CN110819571A - Columbia agar plate and preparation method thereof - Google Patents
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- CN110819571A CN110819571A CN201911185371.0A CN201911185371A CN110819571A CN 110819571 A CN110819571 A CN 110819571A CN 201911185371 A CN201911185371 A CN 201911185371A CN 110819571 A CN110819571 A CN 110819571A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
A Columbia agar plate relates to the technical field of culture medium, which contains various substances such as animal peptone, soybean peptone, yeast extract powder, corn starch, sodium chloride, defibrinated sheep blood, agar and the like. The Columbia plate agar has more comprehensive nutrition, can effectively promote the growth of fastidious bacteria, and improves the separation rate of bacteria. A method for preparing Columbia agar plate, which dissolves maize starch which is not easy to dissolve at high temperature separately, ensures the dissolving efficiency, and avoids the nutrition of other components from being damaged by high temperature. In addition, after the culture solution is sterilized at high temperature, the temperature is firstly reduced to 80-90 ℃, the culture solution is fully exhausted, and bubbles are prevented from being formed in the rapid cooling process. Meanwhile, the defibered sheep blood is fully preheated before being added, so that the problem of agar agglomeration caused by too large temperature difference between the defibered sheep blood and the culture solution is avoided. The method is simple to operate, has low requirements on equipment, can effectively improve the product quality, and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of culture media, and particularly relates to a Columbia agar plate and a preparation method thereof.
Background
Pathogenic bacteria or conditional pathogenic bacteria exist in an infected part to cause common clinical infections, such as respiratory tract infection, genital tract infection, intestinal tract infection and the like, in order to accurately diagnose the bacterial types of the infected part, a patient part is usually adopted to collect samples for culture clinically, and a clinical laboratory technician selects suspicious bacterial strain types for identification according to clinical symptoms, so that the types of the infected bacteria of the patient part are judged, and a basis is provided for clinical treatment. Diagnosis and culture of infected bacterial types at affected parts are the only feasible method in clinical diagnosis at present, and other methods such as immune antigen detection and nucleic acid detection are not suitable for direct detection and judgment of diseases with multiple bacterial types at affected parts. Clinically, a solid culture medium, a liquid culture medium or a selective culture medium is usually adopted to separate suspicious strains so as to carry out further clinical identification. The blood agar plate is prepared by an artificial method, is sterilized and is added with defibered sheep blood, can culture bacteria with higher nutrient content and observe hemolysis, and judges the characteristics of the bacteria, particularly the hemolysis of streptococcus bacteria, by observing the hemolysis.
The Columbia agar culture medium is improved on the basis of blood agar culture medium, and is used for culturing fastidious bacteria and other bacteria with high nutrition requirements. However, the conventional Columbia agar is not completely uniform in nutrient components, and although fastidious bacteria can be cultured, the culture effect is not excellent, and it is difficult to satisfy more and more strict detection. In addition, the existing preparation method of Columbia agar is more suitable for small-scale self-production, and the conditions of agglomeration, bubble inclusion and the like can occur during large-scale production, so that the product quality is influenced.
Disclosure of Invention
The invention aims to provide a Columbia agar plate which has more comprehensive nutrition, can effectively promote the growth of fastidious bacteria and improve the separation rate of the bacteria.
The invention also aims to provide a preparation method of the Columbia agar plate, which has simple operation and low requirement on equipment, can effectively avoid the problems of agglomeration and air bubbles in the preparation process, improves the product quality and is suitable for industrial production.
The embodiment of the invention is realized by the following steps:
a Columbia agar plate is prepared by mixing culture solution and defibrinated sheep blood, and cooling and solidifying; wherein, by weight, the culture solution comprises:
8-12 parts of animal peptone, 2-5 parts of soybean peptone, 1-3 parts of corn starch, 4-6 parts of sodium chloride, 2-4 parts of yeast extract powder, 13-18 parts of agar and 900-1000 parts of water;
the volume ratio of the culture solution to the defibered sheep blood is 1: 0.04 to 0.06.
A preparation method of the Columbia agar plate comprises the following steps:
mixing animal peptone, soybean peptone, sodium chloride, yeast extract powder and agar with part of water, and heating to 80-90 ℃ for dissolution to obtain a premixed solution A;
mixing the corn starch with the rest part of water, and heating to 130-135 ℃ for dissolving to obtain a premixed solution B;
mixing the premixed solution A and the premixed solution B to obtain a culture solution;
sterilizing the culture solution at 120-125 ℃ for 20-40 min;
cooling the sterilized culture solution to 80-90 ℃, continuing to heat for 5-10 min, and cooling to 40-45 ℃;
and uniformly mixing the preheated defibered sheep blood with the cooled culture solution, pouring the mixture into a plate, and further cooling to obtain a Columbia agar plate.
The embodiment of the invention has the beneficial effects that:
the embodiment of the invention provides a Columbia agar plate which contains various substances such as animal peptone, soybean peptone, yeast extract powder, corn starch, sodium chloride, defibered sheep blood, agar and the like. Wherein, the animal peptone, the soybean peptone, the yeast extract powder and the corn starch provide proper carbon source and nitrogen source for the microorganism; the defibered sheep blood contains rich nutritional factors, promotes the growth of common bacteria, and can observe the hemolysis condition of the bacteria; sodium chloride maintains the balance of the osmotic pressure of the bacteria; agar is the medium coagulant. The Columbia agar plate has comprehensive nutrition, and can effectively promote the growth of fastidious bacteria and improve the separation rate of bacteria.
The embodiment of the invention also provides a preparation method of the Columbia agar plate, which separately dissolves the maize starch which is not easy to dissolve at high temperature, thereby ensuring the dissolving efficiency and avoiding the nutrient formation damage of other components caused by high temperature. In addition, after the culture solution is sterilized at high temperature, the temperature is firstly reduced to 80-90 ℃, the culture solution is fully exhausted, and bubbles are prevented from being formed in the rapid cooling process. Meanwhile, the defibered sheep blood is fully preheated before being added, so that the problem of agar agglomeration caused by too large temperature difference between the defibered sheep blood and the culture solution is avoided. The method is simple to operate, has low requirements on equipment, can effectively improve the product quality, and is suitable for industrial production.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The Columbia agar plate and the preparation method thereof according to the embodiment of the present invention will be described in detail below.
The embodiment of the invention provides a Columbia agar plate which is obtained by mixing a culture solution and defibered sheep blood and then cooling and solidifying. Wherein, by weight, the culture solution comprises:
8-12 parts of animal peptone, 2-5 parts of soybean peptone, 1-3 parts of corn starch, 4-6 parts of sodium chloride, 2-4 parts of yeast extract powder, 13-18 parts of agar and 900-1000 parts of water;
the volume ratio of the culture solution to the defibered sheep blood is 1: 0.04 to 0.06.
The animal peptone, the soybean peptone, the yeast extract powder and the corn starch provide proper carbon sources and nitrogen sources for microorganisms, and the substances comprise plant protein and animal protein, so that more abundant amino acid types can be provided for the growth of bacteria. The corn starch is rich in sugar and can provide more sufficient nutrition for the growth of fastidious bacteria. The sodium chloride can maintain the balance of the osmotic pressure of the bacteria and ensure the growth of the bacteria. Agar is the medium coagulant to form a solid phase medium. The Columbia agar plate has comprehensive nutrition, and can effectively promote the growth of fastidious bacteria and improve the separation rate of bacteria.
Preferably, the culture solution comprises the following components in parts by weight:
9-11 parts of animal peptone, 2-4 parts of soybean peptone, 1-3 parts of corn starch, 4-6 parts of sodium chloride, 2-4 parts of yeast extract powder, 14-16 parts of agar and 950-1000 parts of water.
More preferably, the culture solution comprises the following components in parts by weight:
10 parts of animal peptone, 3 parts of soybean peptone, 2 parts of corn starch, 5 parts of sodium chloride, 3 parts of yeast extract powder, 15 parts of agar and 1000 parts of water.
Furthermore, defibered sheep blood not only contains abundant nutritional factors and can promote the growth of common bacteria, but also can be used for observing the hemolysis condition of the bacteria. In this example, the volume ratio of the culture solution to defibrinated sheep blood was 1: 0.04 to 0.06. Preferably, the volume ratio of the culture solution to the defibered sheep blood is 1: 0.05.
the embodiment of the invention also provides a preparation method of the Columbia agar plate, which comprises the following steps:
s1, mixing animal peptone, soybean peptone, sodium chloride, yeast extract powder and agar with part of water, and heating to 80-90 ℃ to dissolve to obtain a premixed solution A.
And S2, mixing the corn starch with the rest part of water, and heating to 130-135 ℃ to dissolve to obtain a premixed solution B.
And S3, mixing the premix solution A and the premix solution B to obtain a culture solution.
S4, sterilizing the culture solution at 120-125 ℃ for 20-40 min.
S5, cooling the sterilized culture solution to 80-90 ℃, continuing to heat for 5-10 min, and cooling to 40-45 ℃;
s6, uniformly mixing the preheated defibered sheep blood with the cooled culture solution, pouring the mixture into a dish, and further cooling to obtain a Columbia agar plate.
Further, animal peptone, soybean peptone, yeast extract, corn starch provide suitable carbon and nitrogen sources for microorganisms. Meanwhile, the corn starch is rich in sugar, so that nutrition is provided for the growth of fastidious bacteria. Sodium chloride can maintain the balance of the osmotic pressure of the bacteria. And agar is the medium coagulant. At normal temperature, the substances are not easy to dissolve, and in order to facilitate the dissolution of the substances and form a uniform solution, the substances can be properly heated during mixing, the heating temperature is preferably 80-90 ℃, and at the temperature, the dissolving efficiency of the substances can be ensured, and the nutrient substances can be prevented from being damaged at high temperature. Meanwhile, the corn starch is difficult to dissolve, and if the corn starch and other materials are dissolved together at the temperature of 80-90 ℃, the dissolving time is longer, and the efficiency is low. If the dissolving temperature is increased, the nutrition of other raw materials is easily destroyed at high temperature. To this end, the applicant dissolved corn starch alone and mixed it with other raw materials.
Preferably, the culture solution is sterilized at 120-125 ℃ for 20-40 min. The sterilization temperature and time have great influence on the quality of the product, and if the sterilization temperature is not enough or the sterilization time is not enough, mixed bacteria residue can be caused, and the use effect of the Columbia agar plate is influenced.
After sterilization, the culture solution needs to be cooled and then mixed with defibered sheep blood to avoid the breakage of sheep blood cells and the damage of nutrition. In the traditional process, the temperature of the culture solution is usually directly reduced to about 40 ℃. The temperature is closer to the body temperature of sheep, and has smaller influence on sheep blood. However, in the industrial production, because the agar has a large consumption, if the one-time cooling mode is directly adopted, air bubbles are easily mixed in the agar, thereby affecting the quality of the product. Therefore, in the process of cooling, the culture solution is firstly cooled to 80-90 ℃, and the air is exhausted for 5-10 min at the temperature, so that the problem is avoided.
Furthermore, in the industrial production, the dosage of the defibered sheep blood is large, and when the defibered sheep blood is directly mixed with the culture solution, the temperature of the defibered sheep blood is low, so that the culture solution contacted with the defibered sheep blood is coagulated, and a large lump is formed. In contrast, in the preparation method, the defibered sheep blood is preheated to 38-40 ℃ and then mixed with the cooled culture solution, so as to solve the problems.
Optionally, the pH of the culture solution is adjusted to 7.0-7.5 before sterilization. The proper pH value is beneficial to the growth of bacteria and improves the separation rate of the bacteria.
Further, the Columbia agar plate is cultured for 24-36 h at 30-35 ℃ for inspection. Only after the culture, the Columbia agar plate without the growth of mixed bacteria can be determined as a qualified product, and can be sealed and put in storage.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
This example provides a Columbia agar plate obtained by mixing a culture solution with defibrinated sheep blood and then cooling and solidifying the mixture. Wherein, by weight, the culture solution comprises:
12 parts of animal peptone, 2 parts of soybean peptone, 4 parts of sodium chloride, 4 parts of yeast extract powder, 1 part of corn starch, 13 parts of agar and 1000 parts of water;
the volume ratio of the culture solution to the defibered sheep blood is 1: 0.06.
the preparation method of the Columbia agar plate comprises the following steps:
s1, mixing animal peptone, soybean peptone, sodium chloride, yeast extract powder, agar and 60% water, and heating to 90 ℃ to dissolve to obtain premix A.
S2, mixing the corn starch with 40% of water, and heating to 135 ℃ to dissolve to obtain a premix B.
And S3, mixing the premix solution A and the premix solution B to obtain a culture solution.
S4, sterilizing the culture solution at 120 ℃ for 40 min.
S5, cooling the sterilized culture solution to 90 ℃, continuing to heat for 5 min, and cooling to 40 ℃;
s6, uniformly mixing the defibered sheep blood preheated to 40 ℃ with the cooled culture solution, pouring the mixture into a dish, and further cooling to obtain a Columbia agar plate.
And S7, after the plate is cooled and solidified, packaging after the water in the plate is steamed in a ten-thousand-level workshop.
And S8, sealing the film, sealing and packaging the film by using a PVC film, and then placing the packaged film on a dish, labeling and placing the dish into a warehouse to be detected.
Example 2
This example provides a Columbia agar plate obtained by mixing a culture solution with defibrinated sheep blood and then cooling and solidifying the mixture. Wherein, by weight, the culture solution comprises:
8 parts of animal peptone, 4 parts of soybean peptone, 6 parts of sodium chloride, 2 parts of yeast extract powder, 3 parts of corn starch, 18 parts of agar and 900 parts of water;
the volume ratio of the culture solution to the defibered sheep blood is 1: 0.04.
the preparation method of the Columbia agar plate comprises the following steps:
s1, mixing animal peptone, soybean peptone, sodium chloride, yeast extract powder, agar and 70% water, and heating to 80 ℃ to dissolve to obtain premix A.
S2, mixing the corn starch with 30% of water, and heating to 130 ℃ to dissolve to obtain a premix B.
And S3, mixing the premix solution A and the premix solution B to obtain a culture solution.
S4, sterilizing the culture solution at 125 ℃ for 20 min.
S5, cooling the sterilized culture solution to 80 ℃, continuing to heat for 10min, and cooling to 45 ℃;
s6, uniformly mixing the defibered sheep blood preheated to 38 ℃ with the cooled culture solution, pouring the mixture into a dish, and further cooling to obtain a Columbia agar plate.
And S7, after the plate is cooled and solidified, packaging after the water in the plate is steamed in a ten-thousand-level workshop.
And S8, sealing the film, sealing and packaging the film by using a PVC film, and then placing the packaged film on a dish, labeling and placing the dish into a warehouse to be detected.
Example 3
This example provides a Columbia agar plate obtained by mixing a culture solution with defibrinated sheep blood and then cooling and solidifying the mixture. Wherein, by weight, the culture solution comprises:
10 parts of animal peptone, 3 parts of soybean peptone, 5 parts of sodium chloride, 3 parts of yeast extract powder, 2 parts of corn starch, 15 parts of agar and 1000 parts of water;
the volume ratio of the culture solution to the defibered sheep blood is 1: 0.05.
the preparation method of the Columbia agar plate comprises the following steps:
s1, mixing animal peptone, soybean peptone, sodium chloride, yeast extract powder, agar and 70% water, and heating to 90 ℃ to dissolve to obtain premix A.
S2, mixing the corn starch with 30% of water, and heating to 130 ℃ to dissolve to obtain a premix B.
And S3, mixing the premix solution A and the premix solution B to obtain a culture solution.
S4, sterilizing the culture solution at 121 ℃ for 30 min.
S5, cooling the sterilized culture solution to 90 ℃, continuing to heat for 5 min, and cooling to 40 ℃;
s6, uniformly mixing the defibered sheep blood preheated to 38 ℃ with the cooled culture solution, pouring the mixture into a dish, and further cooling to obtain a Columbia agar plate.
And S7, after the plate is cooled and solidified, packaging after the water in the plate is steamed in a ten-thousand-level workshop.
And S8, sealing the film, sealing and packaging the film by using a PVC film, and then placing the packaged film on a dish, labeling and placing the dish into a warehouse to be detected.
Test examples
Bacterial culture tests were performed and the growth index (GI value) was evaluated using the colombia agar plates of examples 1 to 3, and a commercially available colombia agar plate of a certain company (comparative example 1) and a commercially available blood agar plate of a certain company (comparative example 2), under the following specific test conditions:
test strains: ATCC49247 and ATCC49226 are standard strains of clinical examination center of Ministry of health, and the rest 3 strains of bacteria are obtained by separating from clinical specimens and determining strains by API card reexamination.
GI value test method: 29 known strains of bacteria were simultaneously inoculated into Columbia agar plates according to examples 1 to 3 and comparative examples, respectively, in the following manner, and the pure bacteria cultured for 18 hours were used as a direct saline suspension (in which Streptococcus was enriched with serum broth) at a concentration of 0.5 M.turbidity as 1: 10 serial dilutions were made for a total of 8 times. 0.1 ml/dish of bacterial liquid of each concentration of each strain is uniformly coated/inoculated on a Columbia agar plate with 5% CO2And (3) after 24 hours of incubation at 35 ℃ in an incubator or a common incubator, counting the number of colonies in a plate with less than 10 colonies, measuring the diameter of the colonies, calculating the average diameter of the colonies, and multiplying the average diameter by the logarithm of the dilution of the strain liquid in the plate to obtain the GI value.
The calculation results are shown in table 1.
TABLE 1 GI value test results
| Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | |
| Haemophilus influenzae | 8.1 | 8.0 | 8.9 | 5.7 | 0 |
| Oral slimy fungus | 8.8 | 8.6 | 9.2 | 5.6 | 0 |
| Gonococci | 9.2 | 8.0 | 9.6 | 5.0 | 0 |
| Pseudotuberculosis of corynebacterium | 8.7 | 8.4 | 9.1 | 6.2 | 0 |
| Coryneform bacterium | 8.3 | 8.1 | 8.7 | 5.8 | 0 |
As can be seen from Table 1, the Columbia agar plates of examples 1 to 3 and comparative example 1 all grew smoothly in the culture of 5 strains, while the Columbia agar plates of examples 1 to 3 all had better growth indexes than those of commercially available Columbia agar plates. However, commercially available blood agar plates cannot grow for the fastidious bacteria. It is fully demonstrated that the Columbia agar plate provided by the embodiment of the present invention has better effect on the culture of bacteria.
In summary, the embodiments of the present invention provide a columbia agar plate, which contains various substances such as animal peptone, soybean peptone, yeast extract powder, corn starch, sodium chloride, defibrinated sheep blood, agar, and the like. Wherein, the animal peptone, the soybean peptone, the yeast extract powder and the corn starch provide proper carbon source and nitrogen source for the microorganism; the defibered sheep blood contains rich nutritional factors, promotes the growth of common bacteria, and can observe the hemolysis condition of the bacteria; sodium chloride maintains the balance of the osmotic pressure of the bacteria; agar is the medium coagulant. The Columbia agar plate has comprehensive nutrition, and can effectively promote the growth of fastidious bacteria and improve the separation rate of bacteria.
The embodiment of the invention also provides a preparation method of the Columbia agar plate, which separately dissolves the maize starch which is not easy to dissolve at high temperature, thereby ensuring the dissolving efficiency and avoiding the nutrient formation damage of other components caused by high temperature. In addition, after the culture solution is sterilized at high temperature, the temperature is firstly reduced to 80-90 ℃, the culture solution is fully exhausted, and bubbles are prevented from being formed in the rapid cooling process. Meanwhile, the defibered sheep blood is fully preheated before being added, so that the problem of agar agglomeration caused by too large temperature difference between the defibered sheep blood and the culture solution is avoided. The method is simple to operate, has low requirements on equipment, can effectively improve the product quality, and is suitable for industrial production.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A Columbia agar plate is characterized in that the Columbia agar plate is obtained by mixing a culture solution and defibered sheep blood and then cooling and solidifying the mixture; wherein, by weight, the culture solution comprises:
8-12 parts of animal peptone, 2-5 parts of soybean peptone, 1-3 parts of corn starch, 4-6 parts of sodium chloride, 2-4 parts of yeast extract powder, 13-18 parts of agar and 900-1000 parts of water;
the volume ratio of the culture solution to the defibered sheep blood is 1: 0.04 to 0.06.
2. The colombia agar plate of claim 1, wherein the culture fluid comprises, in parts by weight:
9-11 parts of animal peptone, 2-4 parts of soybean peptone, 1-3 parts of corn starch, 4-6 parts of sodium chloride, 2-4 parts of yeast extract powder, 14-16 parts of agar and 950-1000 parts of water.
3. The colombia agar plate of claim 2, wherein the culture fluid comprises, in parts by weight:
10 parts of the animal peptone, 3 parts of the soybean peptone, 2 parts of the corn starch, 5 parts of the sodium chloride, 3 parts of the yeast extract powder, 15 parts of the agar and 1000 parts of the water.
4. The colombia agar plate of claim 1, wherein the volume ratio of the culture fluid to the defibered sheep blood is 1: 0.05.
5. a method for preparing a Columbia agar plate according to any one of claims 1 to 4, comprising:
mixing the animal peptone, the soybean peptone, the sodium chloride, the yeast extract powder, the agar and part of the water, and heating to 80-90 ℃ to dissolve to obtain a premixed solution A;
mixing the corn starch with the rest part of the water, and heating to 130-135 ℃ for dissolving to obtain a premixed solution B;
mixing the premixed solution A and the premixed solution B to obtain the culture solution;
sterilizing the culture solution at 120-125 ℃ for 20-40 min;
cooling the sterilized culture solution to 80-90 ℃, continuing to heat for 5-10 min, and cooling to 40-45 ℃;
and uniformly mixing the preheated defibered sheep blood with the cooled culture solution, pouring the mixture into a dish, and further cooling to obtain the Columbia agar plate.
6. The method of claim 5, wherein the water used to dissolve the corn starch is used in an amount of 30% to 40% of the total amount of water.
7. The method according to claim 6, wherein the defibered sheep blood is preheated to 38 to 40 ℃ and then mixed with the cooled culture solution.
8. The method according to claim 7, wherein the pH of the culture medium is adjusted to 7.0 to 7.5 before sterilization.
9. The method according to claim 8, wherein the Columbia agar plate is incubated at 30 to 35 ℃ for 24 to 36 hours for examination.
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