CN111763652B - Culture medium for separating and counting clostridium butyricum - Google Patents
Culture medium for separating and counting clostridium butyricum Download PDFInfo
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- CN111763652B CN111763652B CN202010912802.5A CN202010912802A CN111763652B CN 111763652 B CN111763652 B CN 111763652B CN 202010912802 A CN202010912802 A CN 202010912802A CN 111763652 B CN111763652 B CN 111763652B
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Abstract
The invention relates to a culture medium for separating and counting clostridium butyricum, belonging to the field of microbial separation and counting. Comprises 15.0g of tryptose peptone, 3.0g of beef extract powder, 1.0g of yeast extract powder, 5.0g of sodium chloride, 0.5g of L-cysteine hydrochloride, 0.5g of sodium thioglycolate, 10.0g of xylose, 0.04g of bromothymol blue, 15.0g of agar powder, 900ml of purified water, 0.25g of D-cycloserine, 0.012g of kanamycin sulfate and 25ml of yolk. Compared with the prior art, the invention has the beneficial effects that: firstly, the culture medium analyzes the specific physiological characteristics of clostridium butyricum, and pertinently performs formula combination and adjustment, so that the clostridium butyricum can be obviously distinguished from other bacteria on visual observation, and the aims of separation and counting of the clostridium butyricum are fulfilled.
Description
Technical Field
The invention relates to a culture medium for separating and counting clostridium butyricum, belonging to the field of microbial separation and counting.
Background
Clostridium butyricum (Clostridium butyricum) belongs to the family of Bacillaceae, genus Clostridium, gram-positive, having spores, spore oval, eccentric or subterminal, and is resistant to adverse environments.
The clostridium butyricum is applied to feed as a feed additive, and can embody five biological characteristics in animal intestinal tracts: the feed additive can promote the proliferation and development of beneficial flora (bifidobacteria and lactobacilli) in the intestinal tract of animals, inhibit the growth and reproduction of harmful bacteria and putrefying bacteria in the intestinal tract, correct the disturbance of the flora in the intestinal tract and reduce the occurrence of enterotoxin; secondly, substances such as B vitamins, vitamin K, amylase and the like can be generated in the intestinal tracts of animals, so that the health-care function is realized; thirdly, the main metabolite of the clostridium butyricum, namely butyric acid, is a main nutrient substance for regeneration and repair of intestinal epithelial tissue cells; fourthly, anaerobic or facultative anaerobic bacillus is not affected by gastric acid, bile acid and the like; fifthly, the feed additive has stronger tolerance to various feed antibiotics and can be used in a compatible way. In addition, the clostridium butyricum is used as a new feed additive, and has important significance for reducing abuse of antibiotic products in the current feed, reducing residue of drugs in meat, reducing drug resistance of animal bacteria and guaranteeing animal health.
Although the research and development of clostridium butyricum just starts in China, the development is fast, and clostridium butyricum products appear. Since 1993, the Japanese imported butyric acid bacteria preparation Yimiaya BM tablets and Miyali Mulberry A granules are clinically applied in more than ten famous hospitals such as Shanghai, Guangzhou and Harbin in sequence, and have remarkable curative effects on treating enteritis, diarrhea, dyspepsia and other diseases caused by intestinal flora disturbance. The research of the researchers in China on the clostridium butyricum is mainly in clinical application, but the research is rarely applied as a feed additive.
In addition, clostridium butyricum can also be used as veterinary drugs, and because clostridium butyricum has resistance to various antibiotics such as streptomycin, the biological effect of clostridium butyricum is not affected when the clostridium butyricum is used in combination with the antibiotics, and the treatment effect can be enhanced. The resistance of clostridium butyricum to various antibiotics is of particular significance for the fact that clostridium butyricum still can exert the efficacy of clostridium butyricum under the condition that antibiotics are generally added into feed.
Clostridium butyricum has wide application in the fields of actual production and scientific research, such as the research of feed additives, microecological viable bacteria medicines, food additives, bacteriostatic agents and the like. In the field, clostridium butyricum often coexists with other bacteria such as escherichia coli, clostridium perfringens, lactic acid bacteria and the like, so that quantitative analysis of clostridium butyricum needs to be realized, but the current detection method of clostridium butyricum mainly refers to a clostridium perfringens culture medium, but the detection method has defects, the clostridium butyricum and the clostridium perfringens have unidentifiable consistency on the clostridium perfringens culture medium by naked eyes, so that a series of tedious biochemical identification or 16SrRNA homology comparison needs to be carried out at the later stage, and the method hardly has operability for enterprise inspectors, so that a method capable of effectively distinguishing clostridium butyricum from other bacteria needs to be provided on the market.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a culture medium for separating and counting clostridium butyricum, and the technical scheme for solving the technical problems is as follows:
a culture medium for separating and counting clostridium butyricum comprises 15.0g of tryptone, 3.0g of beef extract powder, 1.0g of yeast extract powder, 5.0g of sodium chloride, 0.5g of L-cysteine hydrochloride, 0.5g of sodium thioglycolate, 10.0g of xylose, 0.04g of bromothymol blue, 15.0g of agar powder, 900ml of purified water, 0.25g of D-cycloserine, 0.012g of kanamycin sulfate, 0.03g of cefoxitin, 10 million U of polymyxin sulfate B, 0.1g of neomycin sulfate and 25ml of yolk.
A method of using a medium for the isolation and enumeration of clostridium butyricum comprising the steps of:
(1) weighing tryptose peptone, beef extract powder, yeast extract powder, sodium chloride, L-cysteine hydrochloride, sodium thioglycolate, xylose, bromothymol blue, agar powder and purified water according to the formula, preparing under the conditions that the pH is 7.3-7.7 and the temperature is 25 ℃, sterilizing at 121 ℃ for 15 minutes, and preserving heat at 50-55 ℃ for later use;
(2) d-cycloserine, kanamycin sulfate, cefoxitin, polymyxin B sulfate and neomycin sulfate are respectively dissolved in 20ml of pure water according to the formula, and are filtered and sterilized for later use;
(3) adopting high-quality free-range egg yolk and carrying out aseptic treatment for later use;
(4) adding the sterilized materials obtained in the steps (2) and (3) into the sterilized materials obtained in the step (1), mixing, pouring the mixture on a flat plate, and placing the flat plate in an anaerobic incubator or an anaerobic bag at the temperature of 33-37 ℃ for anaerobic culture for 48-72 hours;
(5) if the plate is yellow, the surrounding has no double-ring characteristics, the plate is proved to be a clostridium butyricum colony, if the plate is yellow green to dark green (the shade of the color has a certain relation with the pH value of the formed colony and the bacterial content of other bacteria), the surrounding has double-ring characteristics, the plate is proved to be a clostridium perfringens colony, if the plate is yellow green to dark green, the surrounding has no double-ring characteristics, the plate is proved to be a clostridium barrageensis colony, and staphylococcus aureus is inhibited from growing; escherichia coli is inhibited from growing; bacillus subtilis is inhibited from growing.
Compared with the prior art, the invention has the beneficial effects that: according to the culture medium disclosed by the invention, a specific carbon source (xylose) is added to metabolize clostridium butyricum to generate acid, so that the alkalinity of the cultured acid is changed to present a special color; by effectively combining antibiotics and an antibacterial agent (D-cycloserine), the growth of most of clostridium butyricum except clostridium butyricum can be inhibited; by adding the yolk which is used as a special specific metabolic substrate, part of bacteria can generate lecithinase, and metabolize the yolk to generate special double-ring colony characteristics with a turbid inner layer and a transparent outer layer; the combination of L-cysteine hydrochloride and sodium thioglycolate can consume a small amount of oxygen remained in the culture medium, and properly control the anaerobic environment during culture. Finally, the specific physiological characteristics of the clostridium butyricum are analyzed, formula combination and adjustment are performed in a targeted manner, so that the clostridium butyricum can be obviously distinguished from other bacteria on visual observation, and the aim of separating and counting the clostridium butyricum is fulfilled; secondly, the effect is obvious, direct and effective, and the cost is low.
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FIG. 1 shows the growth of various colonies on the medium of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
A culture medium for separating and counting clostridium butyricum comprises 15.0g of tryptone, 3.0g of beef extract powder, 1.0g of yeast extract powder, 5.0g of sodium chloride, 0.5g of L-cysteine hydrochloride, 0.5g of sodium thioglycolate, 10.0g of xylose, 0.04g of bromothymol blue, 15.0g of agar powder, 900ml of purified water, 0.25g of D-cycloserine, 0.012g of kanamycin sulfate, 0.03g of cefoxitin, 10-myristyl B sulfate, 0.1g of neomycin sulfate and 25ml of yolk, wherein a specific carbon source (xylose) is added to ensure that the clostridium butyricum is metabolized to generate acid so as to cause the alkalinity change of cultured amino acid to show a special color, the effective combination of antibiotic and antibacterial agent (D-cycloserine) can inhibit the growth of most of clostridium butyricum except the clostridium butyricum, the yolk is used as a special specific metabolic substrate, and part of bacteria can generate egg phospholipase, the metabolism yolk generates the characteristics of a special double-ring colony with a turbid inner layer and a transparent outer layer, and the combined use of the L-cysteine hydrochloride and the sodium thioglycollate can consume a small amount of residual oxygen in a culture medium and properly control the anaerobic environment during culture.
A method of using a medium for the isolation and enumeration of clostridium butyricum comprising the steps of:
(1) weighing tryptose peptone, beef extract powder, yeast extract powder, sodium chloride, L-cysteine hydrochloride, sodium thioglycolate, xylose, bromothymol blue, agar powder and purified water according to the formula, preparing under the conditions that the pH is 7.3-7.7 and the temperature is 25 ℃, sterilizing at 121 ℃ for 15 minutes, and preserving heat at 50-55 ℃ for later use;
(2) d-cycloserine, kanamycin sulfate, cefoxitin, polymyxin B sulfate and neomycin sulfate are respectively dissolved in 20ml of pure water according to the formula, and are filtered and sterilized for later use;
(3) adopting high-quality free-range egg yolk and carrying out aseptic treatment for later use;
(4) adding the sterilized materials obtained in the steps (2) and (3) into the sterilized materials obtained in the step (1), mixing, pouring the mixed materials on a flat plate, and placing the flat plate into an anaerobic incubator or an anaerobic bag at the temperature of 33-37 ℃ for anaerobic culture for 48-72 hours;
(5) if the plate is yellow, the surrounding has no double-ring characteristics, the plate is proved to be a clostridium butyricum colony, if the plate is yellow green to dark green (the shade of the color has a certain relation with the pH value of the formed colony and the bacterial content of other bacteria), the surrounding has double-ring characteristics, the plate is proved to be a clostridium perfringens colony, if the plate is yellow green to dark green, the surrounding has no double-ring characteristics, the plate is proved to be a clostridium barrageensis colony, and staphylococcus aureus is inhibited from growing; escherichia coli is inhibited from growing; bacillus subtilis is inhibited from growing.
The number of bacterial colonies on the plate is not fixed, the plate only has six bacterial colonies in the experiment, staphylococcus aureus, escherichia coli and bacillus subtilis are all inhibited and cannot grow, only other three bacterial colonies need to be detected, the characteristics of the other three bacterial colonies are different and can be distinguished, the types of the bacterial colonies on the plate can be eight, the number of the bacterial colonies is not fixed, only the bacterial colonies of clostridium butyricum are yellow, the two-ring characteristic is not formed around the bacterial colonies, and the bacterial colonies of clostridium butyricum can be separated through the characteristic.
The picture about the growth of the strain is shown in the attached figure of the specification.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (2)
1. A medium for the isolation and enumeration of clostridium butyricum, comprising: consists of 15.0g of tryptose peptone, 3.0g of beef extract powder, 1.0g of yeast extract powder, 5.0g of sodium chloride, 0.5g of L-cysteine hydrochloride, 0.5g of sodium thioglycolate, 10.0g of xylose, 0.04g of bromothymol blue, 15.0g of agar powder, 900ml of purified water, 0.25g of D-cycloserine, 0.012g of kanamycin sulfate, 0.03g of cefoxitin, 10 million U of polymyxin sulfate B, 0.1g of neomycin sulfate and 25ml of yolk.
2. A method of using the medium according to claim 1 for the isolation and enumeration of clostridium butyricum, wherein the medium comprises: the method comprises the following steps:
(1) the formula of claim 1, wherein the tryptose peptone, beef extract powder, yeast extract powder, sodium chloride, L-cysteine hydrochloride, sodium thioglycolate, xylose, bromothymol blue, agar powder and purified water are weighed as required, prepared at a pH of 7.3-7.7 and a temperature of 25 ℃, sterilized at a temperature of 121 ℃ for 15 minutes, and finally kept at a temperature of 50-55 ℃ for later use;
(2) dissolving D-cycloserine, kanamycin sulfate, cefoxitin, polymyxin B sulfate and neomycin sulfate in 20ml of pure water respectively according to claim 1, and filtering and sterilizing for later use;
(3) adopting high-quality free-range egg yolk and carrying out aseptic treatment for later use;
(4) adding the sterilized materials obtained in the steps (2) and (3) into the sterilized materials obtained in the step (1), mixing, pouring the mixture on a flat plate, and placing the flat plate in an anaerobic incubator or an anaerobic bag at the temperature of 33-37 ℃ for anaerobic culture for 48-72 hours;
(5) if the plate is yellow, the periphery of the plate has no double-ring characteristics, the plate is proved to be a clostridium butyricum colony, if the plate is yellow green to dark green, the periphery of the plate has double-ring characteristics, the plate is proved to be a clostridium perfringens colony, if the plate is yellow green to dark green, the periphery of the plate has no double-ring characteristics, the plate is proved to be a clostridium barrageensis colony, and staphylococcus aureus is inhibited from growing; escherichia coli is inhibited from growing; bacillus subtilis is inhibited from growing.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112481351A (en) * | 2020-12-16 | 2021-03-12 | 湖北华扬科技发展有限公司 | Method for counting spores of clostridium butyricum in composite microecological product |
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| CN117683847A (en) * | 2024-01-31 | 2024-03-12 | 山东拓普生物工程有限公司 | Bacillus megaterium counting agar |
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| CN112481351A (en) * | 2020-12-16 | 2021-03-12 | 湖北华扬科技发展有限公司 | Method for counting spores of clostridium butyricum in composite microecological product |
| CN112481351B (en) * | 2020-12-16 | 2024-03-08 | 湖北华扬科技发展有限公司 | Counting method of clostridium butyricum spores in composite microecological product |
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