CN109880772B - Method for separating and culturing helicobacter pylori strain - Google Patents
Method for separating and culturing helicobacter pylori strain Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, relates to a bacteria isolated culture method, and particularly relates to a helicobacter pylori strain isolated culture method. During the sample transportation process of the method, storing the sample suspected to contain the helicobacter pylori in the transportation solution; the transport solution comprises a Brookfield broth culture medium, an adsorbent, a reducing agent and a helicobacter pylori lipopolysaccharide extract. Wherein the helicobacter pylori lipopolysaccharide extract has inhibiting effect on mixed bacteria and protecting effect on helicobacter pylori thallus. The method can prolong the transport time of the sample to a bacterial culture research unit and keep the activity of the helicobacter pylori, thereby reducing the separation and culture difficulty of the wild helicobacter pylori strain and improving the positive rate of the bacterial culture. The helicobacter pylori culture method can be applied to researches such as bacterial typing, pathogenic mechanism research, animal model construction, pathogenic factor determination and the like.
Description
Technical Field
The invention belongs to the field of biological medicines, relates to a bacteria isolated culture method, and particularly relates to a helicobacter pylori strain isolated culture method.
Background
Helicobacter pylori (h. pylori) is a helical gram-negative microaerophilic bacterium, infects half of the global population, is the main pathogenic bacterium causing gastritis, peptic ulcer and gastric cancer, and the discovery of Helicobacter pylori deepens the cognition of human beings on the relationship between chronic infection, inflammation and cancer, and is classified as a class I carcinogenic factor of human gastric cancer by the world health organization. The clinical strain of helicobacter pylori after separation culture can be used for basic research such as bacterial typing, pathogenic mechanism research, animal model construction, pathogenic factor determination and the like. However, the requirement of helicobacter pylori on growth conditions is very strict, the requirement on culture conditions is similar to the conditions of gastric submucosa in vivo, and the requirement on nutrient conditions is higher, so that the problems of low culture positive rate of helicobacter pylori and the like are caused, and the further research on the clinical strains of helicobacter pylori is influenced.
The inventor published a scientific research paper ' separation culture and identification of helicobacter pylori clinical strains, Liu Zheng Mei and the like, Guiyang medical college school newspaper, 2016 ' in 2016 ', and the paper establishes a separation culture method of helicobacter pylori clinical strains, which specifically comprises the following steps: taking a clinical sample, putting the sample into a Brookfield broth conveying liquid containing antibiotics, and then conveying the sample to a laboratory for separation and detection; a clinical sample suspected of containing helicobacter pylori is inoculated on a Columbia selective medium containing 10% of sheep whole blood, after the sample is cultured for 3-5 days at 37 ℃ in a three-gas culture box, transparent and tiny suspicious colonies on a blood plate are picked for colony detection. However, the above technical solutions have the following problems: during the transport process, the helicobacter pylori is under stress conditions (i.e. environmental conditions unsuitable for the growth of the helicobacter pylori), and the excessively long transport time can cause the death of the bacteria or morphological variation. Therefore, the time for transporting the sample to the laboratory where the isolation culture is performed after the sampling is limited to about 4 hours, which is too strict. In the case of a remote separation of the sampling site and the experimental culture site, the transport of the specimen takes a long time, which leads to massive death or morphological changes of the isolated H.pylori to be cultured, and finally to low positive rates and massive "false negatives" of the bacterial culture.
Disclosure of Invention
The invention mainly solves the technical problem of providing a method for separating and culturing helicobacter pylori strains, which can prolong the transportation time limit of transporting samples to a bacterial culture research unit, thereby reducing the separation and culture difficulty of wild helicobacter pylori strains and improving the positive rate of bacterial culture, so as to carry out various researches on the wild helicobacter pylori strains.
In order to solve the technical problems, the invention provides the following technical scheme:
a method for isolated culture of helicobacter pylori strain comprises the following steps,
(1) a transferring step: during the transfer, the helicobacter pylori sample is stored in a transfer solution, wherein the transfer solution comprises a Brucella broth culture medium, an adsorbent, a reducing agent and a helicobacter pylori lipopolysaccharide extract; (2) an inoculation step; (3) and (5) culturing.
By adopting the technical scheme, the preservation time of the helicobacter pylori in the transport solution can be prolonged, the difficulty of separating and culturing the wild helicobacter pylori strain is reduced, and the culture positive rate of the bacterium is improved. The wild type helicobacter pylori strain is a helicobacter pylori original strain separated from nature or organism. The sample is an object suspected of containing H.pylori and the sample may be in various forms, for example, the sample may be a cotton ball or a sampler dipped or wiped through the site suspected of containing H.pylori. The culture positive rate of helicobacter pylori refers to the ratio of samples identified as positive for helicobacter pylori in culture to the total number of samples. The technical scheme only relates to the operation steps after the sample is obtained, the operation steps are preservation, transportation, separation culture and identification of helicobacter pylori in the sample, and no sampling operation or process is involved.
The technical principle of the technical scheme is as follows: the delivery liquid has a maintenance function on the helicobacter pylori, and the helicobacter pylori can be stored in the delivery liquid for a long time, so that the delivery time limit of the helicobacter pylori to a bacterial culture research unit after the helicobacter pylori sample is collected is increased. Among them, the helicobacter pylori lipopolysaccharide extract has an inhibitory effect on the mixed bacteria (see experimental example 2), can reduce the competitive inhibitory effect of the mixed bacteria on helicobacter pylori, and the helicobacter pylori lipopolysaccharide extract of the present invention has no inhibitory effect on helicobacter pylori itself. The extract does not contain antibiotics, and the phenomenon that the antibiotics inhibit the growth of the helicobacter pylori can not occur. The adsorbent can adsorb the secondary metabolite of bacteria in the transport liquid, and the secondary metabolite is prevented from inhibiting the growth of helicobacter pylori. The reducing agent keeps micro-oxygen environment, which is beneficial to the growth of helicobacter pylori.
The beneficial effects of the technical scheme are specifically described as follows:
(1) helicobacter pylori lipopolysaccharide extract can substitute for antibiotic, and has effect in inhibiting mixed bacteria
Helicobacter pylori is susceptible to deformation or death under environmental stress, wherein deformation includes the change of the strain from campylobacter to globular or filamentous type. The globular morphology is generally in a non-culturable state, whereas the filamentous morphology is generally difficult to re-culture. The stress conditions include conditions unsuitable for the growth of helicobacter pylori, such as antibiotic action, the influence of toxic factors in the environment, and gas conditions that do not meet microaerophilic requirements. Wild-type helicobacter pylori is isolated from the helicobacter pylori growth environment in which there are many mixed bacteria, and antibiotics are used during transportation of the wild-type helicobacter pylori, which has just been isolated from the bacteria-bearing growth environment, to a bacteria culture research unit in order to inhibit the growth of the mixed bacteria. The antibiotic has a great inhibiting effect on the growth and the shape maintenance of the helicobacter pylori. Due to the long-term use of the antibiotics, the resistance of mixed bacteria is enhanced, the use amount of the antibiotics is correspondingly increased, so that the aim of inhibiting the mixed bacteria can be achieved, and the increase of the use amount of the antibiotics can cause stronger environmental stress to the helicobacter pylori, thereby influencing the growth state of the helicobacter pylori and improving the difficulty of subsequent bacterial culture. However, if antibiotics are not used, the growth of mixed bacteria cannot be inhibited, the mixed bacteria and the helicobacter pylori compete for nutrient resources, the mass propagation of the mixed bacteria can release toxic secondary metabolites, the growth state of the helicobacter pylori is poor, and in the subsequent bacterial culture, the problem of high mixed bacteria rate can occur, and the positive rate of the helicobacter pylori culture is influenced.
The technical proposal adopts the helicobacter pylori lipopolysaccharide extract to inhibit the mixed bacteria in the delivery liquid. Compared with the scheme of using combined antibiotics in the prior art, the method has the following advantages: a. the dosage of the antibiotic is greatly reduced, thereby reducing the stress effect of the antibiotic on the helicobacter pylori. For example: in the prior art, about 5mg of antibiotics is usually added into every 100ml of transport liquid, and by adopting the technical scheme, the antibiotics do not need to be added into the transport liquid. b. The helicobacter pylori lipopolysaccharide extract has a good inhibition effect on mixed bacteria and a certain protection effect on the helicobacter pylori per se. Helicobacter pylori lipopolysaccharide is a component of the cell wall of helicobacter pylori, is located at the outermost layer of the cell wall, and covers the peptidoglycan of the cell wall. Helicobacter pylori lipopolysaccharide has been studied as a pathogenic factor for a long time, and most reports on the helicobacter pylori lipopolysaccharide focus on the pathogenic mechanism, the molecular structure and the like. The inventor researches and discovers 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 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.
(2) In the delivery liquid of the scheme, the addition of the adsorbent can remove toxic secondary metabolites of various bacteria in the delivery liquid, reduce toxic ions (such as peroxide) in the delivery liquid, and create conditions for storing the helicobacter pylori in the delivery liquid for a long time and maintaining high activity of the helicobacter pylori in the delivery liquid.
(3) In the transport liquid of the scheme, the reducing agent is added to maintain the growth condition of microaerophilic helicobacter pylori.
(4) The delivery liquid can store helicobacter pylori for a long time, can maintain the activity of the helicobacter pylori at a higher level, and the helicobacter pylori stored in the delivery liquid has a better spiral shape, thereby creating conditions for subsequent subculture and improving the positive rate of the subsequently cultured helicobacter pylori.
Further, in the inoculation step, a Columbia blood agar plate containing a helicobacter pylori lipopolysaccharide extract is prepared, and the sample is inoculated onto the Columbia blood agar plate.
By adopting the technical scheme, the helicobacter pylori lipopolysaccharide extract can inhibit the growth of mixed bacteria on a Columbia blood agar plate and has a protection effect on helicobacter pylori thallus. The Columbia blood agar plate is rich in nutrition, is mainly used for culturing bacteria with higher nutritional requirements, and is a relatively suitable culture medium type for culturing helicobacter pylori.
Further, the Columbia blood agar plate contains fetal bovine serum or defibrinated sheep whole blood.
By adopting the technical scheme, the serum or the whole blood is added into the culture plate, so that the nutrient substances (such as amino acids, vitamins, inorganic substances, lipid substances, nucleic acid derivatives and the like) which are lacked by the culture medium can be provided, and hormones and growth factors which are favorable for the growth and proliferation of cells can be provided. The inventor researches and discovers that 10% of defibrinated sheep whole blood provides more sufficient nutrition, and the growth state of helicobacter pylori in the defibrinated sheep whole blood is better than that in fetal calf serum.
Further, in the culture step, placing the Columbia blood agar plate inoculated with the sample in a culture device, and culturing at the constant temperature of 37 ℃ for 3-5 days to obtain a suspected colony growing on the Columbia blood agar plate; the gas composition in the culture device is 5% of oxygen, 10% of carbon dioxide and 85% of nitrogen.
By adopting the technical scheme, the gas composition in the culture device is 5% of oxygen, 10% of carbon dioxide gas and 85% of nitrogen, so that a suitable air environment is provided for the helicobacter pylori, the helicobacter pylori is an obligate microaerophilic bacterium, the stable growth of the helicobacter pylori needs to depend on a microenvironment containing 5% -8% of oxygen, and the helicobacter pylori cannot grow in the atmosphere and an absolute anaerobic environment. When the culture is carried out for about 3 days, the macroscopic morphology and the microscopic thallus morphology of the helicobacter pylori colony are typical (spiral and gull-shaped), the bacteria are in logarithmic growth phase, and the researches such as passage, storage or drug sensitivity experiments can be carried out. After about 5 days of culture, the beginning of the bacterial spheroidicity is seen after gram staining, the bacterial spheroidicity is changed into a short rod shape, and the shape of the helicobacter pylori is changed into a spheroid shape when the culture is prolonged to 7 days. The deformed helicobacter pylori is in a poor growth state and is difficult to recover successfully after being stored at low temperature.
Further, the culture device is a three-gas incubator or an anaerobic tank with an air generating bag arranged inside.
By adopting the technical scheme, the inventor researches and discovers that the anaerobic tank or the three-gas incubator with the gas generating bag arranged inside can provide a good growth environment for the growth of the helicobacter pylori. Wherein, the growth condition of the helicobacter pylori in the three-gas incubator is better than that of the helicobacter pylori in an anaerobic tank provided with a gas generating bag.
Further, the method also comprises an identification step after the culture step: and identifying the suspected colony obtained in the culturing step.
By adopting the technical scheme, the identification step is to determine whether the suspected bacterial colony is a helicobacter pylori bacterial colony, the suspected bacterial colony with a positive identification result is the helicobacter pylori bacterial colony, and the helicobacter pylori bacterial colony can be used for further basic researches such as bacterial typing, pathogenic mechanism research, animal model construction, pathogenic factor determination and the like.
Further, the identification method adopted in the identification step is as follows: one or a combination of both of a gram stain assay and a urease assay.
By adopting the technical scheme, the gram staining detection method and the urease detection method are effective methods for detecting and identifying the helicobacter pylori. The gram stain assay was: after staining, gram-negative tiny Campylobacter bacteria were observed under a light microscope to be helicobacter pylori-positive. The urease detection method comprises the following steps: and picking transparent tiny suspicious colonies on a Columbia blood agar plate to urease test paper, and determining that the test paper turns red from yellow after 1 minute, wherein the test paper is positive to the helicobacter pylori.
Furthermore, the preparation method of the helicobacter pylori lipopolysaccharide extract is an ultrasonic extraction method.
By adopting the technical scheme, the ultrasonic extraction method is adopted to extract the helicobacter pylori lipopolysaccharide, and the ultrasonic extraction method has the advantages of high efficiency, convenience, rapidness and the like. The cavitation of the ultrasound generates strong impact action on the cell wall of the helicobacter pylori, and the cell wall is damaged to release lipopolysaccharide. The molecular structure and molecular weight of lipopolysaccharide are the key to determine its biological activity, and the difference of extraction methods can lead to the difference of the fine structure and molecular weight of polysaccharide. The inventor researches and discovers that the helicobacter pylori lipopolysaccharide extract extracted by an ultrasonic method has better activity of inhibiting mixed bacteria and can replace antibiotics, thereby reducing the possibility that the helicobacter pylori is in a stressed state under the action of the antibiotics.
Further, the adsorbent in the transporting step is cyclodextrin, activated carbon or soluble starch.
By adopting the technical scheme, the cyclodextrin, the activated carbon or the soluble starch can absorb toxic oxygen ions generated by derivation in the delivery liquid and toxic products generated by bacterial metabolism, so that the helicobacter pylori is prevented from being in a stress condition too early, the storage time of the helicobacter pylori in the delivery liquid is further prolonged, and the difficulty of subsequent bacterial culture is reduced.
Further, the reducing agent in the transporting step is cysteine.
By adopting the technical scheme, the addition of the cysteine can lead the transport solution to be reduced in advance, reduce the oxidation-reduction potential to a certain degree and maintain the preferable microaerophilic environment of the helicobacter pylori.
Drawings
FIG. 1 shows the morphology under light microscopy of the strains identified as positive for H.pylori in example 1: gram stained and observed under the microscope (200 x optical microscope).
FIG. 2 shows the morphology under light microscopy of the strains identified as positive for H.pylori in example 2: gram stained and observed under the microscope (200 x optical microscope).
FIG. 3 shows the morphology under light microscopy of the strains identified as positive for H.pylori in example 3: gram stained and observed under the microscope (200 x optical microscope).
FIG. 4 is a graph showing the results of the delivery fluid effect verification experiment of Experimental example 1, which is a graph showing the positive rate of helicobacter pylori as a function of the treatment time after treatment with different delivery fluids.
FIG. 5 is a morphological diagram of bacteria of helicobacter pylori of group T0 in Experimental example 1 after storage for 5 hours in a transport solution (200X observation under an optical microscope, the bacteria were gram-stained).
FIG. 6 is a morphological diagram of bacteria of helicobacter pylori of group T1 in Experimental example 1 after storage for 10 hours in a shipping solution (200X observation under an optical microscope, the bacteria were gram-stained).
Detailed Description
The following is further detailed by the specific embodiments, wherein:
examples 1-3 are isolated cultures of wild-type H.pylori using the method of the invention; experimental example 1 is a test of the effectiveness of the delivery fluid of the invention using a standard strain of helicobacter pylori; experimental example 2 is an experiment of bacteriostatic action of a helicobacter pylori lipopolysaccharide extract.
Example 1: the method of the invention is adopted to separate and culture the wild helicobacter pylori
(1) Material and instrument
Microaerophilic culture tanks and microaerophilic gas-generating bags, clean benches (Mitsubishi, japan); a three gas incubator, disposable inoculating loop, columbia agar (OXOID, uk); bouillon broths, gram stain (anhia hong ci medical, china), urease test paper (guangdong zhahi kedi science, china); fetal bovine serum, cysteine, soluble starch, activated carbon, cyclodextrin, Phosphate Buffer (PBS), defibered sheep whole blood (friendly biotechnology limited, china); DNase I, RNaseA, Tris-HCl (hydroxymethyl) aminomethane hydrochloride (TAKARA); the standard strain NCTC11639 is given a gift from the China center for the management and preservation of helicobacter pylori.
(1.1) preparation of Hp lipopolysaccharide extract
Firstly, the helicobacter pylori NCTC11639 is activated, screened and expanded to culture, and bacteria in a growth vigorous stage are collected to prepare a 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 for 30min at 3000rpm, and collecting supernatant. 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 (pH8) was 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 at 65 ℃ for 2 h. 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.
(1.2) preparation of transport solution
A solution of hpLPS was prepared by dissolving 15g of hpLPS in 100ml of sterile distilled water and filter-sterilizing using a 0.22 μm filter. Weighing 281g of bouillon powder, heating and stirring to dissolve in distilled water, adding distilled water to a constant volume of 100ml to obtain a bouillon culture medium, adding 1g of soluble starch, dissolving and mixing uniformly, and autoclaving at 121 ℃. After cooling, 10ml of sterile hpLPS solution and 0.1ml of sterile 0.5g/ml cysteine solution were added. The Brookfield broth containing hpLPS was then aseptically dispensed into 1ml EP tubes, 0.8ml per EP tube, and stored in a refrigerator at 4 ℃ until use.
(1.3) Columbia blood agar plate preparation
Weighing 39g of Columbia agar, adding 90ml of double distilled water, sterilizing at 121 ℃ under high pressure, cooling to about 50 ℃, adding 10ml of 10% (V/V) defibered sheep whole blood into an ultra-clean workbench, mixing uniformly, and pouring into a flat plate to obtain the Columbia blood agar culture plate.
(2) Isolated culture of helicobacter pylori
(2.1) respectively preserving the samples in the transport liquid, wherein the time for preserving the samples in the transport liquid is not more than 12 hours. The experiment collected 25 samples in total.
(2.2) 200. mu.l of the sterile-treated hpLPS solution prepared in step (1.2) was spread on a Columbia blood agar plate, and the sample stored in the transport solution in step (2.1) was spread on the Columbia blood agar plate to complete the inoculation step.
(2.3) placing the Columbia blood agar plate with the smeared sample in a three-atmosphere incubator, and culturing at the constant temperature of 37 ℃ for 3 days to obtain a suspected colony growing on the Columbia blood agar plate. The gas composition in the three-gas culture box is 5% of oxygen, 10% of carbon dioxide gas and 85% of nitrogen.
(2.4) detection of suspected colonies on Columbia blood agar plates by gram stain detection and urease detection.
Gram stain assay: placing a ring of physiological saline on a clean glass slide by using an inoculating ring, selecting transparent tiny suspicious bacterial colonies on a Columbia blood agar plate, coating the transparent tiny suspicious bacterial colonies on the physiological saline, drying, heating and fixing on an alcohol lamp, primarily dyeing crystal violet for 1min, mordanting iodine solution for 1min, decoloring alcohol for 30s, counterdyeing pink dye for 30s, observing under a microscope, and determining that tiny campylobacter which is negative in gram dyeing is positive in helicobacter pylori.
Urease detection method: and (3) selecting a transparent tiny suspicious colony on a Columbia blood agar plate to cause urease test paper, and after 1min, if the test paper is changed from yellow to red, the test paper is positive to the helicobacter pylori.
(3) Analysis of results
In the embodiment, 25 samples are collected, and are subjected to separation culture and identification to obtain 19 positive strains (the positive strains are detected and identified by a gram staining detection method and a urease detection method), wherein the positive rate is 76%. The strain which is detected to be positive by helicobacter pylori has the following characteristics: after 3 days of microaerophilic culture on a Columbia blood agar culture plate, the colony is gray-white needle-point-like, the urease test is positive, and gram-negative (purple red) tiny, bent and spiral bacilli can be observed under a microscope (200 times optical microscope) after gram staining (see figure 1).
Example 2: the method of the invention is adopted to separate and culture the wild helicobacter pylori
The material, apparatus and procedure for helicobacter pylori isolation culture of this example were substantially the same as those of example 1, except that:
(1) unlike (1.2) the preparation of the transport solution in example 1, the method employed in this example was:
a solution of hpLPS was prepared by dissolving 15g of hpLPS in 100ml of sterile distilled water and filter-sterilizing using a 0.22 μm filter. Weighing 281g of bouillon powder, heating and stirring to dissolve in distilled water, adding distilled water to a constant volume of 100ml to obtain a bouillon culture medium, adding 0.5g of activated carbon particles, dissolving and mixing uniformly, and sterilizing at 121 ℃ under high pressure. After cooling, 8ml of sterile hpLPS solution and 0.1ml of sterile 0.5g/ml cysteine solution were added. The Brookfield broth containing hpLPS was then aseptically dispensed into 1ml EP tubes, 0.8ml per EP tube, and stored in a freezer at 4 ℃ until use.
(2) In this example, 32 samples were collected in total.
(3) Unlike (1.3) Columbia blood agar plate preparation in example 1, this example adopted the method of
Weighing 39g of Columbia agar, adding 90ml of double distilled water, sterilizing at 121 ℃ under high pressure, cooling to about 50 ℃, adding 10ml of 10% (V/V) fetal calf serum into an ultra-clean workbench, mixing uniformly, and pouring into a flat plate to obtain the Columbia blood agar culture plate.
In the embodiment, 32 samples are collected, and 20 positive strains (positive in gram staining detection method and urease detection method) are obtained through separation, culture and identification, and the positive rate is 62.5%. The strains which are detected to be positive for helicobacter pylori have the following characteristics: after 3 days of microaerophilic culture on a Columbia blood agar culture plate, the colony is gray-white needle-point-like, the urease test is positive, and gram-negative (purple red) tiny, crooked and spiral bacilli can be observed under a microscope (200 times optical microscope) after gram staining (see figure 2).
Example 3: the method of the invention is adopted to separate and culture the wild helicobacter pylori
The material, apparatus and procedure for helicobacter pylori isolation culture of this example were substantially the same as those of example 1, except that:
(1) unlike (1.2) the preparation of the transport solution in example 1, the method employed in this example was:
a solution of hpLPS was prepared by dissolving 15g of hpLPS in 100ml of sterile distilled water and filter-sterilizing using a 0.22 μm filter. Weighing 281g of bouillon powder, heating and stirring to dissolve in distilled water, adding distilled water to a constant volume of 100ml to obtain a bouillon culture medium, adding 1g of cyclodextrin, dissolving and mixing uniformly, and then sterilizing at 121 ℃ under high pressure. After cooling, 3ml of sterile hpLPS solution and 0.1ml of sterile 0.5g/ml cysteine solution were added. The Brookfield broth containing hpLPS was then aseptically dispensed into 1ml EP tubes, 0.8ml per EP tube, and stored in a freezer at 4 ℃ until use.
(2) In this example, a total of 27 samples were collected.
In the embodiment, 27 samples are collected in total, and after separation culture and identification, 15 positive strains are obtained (the positive strains are detected and identified by a gram staining detection method and a urease detection method), and the positive rate is 55.6%. The strains which are detected to be positive for helicobacter pylori have the following characteristics: after 3 days of microaerophilic culture on a Columbia blood agar culture plate, the colony is gray-white needle-point-like, the urease test is positive, and gram-negative (purple red) tiny, crooked and spiral bacilli can be observed under a microscope (200 times optical microscope) after gram staining (see figure 3).
Experimental example 1: transport liquid effect verification experiment
The standard strain NCTC11639 used in the verification experiment is given by the management and preservation center of helicobacter pylori strains in China. Dipping helicobacter pylori in logarithmic growth phase from a Columbia blood agar culture plate by using a pipette tip in an aseptic environment, then dipping the part dipped with thalli of the pipette tip into a conveying liquid (4 conveying liquids in total, shown in table 1), repeatedly blowing and uniformly mixing by using the pipette tip, and adjusting the concentration of bacteria in the conveying liquid to 1.0 x 106one/mL. After the transfer liquid containing helicobacter pylori is preserved for a certain time at 4 ℃ (the preservation time is shown in table 1, 6 preservation times are set for each transfer liquid experimental group in the experiment), 0.2ml of transfer liquid containing helicobacter pylori is taken to coat a Columbia blood agar plate, and after 3 days of culture under microaerophilic conditions (a three-gas incubator, culture at 37 ℃), helicobacter pylori positive identification is carried out, and the positive rate and the form of the helicobacter pylori are detected.
See example 1 (1.3); gram stain assays detect the positive rate and morphology of H.pylori see example 1 (2.4). 30 repeated experiments are set for each preservation time of each delivery liquid, and the helicobacter pylori positive rate refers to the proportion of the repeated experiments which are detected to be positive by a gram stain detection method in the 30 repeated experiments. The experimental design of this experimental example is shown in table 1:
table 1: experimental design for verifying effect of conveying liquid
As shown in FIG. 4, it is understood from the results of the experiments that the transport solutions of T1, T2 and T3 can preserve H.pylori for a long period of time, and even if the cells are preserved for 12 hours in the transport solutions of T1, T2 and T3, the positive rate of H.pylori is 35% or more, which means that H.pylori can survive for a long period of time in the transport solutions of T1, T2 and T3 and can maintain its activity, which provides sufficient time for the preservation and transport of H.pylori. After the delivery liquid in the prior art stores helicobacter pylori for 5 hours, the positive rate of the helicobacter pylori is reduced to 10%, which indicates that the delivery liquid in the prior art causes certain environmental stress to the helicobacter pylori, so that the activity of the helicobacter pylori is reduced and even the helicobacter pylori dies.
The results of the gram stain assay for the detection of the morphology of H.pylori are: after the T0 group preserved the strains for 5h in the delivery liquid in the prior art, a large amount of brevibacterium morphologies and coccoid morphologies (see figure 5) were observed by a light microscope, and the brevibacterium morphologies and the coccoid morphologies are growth morphologies with poor activity of helicobacter pylori; while the cells were preserved in the transport solutions of T1, T2 and T3 for 10h, a large number of gull-shaped and S-shaped bent bacterial forms were observed under a light microscope (see FIG. 6: H. pylori forms were observed under a light microscope after the cells were preserved in the transport solutions of T1 for 10 h). The results show that the transport fluids in T1, T2 and T3 have better effect on maintaining the morphology of helicobacter pylori, namely the transport fluids in T1, T2 and T3 have stronger effect on maintaining the activity of the helicobacter pylori.
Experimental example 2: bacteriostatic experiment of helicobacter pylori lipopolysaccharide extract
The bacteriostatic ring experiment was performed according to "disinfection technical code (2017 edition)". The experimental bacteria adopted in the experiment are three common mixed bacteria of Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis) and Staphylococcus aureus (Staphylococcus aureus) to carry out the bacteriostatic ring experiment.
(1) Preparing LB culture medium plate
To 950ml of deionized water were added 10g of tryptone, 5g of yeast extract, 10g of NaCl, and 15g of agar, and the vessel was shaken until the solute was dissolved. The pH value is adjusted to 7.0 by using 5mol/L NaOH, and the volume is adjusted to 1L by using deionized water. Steam sterilizing at 121 deg.C under high pressure for 20min, heating for sterilizing, and pouring the culture medium into a plate before cooling and solidifying.
(2) Preparation of bacteriostatic tablets
Preparation of lipopolysaccharide sample tablet: a sterile and dried filter paper (diameter 5mm) was taken, 20. mu.l of a sterile distilled water solution of a helicobacter pylori lipopolysaccharide extract (hpLPS) was dropped on each filter paper, and then the filter paper was laid flat in a clean sterile plate, and was left to open the lid and dried in an incubator at 37 ℃ for use.
Preparation of negative control sample piece, sterile dry filter paper (diameter 5mm) is dropped with 20 μ l sterile distilled water for drying.
Preparation of hpLPS see example 1(1.1), hpLPS solid was dissolved in sterile distilled water, and the concentration gradient of the hpLPS solution was set as: 1g/ml, 5g/ml, 8g/ml, 10g/ml,12g/ml, 15 g/ml.
(3) Inoculation of the laboratory bacteria
Dipping with sterile cotton swab to 5 × 10 concentration5cfu/ml-5×106cfu/ml experimental bacterial suspension is evenly smeared on the surface of an LB culture medium flat plate for 3 times. For each application 1 time, the plate was rotated 60 degrees and finally the cotton swab was applied around the edge of the plate for one revolution. The petri dish was covered and left to dry at room temperature for 5 min.
(4) Bacteriostatic agent sample slice plaster
And (3) using sterile forceps to take samples and paste the samples on the surface of the flat plate, wherein the distance between the centers of the samples is more than 25mm, and the distance between the centers of the samples and the periphery of the flat plate is more than 15 mm. After the sample is placed, the sample is lightly pressed by using sterile tweezers to be tightly attached to the surface of the flat plate. The plate was covered, placed in a 37 ℃ incubator, and the results were observed after 18 hours of incubation. The diameter of the antibacterial ring (including the patch) was measured and recorded. Repeating the experiment for 3 times, wherein all the experiments with 3 times of repetition have the result of bacteriostasis, and judging the product to be qualified. The negative control group should have no bacteriostatic ring, otherwise the test is invalid.
(5) Results of the experiment
The bacteriostatic effect is shown in table 3, wherein "-" indicates no inhibition (the bacteriostatic ring is less than 7mm), "+" indicates that the diameter of the bacteriostatic ring is greater than or equal to 7mm but less than 15mm, and "+" indicates that the diameter of the bacteriostatic ring is greater than or equal to 15 mm. According to the experimental results, the hpLPS solution with the concentration of 15g/ml has stronger inhibition effect on three experimental bacteria, and the hpLPS solution with lower concentration (10g/ml and 12g/ml) also has inhibition effect on the three experimental bacteria with different degrees.
Table 2: experimental result of bacteriostatic effect of helicobacter pylori lipopolysaccharide extract
The foregoing is merely an example of the present invention and common general knowledge of known specific structures and features of the embodiments is not described herein in any greater detail. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (9)
1. A method for isolated culture of a strain of helicobacter pylori, comprising the steps of,
(1) a transferring step: during transport, the sample is stored in a transport solution comprising a bouillon broth, an adsorbent, a reducing agent and a helicobacter pylori lipopolysaccharide extract; (2) an inoculation step; (3) a culture step;
the helicobacter pylori lipopolysaccharide extract is prepared by the following method:
ultrasonically treating a suspension containing helicobacter pylori, and centrifuging to obtain a liquid phase to obtain a supernatant I; obtaining a crude extraction sample after dialysis purification and freeze drying of the supernatant I; re-dissolving the crude extract by using a buffer solution to obtain a re-dissolved sample, respectively treating the re-dissolved sample by using DNase I, RNase and proteinase K, adding water saturated phenol, and centrifuging to obtain a liquid phase to obtain a supernatant II; dialyzing and purifying the supernatant II, and then treating the dialyzed supernatant II with ethanol to obtain a precipitate; freeze drying the precipitate to obtain the lipopolysaccharide extract of helicobacter pylori.
2. The method for the isolated culture of a helicobacter pylori strain according to claim 1, wherein in the inoculation step, a Columbia blood agar plate containing a helicobacter pylori lipopolysaccharide extract is prepared first, and the sample is inoculated onto the Columbia blood agar plate.
3. The method for the isolated culture of helicobacter pylori strain according to claim 2, wherein the Columbia blood agar plate contains fetal bovine serum or defibrinated sheep whole blood.
4. The method for the isolated culture of a helicobacter pylori strain according to any one of claims 1 to 3, wherein in the culturing step, the Columbia blood agar plate inoculated with the sample is placed in a culture device and incubated at 37 ℃ for 3 to 5 days to obtain a suspected colony growing on the Columbia blood agar plate; the gas composition in the culture device is 5% of oxygen, 10% of carbon dioxide and 85% of nitrogen.
5. The method for isolated culture of helicobacter pylori strain according to claim 4, wherein the culture device is a three-gas incubator or an anaerobic tank with an air-generating bag inside.
6. The method for the isolated culture of a strain of helicobacter pylori according to claim 4, further comprising, after the culturing step, the step of identifying: and identifying the suspected colony obtained in the culturing step.
7. The method for the isolated culture of a helicobacter pylori strain according to claim 6, wherein the identification step employs an identification method comprising: one or a combination of both of a gram stain assay and a urease assay.
8. The method for the isolated culture of a strain of helicobacter pylori according to any one of claims 1 to 3 and 5 to 7, wherein the adsorbent is cyclodextrin, activated carbon or soluble starch.
9. The method for the isolated culture of a helicobacter pylori strain according to any one of claims 1 to 3 and 5 to 7, wherein the reducing agent is cysteine.
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