CN114854528A - Culture device and culture method suitable for campylobacter microaerophilic - Google Patents
Culture device and culture method suitable for campylobacter microaerophilic Download PDFInfo
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- CN114854528A CN114854528A CN202210508653.5A CN202210508653A CN114854528A CN 114854528 A CN114854528 A CN 114854528A CN 202210508653 A CN202210508653 A CN 202210508653A CN 114854528 A CN114854528 A CN 114854528A
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a culture device and a culture method suitable for a campylobacter microaerophilic, wherein the culture device adopts a simple culture tank with good sealing property, a stable gas environment is provided by a gas bottle, a partition plate is arranged in the culture tank to fix and support a culture dish, and the culture dish can be effectively prevented from toppling over; the invention also can fill the mixed gas required by the campylobacter culture into the tank body by arranging the air inlet valve and the air outlet valve, and timely closes the air outlet valve and the air inlet valve by monitoring the data of the gas concentration sensor, so that the operation is simple and convenient, continuous air inlet is not needed, and the invention is economical and practical. According to the culture method, reasonable culture media are prepared in the links from sample collection and culture to campylobacter separation identification and purification, strict process steps are set, single purified culture colonies can be effectively obtained, and a good sample basis is provided for subsequent continuous research on campylobacter.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a culture device and a culture method suitable for campylobacter microaerophilic.
Background
Campylobacter (Campylobacter) was discovered by Veron et al in 1963, mainly including Campylobacter jejuni (c.jejuni) and Campylobacter coli (c.coli), which is red in gram-staining reaction; the host source of campylobacter is wide, and the campylobacter exists widely in warm-blooded animals, such as intestinal tracts of poultry, pigs, cows and wild birds, wherein the bacteria-carrying rate of the birds is highest; the campylobacter carried by animals can not be directly attacked, but can cause diseases through food chain transmission, the campylobacter of 100-500 CFU can cause infection of people, and can cause headache, fever, nausea, vomiting, abdominal pain, diarrhea and the like, thereby causing Guillain-Barre syndrome seriously and even causing death of children, old people and people with low immunity;
the campylobacter has unique physiological and biochemical characteristics, and the optimal growth condition is 5% O 2 、10％CO 2 And 85% N 2 A microaerophilic environment, wherein the culture temperature is 42 ℃; the growth speed of the strain is slow, and the strain is generally cultured for 36 hours to reach the logarithmic phase of growth; the bacterium is not hemolyzed on a blood plate, two types of bacterial colonies are formed according to the humidity of the blood plate, and when the humidity is high: semi-transparent colonies with low flatness, irregular edges and uneven sizes diffuse along the scribing direction and tend to grow densely and continuously; when the humidity is low: the bacterial colony is a transparent bacterial colony which is smooth, raised, round and regular in edge and has metallic luster.
The culture condition of the campylobacter is microaerophilic environment, so that the campylobacter is very sensitive to gas components, and the stable culture environment is the key to whether the separation culture of the campylobacter is successful or not; at present, the culture method or operation of campylobacter is complicated, or the equipment is expensive, or time and material are consumed; therefore, there is a need for a small micro-aerobic culture apparatus with simple structure, practicality and convenience, which can provide the required gas environment during the whole culture process, so as to overcome the difficulties in the prior art.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects in the prior art, the invention provides the culture device which is simple in structure, practical and convenient, can provide a stable gas environment and is suitable for the micro-aerobic of campylobacter.
The technical scheme is as follows: in order to achieve the above object, the present invention is a culture apparatus suitable for microaerophilic culture of campylobacter, comprising a gas bottle and a culture tank; the culture tank comprises a tank body and a tank cover, and an accommodating space of a culture dish is arranged in the tank body;
an air inlet valve is arranged on the lower side of the tank body, and the air bottle is connected with the air inlet valve through a hose;
the tank cover is provided with an exhaust valve and a gas concentration sensor, and the gas concentration sensor is provided with a part extending into the accommodating space;
also included is a display coupled to the gas concentration sensor.
Preferably, a partition plate is arranged in the tank body, and the partition plate divides the accommodating space into a plurality of cavities matched with the culture dish shelf; the partition plate can enter and exit relative to the tank body.
Preferably, a plurality of vent holes are arranged on the partition plate in an array manner; the partition plate comprises a partition transverse plate and a partition longitudinal plate, and the partition transverse plate and the partition longitudinal plate are respectively arranged in parallel relative to two adjacent side walls of the tank body.
Preferably, the culture dish shelf has a rim, a base, and side baffles;
the base is arranged at the bottom of the frame; the side baffle is arranged on one side of the frame and encloses a semi-cylindrical culture dish accommodating space with the frame.
A culture method suitable for campylobacter microaerophilic is based on the culture device, and comprises the following steps:
step 1), collecting a chicken cloaca or intestinal content sample;
step 2), diluting the sample and inoculating the diluted sample into a culture dish containing a solid culture medium;
step 3), placing a culture dish on the culture dish shelf, placing the culture dish shelf into the tank body, and closing the tank cover to seal the tank body;
step 4), opening the intake valve and the exhaust valve; opening a gas cylinder switch and observing the display, and closing the exhaust valve, the gas cylinder switch and the gas inlet valve in sequence when the concentration of the carbon dioxide reaches 10%;
step 5), placing the sample treated in the step into a culture tank at 42 ℃ for microaerophilic culture for 36 hours to enable the campylobacter to reach logarithmic growth phase;
and 6), separating, identifying and purifying campylobacter.
The preparation method of the solid culture medium in the step 2) is as follows:
step 1.1), preparing cefoperazone stock solution: weighing 3.5g (accurate to 0.01g) of cefoperazone, adding 50mL of ultrapure water, uniformly mixing and dissolving to obtain 70mg/mL cefoperazone stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.2), preparing polymyxin B stock solution: weighing 0.17g (accurate to 0.001g) of polymyxin B, adding 50mL of ultrapure water, uniformly mixing and dissolving to obtain 3.4mg/mL polymyxin B stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.3), preparing amphotericin B stock solution: weighing 1.0g (accurate to 0.01g) of amphotericin B, adding into 40mL of DMSO, uniformly mixing and dissolving to obtain 25mg/mL amphotericin B stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.4), preparing a rifampicin stock solution: weighing 0.05g (accurate to 0.001g) of rifampicin, adding into 10mLDMSO, uniformly mixing and dissolving to obtain 5mg/mL rifampicin stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.5), preparing a mixed stock solution of cycloheximide and trimethoprim: weighing 0.25g (accurate to 0.001g) of trimethoprim and 2.5g (accurate to 0.01g) of cycloheximide, adding 50mL of ethanol, uniformly mixing and dissolving to obtain 5mg/mL and 50mg/mL trimethoprim and cycloheximide mixed stock solutions, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.6), weighing proper amounts of CCDA culture medium powder and MH solid culture medium powder respectively, adding proper volumes of distilled water, carrying out autoclaving at 121 ℃ for 15min, keeping the temperature of a water bath kettle at 55 ℃ for 1h, adding 1mL of 6 stock solutions corresponding to the steps 1.1) to 1.5) into the CCDA culture medium, adding 5% defibered sheep blood into the MH culture medium according to volume, carrying out magnetic stirring uniformly, pouring into sterile plates respectively, drying in a super clean bench for 2h, and carrying out inversion refrigeration at 4 ℃ at low temperature.
The step 1) specifically comprises the following steps:
collecting cloacal cotton swabs, storing the cloacal cotton swabs into a Cary-Blair semisolid transportation culture medium, collecting intestinal contents, storing the intestinal contents into a sterile EP tube, and conveying the intestinal contents to a laboratory at a low temperature by using an ice box within 36 h.
The step 2) specifically comprises the following steps:
taking out cloacal cotton swab or intestinal content, soaking in finger-shaped tube containing 700 μ L of 1 × PBS solution, soaking thoroughly, taking 100 μ L of LPBS diluent, adding into culture dish containing CCDA solid culture medium containing six antibiotics, and coating with sterile L rod;
the step 3) of placing the culture dish on the culture dish shelf specifically comprises the following steps:
placing the petri dish upside down on the petri dish shelf.
The step 6) specifically comprises the following steps:
step 6.1), selecting suspicious colonies (flat colonies with metallic luster and drop-like colonies) and inoculating the suspicious colonies to an MH blood plate for microaerophilic culture at 42 ℃ for 36 hours, and repeating for 2-3 times until single pure culture colonies are obtained;
step 6.2), adopting a PCR method or biochemical identification of campylobacter;
and 6.3) scraping the identified strain by using a cotton swab and storing the strain in a 20% glycerol BHI culture medium.
Has the advantages that: according to the culture device and the culture method for the campylobacter microaerophilic, disclosed by the invention, a simple culture tank with good sealing property is adopted, a stable gas environment is provided through a gas bottle, the partition plate is arranged inside the culture tank, and the culture dish is fixed and supported, so that the culture dish can be effectively prevented from toppling due to shaking in the use process of the culture tank, and the stability of the culture dish when the culture dish is placed is effectively improved;
according to the invention, by arranging the air inlet valve and the air outlet valve, the mixed gas required by the campylobacter culture can be filled into the tank body, and the air outlet valve and the air inlet valve are closed in time by monitoring the data of the gas concentration sensor, so that the operation is simple and convenient, continuous air inlet is not needed, and the device is economical and practical;
the culture method provided by the invention has the advantages that the reasonable culture medium is prepared from the sample collection and culture links to the separation, identification and purification links of the campylobacter, and strict process steps are set, so that a single pure culture bacterial colony can be effectively obtained, and a good sample basis is provided for the subsequent continuous research on the campylobacter.
Drawings
FIG. 1 is an overall view of a culture apparatus suitable for the microaerophilic culture of Campylobacter;
FIG. 2 is an oblique view showing the opened state of the culture tank;
FIG. 3 is a view showing a closed state of a culture tank;
FIG. 4 is a side view of the culture tank in an open state;
FIG. 5 is an overall view of a culture dish shelf;
FIG. 6 is a first state view of the culture dish shelf of the preferred embodiment;
FIG. 7 is a second state view of the culture dish shelf of the preferred embodiment.
In the figure: 1-gas bottle, 2-culture tank, 3-culture dish shelf, 4-test bench, 5-hose, 11-gas bottle switch, 21-tank, 22-tank cover, 23-gas inlet valve, 24-gas outlet valve, 25-gas concentration sensor, 26-display, 27-separation plate, 28-sealing button, 29-sealing ring, 271-separation transverse plate, 272-separation longitudinal plate, 31-frame, 311-bottom frame, 312-upper frame, 32-base, 33-side baffle, 34-movable baffle and 35-connecting rod.
Detailed Description
The present invention will be further described with reference to the accompanying drawings.
The culture device suitable for the microaerophilic culture of campylobacter comprises a gas bottle 1 and a culture tank 2; the gas cylinder 1 is provided with a cylinder switch 11; the culture tank 2 comprises a tank body 21 and a tank cover 22, the tank body 21 is provided with a containing space of a culture dish, and the tank body 21 and the tank cover 22 are made of colorless and transparent amorphous thermoplastic materials; an air inlet valve 23 is arranged on the lower side of the tank body 21, and the gas bottle 1 is connected with the air inlet valve 23 through a hose 5; the tank cover 22 is provided with an exhaust valve 24 and a gas concentration sensor 25, and the gas concentration sensor 25 has a part extending into the accommodating space; a display 26 connected with the gas concentration sensor 25, wherein in the embodiment, the display 26 can receive the data collected by the gas concentration sensor 25 and display the collected data; the cover 22 is rotatably connected with the can body 21, and the cover 22 can be switched between an open state and a closed state relative to the can body 21; when in the closed state (as shown in fig. 3), the can body 21 and the can lid 22 are locked by the sealing button 28.
In this embodiment, the gas bottle 1 is filled with a mixed gas for culturing campylobacter, the molecular mass of the mixed gas is greater than the average molecular mass of air, when the campylobacter needs to be cultured, the cover 22 is closed, the sealing button 28 is locked, the air inlet valve 23, the air outlet valve 24 and the gas bottle switch 11 are opened, the mixed gas enters the culture tank 2 from the air inlet valve 23 located at the lower side of the tank body 21 by the upward air exhaust method, and the air in the culture tank 2 is exhausted from the air outlet valve 24 located at the cover 22, when the gas concentration displayed by the display 26 reaches a preset value, the air outlet valve 24, the gas bottle switch 11 and the air inlet valve 23 are closed in sequence, so that the gas in the culture tank 2 reaches a set value; the arrangement of the sealing button 28 makes the operation of the culture tank 2 more convenient and faster.
Preferably, the gas bottle 1 contains 5% O 2 、10％CO 2 And 85% N 2 The gas concentration sensor 25 is CO 2 A concentration sensor; in this embodiment, the carbon dioxide concentration of the mixed gas is 10%, the carbon dioxide concentration in the air is about 0.04%, and the difference between the two concentrations is large, so that the CO is present 2 The concentration sensor can accurately reflect whether or not the culture tank 2 is filled with the mixed gas.
Preferably, as shown in fig. 2, a partition plate 27 is arranged inside the tank 21, and the partition plate 27 divides the accommodating space into a plurality of cavities matched with the culture dish shelves 3; the partition plate 27 is capable of moving in and out of the tank 21; a plurality of vent holes are formed in the partition plate 27 in an array manner; the partition plate 27 includes a horizontal partition plate 271 and a vertical partition plate 272, which are disposed in parallel with the two adjacent side walls of the tank 21; the culture dish shelf 3 is provided with a frame 31, a base 32 and a side baffle 33; the base 32 is arranged at the bottom of the frame 31; the side baffle 33 is disposed at one side of the frame 31, and encloses a semi-cylindrical dish receiving space (shown in fig. 5) with the frame 31.
In a preferred embodiment, as shown in fig. 6-7, the frame 31 is composed of a bottom frame 311 and an upper frame 312, both of which are U-shaped, and the sides of the two are in sliding connection, and the upper frame 312 can be used as a handle; the side baffles 33 are fixed on one side of the bottom frame 311, and the movable baffles 34 are rotatably mounted at the left edge and the right edge of the other side of the bottom frame 311, namely a total of two movable baffles 34; a connecting rod 35 is connected between each movable baffle 34 and the upper frame 312, and two ends of the connecting rod 35 are respectively connected with the movable baffle 34 and the upper frame 312 through ball hinges; thus, the two movable baffles 34 can be switched between a relatively open state and a mutually closed state, and when the upper frame 312 is at a high position and the two movable baffles 34 are in the mutually closed state, the culture dish cannot enter or exit the culture dish shelf 3; when the upper frame 312 is in the low position and the two movable baffles 34 are in the relatively open position, the culture dish can enter and exit the culture dish shelf 3. Through the above structure, initially, as shown in fig. 6, the upper frame 312 is at a low position, at this time, a user can load a culture dish into the culture dish shelf 3, after loading, the culture dish shelf 3 needs to be loaded into the culture tank 2, during the process that the user lifts the upper frame 312, because the culture dish in the culture dish shelf 3 is heavy, the upper frame 312 initially slides relative to the bottom frame 311, and the two movable baffles 34 are gradually closed with each other through the connecting rod 35 (because both ends of the connecting rod 35 are spherical hinges, the upper frame 312 is lifted to drive the movable baffles 34 to be linked) until the movable baffles 34 are in contact with the culture dish (as shown in fig. 7), thereafter, the movable baffles 34 cannot continue to move, the upper frame 312 cannot continue to slide relative to the bottom frame 311, thereafter, the user can lift the whole culture dish shelf 3, during the moving, the culture dish cannot fall out of the culture dish shelf 3 due to the blocking of the movable baffles 34, the safety of the culture dish can be ensured.
In this embodiment, the partition plate 27 can be taken out from the tank body 21, so that the use is more flexible and convenient, and the accommodating space is divided into a plurality of cavities matched with the culture dish shelves 3 by the partition plate 27, so that the culture dish can be effectively prevented from toppling due to shaking in the use process, and the stability of the culture dish in placement is effectively improved; the arrangement of the vent holes in the partition plate 27 makes it possible to make the gas in the culture tank 2 more uniform.
Preferably, the culture tank 2 further comprises a sealing ring 29, the sealing ring 29 is disposed on the tank cover 22, and when in a closed state, the sealing ring 29 contacts with the upper edge of the tank body 21 to seal the culture tank 2; the surface of the sealing ring 29 is provided with an anti-oxidation coating.
Preferably, the culture tank 2 is placed on a test stand 4, so that the operation is more convenient.
A culture method suitable for the microaerophilic culture of campylobacter, which is based on the culture device, comprises the following steps:
step 1), collecting a chicken cloaca or intestinal content sample;
step 2), diluting the sample and inoculating the diluted sample into a culture dish containing a solid culture medium;
step 3), placing a culture dish on the culture dish shelf 3, placing the culture dish shelf 3 into the tank body 21, and closing the tank cover 22 to seal the tank body 21;
step 4), opening the intake valve 23 and the exhaust valve 24; opening the gas cylinder switch 11 and observing the display 26, and closing the exhaust valve 24, the gas cylinder switch 11 and the intake valve 23 in sequence when the concentration of carbon dioxide reaches 10%;
step 5), placing the sample treated in the step in a culture tank 2 for microaerophilic culture at 42 ℃ for 36 hours to enable the campylobacter to reach the logarithmic phase;
and 6), identifying and purifying campylobacter.
The method for culturing the campylobacter is adopted to culture the campylobacter, and a stable gas environment required by the campylobacter can be provided without repeatedly inflating during the culturing process, so that the campylobacter reaches the logarithmic phase.
Specifically, the preparation method of the solid medium in the step 2) is as follows:
step 1.1), preparing a cefoperazone stock solution: weighing 3.5g (accurate to 0.01g) of cefoperazone, adding 50mL of ultrapure water, uniformly mixing and dissolving to obtain 70mg/mL cefoperazone stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.2), preparing polymyxin B stock solution: weighing 0.17g (accurate to 0.001g) of polymyxin B, adding 50mL of ultrapure water, uniformly mixing and dissolving to obtain 3.4mg/mL polymyxin B stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.3), preparing amphotericin B stock solution: weighing 1.0g (accurate to 0.01g) of amphotericin B, adding into 40mL of DMSO, uniformly mixing and dissolving to obtain 25mg/mL amphotericin B stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.4), preparing a rifampicin stock solution: weighing 0.05g (accurate to 0.001g) of rifampicin, adding into 10mLDMSO, uniformly mixing and dissolving to obtain 5mg/mL rifampicin stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.5), preparing a mixed stock solution of cycloheximide and trimethoprim: weighing 0.25g (accurate to 0.01g) of trimethoprim and 2.5g (accurate to 0.01g) of cycloheximide, adding 50mL of ethanol, uniformly mixing and dissolving to obtain 5mg/mL and 50mg/mL trimethoprim and cycloheximide stock solutions, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.6), weighing proper amounts of CCDA medium powder and MH solid medium powder respectively, adding proper volumes of distilled water (here, the amounts of the CCDA and MH medium powder and the amount of the distilled water are weighed according to the specification of the CCDA medium powder), carrying out autoclaving at 121 ℃ for 15min, keeping the temperature of a water bath kettle at 55 ℃ for 1h, adding CCDA medium into 1.1) -1.5) corresponding 6 stock solutions respectively, adding 5% defibrated sheep blood into the MH solid medium according to the volume, uniformly stirring by magnetic force, pouring into sterile flat dishes respectively, drying in a clean bench for 2h, and carrying out inversion and low-temperature refrigeration at 4 ℃.
The step 1) is specifically as follows: collecting cloaca cotton swabs and storing the cloaca cotton swabs into a Cary-Blair semi-solid transport culture medium, or collecting intestinal contents and storing the intestinal contents into a sterile EP tube, and conveying the intestinal contents to a laboratory at a low temperature by using an ice box within 36 h. The Cary-Blair semi-solid transport medium is prepared in advance, and the specific preparation method comprises the following steps: weighing an appropriate amount of Cary-Blair semisolid transport medium powder according to the specification, adding an appropriate volume of distilled water, autoclaving at 121 ℃ for 15min, subpackaging with 3 mL/tube, and refrigerating at 4 ℃.
The step 2) is specifically as follows: the cloacal cotton swab or intestinal contents were removed and soaked in a finger tube containing 700. mu.L of 1 XPBS (phosphate buffered saline) solution, thoroughly soaked, and 100. mu.L of PBS dilution was placed in a petri dish containing solid medium and spread on a sterile L-bar. Here, the 1 × PBS solution is prepared in advance by a specific preparation method: weighing 20mL of 10 XPBS, adding 180mL of double distilled water, mixing uniformly, autoclaving at 121 ℃ for 15min, and refrigerating at 4 ℃ at low temperature.
Correspondingly, the step 3) of placing the culture dish on the culture dish shelf 3 specifically includes: the petri dish is placed upside down on the petri dish shelf 3.
The step 6) specifically comprises the following steps:
step 6.1), selecting suspicious colonies (flat colonies with metallic luster and drop-like colonies) and inoculating the suspicious colonies to an MH blood plate for microaerophilic culture at 42 ℃ for 36 hours, and repeating for 2-3 times until single pure culture colonies are obtained; here, the MH blood plate is prepared in advance by the specific preparation method: weighing an appropriate amount of MH culture medium powder according to the specification, adding an appropriate amount of distilled water, carrying out autoclaving at 121 ℃ for 15min, keeping the temperature of a water bath kettle at 55 ℃ for 1h, adding 5% defibered sheep blood by volume, carrying out magnetic stirring uniformly, pouring into a sterile plate, drying in a super clean bench for 2h, and carrying out inversion and low-temperature refrigeration at 4 ℃.
Step 6.2), adopting a PCR method or biochemical identification of campylobacter;
step 6.3), scraping the identified strain by using a cotton swab and storing the strain in a 20% glycerol BHI culture medium; the glycerol BHI culture medium is prepared in advance, and the specific preparation method comprises the following steps: weighing a proper amount of BHI culture medium powder according to the specification, mixing 20mL of glycerol and 80mL of distilled water, adding the powder into a 20% glycerol aqueous solution, uniformly mixing, carrying out autoclaving at 121 ℃ for 15min, and storing at 4 ℃.
Effective single pure culture colonies can be obtained through the steps 6.1) to 6.3), and a good basis is provided for the subsequent research on campylobacter.
Description of the effect of the respective stock solutions in step 1.1) -step 1.5): the method for preparing the campylobacter selective culture medium by selecting six antibiotics of cefoperazone, polymyxin B, amphotericin B, rifampicin, cycloheximide and trimethoprim and CCDA is based on natural drug resistance of campylobacter to the six antibiotics, and genes for coding the drug resistance of the antibiotics are determined by chromosome genes of the campylobacter, are vertically inherited and cannot be changed, so that the prepared six-resistance CCDA selective culture medium is beneficial to supporting the growth of intestinal campylobacter and improving the selectivity of the culture medium. The selective culture medium prepared by adopting antibiotics with different sub-bacteriostatic concentrations is beneficial to inhibiting the growth of other microorganisms. Cefoperazone inhibits growth of gram-negative enterobacteria and some gram-positive bacteria, polymyxin B inhibits various gram-negative bacteria such as escherichia coli, amphotericin B inhibits growth of yeast and fungi, rifampicin can inhibit other anaerobic bacteria such as non-campylobacter, cycloheximide inhibits growth of yeast and mold, trimethoprim inhibits various gram-positive bacteria.
The CCDA culture medium contains peptone, casein hydrochloride and beef extract powder, can provide nutrient components necessary for the growth of campylobacter, the casein helps the growth of the nalidixic acid-resistant thermophilic campylobacter, and the campylobacter agar contains ferric sulfate, sodium propionate and activated carbon, can relieve toxins such as peroxide generated by metabolism, and helps the growth of oxygen-sensitive campylobacter.
The campylobacter culture method can effectively separate campylobacter in the whole implementation process, and is simple, convenient and effective to operate by purifying, enriching and culturing; the culture device can ensure that bacteria are in a stable growth environment, and the survival rate of campylobacter is improved.
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.
Claims (10)
1. A culture device suitable for campylobacter microaerophilic comprises a gas bottle (1) and a culture tank (2); the culture tank (2) comprises a tank body (21) and a tank cover (22), and a culture dish accommodating space is arranged in the tank body (21); it is characterized in that the preparation method is characterized in that,
an air inlet valve (23) is arranged on the lower side of the tank body (21), and the air bottle (1) is connected with the air inlet valve (23) through a hose (5);
the tank cover (22) is provided with an exhaust valve (24) and a gas concentration sensor (25), and the gas concentration sensor (25) is provided with a part extending into the accommodating space;
also included is a display (26) connected to the gas concentration sensor (25).
2. The culture device for the microaerophilic growth of campylobacter according to claim 1, characterized in that a partition plate (27) is provided inside the tank (21), said partition plate (27) dividing the housing space into a plurality of cavities matching the culture dish shelves (3); the partition plate (27) can be moved in and out of the tank (21).
3. The culture device for the microaerophilic growth of campylobacter according to claim 2, wherein the partition plate (27) is provided with a plurality of vent holes in an array; the partition plate (27) comprises a partition transverse plate (271) and a partition longitudinal plate (272), which are respectively arranged in parallel relative to two adjacent side walls of the tank body (21).
4. The culture device for the microaerophilic growth of campylobacter according to claim 2, characterized in that said culture dish rack (3) has a frame (31), a base (32) and side baffles (33); the base (32) is arranged at the bottom of the frame (31); the side baffle (33) is arranged on one side of the frame (31) and encloses a semi-cylindrical culture dish accommodating space with the frame (31).
5. The culture device for the microaerophilic growth of campylobacter according to claim 1, characterized in that the tank cover (22) is rotatably connected to the tank (21), the tank cover (22) being switchable between an open state and a closed state with respect to the tank (21);
when in the closed state, the can body (21) and the can cover (22) are locked by a sealing buckle (28).
6. A culture method suitable for microaerophilic culture of campylobacter, which is based on the culture device of claim 1, and which comprises:
step 1), collecting a chicken cloaca or intestinal content sample;
step 2), diluting the sample and inoculating the diluted sample into a culture dish containing a solid culture medium;
step 3), placing a culture dish on the culture dish shelf (3), placing the culture dish shelf (3) into the tank body (21), and closing the tank cover (22) to seal the tank body (21);
step 4), opening the intake valve (23) and the exhaust valve (24); opening the gas cylinder switch (11) and observing the display (26), and closing the exhaust valve (24), the gas cylinder switch (11) and the gas inlet valve (23) in sequence when the concentration of carbon dioxide reaches 10%;
step 5), placing the sample treated in the step into a culture tank (2) for microaerophilic culture at 42 ℃ for 36 hours to enable the campylobacter to reach the logarithmic phase;
and 6), identifying and purifying campylobacter.
7. The method for culturing the microaerophilic strain as claimed in claim 6, wherein the solid medium in step 2) is prepared by the following method:
step 1.1), preparing a cefoperazone stock solution: weighing 3.5g of cefoperazone, adding 50mL of ultrapure water, uniformly mixing and dissolving to obtain 70mg/mL cefoperazone stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.2), preparing polymyxin B stock solution: weighing 0.17g of polymyxin B, adding 50mL of ultrapure water, uniformly mixing and dissolving to obtain 3.4mg/mL polymyxin B stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.3), preparing amphotericin B stock solution: weighing 1.0g of amphotericin B, adding into 40mLDMSO, uniformly mixing and dissolving to obtain 25mg/mL amphotericin B stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.4), preparing a rifampicin stock solution: weighing 0.05g of rifampicin, adding the rifampicin into 10mLDMSO, uniformly mixing and dissolving to obtain 5mg/mL rifampicin stock solution, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.5), preparing a mixed stock solution of cycloheximide and trimethoprim: weighing 0.25g of trimethoprim and 2.5g of cycloheximide, adding 50mL of ethanol, uniformly mixing and dissolving to obtain 5mg/mL and 50mg/mL of trimethoprim and cycloheximide mixed stock solutions, subpackaging 1mL, and freezing at-20 ℃ for later use;
step 1.6), weighing proper amounts of CCDA culture medium powder and MH solid culture medium powder respectively, adding proper volumes of distilled water, carrying out autoclaving at 121 ℃ for 15min, keeping the temperature of a water bath kettle at 55 ℃ for 1h, adding 1mL of 6 stock solutions corresponding to the steps 1.1) to 1.5) into the CCDA culture medium, adding 5% defibered sheep blood into the MH culture medium according to volume, carrying out magnetic stirring uniformly, pouring into sterile plates respectively, drying in a super clean bench for 2h, and carrying out inversion refrigeration at 4 ℃ at low temperature.
8. The culture method suitable for campylobacter microaerophilic according to claim 6, wherein the step 1) comprises in particular:
collecting cloacal cotton swabs, storing the cloacal cotton swabs into a Cary-Blair semisolid transportation culture medium, collecting intestinal contents, storing the intestinal contents into a sterile EP tube, and conveying the intestinal contents to a laboratory at a low temperature by using an ice box within 36 h.
9. The culture method suitable for the microaerophilic culture of campylobacter according to claim 6, wherein said step 2) comprises in particular:
taking out cloaca cotton swab or intestinal content, soaking in finger-shaped tube containing 700 μ L of 1 × PBS solution, soaking thoroughly, taking 100 μ L of LPBS diluent, placing into culture dish containing solid culture medium, and coating with sterile L rod;
the step 3) of placing the culture dish on the culture dish shelf (3) specifically comprises the following steps:
placing the culture dish upside down on the culture dish shelf (3).
10. The culture method suitable for the microaerophilic culture of campylobacter according to claim 6, wherein said step 6) comprises in particular:
step 6.1), selecting suspicious colonies, inoculating the suspicious colonies on an MH blood plate, carrying out microaerophilic culture at 42 ℃ for 36h, and repeating for 2-3 times until single pure culture colonies are obtained;
step 6.2), adopting a PCR method or biochemical identification of campylobacter;
and 6.3) scraping the identified strain by using a cotton swab and storing the strain in a 20% glycerol BHI culture medium.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5891709A (en) * | 1992-07-13 | 1999-04-06 | The United States Of America As Represented By The Secretary Of The Agriculture | Campy-Cefex selective and differential medium for campylobacter |
| CN207362196U (en) * | 2017-07-31 | 2018-05-15 | 华夏源（上海）干细胞技术有限公司 | Tissue Culture Dish frame |
| CN112029679A (en) * | 2020-08-12 | 2020-12-04 | 河北医科大学第四医院（河北省肿瘤医院） | Large-scale culture method of helicobacter pylori |
| CN215924963U (en) * | 2021-06-10 | 2022-03-01 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | Culture device |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5891709A (en) * | 1992-07-13 | 1999-04-06 | The United States Of America As Represented By The Secretary Of The Agriculture | Campy-Cefex selective and differential medium for campylobacter |
| CN207362196U (en) * | 2017-07-31 | 2018-05-15 | 华夏源（上海）干细胞技术有限公司 | Tissue Culture Dish frame |
| CN112029679A (en) * | 2020-08-12 | 2020-12-04 | 河北医科大学第四医院（河北省肿瘤医院） | Large-scale culture method of helicobacter pylori |
| CN215924963U (en) * | 2021-06-10 | 2022-03-01 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | Culture device |
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