CN108277257B - Culture medium - Google Patents
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- CN108277257B CN108277257B CN201710600643.3A CN201710600643A CN108277257B CN 108277257 B CN108277257 B CN 108277257B CN 201710600643 A CN201710600643 A CN 201710600643A CN 108277257 B CN108277257 B CN 108277257B
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Abstract
The invention relates to the field of bioengineering, and particularly relates to a culture medium. The culture medium is prepared from extract of ox heart, extract of ox brain, cysteine, and Na2HPO4Peptone, NaCl and agar powder. The culture sensitivity of the HP can reach 98.88%, the specificity is 100%, the defects of the prior art are greatly improved, and the application of the HP culture method is improved.
Description
Technical Field
The invention relates to the field of bioengineering, and particularly relates to a culture medium.
Background
Gastritis (gastrolitis) is a gastric mucositis caused by various causes and is one of the most common digestive diseases. According to the clinical onset of the disease, the disease can be generally divided into two types of acute gastritis and chronic gastritis; it can be classified into helicobacter pylori-associated gastritis, stress gastritis, autoimmune gastritis, etc. according to the cause of disease. Gastritis, which is caused by different etiologies, varies in its pathological and histological changes, and generally includes three processes, namely epithelial injury, mucosal inflammatory response, and epithelial regeneration. Acute gastritis can be classified into simple gastritis, erosive gastritis, purulent gastritis, etc. according to its pathological changes, and chronic gastritis can be classified into non-atrophic gastritis, atrophic gastritis and special gastritis. The diagnosis and differential diagnosis of various types of gastritis are mainly based on gastroscopy. Wherein, the main pathogenic bacteria of the chronic gastritis is helicobacter pylori, and more than 90 percent of patients with the chronic gastritis are infected by the helicobacter pylori.
Helicobacter pylori is a pathogenic bacterium that colonizes the gastric mucosa, with infection rates as high as 50-70% worldwide. HP infection is the major cause of chronic gastritis. Meanwhile, long-term HP infection can cause severe atrophic gastritis and accompanying intestinal metaplasia, and can also cause gastric cancer seriously. Current assays for HP infection include a variety of methods: histopathology and immunohistochemistry, rapid urease assay, tissue bacteria culture, urea breath assay, fecal HP antigen detection, and serological antibody detection, but there is no absolute gold standard detection method.
The culture of tissue bacteria is still an effective method for directly judging HP infection, and due to the high specificity and the important role of the tissue bacteria in the guidance research of antibiotic resistance, the tissue bacteria occupies an important position in the research of HP infection. The culture of helicobacter pylori (h. pyrori, HP) is an important technical means for diagnosing whether helicobacter pylori is infected, but at present, the method is limited by technology, the culture positive rate of HP is low, the sensitivity is not enough, however, the most defect of the culture of HP at present is low sensitivity, the positive rate of the culture is only 50-70%, even under the sampling and culture of a very experienced laboratory and an operator, the positive rate can be increased to 70-90% only by a marginal way (the standard of taking immunohistochemistry as a reference).
Disclosure of Invention
In view of the above, the present invention provides a culture medium. The invention mainly aims at the defect of low culture positive rate, prepares a dominant culture medium, ensures that the culture positive rate of the HP reaches 98.88 percent, has 100 percent of specificity, greatly improves the defects of the prior art and improves the application of the HP culture method.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a basic culture medium, which is prepared from bovine heart extract, bovine brain extract, cysteine and Na2HPO4Peptone, NaCl and agar powder.
In some embodiments, cysteine, Na, in g/g/g/g/g/mL/mL in the basal medium2HPO4The mass volume ratio of peptone, NaCl and agar to the bovine heart extract and the bovine brain extract is (1-5) to (10-12) to (4-8) to (0.5-2) to (25-30) to 250 to 200.
In some embodiments, each 1000mL agar plate contains:
in some embodiments, each 1000mL agar plate contains:
the invention also provides application of the basic culture medium in culturing helicobacter pylori.
The invention also provides a complete culture medium, which comprises the basic culture medium, antibiotics and animal blood.
In some embodiments of the invention, the antibiotic comprises one or a mixture of two or more of vancomycin, trimethoprim, amphotericin, or polymyxin.
In some embodiments of the invention, the mass ratio of cysteine to vancomycin, trimethoprim, amphotericin to polymyxin is (1000-5000): (9-12): (3-6).
The invention also provides the application of the complete culture medium in culturing helicobacter pylori.
The invention also provides a preparation method of the complete culture medium, which comprises the following steps:
step 1: taking bovine heart extract, bovine brain extract, cysteine, peptone, sodium chloride and disodium hydrogen phosphate according to the formula amount, mixing with water, heating for dissolving, filtering, collecting filtrate, adjusting the pH value to 7.1-7.3, subpackaging, sterilizing, cooling and sealing to obtain a basic culture medium;
step 2: taking the basic culture medium prepared in the step 1, adding agar powder with the formula amount, and sterilizing;
and step 3: cooling to 45-50 ℃, and adding antibiotics with the formula amount;
and 4, step 4: adding 10% (V/V) animal blood (preferably fresh sheep blood), mixing, pouring into flat plate, cooling, solidifying, and sealing.
Specifically, the preparation method of the complete culture medium comprises the following steps:
mixing, heating to dissolve, filtering with filter paper, adjusting pH to 7.2 + -0.1, packaging into 800 mL/bottle, autoclaving in high pressure steam sterilizer at 121 deg.C for 20min, cooling to room temperature, sealing, and storing at 4 deg.C.
When preparing the plate:
1) taking a certain amount of the basal culture medium stored at the temperature of more than 4 ℃, adding 25-30 g of agar powder into every 1L of the basal culture medium, and then sterilizing the basal culture medium for 20min at the temperature of 121 ℃;
2) when the temperature of the culture medium is reduced to about 45-50 ℃, adding stock solutions of various antibiotics according to the concentration of adding 9-12 mg of vancomycin, 3-6 mg of amphotericin, 3-6 mg of polymyxin and 3-6 mg of methoxyampicillin pyrimidine into every 1L of the culture medium;
3) adding fresh sheep blood according to the amount of adding 100mL of fresh sheep blood in each 1L of culture medium;
4) fully and uniformly mixing, and then pouring the mixture into a flat plate; (each plate with a diameter of 90mm needs about 20mL of culture medium, care is taken to avoid air bubbles, the plate is horizontally placed after pouring, and the action is rapid so that the plate cannot be poured after the liquid culture medium is solidified.)
5) After the flat plate is cooled and solidified, the flat plate is put into a sterile sealing bag, sealed in vacuum and stored at 4 ℃.
The detection method comprises the following steps:
1. preparation before the test: the desired medium was removed and allowed to return to room temperature (18 ℃ -25 ℃).
2. Under the direct vision of an endoscope, gastric mucosa tissues at different parts are clamped.
3. Directly coating and inoculating the removed gastric mucosa tissue surface to an HP selective separation culture medium on a super clean bench.
4. Placing the culture medium in an anaerobic jar, slowly injecting mixed gas (5% O)2、10％CO2、85％N2) Continuously culturing for 5-7 days at 37 ℃;
5. the colony morphology was observed.
Preferably, the preparation method of the complete culture medium comprises the following steps:
mixing, heating to dissolve, filtering with filter paper, adjusting pH to 7.2 + -0.1, packaging into 800 mL/bottle, autoclaving in high pressure steam sterilizer at 121 deg.C for 20min, cooling to room temperature, sealing, and storing at 4 deg.C.
When preparing the plate:
6) taking a certain amount of basal culture medium stored at 4 deg.C, adding agar powder in an amount of 25g per 1L, and autoclaving at 121 deg.C for 20 min;
7) when the temperature of the culture medium is reduced to about 45-50 ℃, adding stock solutions of various antibiotics according to the concentration of 10mg of vancomycin, 5mg of amphotericin, 5mg of polymyxin and 5mg of methoxyampicillin per 1L of the culture medium;
8) adding fresh sheep blood according to the amount of adding 100mL of fresh sheep blood in each 1L of culture medium;
9) fully and uniformly mixing, and then pouring the mixture into a flat plate; (each plate with a diameter of 90mm needs about 20mL of culture medium, care is taken to avoid air bubbles, the plate is horizontally placed after pouring, and the action is rapid so that the plate cannot be poured after the liquid culture medium is solidified.)
10) After the flat plate is cooled and solidified, the flat plate is put into a sterile sealing bag, sealed in vacuum and stored at 4 ℃.
The detection method comprises the following steps:
1. preparation before the test: the desired medium was removed and allowed to return to room temperature (18 ℃ -25 ℃).
2. Under the direct vision of an endoscope, gastric mucosa tissues at different parts are clamped.
3. Directly coating and inoculating the removed gastric mucosa tissue surface to an HP selective separation culture medium on a super clean bench.
4. Placing the culture medium in an anaerobic jar, slowly injecting mixed gas (5% O)2、10％CO2、85％N2) Continuously culturing for 5-7 days at 37 ℃;
5. the colony morphology was observed.
Aiming at the defect of low positive rate of helicobacter pylori culture, the invention prepares a culture medium which is prepared from bovine heart extract, bovine brain extract, cysteine and Na2HPO4, peptone, NaCl and agar powder, and adding proper amount of antibiotic and fresh sheep blood. The culture sensitivity of HP can reach 98.88%, the specificity is 100%, and the prior art is greatly improvedThe disadvantages of the technology improve the application of the HP culture method.
Detailed Description
The invention discloses a culture medium, which can be realized by appropriately improving process parameters by a person skilled in the art with reference to the content in the specification. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The raw materials and reagents used in the culture medium provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
The formula of the culture medium for culturing the helicobacter pylori is as follows:
per 1000mL agar plate contain:
the preparation method comprises the following specific steps:
according to the formula:
mixing, heating to dissolve, filtering with filter paper, adjusting pH to 7.2 + -0.1, packaging into 800 mL/bottle, autoclaving in high pressure steam sterilizer at 121 deg.C for 20min, cooling to room temperature, sealing, and storing at 4 deg.C.
When preparing the plate:
1) taking a certain amount of basal culture medium stored at 4 deg.C, adding agar powder in an amount of 25g per 1L, and autoclaving at 121 deg.C for 20 min;
2) when the temperature of the culture medium is reduced to about 45-50 ℃, adding stock solutions of various antibiotics according to the concentration of adding 12mg of vancomycin, 6mg of amphotericin, 3mg of polymyxin and 3mg of methoxyampicillin pyrimidine into every 1L of the culture medium;
3) adding fresh sheep blood according to the amount of adding 100mL of fresh sheep blood in each 1L of culture medium;
4) fully and uniformly mixing, and then pouring the mixture into a flat plate; (each plate with a diameter of 90mm needs about 20mL of culture medium, care is taken to avoid air bubbles, the plate is horizontally placed after pouring, and the action is rapid so that the plate cannot be poured after the liquid culture medium is solidified.)
5) After the flat plate is cooled and solidified, the flat plate is put into a sterile sealing bag, sealed in vacuum and stored at 4 ℃.
Example 2
The formula of the culture medium for culturing the helicobacter pylori is as follows:
per 1000mL agar plate contain:
the preparation method comprises the following specific steps:
according to the formula:
mixing, heating to dissolve, filtering with filter paper, adjusting pH to 7.2 + -0.1, packaging into 800 mL/bottle, autoclaving in high pressure steam sterilizer at 121 deg.C for 20min, cooling to room temperature, sealing, and storing at 4 deg.C.
When preparing the plate:
1) taking a certain amount of basal culture medium stored at 4 deg.C, adding agar powder in an amount of 25g per 1L, and autoclaving at 121 deg.C for 20 min;
2) when the temperature of the culture medium is reduced to about 45-50 ℃, adding stock solutions of various antibiotics according to the concentration of 10mg of vancomycin, 5mg of amphotericin, 5mg of polymyxin and 5mg of methoxyampicillin per 1L of the culture medium;
3) adding fresh sheep blood according to the amount of adding 100mL of fresh sheep blood in each 1L of culture medium;
4) fully and uniformly mixing, and then pouring the mixture into a flat plate; (each plate with a diameter of 90mm needs about 20mL of culture medium, care is taken to avoid air bubbles, the plate is horizontally placed after pouring, and the action is rapid so that the plate cannot be poured after the liquid culture medium is solidified.)
5) After the flat plate is cooled and solidified, the flat plate is put into a sterile sealing bag, sealed and stored at 4 ℃.
Example 3
The formula of the culture medium for culturing the helicobacter pylori is as follows:
per 1000mL agar plate contain:
the preparation method comprises the following specific steps:
according to the formula:
mixing, heating to dissolve, filtering with filter paper, adjusting pH to 7.2 + -0.1, packaging into 800 mL/bottle, autoclaving in high pressure steam sterilizer at 121 deg.C for 20min, cooling to room temperature, sealing, and storing at 4 deg.C.
When preparing the plate:
1) taking a certain amount of basal culture medium stored at 4 deg.C, adding agar powder in an amount of 30g per 1L, and autoclaving at 121 deg.C for 20 min;
2) when the temperature of the culture medium is reduced to about 45-50 ℃, adding stock solutions of various antibiotics according to the concentration of adding 9mg of vancomycin, 3mg of amphotericin, 6mg of polymyxin and 6mg of methoxyampicillin pyrimidine into every 1L of the culture medium;
3) adding fresh sheep blood according to the amount of adding 100mL of fresh sheep blood in each 1L of culture medium;
4) fully and uniformly mixing, and then pouring the mixture into a flat plate; (each plate with a diameter of 90mm needs about 20mL of culture medium, care is taken to avoid air bubbles, the plate is horizontally placed after pouring, and the action is rapid so that the plate cannot be poured after the liquid culture medium is solidified.)
5) After the flat plate is cooled and solidified, the flat plate is put into a sterile sealing bag, sealed in vacuum and stored at 4 ℃.
Example 4
Clinical samples: gastroscopy confirmed positive and negative subjects with H.pylori infection and clamped gastric mucosal tissue samples under direct endoscopic vision.
The experimental method comprises the following steps:
preparation before the test: the culture medium provided by the invention in the embodiment 1-3 is taken out and returned to the room temperature (18 ℃ -25 ℃).
Under the direct vision of an endoscope, gastric mucosa tissues at different parts are clamped.
Directly coating and inoculating the removed gastric mucosa tissue surface to an HP selective separation culture medium on a super clean bench. Then placing the mixture in an anaerobic tank, and slowly injecting mixed gas (5% O)2、10％CO2、85％N2) Continuously culturing at 37 deg.C for 5-7 days;
observing the culture result: if a fine needle-tip, gray-white and translucent colony grows, the helicobacter pylori infection is judged to be positive, and if no bacteria grow for 7 days after continuous culture, the helicobacter pylori infection is judged to be negative.
And (3) data analysis:
the results of 89 positive clinical specimens and 86 negative clinical specimens cultured with the medium provided in example 2 of the present invention are shown in Table 1.
TABLE 1
By taking gastroscope detection as a gold standard, the culture result of the culture medium has a positive rate of 98.88 percent, namely the sensitivity can reach 98.88 percent, and the culture result of a negative sample is consistent with the gastroscope detection result, namely the specificity can reach 100 percent.
The above experiments were carried out using the media prepared in examples 1 and 3, and the results were not significantly different from the results of the detection using the media prepared in example 2 (P > 0.05).
According to the conclusion of the experiment, the culture sensitivity of the culture medium provided by the invention to the HP of a clinical sample is as high as 98.88%; the specificity reaches 100%.
Example 5
Control group: SKIRROW medium, Brain Heart Infusion (BHI w ith 7% calf serum), Columbia blood agar medium, etc.;
experimental groups: the culture media prepared in examples 1 to 3 of the present invention.
The results of culturing the above 175 clinical samples were compared with the HP blood agar medium prepared in example 2 of the present invention, using Columbia blood agar medium as a control, and are shown in Table 2:
TABLE 2
As can be seen from the results of comparative experiments, the culture sensitivity of the HP blood agar culture medium of the invention to HP in clinical samples is as high as 98.88%, which is much higher than that of the control culture medium (70.79%); its specificity was 100%, also better than that of the control medium (98.84%).
The experiment was carried out on SKIRROW medium and Brain Heart Infusion (BHI with 7% calf serum), and the experimental result was not significantly different from that of Columbia blood agar medium (P > 0.05).
The above experiments were carried out using the media prepared in examples 1 and 3, and the results were not significantly different from the results of the detection using the media prepared in example 2 (P > 0.05).
The comprehensive experiment results show that compared with a SKIRROW culture medium, Brain Heart Infusion (BHI with 7% calf serum) and Columbia blood agar culture medium, the sensitivity can be obviously improved (P is less than 0.05), and the isolated culture sensitivity of a clinical sample HP is as high as 98.88%, which is obviously higher than that of a control culture medium; the specificity is 100 percent, and is also obviously improved (P is less than 0.05) compared with the specificity (98.84 percent) of a control culture medium.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. A complete culture medium, comprising a basal medium, antibiotics and animal blood;
every 1000mL of the basal medium contains:
250mL of cow heart extract
200mL of bovine brain extract
Cysteine 2g
Na2HPO4 10g
Peptone 5g
NaCl 1g
Agar powder 25g
Distilled water to 1000mL
Vancomycin 10mg
Trimethoprim 5mg
Amphotericin 5mg
Polymyxin 5mg
Adding fresh sheep blood according to the amount of adding 100mL of fresh sheep blood in every 1L of the basic culture medium.
2. Use of the complete medium according to claim 1 for the cultivation of helicobacter pylori.
3. The method of claim 1, comprising the steps of:
step 1: taking bovine heart extract, bovine brain extract, cysteine, peptone, sodium chloride and disodium hydrogen phosphate according to the formula amount, mixing with water, heating for dissolving, filtering, collecting filtrate, adjusting the pH value to 7.1-7.3, subpackaging, sterilizing, cooling and sealing to obtain a basic culture medium;
step 2: taking the basic culture medium prepared in the step 1, adding agar powder with the formula amount, and sterilizing;
and step 3: cooling to 45-50 ℃, and adding the antibiotic with the formula amount;
and 4, step 4: adding 10% (V/V) of animal blood, mixing, pouring into flat plate, cooling, solidifying, and sealing.
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| CN201710600643.3A CN108277257B (en) | 2017-07-21 | 2017-07-21 | Culture medium |
| PCT/CN2018/081701 WO2019015357A1 (en) | 2017-07-21 | 2018-04-03 | Medium |
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| CN101659979A (en) * | 2008-08-25 | 2010-03-03 | 薄涛 | Brain-heart-infusion neutral nutrient medium |
| CN102888442A (en) * | 2011-07-20 | 2013-01-23 | 农高惠 | Kit for rapid culture, identification and drug sensitivity of gastric helicobacter pylori and inspection method thereof |
| CN104195086A (en) * | 2014-08-27 | 2014-12-10 | 博奥生物集团有限公司 | Composite enrichment medium for five bacteria and preparation method for composite enrichment medium |
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| CN106635862A (en) * | 2015-10-30 | 2017-05-10 | 杭州致远医学检验所有限公司 | Isolation medium for helicobacter pylori |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1077750A (en) * | 1993-05-25 | 1993-10-27 | 海拉尔市卫生防疫站 | Food micro-organism detecting plate and preparation thereof and application |
| JP4811760B2 (en) * | 2004-12-27 | 2011-11-09 | 独立行政法人農業・食品産業技術総合研究機構 | Enterobacteria and its utilization to improve equol production by utilization of daidzein |
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| CN101659979A (en) * | 2008-08-25 | 2010-03-03 | 薄涛 | Brain-heart-infusion neutral nutrient medium |
| CN102888442A (en) * | 2011-07-20 | 2013-01-23 | 农高惠 | Kit for rapid culture, identification and drug sensitivity of gastric helicobacter pylori and inspection method thereof |
| CN104195086A (en) * | 2014-08-27 | 2014-12-10 | 博奥生物集团有限公司 | Composite enrichment medium for five bacteria and preparation method for composite enrichment medium |
| CN106635862A (en) * | 2015-10-30 | 2017-05-10 | 杭州致远医学检验所有限公司 | Isolation medium for helicobacter pylori |
| CN105820975A (en) * | 2016-03-25 | 2016-08-03 | 美利泰格诊断试剂(嘉兴)有限公司 | Oral cavity helicobacter pylori culture method |
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