CN109880772A - A kind of method for isolating and culturing Helicobacter pylori strain - Google Patents
A kind of method for isolating and culturing Helicobacter pylori strain Download PDFInfo
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Abstract
本发明属于生物医药领域，涉及一种细菌分离培养方法，具体涉及一种幽门螺杆菌菌株分离培养的方法。在本方法的样本转运过程中，将疑似含有幽门螺杆菌的样本保存于运送液中；所述运送液包括布氏肉汤培养基、吸附剂、还原剂和幽门螺杆菌脂多糖提取物。其中，幽门螺杆菌脂多糖提取物对杂菌具有抑制作用,对幽门螺杆菌菌体具有保护作用。本发明方法可延长样本转运至细菌培养研究单位的转运时间并保持幽门螺杆菌活性，从而降低野生型幽门螺杆菌菌株分离培养难度并提高细菌培养的阳性率。本发明的幽门螺杆菌培养方法可以应用到细菌分型、致病机制研究、构建动物模型及确定致病因子等研究中。
The invention belongs to the field of biomedicine and relates to a method for separating and culturing bacteria, in particular to a method for separating and culturing Helicobacter pylori strains. During the sample transfer process of the method, the sample suspected of containing Helicobacter pylori is stored in a transport liquid; the transport liquid includes a Brucella broth medium, an adsorbent, a reducing agent and a Helicobacter pylori lipopolysaccharide extract. Among them, Helicobacter pylori lipopolysaccharide extract has inhibitory effect on miscellaneous bacteria, and has protective effect on Helicobacter pylori bacteria. The method of the invention can prolong the transfer time of the sample to the bacterial culture research unit and maintain the activity of Helicobacter pylori, thereby reducing the difficulty of isolating and culturing wild-type Helicobacter pylori strains and improving the positive rate of bacterial culture. The Helicobacter pylori culture method of the present invention can be applied to bacterial typing, pathogenic mechanism research, construction of animal models and determination of pathogenic factors.
Description
Technical field
The invention belongs to biomedicine fields, are related to a kind of bacterium isolated culture method, and in particular to a kind of helicobacter pylorus
The method that bacteria strain is separately cultured.
Background technique
Helicobacter pylori (Helicobacter pylori, H.pylori) is a kind of micro- need of spiral helicine Gram-negative
Oxygen bacterium infects the population of global half, is the main pathogenic fungi for causing gastritis, peptic ulcer, gastric cancer, the hair of helicobacter pylori
Cognition of the mankind to relationship between chronic infection, inflammation and cancer is now deepened, Human Gastric carcinoma is classified as by the World Health Organization
I class carcinogen.The clinical strains for the helicobacter pylori being separately cultured can be used for typing of bacteria, pathogenic mechanism research, building
The basic research such as animal model and determining virulence factor.But helicobacter pylori is very harsh to growth conditions requirement, it is desirable that with
The similar condition of culture of stomach submucosa condition in vivo, and it is more demanding to nutritional condition, to cause helicobacter pylori
The problems such as positive rate is lower is cultivated, the further research to helicobacter pylori clinical strains is affected.
Inventor has delivered in 2016 Scientific Articles, and " helicobacter pylori clinical strains being separately cultured and identifying, Liu Zheng
Beauty etc., Guiyang Medical College journal, 2016 ", which establishes a kind of method of helicobacter pylori clinical strains being separately cultured,
Specifically: after taking clinical sample, sample is put in antibiotic brucella broth and is transported in liquid, then sample is sent to laboratory point
From and detection；The Colombia for taking the doubtful clinical sample containing helicobacter pylori to be inoculated in 10% Sheep Whole Blood selectively cultivates
On base, 37 DEG C after culture 3-5 days in three gas incubators, transparent tiny suspicious bacterium colony progress bacterium colony detection on picking blood plate.
But above-mentioned technical proposal has the following problems: in transport process, helicobacter pylori, which is under stress conditions, (is not suitable for pylorus
The environmental condition of Helicobacter growth), transhipment time is too long to be will lead to bacterial death or modal variation occurs.So after sampling
The time restriction that sample is transported to the laboratory being separately cultured is 4 hours or so, and the time restriction is excessively stringent.For
The case where sampling position and experiment cultivate place relatively far apart, the transhipment of sample takes a long time, this is resulted in wait cultivate
There is modal change in isolated helicobacter pylori mortality, and the positive rate that finally will appear Bacteria Culture is low and a large amount of
The case where " false negative ".
Summary of the invention
The invention mainly solves the technical problem of providing a kind of method that Helicobacter pylori Strains are separately cultured, this method
The transhipment time limitation that sample is transported to Bacteria Culture research unit can be extended, to reduce wild type H. pylori strain point
From culture difficulty and the positive rate of Bacteria Culture is improved, to carry out various researchs to wild type H. pylori strain.
In order to solve the above technical problems, the invention proposes technical solutions once:
A kind of method that Helicobacter pylori Strains are separately cultured, includes the following steps,
(1) step is transported: in transport process, by helicobacter pylori Sample preservation in transport liquid, the transport liquid packet
Include brucella broth culture medium, adsorbent, reducing agent and Helicobacter pylori lipopolysaccharide extract；(2) inoculation step；(3) culture step
Suddenly.
By adopting the above technical scheme, helicobacter pylori can be increased and transporting the holding time in liquid, it is deep and remote to reduce wild type
The difficulty of Helicobacter pylori strain isolation culture improves the culture positive rate of the bacterium.Wild type H. pylori strain is from certainly
Isolated helicobacter pylori original strain in right boundary or organism.Sample is the doubtful object containing helicobacter pylori, sample
It originally can be diversified forms, for example, sample can be cotton balls or the sampling for dipping or wiping across the doubtful aggregate site of helicobacter pylori
Device.The culture positive rate of helicobacter pylori refers to that culture identification is the ratio that the sample of Helicobacter pylori accounts for total number of samples.
The technical program pertains only to obtain the operating procedure after sample, which is the guarantor carried out to the helicobacter pylori in sample
It deposits, transport, be separately cultured and identify, be not related to any sampling operation or process.
The technical principle of the technical program are as follows: transport liquid and maintenance effect played to helicobacter pylori, helicobacter pylori can be with
Long period, which is stored in, transports in liquid, to be transported to Bacteria Culture research unit after increasing acquisition helicobacter pylori sample
Transport the time limit.Wherein, Helicobacter pylori lipopolysaccharide extract is inhibited to miscellaneous bacteria (see experimental example 2), can reduce miscellaneous
Bacterium is to the competitive inhibitory effect of helicobacter pylori, and Helicobacter pylori lipopolysaccharide extract of the invention is to helicobacter pylori sheet
Body does not have inhibiting effect.Extract is free of antibiotic, is not in that antibiotic inhibits the phenomenon that growth of H. pylori.Adsorbent
The secondary metabolite of bacterium in adsorbable transport liquid avoids secondary metabolite from generating growth of H. pylori and inhibits to make
With.Reducing agent keeps micro- oxygen environment, is conducive to growth of H. pylori.
The beneficial effect of the technical program is described in detail below:
(1) Helicobacter pylori lipopolysaccharide extract substitute antibiotics, it is inhibited to miscellaneous bacteria
Helicobacter pylori is easy to happen deformation or death under environment-stress, and wherein deformation includes that thallus becomes from campylobacter
For ball-type, Filamentous type.Ball-type form be generally it is non-can cultivation conditions, Filamentous type form is generally more difficult to be further cultured for.It assists and compels condition packet
Include antibiotic effect, environment Poisoning Effects of Factors and gas condition do not meet it is micro- it is aerobic require etc. to be not suitable for helicobacter pylori it is raw
Long condition.Wild-type H. pylori is isolated from growth of H. pylori environment, in the growing environment exist compared with
The wild-type H. pylori just separated from the growing environment for have bacterium is transported to bacterium to inhibit varied bacteria growing by more miscellaneous bacterias
It will use antibiotic in the transport process of culture studies unit.Antibiotic has the growth of helicobacter pylori and the maintenance of form
There is biggish inhibiting effect.Since the long-time of antibiotic uses, miscellaneous bacteria resistance enhancing, the dosage of antibiotic is also increased accordingly
It can achieve the purpose that inhibit miscellaneous bacteria, the increase of antibiotic dosage can also cause stronger environment-stress to helicobacter pylori, thus
The growth conditions for affecting helicobacter pylori improve the difficulty of subsequent Bacteria Culture.However if not using antibiotic,
It cannot inhibit varied bacteria growing, miscellaneous bacteria and pylorus helical bacillus fight for nutritional resource, and miscellaneous bacteria mass propagation can also discharge toxicity
Secondary metabolite, the growth conditions for also resulting in helicobacter pylori are poor, and in subsequent Bacteria Culture, it may appear that miscellaneous
The higher problem of bacterium rate, affects the positive rate of heiicobacter pylori cultivation.
The technical program uses Helicobacter pylori lipopolysaccharide extract to inhibit to transport the miscellaneous bacteria in liquid.It is existing compared to rising
The scheme that Antibiotics combination is used in technology, has the advantage that the dosage of a. antibiotic greatly reduces, to reduce antibiosis
Coercion of the element to helicobacter pylori.Such as: usually every 100ml transports the antibiotic that 5mg or so is added in liquid in the prior art,
And the technical program is used, it transports and does not need that antibiotic is added in liquid.B. Helicobacter pylori lipopolysaccharide extract to miscellaneous bacteria have compared with
Good inhibiting effect, there is certain protective effect to helicobacter pylori itself.Helicobacter pylori lipopolysaccharide is that helicobacter pylori is thin
One of cell wall ingredient positioned at the outermost layer of cell wall, and is covered on the mucopeptide of cell wall.For a long time, helicobacter pylorus
Bacterium lipopolysaccharides is always to be used as virulence factor to be studied, and has focused largely on it to the report of Helicobacter pylori lipopolysaccharide and causes a disease
Mechanism, molecular structure etc..Inventor the study found that Helicobacter pylori lipopolysaccharide extract have broad-spectrum antibacterial action, can
To substitute part antibiotic, play the role of inhibiting varied bacteria growing in being separately cultured of helicobacter pylori.And helicobacter pylorus
Bacterium lipopolysaccharides extract can form certain attachment on the cell wall of helicobacter pylori thallus, provide helicobacter pylori certain
Protective effect, resist culture environment in antibiotic and toxicity secondary metabolite.
(2) in the transport liquid of this programme, the addition of adsorbent can remove the toxicity secondary generation for transporting various bacteriums in liquid
It thanks to product, reduces the toxic ion (such as peroxide) transported in liquid, be stored in and transported in liquid for the helicobacter pylori long period
Greater activity is maintained to create conditions in transporting liquid with helicobacter pylori.
(3) in the transport liquid of this programme, the addition of reducing agent can maintain the microaerophilic growth conditions of helicobacter pylori.
(4) the transport liquid of this programme is in addition to can store helicobacter pylori the long period, additionally it is possible to maintain helicobacter pylori
For activity in a higher level, the helicobacter pylori being stored in this transport liquid has preferable spiral, is subsequent
Secondary culture creates condition, and also improves the Helicobacter pylori rate of follow-up cultivation.
Further, in the inoculation step, the Columbia Blood Agar of the extract containing Helicobacter pylori lipopolysaccharide is prepared
Plate, then the sample is inoculated on Columbia Blood Agar plate.
By adopting the above technical scheme, Helicobacter pylori lipopolysaccharide extract can inhibit on Columbia Blood Agar plate
Varied bacteria growing, and play a protective role to helicobacter pylori thallus itself.Columbia Blood Agar plate is full of nutrition, main to use
It is the more suitable type of culture medium of heiicobacter pylori cultivation in the culture of the higher bacterium of nutritional requirement.
Further, fetal calf serum or de- fiber Sheep Whole Blood are contained in the Columbia Blood Agar plate.
By adopting the above technical scheme, serum or whole blood are added in culture plate, it is possible to provide the nutrition that culture medium is lacked
Substance (such as amino acid, vitamin, inorganic matter, lipid material, nucleic acid derivative) can also be provided with and grow increasing conducive to cell
Grow required hormone, growth factor.Inventor imprisons the study found that the nutrition that provides of 10% de- fiber Sheep Whole Blood is more abundant
Helicobacter pylori growth conditions in de- fiber Sheep Whole Blood are more preferable compared with its growth conditions in fetal calf serum.
Further, in incubation step, the Colombia's blood agar plate for being inoculated with the sample is placed in culture apparatus
In, 37 DEG C constant temperature incubation 3-5 days, obtain the doubtful bacterium colony that is grown on Columbia Blood Agar plate；In the culture apparatus
Gas composition be oxygen 5%, carbon dioxide gas 10% and nitrogen 85%.
By adopting the above technical scheme, the gas composition in culture apparatus is oxygen 5%, carbon dioxide gas 10% and nitrogen
85% provides suitable air environment for helicobacter pylori, because helicobacter pylori is a kind of obligate microaerobion, it steady
Fixed growth needs the microenvironment by the oxygen containing 5%-8%, it cannot grow in absolute anaerobic environment in an atmosphere.Culture
At 3 days or so, the naked eyes form and mirror hypothallus form of helicobacter pylori bacterium colony are very typical (helical form and sea-gull shape), bacterium
In logarithmic growth phase, it can be passed on, be saved or the research such as Antibiotics resistance test.After culture about 5 days, contaminated through gram
Visible bacterium starts spherical change under mirror after color, is in rod-short, when culture extended culture to 7 days, helicobacter pylori form is spherical in shape
Become.Helicobacter pylori after deformation is in poor growth conditions, is difficult to Successful Resuscitation after cryo-conservation.
Further, the culture apparatus is three gas incubators or the internal anaerobic jar for being equipped with production airbag.
By adopting the above technical scheme, inventor studies the internal anaerobic jar for being equipped with production airbag of discovery or three gas incubators
Good growing environment can be provided for growth of H. pylori.Wherein, the growth of H. pylori situation in three gas incubators
Relatively better than the growth of H. pylori situation being equipped in the anaerobic jar for producing airbag.
Further, further include authentication step after incubation step: to the doubtful bacterium colony obtained in incubation step into
Row identification.
By adopting the above technical scheme, authentication step is the identification in order to determine whether doubtful bacterium colony is helicobacter pylori bacterium colony
As a result be positive doubtful bacterium colony be helicobacter pylori bacterium colony, the helicobacter pylori bacterium colony can be used for further typing of bacteria,
The basic research such as pathogenic mechanism research, building animal model and determining virulence factor.
Further, the identification method that the authentication step uses are as follows: in Gram's staining detection method and urease detection method
One or two kinds of combinations.
By adopting the above technical scheme, Gram's staining detection method and urease detection method are Testing and appraisal helicobacter pylori
Effective ways.Gram's staining detection method are as follows: after dyeing, light is shown in tiny, the bending bar of Gram-negative under the microscope
Bacterium is Helicobacter pylori.Urease detection method are as follows: transparent tiny suspicious bacterium colony is extremely on picking Columbia Blood Agar plate
It is then Helicobacter pylori that test paper, which becomes red from yellow, on urea enzyme test peper, after 1 minute.
Further, the preparation method of the Helicobacter pylori lipopolysaccharide extract is ultrasonic extraction.
By adopting the above technical scheme, Helicobacter pylori lipopolysaccharide is extracted using ultrasonic extraction, is had high-efficient
And the advantages that convenient and efficient.The cavitation of ultrasound generates powerful percussion to the cell wall of helicobacter pylori, destroys thin
Cell wall discharges lipopolysaccharides.The molecular structure and molecular weight of lipopolysaccharides are the key that determine its bioactivity, and extracting method is not
Together, the fine structure of polysaccharide and the difference of molecular weight be can lead to.Inventor is the study found that the helicobacter pylori rouge that ultrasonic method is extracted
Polyoses extract has the preferable activity for inhibiting miscellaneous bacteria, may replace antibiotic, to reduce helicobacter pylori in the work of antibiotic
With lower a possibility that being in stress state.
Further, transporting adsorbent described in step is cyclodextrin, activated carbon or soluble starch.
By adopting the above technical scheme, cyclodextrin, activated carbon or soluble starch, which can be absorbed, transports what derivation in liquid generated
The toxic product that toxicity oxonium ion and bacterial metabolism generate, prevents helicobacter pylori to be under stress conditions too early, and then increases
Helicobacter pylori is transporting the holding time in liquid, reduces the difficulty of subsequent Bacteria Culture.
Further, transporting reducing agent described in step is cysteine.
By adopting the above technical scheme, the addition of cysteine can be such that transport liquid restores in advance, and oxidation-reduction potential is dropped
As low as to a certain degree, micro- aerobic environment for keeping helicobacter pylori preferred.
Detailed description of the invention
Fig. 1 illustrates form of the bacterial strain that Helicobacter pylori is identified as in embodiment 1 under light microscopic: through gram
(200 times of optical microscopies) is observed under the microscope after dyeing.
Fig. 2 illustrates form of the bacterial strain that Helicobacter pylori is identified as in embodiment 2 under light microscopic: through gram
(200 times of optical microscopies) is observed under the microscope after dyeing.
Fig. 3 illustrates form of the bacterial strain that Helicobacter pylori is identified as in embodiment 3 under light microscopic: through gram
(200 times of optical microscopies) is observed under the microscope after dyeing.
Fig. 4 is the transport liquid compliance test result experimental result picture of experimental example 1, which is helicobacter pylorus after different transport liquid processing
Bacterium positive rate is with the curve graph for handling time change.
Fig. 5 is ne ar figure (200 times optics of the T0 group helicobacter pylori in transporting liquid after preservation 5h in experimental example 1
Micro- sem observation, bacterium is through Gram's staining).
Fig. 6 is ne ar figure (200 times optics of the T1 group helicobacter pylori in transporting liquid after preservation 10h in experimental example 1
Micro- sem observation, bacterium is through Gram's staining).
Specific embodiment
It is further described below by specific embodiment, in which:
Embodiment 1-3 is to be separately cultured using the method for the present invention to wild-type H. pylori；Experimental example 1 is to adopt
The compliance test result that liquid carries out is transported to the present invention with helicobacter pylori reference culture；Experimental example 2 mentions for Helicobacter pylori lipopolysaccharide
The bacteriostasis of object is taken to test.
Embodiment 1: wild-type H. pylori is separately cultured using the method for the present invention
(1) material and instrument
Micro- aerobic culture tank and micro- aerobic production airbag, superclean bench (Mitsubishi, Japan)；Three gas incubators, one
Secondary property oese, Columbia agar (OXOID, Britain)；Brucella broth, Gram stain (Chaohu, Anhui Province, China expands kind medical company,
China), urea enzyme test peper (Guangdong Zhuhai Ke Di scientific & technical corporation, China)；Fetal calf serum, cysteine, soluble starch, activity
Charcoal, cyclodextrin, phosphate (PBS) buffer, de- fiber Sheep Whole Blood (You Kang Biotechnology Co., Ltd, China)；Deoxidation core
Ribonuclease T. DNase I, ribalgilase RNaseA, three (methylol) aminomethane hydrochloride Tris-HCL (TAKARA)；Standard
Bacterial strain NCTC11639 is presented by the management of Helicobacter pylori in China bacterial strain and collection.
(1.1) preparation of Helicobacter pylori lipopolysaccharide extract
Helicobacter pylori NCTC11639 is activated first, is screened, expands culture, collects the bacterium of growth animated period,
Bacterial suspension is made.Bacterial suspension is centrifuged 10min through 5000rpm, must precipitate.After precipitating is washed with PBS buffer solution, 5000rpm
It is centrifuged 10min, is repeated 2 times.Sediment (thallus) is resuspended with PBS buffer solution, and adjusting cell concentration is 2.0 × 1010A/mL, system
At dense bacterium solution.It is ultrasonically treated dense bacterium solution 20min, ultrasonic power 300W, ultrasonic treatment interval carries out, stop 10s after ultrasonic 10s,
So circulation is until 20min terminates.Dense bacterium solution after ultrasonic treatment is centrifuged 30min through 3000rpm, collects supernatant.Collect institute
There is supernatant to be placed in bag filter, flowing water is dialysed for 24 hours (every 5-6h changes a water), and freeze-dried to obtain helicobacter pylori rouge more
The sample that sugar slightly mentions.
It takes the 100mmol/L Tris-HCL (pH8) of 10mL to redissolve the sample slightly mentioned, adds final concentration of 100 μ g/mL's
Overnight, the Proteinase K of final concentration of 100 μ g/mL, 65 DEG C of works are then added in the RNase of DNase I and 50 μ g/mL, 37 DEG C of digestion
Use 2h.Then water-saturated phenol is added, mixes, 4000rpm centrifugation 30min takes supernatant, and supernatant is placed in bag filter and is distilled
Water is dialysed 12h (every 5-6h changes a water), and solution in bag filter is collected, and adds 10 times of 95% ethyl alcohol of volume, and -20 DEG C heavy overnight
It forms sediment, then 4000r/min is centrifuged 30min, takes the dry weighing of pellet frozen, obtains Helicobacter pylori lipopolysaccharide extract solid
(hpLPS), it saves backup for -20 DEG C.
(1.2) liquid preparation is transported
HpLPS solution is prepared, 15g hpLPS is dissolved in 100ml sterile distilled water, is removed using 0.22 μm of membrane filtration
Bacterium.Brucella broth powder 281g is weighed, heating stirring is dissolved in distilled water, and adding distilled water to be settled to final volume is 100ml,
Brucella broth culture medium is obtained, 1g soluble starch, 121 DEG C of high pressure sterilizations after dissolution mixes are added.Sterilization is added after cooling
The 0.5g/ml cysteine solution 0.1ml of hpLPS solution 10ml and the sterilization processing of processing.Then by the Bu Shi containing hpLPS
Broth bouillon is aseptic subpackaged in 1ml EP pipe, every EP pipe 0.8ml, is placed in 4 DEG C of refrigerators and saves backup.
(1.3) prepared by Columbia Blood Agar culture plate
It weighs Columbia agar 39g, is added distilled water 90ml, 121 DEG C of high pressure sterilizations, when being cooled to 50 DEG C or so, in
10% (V/V) that 10ml is added in superclean bench takes off fiber Sheep Whole Blood, and plate is poured into after mixing to get Columbia Blood Agar
Culture plate.
(2) helicobacter pylori is separately cultured
(2.1) sample is stored in respectively and is transported in liquid, Sample preservation transported the time in liquid no more than 12 hours.This
Secondary experiment collecting sample totally 25.
(2.2) by the hpLPS solution coating of the sterilization processing of 200 μ l steps (1.2) preparation in Columbia Blood Agar
On plate, reuses the sample saved in the transport liquid in step (2.1) and smeared on Columbia Blood Agar plate, complete to connect
Kind step.
(2.3) Colombia's blood agar plate that sample was smeared is placed in three gas incubators, 37 DEG C of constant temperature incubations 3
It, the doubtful bacterium colony that must be grown on Columbia Blood Agar plate.Gas composition in three gas incubators is oxygen 5%, two
Carbon oxide gas 10% and nitrogen 85%.
(2.4) with Gram's staining detection method and urease detection method to the doubtful bacterium colony on Columbia Blood Agar plate
It is detected.
Gram's staining detection method: a ring physiological saline is taken to be placed on clean glass slide with oese, picking brother's rival
Transparent tiny suspicious bacterium colony is in spreadable on physiological saline on sub- blood agar plate, after dry, fixes in heating on alcolhol burner, crystallization
Purple just to contaminate 1min, iodine solution mordant dyeing 1min, alcohol decoloration 30s, Huang red dye liquor redyes 30s, and microscopically observation is shown in Gram's staining
Negative tiny, bending bacillus is Helicobacter pylori.
Urease detection method: transparent tiny suspicious bacterium colony causes on urea enzyme test peper on picking Columbia Blood Agar plate,
It is then Helicobacter pylori that test paper, which becomes red from yellow, after 1min.
(3) interpretation of result
The present embodiment is total to collecting sample 25, is separately cultured and is identified, (the Gram's staining inspection of 19 plants of positive strain is obtained
Survey method and the equal Testing and appraisal of urease detection method are the positive), positive rate 76%.It is detected as the bacterium of Helicobacter pylori
Strain has a characteristic that micro- aerobic culture was in canescence needle point sample bacterium colony, urine after 3 days on Columbia Blood Agar culture plate
Plain enzyme experiment is the positive, and (200 times of optical microscopies) observes visible Gram-negative (purple under the microscope after Gram's staining
It is red) tiny, bending, helical form bacillus (see Fig. 1).
Embodiment 2: wild-type H. pylori is separately cultured using the method for the present invention
The present embodiment helicobacter pylori is separately cultured material, instrument and process substantially with embodiment 1, and difference is:
(1) it is different from transporting liquid preparation, the method that the present embodiment uses in embodiment 1 (1.2) are as follows:
HpLPS solution is prepared, 15g hpLPS is dissolved in 100ml sterile distilled water, is removed using 0.22 μm of membrane filtration
Bacterium.Brucella broth powder 281g is weighed, heating stirring is dissolved in distilled water, and adding distilled water to be settled to final volume is 100ml,
Brucella broth culture medium is obtained, 0.5g micro activated carbon particle, 121 DEG C of high pressure sterilizations after dissolution mixes are added.It is added after cooling sterile
Change the hpLPS solution 8ml of processing and the 0.5g/ml cysteine solution 0.1ml of sterilization processing.Then by the cloth containing hpLPS
Family name's broth bouillon is aseptic subpackaged in 1mlEP pipe, every EP pipe 0.8ml, is placed in 4 DEG C of refrigerators and saves backup.
(2) total collecting sample 32 in the present embodiment.
(3) it is different from the preparation of (1.3) Columbia Blood Agar culture plate, the method that the present embodiment is taken in embodiment 1
For
It weighs Columbia agar 39g, is added distilled water 90ml, 121 DEG C of high pressure sterilizations, when being cooled to 50 DEG C or so, in
10% (V/V) fetal calf serum of 10ml is added in superclean bench, and it is flat to get Columbia Blood Agar culture that plate is poured into after mixing
Plate.
The present embodiment is total to collecting sample 32, is separately cultured and is identified, (the Gram's staining inspection of 20 plants of positive strain is obtained
Survey method and the equal Testing and appraisal of urease detection method are the positive), positive rate 62.5%.It is detected as the bacterium of Helicobacter pylori
Strain has a characteristic that on Columbia Blood Agar culture plate after micro- aerobic culture 3 days in canescence needle point sample bacterium colony, urea
Enzyme experiment is the positive, and the visible Gram-negative of (200 times of optical microscopies) observation is (purplish red under the microscope after Gram's staining
Color) tiny, bending, helical form bacillus (see Fig. 2).
Embodiment 3: wild-type H. pylori is separately cultured using the method for the present invention
The present embodiment helicobacter pylori is separately cultured material, instrument and process substantially with embodiment 1, and difference is:
(1) it is different from transporting liquid preparation, the method that the present embodiment uses in embodiment 1 (1.2) are as follows:
HpLPS solution is prepared, 15g hpLPS is dissolved in 100ml sterile distilled water, is removed using 0.22 μm of membrane filtration
Bacterium.Brucella broth powder 281g is weighed, heating stirring is dissolved in distilled water, and adding distilled water to be settled to final volume is 100ml,
Brucella broth culture medium is obtained, 1g cyclodextrin, 121 DEG C of high pressure sterilizations after dissolution mixes are added.Sterilization processing is added after cooling
HpLPS solution 3ml and sterilization processing 0.5g/ml cysteine solution 0.1ml.Then by the brucella broth containing hpLPS
Culture medium is aseptic subpackaged in 1mlEP pipe, every EP pipe 0.8ml, is placed in 4 DEG C of refrigerators and saves backup.
(2) total collecting sample 27 in the present embodiment.
The present embodiment is total to collecting sample 27, is separately cultured and is identified, (the Gram's staining inspection of 15 plants of positive strain is obtained
Survey method and the equal Testing and appraisal of urease detection method are the positive), positive rate 55.6%.It is detected as the bacterium of Helicobacter pylori
Strain has a characteristic that on Columbia Blood Agar culture plate after micro- aerobic culture 3 days in canescence needle point sample bacterium colony, urea
Enzyme experiment is the positive, and the visible Gram-negative of (200 times of optical microscopies) observation is (purplish red under the microscope after Gram's staining
Color) tiny, bending, helical form bacillus (see Fig. 3).
Experimental example 1: the experiment of liquid compliance test result is transported
Reference culture NCTC11639 used in this confirmatory experiment is presented by the management of Helicobacter pylori in China bacterial strain and collection
It gives.In an aseptic environment, it is dipped from Columbia Blood Agar culture plate with liquid-transfering gun pipette tips deep and remote in logarithmic growth phase
Then the part for being moistened with thallus of pipette tips is immersed and transports liquid (transporting totally 4 kinds of liquid, be shown in Table 1), uses liquid-transfering gun by Helicobacter pylori
Piping and druming mixes repeatedly, then adjusts and transport in liquid bacterial concentration to 1.0 × 106A/mL.Transport liquid containing helicobacter pylori is existed
(the preservation time is shown in Table 1, this experiment is provided with 6 kinds of preservations to every kind of transport liquid experimental group after a certain period of time for preservation under the conditions of 4 DEG C
Time), the transport liquid that then takes 0.2ml to contain helicobacter pylori coating Columbia Blood Agar plate, (three under micro- aerobic condition
Gas incubator, 37 DEG C of cultures) after culture 3 days, Helicobacter pylori identification is carried out, the positive rate and shape of helicobacter pylori are detected
State.
The preparation method of Columbia Blood Agar plate is referring to embodiment 1 (1.3)；Gram's staining detection method detects pylorus
The positive rate and form of helicobacter are referring to embodiment 1 (2.4).It is real that 30 repetitions are arranged in every kind of preservation time of every kind of transport liquid
It tests, Helicobacter pylori rate refers in 30 repetition experiments, and Gram's staining detection method Testing and appraisal is helicobacter pylori sun
Property the shared ratio of repetition experiment.The experimental design of this experimental example is as shown in table 1:
Table 1: the design of liquid compliance test result experiments experiment is transported
Experimental result is as shown in figure 4, according to the experimental results, the transport liquid in T1, T2 and T3 can be with preservation helicobacter pylorus
Bacterium longer time, though thallus in the transport liquid in T1, T2 and T3 preservation 12h, Helicobacter pylori rate also exist
35% or more, the longer time and can it means that helicobacter pylori can survive in the transport liquid in T1, T2 and T3
To keep its activity, this just provides the sufficient time for the preservation transhipment of helicobacter pylori.And the transport liquid of the prior art exists
After preservation helicobacter pylori 5h, Helicobacter pylori rate has had been reduced to 10%, illustrates transport liquid in the prior art to deep and remote
Helicobacter pylori causes certain environment stress, so that pylorus helical bacillus activity reduces even death.
Gram's staining detection method detects the experimental result of the form of helicobacter pylori are as follows: the fortune of T0 group in the prior art
After liquor charging preservation thallus 5h, om observation to a large amount of brevibacterium forms and spherical morphology (see Fig. 5), brevibacterium form and spherical shape
State is the poor growthform of helicobactor pylori activity；And in T1, T2 and T3, liquid preservation thallus 10h is transported, it can also be in light microscopic
Under observe a large amount of sea-gull shapes and the forniciform ne ar of S type (after transporting liquid preservation thallus 10h see Fig. 6: T1 group, under light microscopic
Observe helicobacter pylori form).Illustrate that the transport liquid in T1, T2 and T3 is preferable to the form maintenance effect of helicobacter pylori,
It is stronger with regard to the active maintenance effect of transport liquid against helicobacter pylori in meaning T1, T2 and T3.
Experimental example 2: the bacteriostatic experiment of Helicobacter pylori lipopolysaccharide extract
Antibacterial ring size experiment is carried out referring to " disinfection technology standard (version in 2017) ".The experimental bacteria used is tested as Escherichia coli
(Escherichia coli), hay bacillus (Bacillus subtilis) and staphylococcus aureus (Staphylococcus
Aureus) three kinds of common miscellaneous bacterias carry out antibacterial ring size experiment.
(1) LB culture medium flat plate is prepared
Tryptone 10g, yeast extract 5g, NaCl 10g, agar 15g are added in 950ml deionized water, shakes and holds
Device is until solute dissolves.With 5mol/L NaOH tune pH to 7.0,1L is settled to deionized water.Steam goes out under 121 DEG C of high pressures
Culture medium after heat sterilization, before culture medium cooled and solidified, is poured into plate by bacterium 20min.
(2) preparation of bacteria inhibition tablet
The preparation of lipopolysaccharides print: taking sterile and dry filter paper (diameter 5mm), and every dropwise addition helicobacter pylori rouge is more
The 20 μ l of sterile distilled water solution of sugar extract (hpLPS), then filter paper is lain against in clean sterilized petri dishes, uncap and set
It is dried in 37 DEG C of incubators rear spare.
The preparation of negative control print: taking sterile dry filter paper piece (diameter 5mm), every 20 μ l of dropwise addition sterile distilled water,
It is spare after drying.
HpLPS solid is dissolved in sterile distilled water by the preparation method of hpLPS referring to embodiment 1 (1.1), setting
The concentration gradient of hpLPS solution are as follows: 1g/ml, 5g/ml, 8g/ml, 10g/ml, 12g/ml, 15g/ml.
(3) inoculation of experimental bacteria
Dipping concentration with sterile cotton swab is 5 × 105cfu/ml-5×106Cfu/ml tests bacteria suspension, flat in LB culture medium
Plate surface is uniformly smeared 3 times.Every to smear 1 time, plate should rotate 60 degree, finally smear cotton swab one week around plate edge.Lid
Good culture dish, sets drying at room temperature 5min.
(4) bacteriostatic agent print is placed with
It is placed with aseptic nipper coupongs in planar surface, at a distance of 25mm or more between each print center, the week with plate
Edge is at a distance of 15mm or more.After being placed with, with the light pressure-like piece of aseptic nipper, it is made to be tightly attached to planar surface.Plate is covered, sets 37 DEG C
Incubator observes result after cultivating 18h.It measures the diameter (including patch) of antibacterial ring size and records.Experiment is repeated 3 times, and 3 repetitions try
Testing has bacteriostasis result person, is judged to qualification.Negative control group should be generated without antibacterial ring size, and otherwise test is invalid.
(5) experimental result
Fungistatic effect is as shown in table 3, and wherein "-" expression does not inhibit (antibacterial ring size is less than 7mm), and "+" indicates inhibition zone diameter
More than or equal to 7mm but it is less than 15mm, " ++ " indicates that inhibition zone diameter is more than or equal to 15mm.According to the experimental results, 15g/ml
HpLPS solution has three kinds of experimental bacterias to be acted on compared with high inhibition, and the hpLPS solution (10g/ml, 12g/ml) of low concentration is also right
Three kinds of experimental bacterias have different degrees of inhibiting effect.
Table 2: Helicobacter pylori lipopolysaccharide extract fungistatic effect experimental result
What has been described above is only an embodiment of the present invention, and the common sense such as well known specific structure and characteristic are not made herein in scheme
Excessive description.It, without departing from the structure of the invention, can be with it should be pointed out that for those skilled in the art
Several modifications and improvements are made, these also should be considered as protection scope of the present invention, these all will not influence what the present invention was implemented
Effect and patent practicability.The scope of protection required by this application should be based on the content of the claims, in specification
The records such as specific embodiment can be used for explaining the content of claim.
Claims (10)
1. a kind of method that Helicobacter pylori Strains are separately cultured, which is characterized in that include the following steps,
(1) transport step: in transport process, by Sample preservation in transporting in liquid, the transport liquid includes brucella broth culture
Base, adsorbent, reducing agent and Helicobacter pylori lipopolysaccharide extract；(2) inoculation step；(3) incubation step.
2. the method that a kind of Helicobacter pylori Strains according to claim 1 are separately cultured, which is characterized in that connect described
In kind step, Colombia's blood agar plate of the extract containing Helicobacter pylori lipopolysaccharide is prepared first, then the sample is connect
In kind to Columbia Blood Agar plate.
3. the method that a kind of Helicobacter pylori Strains according to claim 2 are separately cultured, which is characterized in that brother's human relations
Than containing fetal calf serum or de- fiber Sheep Whole Blood in sub- blood agar plate.
4. a kind of method that Helicobacter pylori Strains are separately cultured described in any one of -3 claims according to claim 1,
It is characterized in that, the Colombia's blood agar plate for being inoculated with the sample is placed in culture apparatus, 37 in incubation step
DEG C constant temperature incubation 3-5 days, obtain the doubtful bacterium colony being grown on Columbia Blood Agar plate；Gas in the culture apparatus
Group becomes oxygen 5%, carbon dioxide gas 10% and nitrogen 85%.
5. the method that a kind of Helicobacter pylori Strains according to claim 4 are separately cultured, which is characterized in that the culture
Device is three gas incubators or the internal anaerobic jar for being equipped with production airbag.
6. the method that a kind of Helicobacter pylori Strains according to claim 4 are separately cultured, which is characterized in that walked in culture
Further include authentication step after rapid: the doubtful bacterium colony obtained in incubation step is identified.
7. the method that a kind of Helicobacter pylori Strains according to claim 6 are separately cultured, which is characterized in that the identification
The identification method that step uses are as follows: the combination of one or both of Gram's staining detection method and urease detection method.
8. a kind of according to claim 1-3, side that Helicobacter pylori Strains are separately cultured described in any one of 5-7 claim
Method, which is characterized in that the preparation method of the Helicobacter pylori lipopolysaccharide extract is ultrasonic extraction.
9. a kind of according to claim 1-3, side that Helicobacter pylori Strains are separately cultured described in any one of 5-7 claim
Method, which is characterized in that the adsorbent is cyclodextrin, activated carbon or soluble starch.
10. according to claim 1-3, a kind of Helicobacter pylori Strains described in any one of 5-7 claim are separately cultured
Method, which is characterized in that the reducing agent is cysteine.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394286A (en) * | 2020-04-29 | 2020-07-10 | 北京大学第三医院（北京大学第三临床医学院） | Method for culturing helicobacter pylori in vitro |
| CN112877245A (en) * | 2021-02-23 | 2021-06-01 | 海南省临床微生物学检测与研究中心 | Sensitive and rapid culture colony formation and efficiency evaluation method of helicobacter pylori |
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| CN113699084A (en) * | 2021-10-19 | 2021-11-26 | 大连先锋生物科技有限公司 | Noninvasive separation culture method and application of oral helicobacter pylori |
| CN114045231A (en) * | 2021-10-22 | 2022-02-15 | 吉林市国科医工科技发展有限公司 | Culture medium for helicobacter pylori and preparation method and application thereof |
| CN116064273A (en) * | 2022-07-14 | 2023-05-05 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Helicobacter pylori standard strains JMF0 and FMA6 containing specific molecular targets and their detection and application |
| CN116064273B (en) * | 2022-07-14 | 2023-08-22 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Helicobacter pylori standard strain JMF0 and FMA6 containing specific molecular targets, detection and application thereof |
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