicating that the antibiotic is ineffective against the bacteria.

[0149] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A bacterial enrichment medium, characterized in that The raw materials include, by weight, 15.0-20.0 parts of tryptone, 3.5-5.5 parts of peptone, 1.5-3.5 parts of glucose, 5.0-6.5 parts of yeast extract, 2.0-2.5 parts of dipotassium hydrogen phosphate, 2.0-3.5 parts of disodium hydrogen phosphate, 1.0-1.5 parts of potassium dihydrogen phosphate, 2.5-7.5 parts of sodium chloride, 12.0-18.0 parts of peptone, 15.0-22.0 parts of beef heart extract powder, 8. 0-12.0 parts, 0.1% NAD nicotinamide adenine dinucleotide 1.0-5.0 parts, 30-100 parts of newborn calf serum inactivated at 56°C for 30 minutes, 2.0-4.0 parts of mannitol, 3.0-5.0 parts of sodium pyruvate, 0.23-0.45 parts of cycloheximide, 0.8-2.0 parts of 0.05% bromothymol blue, 2.5-4.5 parts of 0.1% methylene blue, 1000 parts of distilled water, pH value is 6.8-7.

5.

2. A bacterial enrichment medium according to claim 1, characterized in that The raw materials include, by mass: 18.0 parts of tryptone, 4.3 parts of peptone, 2.0 parts of glucose, 5.5 parts of yeast extract, 2.2 parts of dipotassium hydrogen phosphate, 2.5 parts of disodium hydrogen phosphate, 1.2 parts of potassium dihydrogen phosphate, 5.0 parts of sodium chloride, 15.0 parts of peptone, 17.5 parts of beef heart extract powder, 10.0 parts of soy peptone, 2.0 parts of 0.1% NAD (nicotinamide adenine dinucleotide), 50 parts of newborn calf serum inactivated at 56°C for 30 minutes, 2.6 parts of mannitol, 4.4 parts of sodium pyruvate, 0.25 parts of cycloheximide, 0.8-2.0 parts of 0.05% bromothymol blue, 2.5-4.5 parts of 0.1% methylene blue, 1000 parts of distilled water, and the pH value is 7.

2.

3. The method for preparing a bacterial enrichment medium according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Add tryptone, peptone, glucose, yeast extract, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, peptone, beef heart extract powder, soy peptone, mannitol, sodium pyruvate, and cycloheximide to distilled water in specific proportions, heat and boil until completely dissolved, cool to room temperature, and adjust the pH to 6.8-7.5 to obtain a prepared solution; sterilize the prepared solution under high pressure and cool to room temperature to obtain a culture medium liquid; S2. Weigh nicotinamide adenine dinucleotide, completely dissolve it in sterile purified water, and filter it through a 0.22 μm filter membrane; add the filtrate to the culture medium obtained in step S1 and mix thoroughly; S3. Inactivate the newborn calf serum and filter it through a 0.22 μm filter membrane; add the filtered serum to the culture medium obtained in step S2 and mix thoroughly; S4. Weigh bromothymol blue, dissolve it in 0.05 M sodium hydroxide, and dilute to volume with sterile purified water to prepare a 0.05% solution; filter with a 0.22-μm filter membrane; weigh methylene blue, dissolve it in sterile purified water, and dilute to volume with sterile purified water to prepare a 0.1% solution; filter with a 0.22-μm filter membrane; separately draw the bromothymol blue filtrate and the methylene blue filtrate and add them to the culture medium liquid obtained in step S3, mix well, and store at 4°C.

4. Use of an enrichment culture medium according to any one of claims 1-2 in screening effective therapeutic drugs for poultry bacterial diseases.

5. Use of a bacterial enrichment culture medium according to claim 4 in screening effective therapeutic drugs for poultry bacterial diseases, characterized in that: The following steps are involved: S1. Sampling: Collect samples of diseased material according to clinical symptoms; S2. Add drugs. Select several therapeutic drugs to be screened and label them separately. After treatment, add them to the corresponding drug screening tubes. S3. Add samples. Various samples are added to the screening tube in an appropriate manner. S4. Culture test: Place the screening tube in a 37°C constant temperature incubator and culture for 16 hours, then observe the results. The negative control tube without sample and drug and the sample control tube with only sample but no drug are cultured simultaneously.

6. Use of an enrichment culture medium according to claim 5 in screening effective therapeutic drugs for poultry bacterial diseases, characterized in that: In step S1, anal swabs or feces are collected for digestive tract symptoms; nasopharyngeal swabs are collected for respiratory tract symptoms; and tissues or organs with obvious lesions are collected from dead poultry.

7. Use of a bacterial enrichment culture medium according to claim 5 in screening effective therapeutic drugs for poultry bacterial diseases, characterized in that: In step S2, the powdered drug is first diluted with purified water to 10 times the drug treatment dose, and then 1.0 mL is drawn into the corresponding drug screening tube; 1.0 mL of the liquid drug is directly drawn into the corresponding drug screening tube.

8. Use of a bacterial enrichment culture medium according to claim 5 in screening effective therapeutic drugs for poultry bacterial diseases, characterized in that: In step S3, 0.1-0.2 mL of tissue exudate sample is drawn and added to the screening tube; soybean-sized amounts of feces, organs, and tissues are taken and added to the screening tube.

9. Use of an enrichment culture medium according to claim 6 in screening effective therapeutic drugs for poultry bacterial diseases, characterized in that: The negative control tube did not fade or change color; the sample control tube faded and was turbid, indicating that pathogens were present in the sample; the color of the effective drug screening tube was consistent with the negative control tube, without fading or changing color.

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