A selective separation medium for Rhodococcus equi and its preparation method and application

Abstract

The invention discloses a selective separation medium for Rhodococcus equi, a preparation method and an application thereof, and belongs to the fields of microbial technology and environmental microbiology. The medium is composed of soluble starch, pyruvic acid, sodium acetate, acid hydrolyzed casein, beef extract powder, yeast extract powder, calcium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium nitrate, zinc sulfate, manganese sulfate, magnesium sulfate heptahydrate, ammonium ferric citrate, potassium tellurite, agar, trimethoprim, cefoperazone, polymyxin B and cycloheximide. The selective separation medium of the invention has a good selective separation effect on Rhodococcus equi, can not only increase the number of Rhodococcus equi, but also reduce the number and types of other microorganisms on the plate, and also includes the application of the selective separation medium for Rhodococcus equi.

Description

Technical Field

[0001] The invention belongs to the fields of microbial technology and environmental microbiology, and in particular relates to a selective separation culture medium for Rhodococcus equi and a preparation method and application thereof. Background Art

[0002] Rhodococcus equi Rhodococcus equi ) is a recognized bacterial pathogen in veterinary medicine and is a member of the actinomycete family Nocardiaceae ( Nocardiaceae ) Rhodococcus Rhodococcus sp. ) is a Gram-positive obligate aerobic bacterium, and its growth morphology changes with the growth conditions and growth cycle. It is widely distributed in nature, especially in soil and feces of herbivorous animals. There are a large number of Rhodococcus equi strains in horse breeding environments. Foals infected with it will develop severe chronic suppurative bronchopneumonia, and the mortality rate after onset is high, resulting in huge losses to the horse breeding industry. Although rare, it occasionally occurs in infections of other mammals, and is more common in immunocompromised people such as patients infected with human immunodeficiency virus. The main clinical symptoms include fever, headache, fatigue, etc.

[0003] When Rhodococcus equi is cultured on blood agar plates at 37°C, the bacteria proliferate slowly, and its colonies are small, moist, smooth, translucent, and mucous, and can produce characteristic orange-red pigments. Rhodococcus equi cultured on sheep blood agar plates can produce synergistic hemolysis with Staphylococcus aureus and Listeria monocytogenes (positive CAMP test), and antagonism between thioimidate and other β-lactam antibiotics is widely present in Rhodococcus equi isolates.

[0004] Rhodococcus equi generally persists in the soil and the intestines of foals. How to accurately screen Rhodococcus equi from the soil is an important means to effectively monitor Rhodococcus equi infection in horse farms. At present, Columbia blood agar plates are generally used to culture Rhodococcus equi and observe its colony morphology. However, Rhodococcus equi grows slowly on this culture medium and the colonies are small. There are a large number of microorganisms and humus such as bacteria and fungi in the soil of animal breeding areas. There is currently no selective isolation culture medium for Rhodococcus equi on the domestic market, and it is impossible to accurately isolate Rhodococcus equi from test samples with a large number of other bacteria and other microorganisms. In order to further strengthen the laboratory's understanding of the pathogen and reduce Rhodococcus equi infection in horse farms, it is urgent to develop a culture medium that effectively meets the growth, metabolism, and reproduction of Rhodococcus equi. This is of great significance for timely monitoring the changes of Rhodococcus equi in the horse farm environment, helping to discover new therapeutic drugs, and thus strengthening the ecological research on Rhodococcus equi. Summary of the invention

[0005] The purpose of the present invention is to provide a selective separation medium for Rhodococcus equi that improves the separation rate of Rhodococcus equi in the soil of animal breeding areas, and a preparation method and application thereof. The medium is prepared by optimizing the nutrients such as carbon source, nitrogen source, antibiotics, etc. in the selective separation medium for Rhodococcus equi, and is suitable for screening and isolating Rhodococcus equi from the soil. To achieve the above purpose, the technical solution adopted by the present invention is: a selective separation medium for Rhodococcus equi, the selective separation medium for Rhodococcus equi comprising: carbon source, nitrogen source, antibiotics, etc. Source, nitrogen source, antibiotics, calcium chloride 0.005-0.015g / L, potassium dihydrogen phosphate 0.3-0.5g / L, disodium hydrogen phosphate 0.5-1.0g / L, potassium nitrate 0.05-0.15g / L, zinc sulfate 0.01-0.02g / L, manganese sulfate 0.01-0.03g / L, magnesium sulfate heptahydrate 0.1-0.2g / L, ammonium ferric citrate 0.03-0.07g / L, potassium tellurite 0.01-0.02g / L, agar 16-18g / L;

[0006] The carbon source comprises: 1-5 g / L soluble starch, 1-5 g / L pyruvic acid, and 1-5 g / L sodium acetate;

[0007] The nitrogen source comprises: 10-25 g / L of acid hydrolyzed casein, 1-10 g / L of beef extract powder, and 1-5 g / L of yeast extract powder;

[0008] The antibiotic composition comprises: 1-10 mg / L of trimethoprim, 1-10 mg / L of cefoperazone, 1-10 mg / L of polymyxin B, and 50-200 mg / L of cycloheximide.

[0009] A method for preparing a selective separation medium for Rhodococcus equi, characterized in that 1-5 g soluble starch, 1-5 g pyruvic acid, 1-5 g sodium acetate, 10-25 g acid hydrolyzed casein, 1-10 g beef extract, 1-5 g yeast extract, 0.005-0.015 g calcium chloride, 0.3-0.5 g potassium dihydrogen phosphate, 0.5-1.0 g disodium hydrogen phosphate, 0.05-0.15 g potassium nitrate, 0.01-0.02 g zinc sulfate, 0.01-0.03 g manganese sulfate, 0.1-0.2 g magnesium sulfate heptahydrate, 0.03-0.07 g ammonium ferric citrate, 0.01-0.02 g potassium tellurite, and 16-18 g agar are weighed, dissolved in deionized water, fixed to 1000 mL, and adjusted to pH 7.5-7.6 with sodium carbonate and citric acid, 121 ℃ for 15 minutes, cooled to 50 ℃, added with 1-10 mg of trimethoprim, 1-10 mg of cefoperazone, 1-10 mg of polymyxin B and 50-200 mg of cycloheximide and mixed, aseptically filled into a 90 mm sterile culture dish to prepare the Rhodococcus equi selective isolation medium.

[0010] The selective isolation medium for Rhodococcus equi is used for the isolation of Rhodococcus equi in the soil of animal breeding areas, that is, the microorganism is Rhodococcus equi ( Rhodococcus equi ).

[0011] The culture temperature was 30 ℃.

[0012] The culture time is 48 to 72 hours.

[0013] The application method of the selective isolation medium for Rhodococcus equi is as follows:

[0014] (1) Place the collected soil sample in a sterile dish to dry naturally, and grind it into powder using a sterile pestle;

[0015] (2) Weigh 1 g of soil sample, add it to 10 mL of 2% oxalic acid, and rotate at 180 r·min. -1 The mixture was shaken in a shaker for 20 min and allowed to settle naturally for 5 min.

[0016] (3) Pour off the supernatant and centrifuge at 3000 rpm for 5 min;

[0017] (4) Suspend the sediment in 5 mL of 1% sterile peptone water, pipette 1 mL of soil suspension into 9 mL of sterile water, and perform a 10-fold gradient dilution to a concentration of 1 g·L -1 ;

[0018] (5) Take 200 μL and spread it on the separation culture medium plate and place it in a constant temperature incubator at 30 ℃.

[0019] The Rhodococcus equi selective separation culture medium provided by the invention is used for enrichment culture and separation screening of Rhodococcus equi for non-disease diagnosis purposes.

[0020] The beneficial effects of the present invention are:

[0021] (1) The raw materials used in the culture medium provided by the present invention are cheap and easy to obtain, and the economic cost is low. Moreover, after multiple experiments, the nutritional components of the culture medium have been optimized, and appropriate trace elements required for the normal growth of Rhodococcus equi have been added. This not only meets the nutritional needs of Rhodococcus equi, making the colony morphology of Rhodococcus equi more characteristic, but also increases the growth rate of specific strains in the strain separation process, shortens the separation time, and improves the separation efficiency. It can achieve enrichment culture of Rhodococcus equi, thereby meeting the growth, metabolism and reproduction of Rhodococcus equi in the horse farm environment, helping to improve the detection and monitoring capabilities of Rhodococcus equi in the breeding environment, and is beneficial to the prevention and control of Rhodococcus equi infection problems in animal breeding areas such as horse farms;

[0022] (2) Taking advantage of the differences in the sensitivity of different microorganisms in the soil to different drugs, antibiotics that have a weak inhibitory effect on Rhodococcus equi but a strong inhibitory effect on a variety of bacteria such as Enterobacter, Staphylococcus, Streptococcus, Salmonella, and certain fungi such as Penicillium and Aspergillus are added to the culture medium. This allows Rhodococcus equi to grow while the miscellaneous bacteria in the environment grow slowly or not at all. This can effectively improve the success rate of isolating Rhodococcus equi from the soil and greatly save operation time.

[0023] (3) When preparing the selective isolation medium for Rhodococcus equi described in the present invention, the components other than antibiotics are mixed, heated and sterilized, and then cooled before adding antibiotics such as trimethoprim, which will not destroy the basic structure and properties of the antibiotics, thereby enabling the medium to better inhibit other bacteria;

[0024] (4) The culture medium preparation process of the present invention is simple to operate, takes a short time to prepare, does not require complicated supporting equipment, can reduce manpower and material resources in the actual production process, has strong practical feasibility, and is easy to use;

[0025] (5) When the culture medium of the present invention is used to separate Rhodococcus equi from soil samples, the sample to be tested is initially treated with 2% oxalic acid, which affects the acid-base balance of the soil and can preliminarily eliminate foreign bacteria, metal ions and humus in the soil. Rhodococcus equi has a strong tolerance to oxalic acid environment, thereby increasing the biomass of Rhodococcus equi in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a comparison chart of the recovery rates of Rhodococcus equi by the selective separation medium in Examples 1-4;

[0027] Figure 2 It is a comparison diagram of the growth curves of Rhodococcus equi in the selective culture medium in Examples 1-4;

[0028] Figure 3 This is a comparison chart of the growth inhibition ability of the selective culture medium of Examples 1-4 on non-equine Rhodococcus;

[0029] Figure 4 This is a comparison chart of the effects of using selective culture medium to screen Rhodococcus equi from soil in Examples 1-4. DETAILED DESCRIPTION

[0030] Three embodiments are described below to more clearly illustrate the technical solutions and advantages of the present invention. By adjusting the nutritional composition of the culture medium of the present invention, counting recovery, growth curve test and other methods are used to evaluate the selective separation effect of the culture medium from the aspects of growth promotion ability and inhibition ability of non-equine Rhodococcus.

[0031] The soil samples in the following examples were all from the same local horse farm. The surface soil samples were collected from the area where the horses lived in the farm. After the soil samples were collected, they were stored in ice bags and ice boxes and immediately brought back to the laboratory for processing.

[0032] The standard strains used in the following examples were purchased from China General Microbiological Culture Collection Center, and the instruments and reagents used were all commercially available conventional products.

[0033] Table 1 Reference strains used in the experiment

[0034] Fungus name Bacteria No. Rhodococcus equi CGMCC1.6322 Rhodococcus coprophilus CGMCC4.1813 Streptococcus equi CGMCC1.10838 Enterococcus faecalis CGMCC1.125 Staphylococcus aureus CGMCC1.4519 Pseudomonas aeruginosa CGMCC1.1785 Bacillus subtilis CGMCC1.439 Streptomyces albus CGMCC4.7658 Aspergillus terreus CGMCC3.3934 Penicillium chrysogenum CGMCC3.15539 Pythium CGMCC3.17815

[0035] 1. Culture medium formula:

[0036] Each liter of culture medium contains: carbon source, nitrogen source, antibiotics, calcium chloride 0.005-0.015g, potassium dihydrogen phosphate 0.3-0.5g, disodium hydrogen phosphate 0.5-1.0g, potassium nitrate 0.05-0.15g, zinc sulfate 0.01-0.02g, manganese sulfate 0.01-0.03g, magnesium sulfate heptahydrate 0.1-0.2g, ammonium ferric citrate 0.03-0.07g, potassium tellurite 0.01-0.02g, agar 16-18g;

[0037] The carbon source composition is: soluble starch 1-5 g / L, pyruvic acid 1-5 g / L, sodium acetate 1-5 g / L; soluble starch, pyruvic acid and sodium acetate provide the carbon source required for the growth of Rhodococcus equi;

[0038] The nitrogen source composition is: acid hydrolyzed casein 10-25g / L, beef extract powder 1-10g / L, yeast extract powder 1-5g / L; acid hydrolyzed casein, beef extract powder and yeast extract powder provide nitrogen source, vitamins and amino acids for the growth of Rhodococcus equi;

[0039] The antibiotic composition is: trimethoprim 1-10 mg / L, cefoperazone 1-10 mg / L, polymyxin B 1-10 mg / L, cycloheximide 50-200 mg / L. Trimethoprim can interfere with the folic acid metabolism of bacteria and has inhibitory effects on a variety of Gram-positive and Gram-negative bacteria. Cefoperazone is a broad-spectrum antibiotic that has good antibacterial activity against common bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, β-hemolytic streptococci and Escherichia coli. Polymyxin B has inhibitory effects on Gram-negative bacteria such as Pseudomonas aeruginosa and Klebsiella, and cycloheximide has a significant inhibitory effect on fungi.

[0040] 2. Preparation of culture medium

[0041] Preparation of the solid culture medium of the present invention: weigh 1-5g soluble starch, 1-5g pyruvic acid, 1-5g sodium acetate, 10-25g acid hydrolyzed casein, 1-10g beef extract powder, 1-5g yeast extract powder, 0.005-0.015g calcium chloride, 0.3-0.5g potassium dihydrogen phosphate, 0.5-1.0g disodium hydrogen phosphate, 0.05-0.15g potassium nitrate, 0.01-0.02g zinc sulfate, 0.01-0.03g manganese sulfate, 0.1-0.2g magnesium sulfate heptahydrate, 0.03-0.07g ammonium ferric citrate, 0.01-0.02g potassium tellurite, 16-18g agar, dissolve in deionized water, dilute to 1000mL, adjust pH to 7.5-7.6 with sodium carbonate and citric acid, and incubate at 121°C, 105 kPa sterilization for 15 minutes, cooling to 50 ℃, adding 1-10 mg trimethoprim, 1-10 mg cefoperazone, 1-10 mg polymyxin B and 50-200 mg cycloheximide and mixing, aseptically filling into a 90 mm sterile culture dish to prepare a solid Rhodococcus equi selective isolation medium.

[0042] Preparation of the liquid culture medium of the present invention: weigh 1-5g soluble starch, 1-5g pyruvic acid, 1-5g sodium acetate, 10-25g acid hydrolyzed casein, 1-10g beef extract powder, 1-5g yeast extract powder, 0.005-0.015g calcium chloride, 0.3-0.5g potassium dihydrogen phosphate, 0.5-1.0g disodium hydrogen phosphate, 0.05-0.15g potassium nitrate, 0.01-0.02g zinc sulfate, 0.01-0.03g manganese sulfate, 0.1-0.2g magnesium sulfate heptahydrate, 0.03-0.07g ammonium ferric citrate, 0.01-0.02g potassium tellurite, dissolve in deionized water, dilute to 1000mL, adjust pH to 7.5-7.6 with sodium carbonate and citric acid, sterilize at 121°C and 105kPa for 15 minutes, cool to 50 ℃, add 1-10 mg of trimethoprim, 1-10 mg of cefoperazone, 1-10 mg of polymyxin B and 50-200 mg of cycloheximide and mix well to prepare a liquid Rhodococcus equi selective separation medium.

[0043] 3. Inoculation and cultivation

[0044] (1) Standard strain recovery experiment: The activated Rhodococcus equi strain was diluted step by step with sterile NaCl solution to no more than 100 cfu·mL -1 , spread on TSA and the selective isolation medium for Rhodococcus equi, spread 3 plates on the two mediums respectively, culture at 30°C for 48-72 hours, observe the growth of the bacteria, count the colonies, and calculate the recovery rate;

[0045] Recovery rate = (selective separation medium colony count / TSA colony count) × 100%

[0046] (2) Dilute the activated Rhodococcus equi strain in stages with 0.9% sterile NaCl solution to about 10 5 cfu·mL -1 , mix 20 μL of bacterial solution with 180 μL of selective liquid culture medium, place in a growth curve instrument, culture at 30 °C for 72 h, read the OD 600 value of the culture well, and draw a growth curve;

[0047] (3) Inhibition ability of selective culture medium: Common soil microorganisms such as Rhodococcus faecalis, Streptococcus equi, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Streptomyces albus, Moraxella solani, Penicillium chrysogenum and Pythium heterotrichum were used as test strains to detect the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 strains of each of the activated strains were added to the culture medium. 4 cfu were inoculated on the selective isolation medium and cultured in a 30 °C constant temperature incubator for 72 h to observe whether colonies appeared;

[0048] (4) Practical application of selective medium for isolation of Rhodococcus equi: Place the collected soil sample in a sterile plate to dry naturally, grind it into powder with a sterile pestle, weigh 1 g of soil sample, add it to 10 mL of 2% oxalic acid, and rotate at 180 r·min. -1 The mixture was shaken in a shaker for 20 min, and allowed to settle naturally for 5 min. The supernatant was poured off and centrifuged at 3000 rpm for 5 min. The sediment was suspended in 5 mL of 1% sterile peptone water. 1 mL of the soil suspension was pipetted into 9 mL of sterile water and diluted 10-fold to a concentration of 1 g·L -1 , spread 200 μL on the separation culture medium plate and culture in a 30 ℃ constant temperature incubator.

[0049] When the selective separation medium for Rhodococcus equi described in the present invention is used in practice, the collected soil sample is ground with a sterile pestle, so that the soil sample can be fully contacted with oxalic acid. In the subsequent oscillation process, the oxalic acid can preliminarily eliminate miscellaneous bacteria, metal ions and humus in the soil by affecting the acid-base balance of the soil. Rhodococcus equi has a strong tolerance to the oxalic acid environment and can increase the biomass of Rhodococcus equi in the sample. After the soil suspension is centrifuged and diluted to a certain concentration, the concentration of Rhodococcus equi is relatively moderate, the number of growths coated on the selective separation medium is convenient to count, and the colonies are easy to observe.

[0050] The selective separation medium for Rhodococcus equi can be prepared as a solid or a liquid. The following embodiments are described in terms of the preparation method of a solid. Example 1

[0051] This embodiment provides a selective separation medium for Rhodococcus equi.

[0052] 1. The culture medium formula is as follows:

[0053] Each liter of culture medium contains: 1.5g soluble starch, 2g pyruvic acid, 1.85g sodium acetate, 17.5g acid hydrolyzed casein, 6g beef extract powder, 4g yeast extract powder, 0.01g calcium chloride, 0.46g potassium dihydrogen phosphate, 0.73g disodium hydrogen phosphate, 0.1g potassium nitrate, 0.016g zinc sulfate, 0.022g manganese sulfate, 0.12g magnesium sulfate heptahydrate, 0.054g ammonium ferric citrate, 0.015g potassium tellurite, 17g agar; additives are 5.8mg trimethoprim, 6.75mg cefoperazone, 6.45mg polymyxin B, and 90mg cycloheximide.

[0054] 2. Preparation of culture medium

[0055] Weigh 1.5 g soluble starch, 2 g pyruvic acid, 1.85 g sodium acetate, 17.5 g acid hydrolyzed casein, 6 g beef extract powder, 4 g yeast extract powder, 0.01 g calcium chloride, 0.46 g potassium dihydrogen phosphate, 0.73 g disodium hydrogen phosphate, 0.1 g potassium nitrate, 0.016 g zinc sulfate, 0.022 g manganese sulfate, 0.12 g magnesium sulfate heptahydrate, 0.054 g ammonium ferric citrate, 0.015 g potassium tellurite, and 17 g agar, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.5-7.6 with sodium carbonate and citric acid, sterilize at 121 °C and 105 kPa for 15 min, cool to 50 °C, add 5.8 mg trimethoprim, 6.75 mg cefoperazone, and polymyxin B. 6.45 mg of chloramphenicol and 90 mg of cycloheximide were mixed and aseptically filled into a 90 mm sterile culture dish to prepare a selective isolation medium for Rhodococcus equi.

[0056] 3. Inoculation and cultivation

[0057] (1) Dilute the activated Rhodococcus equi strain in sterile NaCl solution to a concentration of no more than 100 cfu / mL. -1 , spread on TSA and separation medium, spread 3 plates on each medium, culture at 30 ℃ for 48-72 h, observe the growth of bacteria, count the colonies, and take the average value to calculate the recovery rate;

[0058] Recovery rate = (selective separation medium colony count / TSA colony count) × 100%

[0059] (2) Dilute the activated Rhodococcus equi strain in stages with 0.9% sterile NaCl solution to about 10 5 cfu·mL -1, mix 20 μL of bacterial solution with 180 μL of selective liquid culture medium, place in a growth curve instrument, culture at 30 °C for 72 h, read the OD 600 value of the culture well, and draw a growth curve;

[0060] (3) Rhodococcus faecalis, Streptococcus equi, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Streptomyces albus, Moraxella solani, Penicillium chrysogenum and Pythium heterotrichum were used as test strains to detect the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 of each of the activated strains were added. 4 cfu were inoculated on the selective isolation medium and cultured in a 30 °C constant temperature incubator for 72 h to observe whether colonies appeared;

[0061] (4) Practical application of selective medium for isolation of Rhodococcus equi: Place the collected soil sample in a sterile plate to dry naturally, grind it into powder with a sterile pestle, weigh 1 g of soil sample, add it to 10 mL of 2% oxalic acid, and rotate at 180 r·min. -1 The mixture was shaken in a shaker for 20 min, and allowed to settle naturally for 5 min. The supernatant was poured off and centrifuged at 3000 rpm for 5 min. The sediment was suspended in 5 mL of 1% sterile peptone water. 1 mL of the soil suspension was pipetted into 9 mL of sterile water and diluted 10-fold to a concentration of 1 g·L -1 , spread 200 μL on the separation culture medium plate and culture in a 30 ℃ constant temperature incubator. Example 2

[0062] This embodiment provides a selective separation medium for Rhodococcus equi.

[0063] 1. The culture medium formula is as follows:

[0064] Each liter of culture medium contains: 4.5g soluble starch, 4.46g pyruvic acid, 4.85g sodium acetate, 24g acid hydrolyzed casein, 9.2g beef extract powder, 4.9g yeast extract powder, 0.015g calcium chloride, 0.46g potassium dihydrogen phosphate, 0.95g disodium hydrogen phosphate, 0.148g potassium nitrate, 0.019g zinc sulfate, 0.028g manganese sulfate, 0.195g magnesium sulfate heptahydrate, 0.06g ammonium ferric citrate, 0.018g potassium tellurite, 17g agar; additives are 10mg trimethoprim, 9.25mg cefoperazone, 9.5mg polymyxin B, and 190mg cycloheximide.

[0065] 2. Preparation of culture medium

[0066] Weigh 4.5 g soluble starch, 4.46 g pyruvic acid, 4.85 g sodium acetate, 24 g acid hydrolyzed casein, 9.2 g beef extract, 4.9 g yeast extract, 0.015 g calcium chloride, 0.46 g potassium dihydrogen phosphate, 0.95 g disodium hydrogen phosphate, 0.148 g potassium nitrate, 0.019 g zinc sulfate, 0.028 g manganese sulfate, 0.195 g magnesium sulfate heptahydrate, 0.06 g ammonium ferric citrate, 0.018 g potassium tellurite, and 17 g agar, dissolve in deionized water, make up to 1000 mL, adjust the pH to 7.5-7.6 with sodium carbonate and citric acid, sterilize at 121°C and 105 kPa for 15 min, and cool to 50 ℃, add 10 mg of trimethoprim, 9.25 mg of cefoperazone, 9.5 mg of polymyxin B, and 190 mg of cycloheximide and mix well, and aseptically fill into a 90 mm sterile culture dish to prepare a selective isolation medium for Rhodococcus equi.

[0067] 3. Inoculation and cultivation

[0068] (1) Dilute the activated Rhodococcus equi strain in sterile NaCl solution to a concentration of no more than 100 cfu / mL. -1 , spread on TSA and separation medium, spread 3 plates on each medium, culture at 30 ℃ for 48-72 h, observe the growth of bacteria, count the colonies, and take the average value to calculate the recovery rate;

[0069] Recovery rate = (selective separation medium colony count / TSA colony count) × 100%

[0070] (2) Dilute the activated Rhodococcus equi strain in stages with 0.9% sterile NaCl solution to about 10 5 cfu·mL -1 , mix 20 μL of bacterial solution with 180 μL of selective liquid culture medium, place in a growth curve instrument, culture at 30 °C for 72 h, read the OD 600 value of the culture well, and draw a growth curve;

[0071] (3) Rhodococcus faecalis, Streptococcus equi, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Streptomyces albus, Moraxella solani, Penicillium chrysogenum and Pythium heterotrichum were used as test strains to detect the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 of each of the activated strains were added. 4 cfu were inoculated on the selective isolation medium and cultured in a 30 °C constant temperature incubator for 72 h to observe whether colonies appeared;

[0072] (4) Practical application of selective medium for isolation of Rhodococcus equi: Place the collected soil sample in a sterile plate to dry naturally, grind it into powder with a sterile pestle, weigh 1 g of soil sample, add it to 10 mL of 2% oxalic acid, and rotate at 180 r·min. -1 The mixture was shaken in a shaker for 20 min, and allowed to settle naturally for 5 min. The supernatant was poured off and centrifuged at 3000 rpm for 5 min. The sediment was suspended in 5 mL of 1% sterile peptone water. 1 mL of the soil suspension was pipetted into 9 mL of sterile water and diluted 10-fold to a concentration of 1 g·L -1 , spread 200 μL on the separation culture medium plate and culture in a 30 ℃ constant temperature incubator. Example 3

[0073] This embodiment provides a selective separation medium for Rhodococcus equi.

[0074] 1. The culture medium formula is as follows:

[0075] Each liter of culture medium contains: 3g soluble starch, 4g pyruvic acid, 3.15g sodium acetate, 11.5g acid hydrolyzed casein, 9g beef extract powder, 2.5g yeast extract powder, 0.01g calcium chloride, 0.4g potassium dihydrogen phosphate, 0.65g disodium hydrogen phosphate, 0.1g potassium nitrate, 0.015g zinc sulfate, 0.02g manganese sulfate, 0.14g magnesium sulfate heptahydrate, 0.05g ammonium ferric citrate, 0.014g potassium tellurite, 17g agar; additives are 2.15mg trimethoprim, 9.45mg cefoperazone, 8.75mg polymyxin B, and 60mg cycloheximide.

[0076] 2. Preparation of culture medium

[0077] Weigh 3 g soluble starch, 4 g pyruvic acid, 3.15 g sodium acetate, 11.5 g acid hydrolyzed casein, 9 g beef extract powder, 2.5 g yeast extract powder, 0.01 g calcium chloride, 0.4 g potassium dihydrogen phosphate, 0.65 g disodium hydrogen phosphate, 0.1 g potassium nitrate, 0.015 g zinc sulfate, 0.02 g manganese sulfate, 0.14 g magnesium sulfate heptahydrate, 0.05 g ammonium ferric citrate, 0.014 g potassium tellurite, and 17 g agar, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.5-7.6 with sodium carbonate and citric acid, sterilize at 121 °C and 105 kPa for 15 min, cool to 50 °C, add 2.15 mg trimethoprim, 9.45 mg cefoperazone, and polymyxin B. 8.75 mg of chloramphenicol and 60 mg of cycloheximide were mixed and aseptically filled into a 90 mm sterile culture dish to prepare a selective isolation medium for Rhodococcus equi.

[0078] 3. Inoculation and cultivation

[0079] (1) Dilute the activated Rhodococcus equi strain in sterile NaCl solution to a concentration of no more than 100 cfu / mL. -1 , spread on TSA and separation medium, spread 3 plates on each medium, culture at 30 ℃ for 48-72 h, observe the growth of bacteria, count the colonies, and take the average value to calculate the recovery rate;

[0080] Recovery rate = (selective separation medium colony count / TSA colony count) × 100%

[0081] (2) Dilute the activated Rhodococcus equi strain in stages with 0.9% sterile NaCl solution to about 10 5 cfu·mL -1 , mix 20 μL of bacterial solution with 180 μL of selective liquid culture medium, place in a growth curve instrument, culture at 30 °C for 72 h, read the OD 600 value of the culture well, and draw a growth curve;

[0082] (3) Rhodococcus faecalis, Streptococcus equi, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Streptomyces albus, Moraxella solani, Penicillium chrysogenum and Pythium heterotrichum were used as test strains to detect the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 of each of the activated strains were added. 4 cfu were inoculated on the selective isolation medium and cultured in a 30 °C constant temperature incubator for 72 h to observe whether colonies appeared;

[0083] (4) Practical application of selective medium for isolation of Rhodococcus equi: Place the collected soil sample in a sterile plate to dry naturally, grind it into powder with a sterile pestle, weigh 1 g of soil sample, add it to 10 mL of 2% oxalic acid, and rotate at 180 r·min. -1 The mixture was shaken in a shaker for 20 min, and allowed to settle naturally for 5 min. The supernatant was poured off and centrifuged at 3000 rpm for 5 min. The sediment was suspended in 5 mL of 1% sterile peptone water. 1 mL of the soil suspension was pipetted into 9 mL of sterile water and diluted 10-fold to a concentration of 1 g·L -1 , spread 200 μL on the separation culture medium plate and culture in a 30 ℃ constant temperature incubator. Example 4

[0084] This embodiment provides a selective separation medium for Rhodococcus equi.

[0085] 1. The culture medium formula is as follows:

[0086] Each liter of culture medium contains: 1.25g soluble starch, 1.2g pyruvic acid, 1.1g sodium acetate, 11.8g acid hydrolyzed casein, 1.75g ​​beef extract powder, 1.5g yeast extract powder, 0.006g calcium chloride, 0.32g potassium dihydrogen phosphate, 0.55g disodium hydrogen phosphate, 0.06g potassium nitrate, 0.011g zinc sulfate, 0.012g manganese sulfate, 0.114g magnesium sulfate heptahydrate, 0.035g ammonium ferric citrate, 0.012g potassium tellurite, 17g agar; additives are 1.5mg trimethoprim, 1.8mg cefoperazone, 2mg polymyxin B, and 70mg cycloheximide.

[0087] 2. Preparation of culture medium

[0088] Weigh 1.25 g soluble starch, 1.2 g pyruvic acid, 1.1 g sodium acetate, 11.8 g acid hydrolyzed casein, 1.75 g beef extract powder, 1.5 g yeast extract powder, 0.006 g calcium chloride, 0.32 g potassium dihydrogen phosphate, 0.55 g disodium hydrogen phosphate, 0.06 g potassium nitrate, 0.011 g zinc sulfate, 0.012 g manganese sulfate, 0.114 g magnesium sulfate heptahydrate, 0.035 g ammonium ferric citrate, 0.012 g potassium tellurite, and 17 g agar, dissolve in deionized water, make up to 1000 mL, adjust the pH to 7.5-7.6 with sodium carbonate and citric acid, sterilize at 121°C and 105 kPa for 15 min, and cool to 50 ℃, add 1.5 mg of trimethoprim, 1.8 mg of cefoperazone, 2 mg of polymyxin B, and 70 mg of cycloheximide and mix well, and aseptically fill into a 90 mm sterile culture dish to prepare a selective isolation medium for Rhodococcus equi.

[0089] 3. Inoculation and cultivation

[0090] (1) Dilute the activated Rhodococcus equi strain in sterile NaCl solution to a concentration of no more than 100 cfu / mL. -1 , spread on TSA and separation medium, spread 3 plates on each medium, culture at 30 ℃ for 48-72 h, observe the growth of bacteria, count the colonies, and take the average value to calculate the recovery rate;

[0091] Recovery rate = (selective separation medium colony count / TSA colony count) × 100%

[0092] (2) Dilute the activated Rhodococcus equi strain in stages with 0.9% sterile NaCl solution to about 10 5 cfu·mL -1 , mix 20 μL of bacterial solution with 180 μL of selective liquid culture medium, place in a growth curve instrument, culture at 30 °C for 72 h, read the OD 600 value of the culture well, and draw a growth curve;

[0093] (3) Rhodococcus faecalis, Streptococcus equi, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Streptomyces albus, Moraxella solani, Penicillium chrysogenum and Pythium heterotrichum were used as test strains to detect the ability of the culture medium to inhibit the growth of other microorganisms. Approximately 10 of each of the activated strains were added. 4 cfu were inoculated on the selective isolation medium and cultured in a 30 °C constant temperature incubator for 72 h to observe whether colonies appeared;

[0094] (4) Practical application of selective medium for isolation of Rhodococcus equi: Place the collected soil sample in a sterile plate to dry naturally, grind it into powder with a sterile pestle, weigh 1 g of soil sample, add it to 10 mL of 2% oxalic acid, and rotate at 180 r·min. -1 The mixture was shaken in a shaker for 20 min, and allowed to settle naturally for 5 min. The supernatant was poured off and centrifuged at 3000 rpm for 5 min. The sediment was suspended in 5 mL of 1% sterile peptone water. 1 mL of the soil suspension was pipetted into 9 mL of sterile water and diluted 10-fold to a concentration of 1 g·L -1 , spread 200 μL on the separation culture medium plate and culture in a 30 ℃ constant temperature incubator.

[0095] like Figure 1 As shown, the average recovery rates of the selective separation culture media of Examples 1-4 were 91%, 66%, 81%, and 62%, respectively. The above results show that the recovery rates of the selective separation culture media of Examples 1-4 for Rhodococcus equi were all higher than 50%, but compared with Examples 2-4, the recovery rate of the culture media in Example 1 was significantly superior. Rhodococcus equi grew well in the culture media of Examples 1-4, and the slopes of the growth curves of the liquid culture media in Example 1 were slightly greater than those of Examples 2-4, indicating that the growth rate of Rhodococcus equi in the liquid culture media of Example 1 was faster and reached a stable period in a shorter period of time. The test strains Rhodococcus coprophilus, Streptococcus equi, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Streptomyces albus, Moraxella solani, Penicillium chrysogenum, and Pythium heterotrichum did not grow on the selective culture media, indicating that the selective culture media of Examples 1-4 can meet the inhibition requirements to a certain extent. When isolating Rhodococcus equi from highly contaminated sources, the use of selective media is very important, such as Figure 4As shown, on the selective culture media of Examples 1-4, the culture media of Example 1 can well inhibit the contaminating bacteria, and the colony morphology of Rhodococcus equi on the culture media is more characteristic (glossy, smooth, and moist), and a larger number of Rhodococcus equi grow, while only weak growth of Rhodococcus equi is observed on the culture media of Example 3. The culture media of Examples 2 and 4 have a smaller number of Rhodococcus equi growing than the culture media of Example 3, and the recovery rate of Rhodococcus equi on the culture media of these Examples is lower.

[0096] In summary, the present invention adjusts and optimizes the culture medium formula according to the growth requirements and characteristics of Rhodococcus equi to form a selective separation medium for Rhodococcus equi, and compares the selective culture medium in given Examples 1-4 through the recovery, growth curve and growth colony phenotypic characteristics of Rhodococcus equi on the selective culture medium, as well as the growth of non-Rhodococcus equi on the selective culture medium. The selective culture medium in Example 1 has a better ability to promote the growth of Rhodococcus equi and can achieve the corresponding inhibitory ability, so Example 1 is the best embodiment of the present invention. The selective separation medium for Rhodococcus equi of the present invention can effectively detect the presence of Rhodococcus equi in the horse farm breeding environment, and can be applied to the enrichment culture, separation screening, and quantity detection of Rhodococcus equi, and has important value for the prevention and control of Rhodococcus equi infection problems in animal breeding areas such as horse farms.

[0097] Finally, it should be noted that the above embodiments are only partial embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. Any improvements made by ordinary technicians in this field without departing from the principles of the present invention should also be regarded as within the scope of protection of the present invention.

Claims

1. A selective isolation medium for Rhodococcus equi, characterized in that: The selective isolation medium for Rhodococcus equi consists of: a carbon source, a nitrogen source, an antibiotic, 0.005-0.015 g / L of calcium chloride, 0.3-0.5 g / L of potassium dihydrogen phosphate, 0.5-1.0 g / L of disodium hydrogen phosphate, 0.05-0.15 g / L of potassium nitrate, 0.01-0.02 g / L of zinc sulfate, 0.01-0.03 g / L of manganese sulfate, 0.1-0.2 g / L of magnesium sulfate heptahydrate, 0.03-0.07 g / L of ammonium ferric citrate, 0.01-0.02 g / L of potassium tellurite, and 16-18 g / L of agar; The carbon source comprises: 1-5 g / L soluble starch, 1-5 g / L pyruvic acid, and 1-5 g / L sodium acetate; The nitrogen source comprises: 10-25 g / L of acid hydrolyzed casein, 1-10 g / L of beef extract powder, and 1-5 g / L of yeast extract powder; The antibiotic composition comprises: 1-10 mg / L of trimethoprim, 1-10 mg / L of cefoperazone, 1-10 mg / L of polymyxin B, and 50-200 mg / L of cycloheximide.

2. A method for preparing the selective isolation medium for Rhodococcus equi as claimed in claim 1, characterized in that: Weigh 1-5 g soluble starch, 1-5 g pyruvic acid, 1-5 g sodium acetate, 10-25 g acid hydrolyzed casein, 1-10 g beef extract powder, 1-5 g yeast extract powder, 0.005-0.015 g / L calcium chloride, 0.3-0.5 g / L potassium dihydrogen phosphate, 0.5-1.0 g / L disodium hydrogen phosphate, 0.05-0.15 g / L potassium nitrate, 0.01-0.02 g / L zinc sulfate, 0.01-0.03 g / L manganese sulfate, 0.1-0.2 g / L magnesium sulfate heptahydrate, 0.03-0.07 g / L ammonium ferric citrate, 0.01-0.02 g / L potassium tellurite, 16-18 g / L agar, dissolve in deionized water, dilute to 1000 mL, adjust pH to 7.5-7.6 with sodium carbonate, 121 The mixture was sterilized by high pressure at 50°C for 15 minutes, cooled to 50°C, 1-10 mg of trimethoprim, 1-10 mg of cefoperazone, 1-10 mg of polymyxin B and 50-200 mg of cycloheximide were added and mixed, and aseptically filled into a 90 mm sterile culture dish to prepare the Rhodococcus equi selective isolation medium.

3. The use of the selective isolation medium for Rhodococcus equi according to claim 1, characterized in that: The selective separation medium for Rhodococcus equi is used for separating Rhodococcus equi in soil of animal breeding areas.

4. The use of the selective isolation medium for Rhodococcus equi according to claim 3, characterized in that: The culture temperature of the isolation medium for culturing Rhodococcus equi is 30°C.

5. The use of the selective isolation medium for Rhodococcus equi according to claim 3, characterized in that: The culture time of culturing Rhodococcus equi in the separation medium is 48 to 72 hours.

6. A method for using the selective isolation medium for Rhodococcus equi as claimed in claim 1, characterized in that: (1) Place the collected soil sample in a sterile dish to dry naturally, and grind it into powder using a sterile pestle; (2) Weigh 1 g of soil sample, add it to 10 mL of 2% oxalic acid, and rotate at 180 r·min. -1 The mixture was shaken in a shaker for 20 min and allowed to settle naturally for 5 min. (3) Pour off the supernatant and centrifuge at 3000 rpm for 5 min; (4) Suspend the sediment in 5 mL of 1% sterile peptone water, pipette 1 mL of soil suspension into 9 mL of sterile water, and perform a 10-fold gradient dilution to a concentration of 1 g·L -1 ; (5) Take 200 μL and spread it on the separation culture medium plate and place it in a constant temperature incubator at 30 ℃.

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