Legionella culture detection kit and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a legionella culture detection kit and application thereof. The invention provides a sample pretreatment solution and a culture medium. The sample pretreatment solution and the culture medium are good in legionella separation effect, high in sensitivity, short in culture time, high in antibacterial activity and less in infectious microbe influence. Activated carbon and animal blood or serum do not need to be added, bacterial colonies are colored, blue gray or blue, observation is easy, and the bacterial colonies are easy to distinguish.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a Legionella culture detection kit and application thereof. Background Art

[0002] Legionella is a Gram-negative bacillus that is widely found in water and soil in natural and artificial environments. It is a highly epidemic respiratory pathogen. Inhalation of Legionella-contaminated aerosols, drinking water contaminated with Legionella, or contact with contaminated water sources on wounds may cause Legionnaires' disease.

[0003] Legionnaires' disease is a new infectious disease that has emerged with the progress of human society and economic development. It has become an important public health issue faced by modern urban development. Many countries and regions in the world have reported sporadic cases of the disease. Most parts of my country have reported cases of Legionnaires' disease. Due to the lack of systematic etiology and epidemiological survey data, the current status of Legionnaires' infection and the prevalence of Legionnaires' disease are unclear. Recent research results show that the infection rate of Legionnaires' disease in people with respiratory or lung diseases is between 6.0% and 17.5%.

[0004] According to the IgM antibody test of the target population in public places in various places, the antibody positive rate of the target population in public places is 4.63%, and that of employees in key industries is 12.70%. There are even reports that the positive rate of the exposed population can be as high as 53%, indicating that Legionella has posed a potential threat to employees in key industries. Regular cleaning and supervision of public places and control of biological pollution from the source are of great significance to reducing the risk of Legionella infection.

[0005] The methods currently used for laboratory diagnosis of Legionnaires' disease are: isolation of the microorganism on culture medium, antigen detection in urine, microscopy, detection of bacterial deoxyribonucleic acid (DNA), and use of paired serology.

[0006] Urine antigen testing and sputum culture or bronchoalveolar lavage fluid are the most appropriate clinical tests for Legionella. Patients who may have been exposed to Legionella during an outbreak should also be tested for Legionnaires' disease, including culture.

[0007] Legionella culture is usually the most specified test method and has a high specificity, although the sensitivity of culture varies from 50% to 80%. Identification of Legionella after culture is the definitive method for diagnosing Legionnaires' disease and is therefore the "gold standard". Iron, L-cysteine, alpha-ketoglutarate, and charcoal yeast extract agar buffered with organic buffer (ACES) are the preferred growth media for isolation. Antibiotics can be added to BCYE alpha-medium to avoid overgrowth of other bacteria to increase the selectivity of the medium.

[0008] The current gold standard for diagnosing Legionnaires' pneumonia is positive bacterial isolation and culture, with a specificity of 100% and a sensitivity of 50% to 70%. Legionnaires can only grow on special culture media, and the isolation rate of environmental samples is as high as 50% or more. The positive rate of biological sample culture detection is related to the severity of the disease. The detection rate of Legionnaires in mild patients is 15% to 25%; the detection rate of Legionnaires in severe patients with respiratory failure is >90%.

[0009] The disadvantages of the current culture method are: it is easily affected by the quality of specimen collection and operation technology, the separation effect is poor, the positive rate is inconsistent; it takes a long time, about 7 to 10 days; the culture medium is expensive. These drawbacks lead to certain difficulties in the clinical diagnosis of Legionella pneumonia. Specifically:

[0010] 1) JP2021090403A introduces a culture medium with added cyclodextrin, which can replace the role of activated carbon or serum, but has no promoting effect on the growth of target bacteria and the detection of target bacteria in samples;

[0011] 2) Although an indicator is added to the Legionella selective isolation medium of CN101643713, it is added on the basis of the activated carbon plate, which does not promote the growth of the target bacteria and has no promoting effect on detection;

[0012] 3) CN112888790A introduces a carbon-free culture medium, but the culture medium includes: serum, wherein the concentration of the serum is 1% to 35%.

[0013] For culture, the culture medium of the existing culture technology is a plate with added activated carbon, which has no color development function, low clinical isolation rate, and poor inhibition of non-target bacteria. The cost of the plate without activated carbon with added serum or BSA is relatively high. The plate with cyclodextrin replacing serum or activated carbon only utilizes the adsorption effect of cyclodextrin to relieve the inhibition of metabolites on Legionella, and has no obvious effect on sample separation, especially the inhibition of substances in respiratory samples on Legionella.

[0014] On the other hand, for sample pretreatment, although hydrochloric acid sample pretreatment reagent is included in the national standard, hydrochloric acid treatment has a greater impact on Legionella, which will affect the detection of Legionella. It also has no digestive function, and the Legionella contained in mucus is not easily released, affecting the detection of the target bacteria. Summary of the invention

[0015] In view of this, the present invention provides a Legionella culture detection kit and its application, which has good Legionella separation effect, high sensitivity, short culture time, strong antibacterial property, and little influence of miscellaneous bacteria. The present invention does not need to add activated carbon and animal blood or serum, and the colony color is blue-gray or blue, which is easy to observe and the colonies are easy to distinguish.

[0016] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0017] The invention provides a Legionella culture medium, comprising a buffer reagent, an extract powder, amino acids, metal ions, carbohydrates, peptones, enzymes, antibiotics, and indicators;

[0018] Excludes:

[0019] (1) blood; and

[0020] (2) serum; and

[0021] (3), carbon; and

[0022] (4) Activated carbon;

[0023] The carbohydrate comprises at least cyclodextrin;

[0024] The amino acids include at least cysteine.

[0025] In some specific embodiments of the present invention, the buffer reagent of the Legionella culture medium includes at least one of ACES, MOPS, dipotassium hydrogen phosphate, and potassium citrate.

[0026] In some specific embodiments of the present invention, the extract powder of the Legionella culture medium comprises yeast extract powder.

[0027] In some specific embodiments of the present invention, the amino acids in the Legionella culture medium include at least one of glycine, serine, tyrosine, and phenylalanine.

[0028] In some specific embodiments of the present invention, the metal ions in the Legionella culture medium include at least one of iron ions and magnesium ions.

[0029] In some specific embodiments of the present invention, the carbohydrate in the Legionella culture medium further comprises at least one of sodium pyruvate, α-ketoglutaric acid, starch and mannitol.

[0030] In some specific embodiments of the present invention, the peptone in the Legionella culture medium includes at least one of animal peptone and plant peptone.

[0031] In some specific embodiments of the present invention, the enzyme in the Legionella culture medium includes at least one of coenzyme I, catalase, and superoxide dismutase.

[0032] In some specific embodiments of the present invention, the antibiotics in the Legionella culture medium include at least one of vancomycin, teicoplanin, lincomycin, bacitracin, cefsulodin sodium, polymyxin, and amphotericin.

[0033] In some specific embodiments of the present invention, the indicator of the Legionella culture medium includes at least one of bromocresol purple and bromothymol blue.

[0034] In some specific embodiments of the present invention, in the Legionella culture medium, the ACES is 5-15 parts, the MOPS is 5-15 parts, the dipotassium hydrogen phosphate is 0.05-2 parts, the potassium citrate is 0.1-0.5 parts, the yeast extract is 5-15 parts, the cysteine ​​is 0.1-1 parts, the glycine is 1-10 parts, the serine is 0.2-5 parts, the tyrosine is 0.2-5 parts, the phenylalanine is 0.2-5 parts, the iron ion is 0.06-0.4 parts of ferric nitrate, the magnesium ion is 0.05-0.2 parts of magnesium chloride, and the pyruvate is 0. The sodium is 1 to 5 parts, the α-ketoglutaric acid is 0.2 to 2 parts, the starch is 1 to 10 parts, the mannitol is 1 to 5 parts, the peptone is 1 to 10 parts, the coenzyme I is 0.002 to 0.02 parts, the vancomycin is 0.0001 to 0.01 parts, the teicoplanin is 0.0001 to 0.01 parts, the lincomycin is 0.0001 to 0.01 parts, the cefsulodin sodium is 0.001 to 0.01 parts, the amphotericin is 0.001 to 0.02 parts, the bromocresol purple is 0.01 to 0.1 parts, and the bromothymol blue is 0.001 to 0.05 parts;

[0035] In terms of enzyme activity, the catalase is 5000-50000 IU, the superoxide dismutase is 5000-50000 IU, the bacitracin is 500-3000 IU, and the polymyxin is 1000-20000 IU.

[0036] In some specific embodiments of the present invention, the Legionella culture medium further comprises agar, which is a coagulant, and the amount of agar used can be adjusted according to the difference in gel strength.

[0037] In some specific embodiments of the present invention, the pH value of the Legionella culture medium is 6.0, 6.1, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.

[0038] The present invention also provides a method for preparing the above-mentioned Legionella culture medium, comprising mixing the buffer reagent, the extract powder, the amino acid, the metal ion, the carbohydrate, the peptone, the enzyme, the antibiotic and the indicator to prepare a solid culture medium to obtain the Legionella culture medium.

[0039] The present invention also provides a kit, comprising a sample pretreatment reagent and the above-mentioned Legionella culture medium;

[0040] The sample pretreatment reagent has a pH value of 1.8 to 3.0 and contains an organic acid, potassium ions and a reducing agent.

[0041] In some specific embodiments of the present invention, the organic acid in the above kit includes at least one of citric acid and lactic acid.

[0042] In some specific embodiments of the present invention, the potassium ions in the above-mentioned kit include potassium chloride.

[0043] In some specific embodiments of the present invention, the reducing agent in the above-mentioned kit includes at least one of TCEP, DTT and thiourea.

[0044] In some specific embodiments of the present invention, in parts by weight, the citric acid in the above-mentioned kit is 10 to 80 parts, the lactic acid is 10 to 40 parts, the potassium chloride is 10 to 15 parts, the TCEP is 1 to 4 parts, the DTT is 0.1 to 1 part, and the thiourea is 0.2 to 4 parts.

[0045] The present invention also provides a method for isolating and culturing Legionella, which is based on the above-mentioned Legionella culture medium or the above-mentioned kit for cultivation.

[0046] The present invention also provides a method for detecting Legionella, characterized in that the method is based on the above-mentioned Legionella culture medium or the above-mentioned kit for detection.

[0047] The present invention also provides a method for diagnosing Legionnaires' disease, characterized in that the diagnosis is based on the above-mentioned Legionnaires' culture medium or the above-mentioned kit.

[0048] The present invention also provides a method for treating Legionnaires' disease, characterized in that the Legionnaires' infection status is obtained based on the Legionnaires' culture medium or the kit, and treatment is performed according to the status.

[0049] The culture medium and pre-treatment solution of the present invention have the following effects:

[0050] The culture medium of the present invention is a transparent culture medium without activated carbon and serum. Although cyclodextrin is added, the addition of cyclodextrin alone cannot achieve the ideal effect. Catalase, superoxide dismutase (SOD), sodium pyruvate and amino acids need to be added to promote the growth of colonies and relieve the inhibitory effect of substances in the sample or non-target bacterial metabolites on Legionella. At the same time, the target bacteria and non-target bacteria can be effectively distinguished through indicators, thereby improving the detection rate of the target bacteria.

[0051] The culture medium of the present invention has the following advantages: no activated carbon, animal blood and serum are added, the process is simple, the problem of short product shelf life caused by unstable animal blood is avoided, and the problem of activated carbon complicating the production process is avoided; carbon dioxide culture greatly improves the clinical separation sensitivity; it has a color development function, can distinguish target bacteria from non-target bacteria, and the cultured bacteria can be directly used to identify drug sensitivity.

[0052] The present invention also provides a sample pretreatment reagent, which uses an organic acid and has little effect on Legionella, and adds a digestion reagent, which has a sample digestion function, reduces the sample viscosity to release the Legionella and non-target bacteria included in the mucus, improves the detection of Legionella, and also improves the killing effect of non-target bacteria, combining digestion and impurity removal in one.

[0053] The present invention can effectively solve the shortcomings of the current culture medium that is easily affected by the quality of specimen collection and the performance of the culture medium, has poor separation effect, inconsistent positive rate, and takes 7 to 10 days. The culture detection kit provided by the present invention has a sample pretreatment reagent that can liquefy the sample, eliminate non-target bacteria, greatly improve the clinical isolation rate, shorten the culture time to 2 to 5 days, and greatly shorten the clinical TAT time. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0055] Figure 1 The results of inoculating samples with commercially available products show that there are no target bacteria, and all of them are non-target bacteria;

[0056] Figure 2 The sample was directly inoculated on the plate of the present invention, and the target bacteria grew well, the color was good, and some non-target bacteria were present;

[0057] Figure 3 It shows that when the plate of the present invention is used in combination with the sample pretreatment reagent, the target bacteria grow well, the color development is good, and there are no non-target bacteria. DETAILED DESCRIPTION

[0058] The present invention discloses a Legionella culture detection kit and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0059] The technical solution of the present invention has the following purposes:

[0060] First, by adding growth factors required for the growth of Legionella into the culture medium, its growth performance can be improved. By adding toxin adsorbents, the separation rate of Legionella can be improved. By adding indicators with certain color development functions and antibiotics, the inhibitory ability against non-target bacteria can be optimized.

[0061] Second, a sample pretreatment reagent with digestion function is provided, which can liquefy the sample and release the contained microorganisms, which is beneficial to the detection of target bacteria and the inhibition of non-target bacteria; the pretreatment reagent is acidic and has a certain function of inhibiting miscellaneous bacteria. Legionella is acid-resistant, and the pretreatment reagent has no effect on Legionella. After the sample is treated with the pretreatment reagent, the growth of non-target bacteria can be inhibited to a great extent, and the detection rate of Legionella can be significantly increased when inoculated on the culture medium.

[0062] Specifically, the present invention provides a Legionella culture detection kit, which can significantly improve the detection rate of Legionella. The kit includes at least component 1 and may also include component 2.

[0063] Component 1 is an agar culture medium without activated carbon, animal blood or serum. It contains growth-promoting factors for Legionella, toxin adsorbents, etc. It also contains indicators to make the colonies blue or blue-gray, thereby improving the detection rate and recognition rate of target bacteria. It also contains an optimized antibiotic combination to enhance the inhibitory ability of non-target bacteria and improve the detection rate of Legionella.

[0064] Component 2 is a sample pretreatment reagent, which has a certain liquefaction function, can liquefy the sample, promote the release of included microorganisms, is beneficial to the detection of target bacteria, and is also beneficial to the inhibition of non-target bacteria, especially drug-resistant bacteria; the pretreatment reagent is acidic and has a certain function of inhibiting miscellaneous bacteria. Legionella is acid-resistant, and the pretreatment reagent has no effect on Legionella.

[0065] The plate (i.e. agar culture medium) of the present invention is composed of peptones, extract powders, carbohydrates, amino acids, metal ions, enzymes, indicators, antibiotics, buffers and the like, with a pH of 6.5 to 7.5;

[0066] The peptones include animal peptones such as trypticase and bacteriological peptone, and plant peptones such as soy peptone;

[0067] The powdered extracts include yeast powdered extracts;

[0068] The carbohydrates include cyclodextrin, sodium pyruvate, α-ketoglutaric acid, starch, and mannitol;

[0069] The amino acids include cysteine, glycine, serine, tyrosine, and phenylalanine;

[0070] The metal ions include iron ions and magnesium ions;

[0071] The enzymes include coenzyme I, catalase, SOD;

[0072] The indicators include bromocresol purple and bromothymol blue;

[0073] The antibiotics include vancomycin, teicoplanin, lincomycin, bacitracin, cefsulodin sodium, polymyxin, and amphotericin;

[0074] The buffer includes ACES buffer and MOPS buffer.

[0075] In some specific embodiments, the peptones include one or more of trypticase, bacteriological peptone, and soy peptone, and the contents are all g / L:

[0076] The trypticase content is 1 to 10, and the optimal content is 2;

[0077] The bacteriological peptone content is 1 to 10, and the optimal value is 8;

[0078] The soy peptone content is 1-10, and the optimal content is 2.

[0079] In some specific embodiments, the extract powder is composed of yeast extract powder, and the content is g / L:

[0080] The yeast extract powder content is 5-15, and the optimal content is 10.

[0081] In some specific embodiments, the carbohydrates include one or more of cyclodextrin, sodium pyruvate, α-ketoglutaric acid, starch, and mannitol, and the content is g / L;

[0082] The cyclodextrin content is 1 to 10, and the optimal content is 3;

[0083] The starch content is 1 to 10, and the optimal value is 3;

[0084] The sodium pyruvate content is 1 to 5, and the optimal content is 2;

[0085] The α-ketoglutaric acid content is 0.2 to 2, and the optimal content is 1;

[0086] The mannitol content is 1-5, and the optimal content is 2.

[0087] In some specific embodiments, the amino acids include one or more of cysteine, glycine, serine, tyrosine, and phenylalanine, and the content is g / L:

[0088] The cysteine ​​content is 0.1-1, and the optimal content is 0.4;

[0089] The serine content is 0.2-5, and the optimal content is 1;

[0090] The glycine content is 1 to 10, and the optimal content is 3;

[0091] The tyrosine content is 0.2-5, and the optimal content is 1;

[0092] The phenylalanine content is 0.2-5, and the optimal content is 1.

[0093] In some specific embodiments, the metal ions include one or more of iron ions and magnesium ions, and the content is mg / L:

[0094] The iron nitrate content is 60-400, and the optimal content is 135;

[0095] The magnesium chloride content is 50-200, and optimally 100.

[0096] In some specific embodiments, the enzymes include one or more of coenzyme I, catalase, and SOD:

[0097] The catalase content is 5000-50000 IU / L, and the optimal content is 10000 IU / L;

[0098] The SOD content is 5000-50000 IU / L, and the optimal content is 16000 IU / L;

[0099] The coenzyme I content is 2-20 mg / L, and the optimal content is 10 mg / L.

[0100] In some specific embodiments, the indicator includes one or more of bromothymol blue and bromocresol purple, and the content is mg / L:

[0101] The content of bromothymol blue is 1 to 50, and the optimal content is 10;

[0102] The content of bromocresol purple is 10-100, and the optimal content is 50.

[0103] In some specific embodiments, the antibiotics include one or more of vancomycin, teicoplanin, lincomycin, bacitracin, cefsulodin sodium, polymyxin, and amphotericin:

[0104] The vancomycin content is 0.1 to 10 mg / L, and the optimal content is 1 mg / L;

[0105] The teicoplanin content is 0.1 to 10 mg / L, and the optimal content is 1 mg / L;

[0106] The lincomycin content is 0.1-10 mg / L, and the optimal content is 1 mg / L;

[0107] The bacitracin content is 500 to 3000 IU / L, and the optimal content is 1500 IU / L;

[0108] The content of cefsulodin sodium is 1 to 10 mg / L, and the optimal content is 3 mg / L;

[0109] The polymyxin is 1000 IU / L to 20000 IU / L, and the optimal amount is 10000 IU / L;

[0110] The content of amphotericin is 1 mg / L to 20 mg / L, and the optimal content is 10 mg / L.

[0111] In some specific embodiments, the buffer is one or more of ACES or MOPS, and potassium hydrogen phosphate and potassium citrate are added at the same time:

[0112] The content of dipotassium hydrogen phosphate is 0.05-2 g / L, and the optimal content is 0.076 g / L;

[0113] The potassium citrate content is 0.1-0.5 g / L, and the optimal content is 0.16 g / L;

[0114] The ACES content is 5 to 15 g / L, and the optimal content is 10 g / L;

[0115] The MOPS content is 5-15 g / L, and optimally 10 g / L.

[0116] In some specific embodiments, the pH value ranges from 6.5 to 7.5.

[0117] The pretreatment reagent of the present invention is composed of organic acid, potassium ions, reducing agent, etc., with a pH of 1.8 to 3.0;

[0118] The organic acids include citric acid and lactic acid;

[0119] The potassium ions include potassium chloride;

[0120] The reducing agent includes TCEP, DTT, and thiourea.

[0121] In some specific embodiments, the organic acids include one or more of citric acid and lactic acid, and the content is g / L:

[0122] The citric acid content is 10-80, and the optimal content is 40;

[0123] The lactic acid content is 10-40, and optimally 20.

[0124] In some specific embodiments, the potassium ion includes potassium chloride, and the content is g / L:

[0125] The potassium chloride content is 10-15, and the optimal content is 12.88.

[0126] In some specific embodiments, the reducing agent includes one or more of TCEP, DTT, and thiourea, and the content is g / L:

[0127] The TCEP content is 1 to 4, and the optimal content is 2;

[0128] The DTT content is 0.1-1, and the optimal content is 0.2;

[0129] The thiourea content is 0.2-4, and the optimal content is 2.

[0130] The product of the present invention has good separation effect on Legionella, high sensitivity, short culture time, strong antibacterial property, and little influence of miscellaneous bacteria. The product of the present invention does not need to add activated carbon and animal blood or serum, the product is transparent, and the colony is easy to observe and the colony is color. The colony is blue or blue-gray, and it is easy to distinguish.

[0131] In some embodiments, the flat panel effect diagram of the present invention is as follows Figure 2 , Figure 3 shown. Figure 2 It shows that when the sample is directly inoculated on the plate of the present invention, although miscellaneous bacteria grow, some target bacteria still grow. Figure 3 The results show that the plate of the present invention and the sample pretreatment reagent are used together, and no bacteria grow, and the target bacteria grow well. Figure 1 The results show that the inoculation sample of the existing commercial products has no growth of target bacteria, but more miscellaneous bacteria grow. Compared with the existing commercial products, the present invention has significant effects.

[0132] The agar powder in the present invention mainly plays a role of coagulation, and the usage amount of different types of agar powder can be adjusted according to specific circumstances.

[0133] In the embodiment, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus, and Neisseria oralis are Gram-negative bacteria; Staphylococcus aureus, Enterococcus faecalis, Leuconostoc lactis, and Lactobacillus salivarius are Gram-positive bacteria; Legionella pneumophila and Nocardia are Gram-negative bacteria; and Candida albicans is a fungus, which are common pathogens of the respiratory tract.

[0134] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0135] The present invention is further described below in conjunction with the examples, wherein Examples 1 to 63 and Comparative Examples 1 to 4 describe Legionella agar culture medium (component 1); and Examples 64 to 85 and Comparative Example 5 describe sample pretreatment reagents (component 2).

[0136] Example 1: Formulation 1

[0137] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, horse serum 50mL / L, agar powder 12g / L, pH 6.8.

[0138] Example 2: Formulation 2

[0139] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, horse serum 50mL / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0140] Example 3: Formulation 3

[0141] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0142] Example 4: Formulation 4

[0143] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0144] Example 5: Formulation 5

[0145] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0146] Example 6: Formulation 6

[0147] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0148] Example 7: Formulation 7

[0149] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0150] Example 8: Formulation 8

[0151] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0152] Example 9: Formulation 9

[0153] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0154] Example 10: Formulation 10

[0155] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0156] Example 11: Formulation 11

[0157] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0158] Example 12: Formulation 12

[0159] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0160] Example 13: Formulation 13

[0161] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0162] Example 14: Formulation 14

[0163] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin 1mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0164] Example 15: Formulation 15

[0165] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin 1mg / L, teicoplanin 1mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0166] Example 16: Formulation 16

[0167] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0168] Example 17: Formulation 17

[0169] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0170] Example 18: Formulation 18

[0171] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0172] Example 19: Formulation 19

[0173] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0174] Example 20: Formulation 20

[0175] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0176] Example 21: Formulation 21

[0177] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.5.

[0178] Example 22: Formulation 22

[0179] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 7.5.

[0180] Example 23: Formulation 23

[0181] Purified water 950g / L, yeast extract powder 10g / L, MOPS 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0182] Example 24: Formulation 24

[0183] Purified water 950g / L, yeast extract powder 10g / L, MOPS 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.5.

[0184] Example 25: Formulation 25

[0185] Purified water 950g / L, yeast extract powder 10g / L, MOPS 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 7.5.

[0186] Example 26: Formulation 26

[0187] Purified water 950g / L, yeast extract powder 10g / L, MOPS 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, cefsulodin sodium 3mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0188] Example 27: Formulation 27

[0189] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, cefsulodin sodium 3mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0190] Example 28: Formulation 28

[0191] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 1g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0192] Example 29: Formulation 29

[0193] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 10g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0194] Example 30: Formulation 30

[0195] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, soy peptone 1g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0196] Example 31: Formulation 31

[0197] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, soy peptone 10g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0198] Example 32: Formulation 32

[0199] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, peptone 1g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0200] Example 33: Formulation 33

[0201] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, peptone 10g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0202] Example 34: Formulation 34

[0203] Purified water 950g / L, yeast extract powder 5g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0204] Example 35: Formulation 35

[0205] Purified water 950g / L, yeast extract powder 15g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0206] Example 36: Formulation 36

[0207] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0208] Example 37: Formulation 37

[0209] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 1g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0210] Example 38: Formulation 38

[0211] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 10g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0212] Example 39: Formulation 39

[0213] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 1g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0214] Example 40: Formulation 40

[0215] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 10g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0216] Example 41: Formulation 41

[0217] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 1g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0218] Example 42: Formulation 42

[0219] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 5g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0220] Example 43: Formulation 43

[0221] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 5g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0222] Example 44: Formulation 44

[0223] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 1g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0224] Example 45: Formulation 45

[0225] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, tyrosine 0.2g / L, phenylalanine 0.2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0226] Example 46: Formulation 46

[0227] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, tyrosine 1g / L, phenylalanine 1g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0228] Example 47: Formulation 47

[0229] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.06g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.05g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0230] Example 48: Formulation 48

[0231] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.4g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.2g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0232] Example 49: Formulation 49

[0233] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 2mg / L, catalase 5000IU / L, superoxide dismutase 5000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0234] Example 50: Formulation 50

[0235] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 20mg / L, catalase 50000IU / L, superoxide dismutase 50000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH6.8.

[0236] Example 51: Formulation 51

[0237] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 10mg / L, bromothymol blue 1mg / L, pH 6.8.

[0238] Example 52: Formulation 52

[0239] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 100mg / L, bromothymol blue 50mg / L, pH 6.8.

[0240] Example 53: Formulation 53

[0241] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 0.1mg / L, teicoplanin 0.1mg / L, lincomycin hydrochloride 0.1mg / L, polymyxin 1000IU / L, bacitracin 500IU / L, amphotericin B 1mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0242] Example 54: Formulation 54

[0243] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 10mg / L, teicoplanin 10mg / L, lincomycin hydrochloride 10mg / L, polymyxin 20000IU / L, bacitracin 3000IU / L, amphotericin B 20mg / L, mannitol 2g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0244] Example 55: Formulation 55

[0245] Purified water 950g / L, yeast extract powder 5g / L, ACES 5g / L, glycine 1g / L, α-ketoglutaric acid 0.2g / L, L-cysteine ​​hydrochloride 0.1g / L, ferric nitrate 0.06g / L, cyclodextrin 1g / L, K2HPO4 0.05g / L, potassium citrate 0.1g / L, starch 1g / L, serine 0.2g / L, trypticase 1g / L, sodium pyruvate 1g / L, magnesium chloride 0.05g / L, coenzyme I 2mg / L, catalase 5000IU / L, superoxide dismutase 5000IU / L, vancomycin hydrochloride 0.1mg / L, teicoplanin 10mg / L, lincomycin hydrochloride 0.1mg / L, polymyxin 1000IU / L, bacitracin 500IU / L, amphotericin B 1mg / L, mannitol 1g / L, agar powder 12g / L, bromocresol purple 10mg / L, bromothymol blue 1mg / L, pH 6.8.

[0246] Example 56: Formulation 56

[0247] Purified water 950g / L, yeast extract powder 15g / L, ACES 15g / L, glycine 10g / L, α-ketoglutaric acid 2g / L, L-cysteine ​​hydrochloride 1g / L, ferric nitrate 0.4g / L, cyclodextrin 10g / L, K2HPO4 0.2g / L, potassium citrate 0.5g / L, starch 10g / L, serine 5g / L, trypticase 10g / L, sodium pyruvate 5g / L, magnesium chloride 0.2g / L, coenzyme I 20mg / L, catalase 50000IU / L, superoxide dismutase 50000IU / L, vancomycin hydrochloride 10mg / L, teicoplanin 10mg / L, lincomycin hydrochloride 10mg / L, polymyxin 20000IU / L, bacitracin 3000IU / L, amphotericin B 20mg / L, mannitol 5g / L, agar powder 12g / L, bromocresol purple 100mg / L, bromothymol blue 50mg / L, pH 6.8.

[0248] Example 57: Formulation 57

[0249] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, cefsulodin sodium 1mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0250] Example 58: Formulation 58

[0251] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, cefsulodin sodium 10mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0252] Example 59: Formulation 59

[0253] Purified water 950g / L, yeast extract powder 10g / L, MOPS 5g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, cefsulodin sodium 3mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0254] Example 60: Formulation 60

[0255] Purified water 950g / L, yeast extract powder 10g / L, MOPS 15g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 2g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, cefsulodin sodium 3mg / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0256] Example 61: Formulation 61

[0257] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferric nitrate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, tyrosine 5g / L, phenylalanine 5g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0258] Example 62: Formulation 62

[0259] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, ferrous sulfate 0.135g / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, tyrosine 5g / L, phenylalanine 5g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8.

[0260] Example 63: Formulation 63

[0261] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / L, L-cysteine ​​hydrochloride 0.4g / L, heme 10mg / L, cyclodextrin 3g / L, K2HPO4 0.076g / L, potassium citrate 0.16g / L, starch 3g / L, serine 1g / L, trypticase 3g / L, sodium pyruvate 2g / L, magnesium chloride 0.1g / L, coenzyme I 10mg / L, catalase 10000IU / L, superoxide dismutase 16000IU / L, vancomycin hydrochloride 1mg / L, teicoplanin 1mg / L, lincomycin hydrochloride 1mg / L, polymyxin 9500IU / L, bacitracin 1500IU / L, amphotericin B 10mg / L, mannitol 2g / L, tyrosine 5g / L, phenylalanine 5g / L, agar powder 12g / L, bromocresol purple 50mg / L, bromothymol blue 10mg / L, pH 6.8. Comparative Example 1: The culture medium currently used for Legionella, GVPC standard formula

[0262] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / , L-cysteine ​​hydrochloride 0.4g / L, ferric pyrophosphate 0.25g / L, activated carbon 2g / L, agar powder 13g / L, pH 6.8±0.2.

[0263] Comparative Example 2: Current culture medium for Legionella, GVPC standard formula

[0264] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / , L-cysteine ​​hydrochloride 0.4g / L, ferric pyrophosphate 0.25g / L, activated carbon 2g / L, polymyxin 80000IU / L, vancomycin 10mg / L, cycloheximide 80mg / L, agar powder 13g / L, pH 6.8±0.2.

[0265] Comparative Example 3: Current culture medium for Legionella without activated carbon formulation

[0266] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / , L-cysteine ​​hydrochloride 0.4g / L, ferric pyrophosphate 0.25g / L, horse serum 50mL / L, agar powder 13g / L, pH 6.8±0.2.

[0267] Comparative Example 4: Current formulation for Legionella culture medium without activated carbon

[0268] Purified water 950g / L, yeast extract powder 10g / L, ACES 10g / L, glycine 3g / L, α-ketoglutaric acid 1g / , L-cysteine ​​hydrochloride 0.4g / L, ferric pyrophosphate 0.25g / L, horse serum 50mL / L, polymyxin 80000IU / L, vancomycin 10mg / L, cycloheximide 80mg / L, agar powder 13g / L, pH 6.8±0.2.

[0269] Example 64: Formulation 2-1

[0270] Purified water 960g / L, potassium chloride 12.88g / L, citric acid 40g / L, adjust pH to 2.2.

[0271] Example 65: Formulation 2-2

[0272] Purified water 980g / L, potassium chloride 12.88g / L, lactic acid 20g / L, adjust pH to 2.2.

[0273] Example 66: Formulation 2-3

[0274] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, adjust pH to 2.2.

[0275] Example 67: Formulation 2-4

[0276] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 2g / L, adjust pH to 2.2.

[0277] Example 68: Formulation 2-5

[0278] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, DTT 1g / L, adjust pH to 2.2.

[0279] Example 69: Formulation 2-6

[0280] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, DTT 1g / L, TCEP 2g / L, adjust pH to 2.2.

[0281] Example 70: Formulation 2-7

[0282] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 2g / L, thiourea 2g / L, adjust pH to 2.2.

[0283] Example 71: Formulation 2-8

[0284] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, DTT 1g / L, TCEP 2g / L, thiourea 2g / L, adjust pH to 2.2.

[0285] Example 72: Formulation 2-9

[0286] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 2g / L, thiourea 2g / L, adjust pH to 1.8.

[0287] Example 73: Formulation 2-10

[0288] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 2g / L, thiourea 2g / L, adjust pH to 3.0.

[0289] Example 74: Formulation 2-11

[0290] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, DTT 1g / L, TCEP 2g / L, thiourea 2g / L, adjust pH to 1.8.

[0291] Example 75: Formulation 2-12

[0292] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, DTT 1g / L, TCEP 2g / L, thiourea 2g / L, adjust pH to 3.0.

[0293] Example 76: Formulation 2-13

[0294] Purified water 970g / L, potassium chloride 12.88g / L, citric acid 10g / L, lactic acid 20g / L, TCEP 2g / L, thiourea 2g / L, adjust pH to 2.2.

[0295] Example 77: Formulation 2-14

[0296] Purified water 900g / L, potassium chloride 12.88g / L, citric acid 80g / L, lactic acid 20g / L, TCEP 2g / L, thiourea 2g / L, adjust the pH to 2.2.

[0297] Example 78: Formulation 2-15

[0298] Purified water 950g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 10g / L, TCEP 2g / L, thiourea 2g / L, adjust pH to 2.2.

[0299] Example 79: Formulation 2-16

[0300] Purified water 920g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 40g / L, TCEP 2g / L, thiourea 2g / L, adjust pH to 2.2.

[0301] Example 80: Formulation 2-17

[0302] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 1g / L, thiourea 2g / L, adjust pH to 2.2.

[0303] Example 81: Formulation 2-18

[0304] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 4g / L, thiourea 2g / L, adjust pH to 2.2.

[0305] Example 82: Formulation 2-19

[0306] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 2g / L, DTT 0.1g / L, thiourea 2g / L, adjust the pH to 2.2.

[0307] Example 83: Formulation 2-20

[0308] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 2g / L, DTT 0.2g / L, thiourea 2g / L, adjust pH to 2.2.

[0309] Example 84: Formulation 2-21

[0310] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 2g / L, DTT 0.2g / L, thiourea 0.2g / L, adjust pH to 2.2.

[0311] Example 85: Formulation 2-22

[0312] Purified water 940g / L, potassium chloride 12.88g / L, citric acid 40g / L, lactic acid 20g / L, TCEP 2g / L, DTT 0.2g / L, thiourea 4g / L, adjust pH to 2.2.

[0313] Comparative Example 5: Formula 2-23

[0314] Purified water 960g / L, 0.2mol / L potassium chloride (14.9g / L), 0.2mol / L hydrochloric acid (17.2mL / L), adjust pH to 2.2. Effect Example 1: Growth effect of target bacteria on different plates

[0315] The cultured Legionella were inoculated on plates with formulations as shown in Examples 1 to 63 and Comparative Examples 1 to 4, respectively, and cultured at 35 to 37° C., 5% to 10% CO 2 for 48 to 72 hours. The results were observed and shown in Table 1.

[0316] Table 1

[0317]

[0318]

[0319] From the results, adding reagents can promote the growth of target bacteria, and the plate provided by the present invention can significantly improve the growth of target bacteria. Removing or replacing some reagents will affect the growth of target bacteria.

[0320] Effect Example 2: Inhibition effect of non-target bacteria on different plates

[0321] The cultured non-target bacteria were inoculated on plates with formulations as shown in Examples 13 to 63, Comparative Example 2, and Comparative Example 4, and cultured at 35 to 37° C., 5% to 10% CO2 for 48 to 72 hours. The results were observed and shown in Table 2.

[0322] Table 2

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329] From the results, it can be seen that the plate provided by the present invention has a more obvious inhibitory ability on non-target bacteria, can reduce the coverage and inhibition of target bacteria, and can improve the detection rate of target bacteria.

[0330] Effect Example 3: Digestion Effects of Different Pretreatment Reagents

[0331] The viscous sputum samples were diluted 1:1 or 1:2 using the pretreatment reagents of Examples 64 to 85 and Comparative Example 5, respectively, and treated for 5 minutes or 15 minutes to observe the dilution digestion effect. The results are shown in Table 3.

[0332] Table 3

[0333]

[0334]

[0335]

[0336] From the results, it can be seen that the viscosity of the reagent can be significantly reduced after the sample is treated with the pre-treatment reagent of the present invention.

[0337] Effect Example 4: Effect of sample pretreatment on the growth of Legionella

[0338] Freshly cultured Legionella was prepared into a bacterial suspension with a McFarland turbidity of 0.5 (about 1.5×10 8 ), and then diluted to (10 4 ), the diluted bacterial solution was diluted 1:1 or 1:2 with the pretreatment reagents as shown in Examples 64 to 85 and Comparative Example 5, and left for 5 minutes or 15 minutes, and 100 microliters were taken to coat two plates after the time was up, and the average number of colonies was taken for comparison. The control was diluted 1:1 or 1:2 with physiological saline, and left for 5 minutes or 15 minutes, and 100 microliters were taken to coat two plates after the time was up, and the average number of colonies was taken for comparison. The results are shown in Table 4.

[0339] Table 4

[0340]

[0341]

[0342] From the pure bacteria results, the sample pretreatment reagent treatment at 1:2 for 15 minutes had no significant effect on the growth of the target bacteria, indicating that the sample pretreatment had no effect on the growth of the target bacteria.

[0343] Effect Example 5: Inhibitory effect of pretreatment reagents on non-target bacteria

[0344] The cultured non-target bacteria were prepared into a bacterial suspension with a McFarland turbidity of 0.5 (about 1.5×10 8 ), dilute 1:1 with the sample pretreatment reagent as formulated in Example 64 to Example 85 and Comparative Example 5, and leave it for 5 minutes. After the time is up, take 100 μl of the three-zone inoculation plate to observe the growth of the target bacteria.

[0345] The control was treated with the same procedure by diluting the bacterial solution with physiological saline. After the time was up, 100 μl was taken and inoculated on a three-zone plate to observe the growth of the target bacteria. The results are shown in Table 5.

[0346] Table 5

[0347]

[0348]

[0349] From the results, it can be seen that the pretreatment reagent has a good inhibitory effect on non-target bacteria.

[0350] Effect Example 6: Detection rate of target bacteria after treatment with different pre-treatment reagents

[0351] Freshly cultured Legionella was prepared into a bacterial suspension with a McFarland turbidity of 0.5 (about 1.5×10 8 ), and then diluted to (10 6 ), take 100 μl and add it to 900 μl sample (10 5 ), dilute the diluted sample with the pretreatment reagents as shown in Examples 64 to 85 and Comparative Example 5 at a ratio of 1:1 and 1:2, respectively, and place for 5 minutes and 15 minutes, respectively. After the time is up, take 100 μl of three-zone lines and inoculate them on a plate with a formula as shown in Example 20 to observe the growth of the target bacteria.

[0352] The control was a sample diluted with physiological saline and treated in the same manner. After the time was up, 100 μl of the sample was inoculated in three zones on a plate having the formula of Example 20 to observe the growth of the target bacteria. The results are shown in Table 6.

[0353] Table 6

[0354]

[0355]

[0356] From the test results of the simulated samples, it can be seen that the detection rate of Legionella is significantly improved by using the sample inoculation plate treated with the pre-treatment reagent of the present invention.

[0357] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Legionella culture medium, characterized in that Including buffer reagents, extract powder, amino acids, metal ions, carbohydrates, peptones, enzymes, antibiotics, indicators; Excludes: (1) blood; and (2) serum; and (3), carbon; and (4) Activated carbon; The carbohydrate comprises at least cyclodextrin; The amino acids include at least cysteine.

2. The Legionella culture medium according to claim 1, characterized in that include: The buffer reagent comprises at least one of ACES, MOPS, dipotassium hydrogen phosphate, and potassium citrate; and / or The powder comprises yeast powder; and / or The amino acids include at least one of glycine, serine, tyrosine and phenylalanine; and / or The metal ions include at least one of iron ions and magnesium ions; and / or The carbohydrates further include at least one of sodium pyruvate, α-ketoglutaric acid, starch, and mannitol; and / or The peptone comprises at least one of animal peptone and plant peptone; and / or The enzyme comprises at least one of coenzyme I, catalase, and superoxide dismutase; and / or The antibiotics include at least one of vancomycin, teicoplanin, lincomycin, bacitracin, cefsulodin sodium, polymyxin, and amphotericin; and / or The indicator comprises at least one of bromocresol purple and bromothymol blue.

3. The Legionella culture medium according to claim 2, characterized in that In parts by weight, the ACES is 5 to 15 parts, the MOPS is 5 to 15 parts, the dipotassium hydrogen phosphate is 0.05 to 2 parts, the potassium citrate is 0.1 to 0.5 parts, the yeast extract is 5 to 15 parts, the cysteine ​​is 0.1 to 1 parts, the glycine is 1 to 10 parts, the serine is 0.2 to 5 parts, the tyrosine is 0.2 to 5 parts, the phenylalanine is 0.2 to 5 parts, the iron ion is 0.06 to 0.4 parts of ferric nitrate, the magnesium ion is 0.05 to 0.2 parts of magnesium chloride, the sodium pyruvate is 1 to 5 parts, the α-keto 0.2-2 parts of glutaric acid, 1-10 parts of starch, 1-5 parts of mannitol, 1-10 parts of peptone, 0.002-0.02 parts of coenzyme I, 0.0001-0.01 parts of vancomycin, 0.0001-0.01 parts of teicoplanin, 0.0001-0.01 parts of lincomycin, 0.0001-0.01 parts of cefsulodin sodium, 0.001-0.01 parts of amphotericin, 0.001-0.02 parts of bromocresol purple, 0.01-0.1 parts of bromothymol blue, 0.001-0.05 parts of bromothymol blue; In terms of enzyme activity, the catalase is 5000-50000 IU, the superoxide dismutase is 5000-50000 IU, the bacitracin is 500-3000 IU, and the polymyxin is 1000-20000 IU.

4. The Legionella culture medium according to any one of claims 1 to 3, characterized in that Agar is also included.

5. The Legionella culture medium according to any one of claims 1 to 4, characterized in that The pH value is 6.5~7.

5.

6. The method for preparing a Legionella culture medium according to any one of claims 1 to 5, characterized in that: The method comprises mixing the buffer reagent, the extract powder, the amino acid, the metal ion, the carbohydrate, the peptone, the enzyme, the antibiotic and the indicator to prepare a solid culture medium to obtain a Legionella culture medium.

7. A kit, characterized in that Comprising a sample pretreatment reagent and the Legionella culture medium according to any one of claims 1 to 5; The sample pretreatment reagent has a pH value of 1.8 to 3.0 and contains an organic acid, potassium ions, and a reducing agent.

8. The kit according to claim 7, characterized in that include: The organic acid comprises at least one of citric acid and lactic acid; and / or The potassium ions include potassium chloride; and / or The reducing agent includes at least one of TCEP, DTT and thiourea.

9. The kit according to claim 8, characterized in that In parts by weight, the citric acid is 10 to 80 parts, the lactic acid is 10 to 40 parts, the potassium chloride is 10 to 15 parts, the TCEP is 1 to 4 parts, the DTT is 0.1 to 1 part, and the thiourea is 0.2 to 4 parts.

10. A method for isolating and culturing Legionella for non-disease diagnosis or diagnostic purposes, characterized in that: Cultivated based on the Legionella culture medium according to any one of claims 1 to 5 or the kit according to any one of claims 7 to 9.

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