CN114045231A - Culture medium for helicobacter pylori and preparation method and application thereof - Google Patents
Culture medium for helicobacter pylori and preparation method and application thereof Download PDFInfo
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- CN114045231A CN114045231A CN202111237527.2A CN202111237527A CN114045231A CN 114045231 A CN114045231 A CN 114045231A CN 202111237527 A CN202111237527 A CN 202111237527A CN 114045231 A CN114045231 A CN 114045231A
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- helicobacter pylori
- amphotericin
- vancomycin
- polymyxin
- antibiotics
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- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 70
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 70
- 239000001963 growth medium Substances 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 239000003242 anti bacterial agent Substances 0.000 claims abstract description 30
- 229940088710 antibiotic agent Drugs 0.000 claims abstract description 30
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 108010040201 Polymyxins Proteins 0.000 claims abstract description 27
- 108010059993 Vancomycin Proteins 0.000 claims abstract description 27
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims abstract description 27
- 229960003165 vancomycin Drugs 0.000 claims abstract description 27
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- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
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- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
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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
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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Abstract
The invention discloses a culture medium for helicobacter pylori and a preparation method and application thereof, belonging to the technical field of microorganisms. Specifically, the present invention provides a culture medium for helicobacter pylori, wherein the culture medium for helicobacter pylori contains, per 1000 mL: 3-10g of agar, 5-10g of cysteine, 0.3-0.8mg of antibiotics, 3-10g of soluble starch, 0.1-1mL of lactic acid and 1-10g of horse whole blood; wherein: the antibiotic consists of vancomycin, amphotericin B and polymyxin; in the antibiotics, the mass fractions of vancomycin, amphotericin B and polymyxin are respectively as follows: vancomycin: 30% -60%; amphotericin B: 30% -80%; polymyxin: 0.1 to 10 percent. The invention also provides a preparation method and application of the culture medium. The culture medium provided by the invention is suitable for the rapid growth and culture of helicobacter pylori, can effectively inhibit the growth of mixed bacteria, reduces the use of antibiotics, and has a great use value.
Description
Technical Field
The invention belongs to the technical field of microorganisms. Specifically, the invention relates to a culture medium for helicobacter pylori, and a preparation method and application thereof.
Background
Helicobacter Pylori (HP) is a gram-negative helicobacter pylori colonized between gastric mucosal epithelium and mucus, and a large number of researches show that the HP is closely related to the occurrence of chronic gastritis, peptic ulcer and lymphoproliferative gastric lymphoma, and has important significance in etiology. Since the first separation in 1983, the technology for detecting helicobacter pylori has been greatly developed and mainly divided into two main categories, one is an invasive detection technology, including a biopsy staining method, a rapid urease method, a bacterial culture method and a PCR method; another class is non-invasive detection techniques, including13C or14C-urease breath test, serum immunological test, fecal antigen test, etc. Wherein the bacterial culture method is the most accurate and reliable detection technology,is considered as the 'gold standard' of diagnosis, is a prerequisite for subsequent research, can develop pathogenicity, drug sensitivity experiments, virulence grade classification of different strains and other researches, and further screens out an effective helicobacter pylori treatment scheme. Therefore, it is important to study the isolated culture technique of helicobacter pylori and to improve the positive detection rate of helicobacter pylori isolated from clinical specimens.
However, helicobacter pylori is a critical micro-aerobic bacterium, and the requirements for a culture medium are quite strict in vitro culture in addition to the strict requirements for a gas environment. The culture medium is required not only to meet the nutritional requirements of helicobacter pylori, but also to provide local microaerophilic conditions and a suitable pH tolerance range. The traditional separation culture medium cannot meet the separation culture requirement of helicobacter pylori, and common culture media such as Columbia agar culture medium and brain heart infusion agar culture have no advantages in culturing the helicobacter pylori, and the positive detection rate is extremely low. In addition, in practical work, the quality of the culture medium has a direct influence on the reliability of the detection result, and therefore, a culture medium which has a good culture effect and can promote the rapid proliferation of helicobacter pylori and avoid the infection of infectious microbes is needed.
Disclosure of Invention
The invention aims to provide a culture medium which has good effect, can promote the rapid propagation of helicobacter pylori and can avoid the infection of mixed bacteria.
In order to achieve the purpose, the invention provides the following technical scheme:
a medium for helicobacter pylori, wherein per 1000mL of the medium for helicobacter pylori comprises:
3-10g of agar, 5-10g of cysteine, 0.3-0.8mg of antibiotics, 3-10mg of soluble starch, 0.1-1mL of lactic acid and 1-10g of horse whole blood; wherein:
the antibiotic consists of vancomycin, amphotericin B and polymyxin; in the antibiotics, the mass fractions of vancomycin, amphotericin B and polymyxin are respectively as follows:
vancomycin: 30% -60%;
amphotericin B: 30% -80%;
polymyxin: 0.1 to 10 percent.
Preferably, the culture medium for helicobacter pylori contains per 1000 mL:
5-8g of agar, 6-8g of cysteine, 0.5-0.8mg of antibiotics, 3-6mg of soluble starch, 0.1-0.3mL of lactic acid and 3-8g of horse whole blood;
in the antibiotics, the mass fractions of vancomycin, amphotericin B and polymyxin are respectively as follows:
vancomycin: 30% -60%;
amphotericin B: 30% -80%;
polymyxin: 0.1 to 10 percent.
Further preferably, the medium for helicobacter pylori contains per 1000 mL:
5g of agar, 8g of cysteine, 0.5mg of antibiotics, 6mg of soluble starch, 0.1mL of lactic acid and 8g of horse whole blood;
in the antibiotics, the mass fractions of vancomycin, amphotericin B and polymyxin are respectively as follows:
vancomycin: 49 percent;
amphotericin B: 49 percent;
polymyxin: 2 percent.
According to a particular embodiment of the invention, the culture medium further comprises a helicobacter pylori lipopolysaccharide extract.
Further preferably, the culture medium for helicobacter pylori contains 1 to 3mg of the helicobacter pylori lipopolysaccharide extract per 1000 mL.
In addition, the invention also provides a preparation method of the culture medium, which comprises the following steps:
s1: preparing antibiotics for later use;
s2: preparing a soluble starch water solution for later use;
s3: adding cysteine into agar, sterilizing, and adding horse whole blood, antibiotic and soluble starch water solution to obtain the culture medium.
According to a specific embodiment of the present invention, the present invention provides the preparation method further comprising a process of adding the helicobacter pylori lipopolysaccharide extract to agar.
According to a specific embodiment of the present invention, further, the preparation method provided by the present invention comprises the steps of:
s1: weighing vancomycin, amphotericin B and polymyxin according to a formula, dissolving in a proper amount of sterile water, uniformly mixing, and irradiating for 1-3h under ultraviolet rays for later use;
s2: weighing soluble starch according to the formula amount, dissolving in an appropriate amount of sterile water, adding lactic acid according to the formula amount, and boiling for 10-20min for later use;
s3: weighing agar according to the formula amount, adding cysteine and helicobacter pylori lipopolysaccharide extract when the temperature is reduced to 45-50 ℃, sequentially adding horse whole blood and the antibiotic and soluble starch aqueous solution prepared in the step S1 and the step S2 after sterilization, uniformly stirring after adding, and cooling to obtain the culture medium.
The invention also provides application of the culture medium in culturing the helicobacter pylori.
In conclusion, the invention provides a novel culture medium for helicobacter pylori, a preparation method and application thereof. In the culture medium provided by the invention, the horse whole blood can provide required nutrient substances (such as amino acids, vitamins, inorganic substances, lipid substances, nucleic acid derivatives and the like) for the helicobacter pylori, can also provide hormones and growth factors which are favorable for the growth and proliferation of cells, and can promote the rapid growth of the helicobacter pylori. The addition of antibiotics can not only reduce the pollution of bacillus subtilis, gram-positive coccus, mould and other bacteria, but also provide thymidine required for HP growth. The addition of the soluble starch can absorb toxic ions generated by the helicobacter pylori and toxic products generated by bacterial metabolism in the culture process, prevent the helicobacter pylori from being in a stress condition too early, further increase the rapid growth of the helicobacter pylori in the culture process and reduce the culture difficulty.
The addition of cysteine can reduce the oxidation-reduction potential to a certain extent, and maintain the microaerophilic environment preferred by helicobacter pylori.
Meanwhile, the culture medium provided by the invention is also added with the helicobacter pylori lipopolysaccharide extract, which has a good inhibition effect on mixed bacteria and can reduce the addition of antibiotics, thereby reducing the stress effect of the antibiotics on the helicobacter pylori. In addition, the H.pylori lipopolysaccharide extract is a component of the H.pylori cell wall, is located at the outermost layer of the cell wall, and covers the peptide-bound part of the cell wall. The inventor finds that the helicobacter pylori lipopolysaccharide extract has a broad-spectrum antibacterial effect, can replace part of antibiotics, and plays a role in inhibiting the growth of mixed bacteria in the isolated culture of the helicobacter pylori. Moreover, the helicobacter pylori lipopolysaccharide extract can form certain adhesion on the cell wall of the helicobacter pylori thallus, provide certain protection effect on the helicobacter pylori and resist antibiotics and toxic secondary metabolites in the culture environment.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.
Example 1 this example provides a medium for helicobacter pylori and a method for preparing the same
1.1 culture medium formula:
5g of agar, 8g of cysteine, 0.5mg of antibiotics, 6g of soluble starch, 0.1mL of lactic acid and 8g of horse whole blood;
in the antibiotics, the mass fractions of vancomycin, amphotericin B and polymyxin are respectively as follows: vancomycin: 49 percent; amphotericin B: 49 percent; polymyxin: 2 percent.
1.2 sources of reagent materials:
agar: beijing Luqiao technology, Inc.; cysteine, soluble starch: annagi chemical Co., Ltd; vancomycin, polymyxin, amphotericin B: BioSharp corporation; lactic acid: a group of national drugs; horse whole blood: jilin university medical college animal center.
1.3 the preparation method comprises the following steps:
s1: weighing vancomycin 0.245mg, amphotericin B0.245 mg and polymyxin 0.01mg according to a formula, dissolving in a proper amount of sterile water, uniformly mixing, and irradiating for 1h under ultraviolet rays for later use;
s2: weighing 6g of soluble starch according to the formula amount, dissolving in a proper amount of sterile water, adding 0.1mL of lactic acid according to the formula amount, and boiling for 20min for later use;
s3: weighing 5g of agar according to the formula amount, heating to dissolve, adding 8g of cysteine when the temperature is reduced to 45-50 ℃, sequentially adding 8g of horse whole blood and the antibiotic and soluble starch aqueous solution prepared in the step S1 and the step S2 after sterilization, uniformly stirring after adding, and cooling to obtain the culture medium.
Example 2 this example provides a culture medium for helicobacter pylori and a method for preparing the same
1.1 culture medium formula:
5g of agar, 8g of cysteine, 0.5mg of antibiotics, 6g of soluble starch, 0.1mL of lactic acid, 3mg of helicobacter pylori lipopolysaccharide extract and 8g of horse whole blood;
in the antibiotics, the mass fractions of vancomycin, amphotericin B and polymyxin are respectively as follows: vancomycin: 49 percent; amphotericin B: 49 percent; polymyxin: 2 percent.
1.2 preparation of Hp lipopolysaccharide extract
Firstly, helicobacter pylori NCTC11639 (gift from the management and preservation center of helicobacter pylori strain, China) is activated, screened and cultured in an enlarged way, and bacteria in a growth vigorous period are collected to prepare bacterial suspension. The bacterial suspension was centrifuged at 5000rpm for 10min to obtain a pellet. After washing the pellet with PBS buffer, it was centrifuged at 5000rpm for 10min and repeated 2 times. The precipitate (cells) was resuspended in PBS buffer to adjust the cell concentration to 2.0X 1010And (4) preparing concentrated bacterial liquid per mL. Carrying out ultrasonic treatment on the concentrated bacteria liquid for 20min at the ultrasonic power of 300W, carrying out ultrasonic treatment intermittently, stopping ultrasonic treatment for 10s, and circulating the steps until 20min is finished. Centrifuging the concentrated bacterial liquid after ultrasonic treatment at 3000rpm for 30min, and collecting the supernatantAnd (4) liquid. Collecting all the supernatant, placing in a dialysis bag, performing running water dialysis for 24h (changing water every 5-6 h), and freeze drying to obtain a sample of crude helicobacter pylori lipopolysaccharide.
10mL of 100mmol/L Tris-HCl (pH 8) were used to reconstitute the crude extract, DNase I was added to a final concentration of 100. mu.g/mL and RNase was added to a final concentration of 50. mu.g/mL, digestion was carried out overnight at 37 ℃ followed by addition of proteinase K to a final concentration of 100. mu.g/mL, and incubation was carried out for 2h at 65 ℃. Adding water saturated phenol, mixing, centrifuging at 4000rpm for 30min to obtain supernatant, dialyzing the supernatant in distilled water in a dialysis bag for 12h (changing water every 5-6 h), collecting the solution in the dialysis bag, adding 10 times volume of 95% ethanol, precipitating at-20 deg.C overnight, centrifuging at 4000r/min for 30min, freeze-drying the precipitate, and weighing to obtain helicobacter pylori lipopolysaccharide extract solid (hpLPS), and storing at-20 deg.C for later use.
1.3 preparation of the culture Medium
S1: weighing vancomycin 0.245mg, amphotericin B0.245 mg and polymyxin 0.01mg according to a formula, dissolving in a proper amount of sterile water, uniformly mixing, and irradiating for 1h under ultraviolet rays for later use;
s2: weighing 6g of soluble starch according to the formula amount, dissolving in a proper amount of sterile water, adding 0.1mL of lactic acid according to the formula amount, and boiling for 20min for later use;
s3: weighing 5g of agar according to the formula amount, heating and dissolving, adding 8g of cysteine and 3mg of helicobacter pylori lipopolysaccharide extract when the temperature is reduced to 45-50 ℃, sequentially adding 8g of horse whole blood and the antibiotic and soluble starch aqueous solution prepared in the step S1 and the step S2 after sterilization, uniformly stirring after adding, and cooling to obtain the culture medium.
Comparative example 1 this comparative example provides a medium commonly used for helicobacter pylori and a method for preparing the same
1.1 culture medium formula:
1.2 preparation method
Taking 250mL of beef heart extract according to the formula; 200mL of bovine brain extract; na (Na)2HPO410g of a mixture; 5g of peptone; 1g of NaCl, mixing, heating for dissolving, filtering by using filter paper, adjusting the pH value to about 7.2, sterilizing, adding 25g of agar powder, adding stock solutions of various antibiotics in the concentrations of 10mg of vancomycin, 5mg of amphotericin, 5mg of polymyxin and 5mg of methoxyampicillin after the temperature of the culture medium is reduced to about 45-50 ℃, adding the mixture, uniformly mixing, and cooling to obtain the culture medium.
And (3) verification experiment:
three plates of the medium prepared in examples 1-2 and comparative example 1 were used, and 10 plates were added to each plate40.1mL of the diluted bacterial suspension was applied to the surface of the plate by means of an L-bar (the bacterial suspension was dried).
The plate was placed at 37 ℃ in a mixed gas (85% N)2、10％CO2、5％O2) And detecting and counting colonies, measuring the diameter of the colonies and the number of the mixed bacteria colonies after culturing for 72 hours in an environment with the humidity of 90%. The results of the experiment are shown in table 1 below:
TABLE 1 Experimental results Table
| Number of colonies | Colony diameter (mm) | Number of colonies of miscellaneous bacteria | |
| Example 1 | 96 | 2.026 | 10 |
| Example 2 | 98 | 2.099 | - |
| Comparative example 1 | 88 | 1.99 | 6 |
The inventor speculates that the reason for the fact that the colony number and the colony diameter of the helicobacter pylori in the example 1 are larger than those in the comparative example 1 is probably because the whole blood of the animal can provide the necessary nutrients (such as amino acids, vitamins, inorganic substances, lipid substances, nucleic acid derivatives and the like) for the helicobacter pylori, can also provide hormones and growth factors which are necessary for the growth and proliferation of cells and can promote the rapid growth of the helicobacter pylori because the horse whole blood is added in the example 1. However, the number of colonies of the mixed bacteria in the culture medium in example 1 is also significantly greater than that in comparative example 1, because the culture medium in comparative example 1 is added with significantly more antibiotics than that in example 1, the growth of the mixed bacteria can be better inhibited, and simultaneously, because of the presence of a large amount of antibiotics, the growth of helicobacter pylori is also inhibited, which is also the reason why the number of colonies and the diameter of the colonies of helicobacter pylori in comparative example 1 are smaller than those in examples 1 and 2.
Comparing example 2 with comparative example 1, it was found that there was no growth of mixed bacteria in example 2, indicating that the helicobacter pylori lipopolysaccharide extract can further inhibit the growth of mixed bacteria instead of antibiotics. Meanwhile, the comparison between the example 1 and the example 2 shows that the number and the diameter of the colonies in the example 2 are improved compared with the example 1, which indicates that the helicobacter pylori lipopolysaccharide has a certain promotion effect on the growth of the helicobacter pylori.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Various alternatives, modifications and combinations of the features of the invention can be made without departing from the spirit and nature of the invention as claimed, and such simple variations and combinations should also be considered as disclosed in the present application, all falling within the scope of the invention.
Claims (9)
1. A medium for helicobacter pylori, wherein per 1000mL of the medium for helicobacter pylori comprises:
3-10g of agar, 5-10g of cysteine, 0.3-0.8mg of antibiotics, 3-10g of soluble starch, 0.1-1mL of lactic acid and 1-10g of horse whole blood; wherein:
the antibiotic consists of vancomycin, amphotericin B and polymyxin; in the antibiotics, the mass fractions of vancomycin, amphotericin B and polymyxin are respectively as follows:
vancomycin: 30% -60%;
amphotericin B: 30% -80%;
polymyxin: 0.1 to 10 percent.
2. The medium according to claim 1, wherein the medium for helicobacter pylori contains per 1000 mL:
5-8g of agar, 6-8g of cysteine, 0.5-0.8mg of antibiotics, 3-6g of soluble starch, 0.1-0.3mL of lactic acid and 3-8g of horse whole blood;
in the antibiotics, the mass fractions of vancomycin, amphotericin B and polymyxin are respectively as follows:
vancomycin: 30% -60%;
amphotericin B: 30% -80%;
polymyxin: 0.1 to 10 percent.
3. The medium according to claim 2, wherein the medium for helicobacter pylori contains per 1000 mL:
5g of agar, 8g of cysteine, 0.5mg of antibiotics, 6g of soluble starch, 0.1mL of lactic acid and 8g of horse whole blood;
in the antibiotics, the mass fractions of vancomycin, amphotericin B and polymyxin are respectively as follows:
vancomycin: 49 percent;
amphotericin B: 49 percent;
polymyxin: 2 percent.
4. The culture medium according to any one of claims 1 to 3, further comprising a helicobacter pylori lipopolysaccharide extract.
5. The medium according to claim 4, wherein 1 to 3mg of the H.pylori lipopolysaccharide extract is contained per 1000mL of the medium for H.pylori.
6. A method of preparing the culture medium of any one of claims 1-5, comprising the steps of:
s1: preparing antibiotics for later use;
s2: preparing a soluble starch water solution for later use;
s3: adding cysteine into agar, sterilizing, and adding horse whole blood, antibiotic and soluble starch water solution to obtain the culture medium.
7. The method according to claim 6, further comprising a process of adding a helicobacter pylori lipopolysaccharide extract to agar.
8. The method for preparing the medium according to claim 6 or 7, comprising the steps of:
s1: weighing vancomycin, amphotericin B and polymyxin according to a formula, dissolving in a proper amount of sterile water, uniformly mixing, and irradiating for 1-3h under ultraviolet rays for later use;
s2: weighing soluble starch according to the formula amount, dissolving in an appropriate amount of sterile water, adding lactic acid according to the formula amount, and boiling for 10-20min for later use;
s3: weighing agar according to the formula amount, adding cysteine and helicobacter pylori lipopolysaccharide extract when the temperature is reduced to 45-50 ℃, sequentially adding horse whole blood and the antibiotic and soluble starch aqueous solution prepared in the step S1 and the step S2 after sterilization, uniformly stirring after adding, and cooling to obtain the culture medium.
9. Use of the medium according to any one of claims 1 to 5 for the cultivation of helicobacter pylori.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115369061A (en) * | 2022-08-24 | 2022-11-22 | 山东博科生物产业有限公司 | Selective helicobacter pylori culture medium and preparation method thereof |
| CN116590192A (en) * | 2023-06-09 | 2023-08-15 | 淄博市临淄区人民医院(淄博市市立医院) | Helicobacter pylori solid separation culture medium and preparation method and application thereof |
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| CN115369061A (en) * | 2022-08-24 | 2022-11-22 | 山东博科生物产业有限公司 | Selective helicobacter pylori culture medium and preparation method thereof |
| CN116590192A (en) * | 2023-06-09 | 2023-08-15 | 淄博市临淄区人民医院(淄博市市立医院) | Helicobacter pylori solid separation culture medium and preparation method and application thereof |
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