CN101392228A - Microaerobic isolated culture method of campylobacter jejuni - Google Patents
Microaerobic isolated culture method of campylobacter jejuni Download PDFInfo
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- CN101392228A CN101392228A CNA200810202320XA CN200810202320A CN101392228A CN 101392228 A CN101392228 A CN 101392228A CN A200810202320X A CNA200810202320X A CN A200810202320XA CN 200810202320 A CN200810202320 A CN 200810202320A CN 101392228 A CN101392228 A CN 101392228A
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Abstract
The invention relates to a microaerophilic separation culture method of Campylobacter jejuni, which adopts the combination of a general candle-cylinder method and a pyrogallic acid method to create a microaerophilic environment. A directive streak cultivation method is adopted against the separation culture of the Campylobacter jejuni, a small amount of animal tissue detection samples are picked and directly inoculated on the selected culture medium by using connection annulus, the concentration of oxygen in the air is reduced by the candle-cylinder method with the combination of pyrogallic acid and anhydrous sodium carbonate mixture, an alkaline environment is formed by using anhydrous sodium carbonate so as to lead the pyrogallic acid to react with oxygen for absorbing the oxygen, therefore the culture environment meets the microaerophilic conditions required by the Campylobacter jejuni. Typical colonies obtained from separation culture are picked for PCR identification so as to further determine the Campylobacter jejuni. The microaerophilic separation culture method of Campylobacter jejuni has the advantages of low cost, simple operation, high detectable rate, good culture efficiency and the like, thus improving the separation culture efficiency of the Campylobacter jejuni and resolving the difficulty for cultivating the Campylobacter jejuni under microaerophilic condition.
Description
Technical field
The present invention relates to a kind of isolation cultivation method of campylobacter jejuni, be specifically related to a kind of microaerobic isolated culture method of Amphixenosis's cause of disease-campylobacter jejuni.Belong to the medical microbial technical field.
Background technology
Campylobacter jejuni (Campylobader jejuni) is a kind of of campylobacter.Campylobacter jejuni extensively exists at nature, and the serious threat human and livestock health is to be subjected to the former bacterium of a kind of Amphixenosis that Chinese scholars is extensively paid attention to the nearly more than ten years.Campylobacter jejuni infects has now become one of modal cause of disease of acute gastroenteritis in the global range, in developing country, campylobacter jejuni is the important cause of disease of children's diarrhae death, also be the common cause of developing country's traveler's diarrhea, severe complications is the acute demyelination-Guillain Barre syndrome of peripheral nervous system.It is significant for the Infection Status of determining campylobacter jejuni to set up an effective separation and Culture of cover and authentication method.
Campylobacter jejuni is a microaerobe, comparatively fragile, air to dry, heating, sterilization, acidity and 21% oxygen is all responsive, therefore its culture condition is also comparatively harsh, especially need little aerobic environment of 5% oxygen, 10% carbonic acid gas, 85% nitrogen, and have " but non-cultivation conditions ", can when envrionment conditions is bad, enter the dormant state of low metabolic activity, the survival but do not breed, recall rate is very low in the separation and Culture of routine.The creation difficulty of little aerobic condition is higher, and the specific equipment of domestic present this bacterium of cultivation is few and cost an arm and a leg, and is difficult for basic unit and promotes.The general candle jar method that adopts falls low oxygen pressure and cultivates this bacterium, and effect is undesirable, and recall rate is on the low side.Therefore the optimization to little aerobic condition has become campylobacter jejuni cultivation urgent problem.
The general poor growth of campylobacter jejuni on the ordinary culture medium, growth needs contain the blood agar culture-medium of antibacterials such as vancomycin, polymyxin B and three methylbenzyl Aminometradines.Now domestic generally with improveing Camp-BAP substratum and SkirrowShi substratum selective medium as campylobacter jejuni.The cultural method of domestic report has candle jar method, anaerobism bag, anaerobism glove box, anaerobism box, biological oxygen consumption method and pyrogallol method etc.Above method exists or to requirement for experiment condition height or problem such as material is difficult to obtain or recall rate is low.Therefore, seek a kind of low cost and the cultural method not high to requirement for experiment condition, that culture effect is good, cultivation and monitoring, the control of campylobacter jejuni is all had significance.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of microaerobic isolated culture method of campylobacter jejuni is provided, with low cost, simple to operate, recall rate height, the culture effect of campylobacter jejuni are good.
For achieving the above object, the method that the present invention adopts common candle jar method to combine with the pyrogallol method is created little aerobic environment, the campylobacter jejuni separation and Culture is adopted direct streak plating, selecting on the substratum with connecing a small amount of animal tissues of collarium picking test sample direct inoculation, cooperate pyrogallol and anhydrous sodium carbonate mixture to reduce concentration of oxygen in the air with candle jar method, utilize anhydrous sodium carbonate to form alkaline environment, impel pyrogallol and oxygen reaction to absorb oxygen, make culture environment reach campylobacter jejuni and cultivate required little aerobic condition.The colonies typical that the picking separation and Culture obtains is PCR and is identified, further is defined as campylobacter jejuni.
Method of the present invention mainly divides following a few step to carry out:
1, the collection of material: aseptic get animal tissues pack into the sterilization vessel in, as sample to be checked.
2, the separation and Culture of campylobacter jejuni: with the test sample of gathering, directly streak inoculation is being selected on the culture medium flat plate, place in the ground cylinder that adds pyrogallol and anhydrous sodium carbonate by a certain percentage, put a lighted candle again, cover close cylinder cap in cylinder central authorities.The combustion candle places incubator to cultivate for 42 ℃ in the lump together with cylinder and observes after horizontal blanking.Wherein, the consumption of pyrogallol and anhydrous sodium carbonate respectively is 2.4-2.6g/L, and both weight ratios are 1:1.
3, observe suspicious bacterium colony: the suspicious bacterium colony of picking from the bacterium colony of sample cultivation gained, wherein: the suspicious bacterium colony of first type is haemolysis not, canescence, pale brown look, flat, moistening, glossy, translucent drops, protruding, smooth, the edge is not whole to be had from the tendency of inoculation alignment external diffusion; The suspicious bacterium colony of second type is haemolysis not also, and is circular, glossy, translucent, often is the single bacterium colony that disperses projection, and the edge is complete, shinny.
4, the preliminary evaluation of campylobacter jejuni: with the suspicious bacterium colony of first type or the suspicious bacterium colony of the 2nd I type of picking, smear is made gram stain microscopy, get the Gram-negative bacterium colony according to the thalline dyeing property and do oxydase and catalase test, oxydase and catalase test positive person tentatively are defined as suspicious Campylobacter bacterium colony.
5, determining of campylobacter jejuni: adopt the campylobacter jejuni PCR method for detecting specificity, the preliminary suspicious Campylobacter bacterium colony of determining is detected, the 358bpDNA fragment that amplifies is checked order, and sequencing result is that the bacterium colony of campylobacter jejuni specific fragment promptly is defined as campylobacter jejuni.
The present invention adopts candle jar method to cooperate pyrogallol and anhydrous sodium carbonate mixture to reduce concentration of oxygen in the air, utilize anhydrous sodium carbonate to form alkaline environment, impel pyrogallol and oxygen reaction to absorb oxygen, make culture environment reach campylobacter jejuni and cultivate required little aerobic condition.
The present invention is lower to the requirement of experiment condition, and experiment material obtains easily, has advantages such as with low cost, simple to operate, that recall rate is high, culture effect is good, and its culture effect is better than cultural methods such as single candle jar method and pyrogallol method.With the inventive method separation and Culture campylobacter jejuni, go out bacterium time weak point, it is many to obtain colony counts, has improved the separation and Culture efficient of campylobacter jejuni, has solved the difficult point that little aerobic condition is cultivated campylobacter jejuni, and suitable grass-roots unit applies.
Embodiment
Below in conjunction with embodiment technical scheme seat of the present invention is further described.Following examples do not constitute limitation of the invention.
Embodiment 1
1, the collection of material: get chicken liver and pack into and add in the penicillin bottle of an amount of sterile saline the about 0.8～1.0cm of aseptic clip liver
3, 4C preserves standby.
2, the separation and Culture of campylobacter jejuni: adopt directly line plate culture, the chicken liver of gathering is ground,, get filtrate then and draw ware and be seeded in and improve on Camp-BAP substratum earlier with the filtering with microporous membrane of 0.65um; The flat board of inoculated bacteria is placed in the ground moisture eliminator of capacity 3000mL.Cylinder cap and cylinder mouth are coated with Vaseline, and consumption (1:1 ratio) adding pyrogallol and anhydrous sodium carbonate in every each 2.6g of 1L volume mix, and put into the segment lighted candle in cylinder central authorities, cover close cylinder cap.From horizontal blanking, place incubator to cultivate 24h for 42 ℃ in the lump behind the about 0.5～1min of combustion candle together with cylinder.
3, observe suspicious bacterium colony: the first type bacterium colony is haemolysis not, canescence, pale brown look, flat, moistening, glossy, and translucent drops, protruding, smooth, the edge is not whole to be had from the tendency of inoculation alignment external diffusion; The second type bacterium colony is haemolysis not also, and is circular, glossy, translucent, and (diameter is 1～2mm), and the edge is complete, shinny often to be the single bacterium colony that disperses projection.
4, the preliminary evaluation of campylobacter jejuni: the suspicious bacterium colony of first type that grows on picking improvement Camp-BAP substratum, smear is made gram stain microscopy.According to the thalline dyeing property, get the Gram-negative bacterium colony and do oxydase and catalase test.Gram-negative bacteria, size are 0.3～0.4 μ m * 1.5～3 μ m, as little funny point-like, and when dithallic end links to each other, S-shaped, gull-shaped, spirrillum or fusiform.Oxydase and catalase test positive person can tentatively be defined as suspicious Campylobacter bacterium colony.
5, determining of campylobacter jejuni: a kind of campylobacter jejuni PCR method for detecting specificity (the Chinese Amphixenosis's magazine that adopts the foundation of having delivered such as Yangcheng ripple, 2003,19 (1): 91-94), the preliminary suspicious Campylobacter bacterium colony of determining is detected, determine whether to be campylobacter jejuni.
Primer sequence is:
VS-F：5’GAT?ATC?TAT?GAT?TTT?ATC?CTG?C?3’
VS-R：5’GAA?TGA?AAT?TTT?AGA?ATG?GGG?3’
Masterplate: get above-mentioned suspicious bacterium colony, inoculation MH liquid nutrient medium, centrifugal collection bacterium mud extracts DNA, as the template of PCR.
Reaction system is: 2 * Taq PCR Master Mix, and 12.5 μ l, upstream primer (25 μ M), 1 μ l, downstream primer, 1 μ l, template DNA (1U/ μ L) 2 μ l, aseptic deionized water, 8.5 μ l, reaction volume are 25 μ l
The pcr amplification condition is: loop parameter is 94 ℃ of pre-sex change 5min, 94 ℃ of 30S, and 58 ℃ of 60S, 72 ℃ of 60S, totally 35 circulations, last 72 ℃ are extended 10min, preserve 1h for 4 ℃.
The evaluation of pcr amplification product: the sepharose of configuration 1%.Add ethidium bromide (EB) glue by 0.5mg/l.Get 5 μ l PCR products and 1 μ l, 10 * loading buffer mixing application of sample, with DL2000 Marker contrast.90V voltage electrophoresis 40min gets glue and places gel imaging system to take and the analytical electrophoresis result.A clear band appears in the result at the 358bp place, get the PCR product and send Shanghai Jierui Biology Engineering Co., Ltd to check order.The result proves that this PCR product is the campylobacter jejuni DNA fragment specific.Therefore, the sample that this band occurs promptly is confirmed as campylobacter jejuni.
Embodiment 2
1, the collection of material: get pig liver and pack into and add in the penicillin bottle of an amount of sterile saline the about 0.8～1.0cm of aseptic clip liver
3, 4C preserves standby.
2, the separation and Culture of campylobacter jejuni: adopt directly line plate culture, the pig liver of gathering is ground,, get filtrate then and draw ware and be seeded in and improve on Camp-BAP substratum earlier with the filtering with microporous membrane of 0.65um; The flat board of inoculated bacteria is placed in the ground moisture eliminator of capacity 3000mL.Cylinder cap and cylinder mouth are coated with Vaseline, and consumption (1:1 ratio) adding pyrogallol and anhydrous sodium carbonate in every each 2.4g of 1L volume mix, and put into the segment lighted candle in cylinder central authorities, cover close cylinder cap.From horizontal blanking, place incubator to cultivate 48h for 42 ℃ in the lump behind the about 0.5～1min of combustion candle together with container.
3, observe suspicious bacterium colony: the first type bacterium colony is haemolysis not, canescence, pale brown look, flat, moistening, glossy, and translucent drops, protruding, smooth, the edge is not whole to be had from the tendency of inoculation alignment external diffusion; The second type bacterium colony is haemolysis not also, and is circular, glossy, translucent, and (diameter is 1～2mm), and the edge is complete, shinny often to be the single bacterium colony that disperses projection.
4, the preliminary evaluation of campylobacter jejuni: the suspicious bacterium colony of second type that grows on picking improvement Camp-BAP substratum, smear is made gram stain microscopy.According to the thalline dyeing property, get the Gram-negative bacterium colony and do oxydase and catalase test.Gram-negative bacteria, size are 0.3～0.4 μ m * 1.5～3 μ m, as little funny point-like, and when dithallic end links to each other, S-shaped, gull-shaped, spirrillum or fusiform.Oxydase and catalase test positive person can tentatively be defined as suspicious Campylobacter bacterium colony.
5, determining of campylobacter jejuni: adopt the campylobacter jejuni PCR method for detecting specificity identical to determine whether to be campylobacter jejuni with embodiment 1.
Pcr amplification product checks order through Shanghai Jierui Biology Engineering Co., Ltd, and the result proves that this PCR product is the campylobacter jejuni DNA fragment specific.Therefore, the sample that this band occurs promptly is determined campylobacter jejuni.
Embodiment 3
1, the collection of material: get chicken liver and pack into and add in the penicillin bottle of an amount of sterile saline the about 0.8～1.0cm of aseptic clip liver
3, 4C preserves standby.
2, the separation and Culture of campylobacter jejuni: adopt directly line plate culture, the chicken liver of gathering is ground,, get filtrate then and draw ware and be seeded in and improve on Camp-BAP substratum earlier with the filtering with microporous membrane of 0.65um; The flat board of inoculated bacteria is placed in the ground moisture eliminator of capacity 3000mL.Cylinder cap and cylinder mouth are coated with Vaseline, and consumption (1:1 ratio) adding pyrogallol and anhydrous sodium carbonate in every each 3.0g of 1L volume mix, and put into the segment lighted candle in cylinder central authorities, cover close cylinder cap.From horizontal blanking, place incubator to cultivate 36h for 42 ℃ in the lump behind the about 0.5～1min of combustion candle together with cylinder.
3, observe suspicious bacterium colony: the first type bacterium colony is haemolysis not, canescence, pale brown look, flat, moistening, glossy, and translucent looking looks like water droplet, and be protruding, smooth, and the edge is not whole to be had from the tendency of inoculation alignment external diffusion; The second type bacterium colony is haemolysis not also, and is circular, glossy, translucent, and (diameter is 1～2mm), and the edge is complete, shinny often to be the single bacterium colony that disperses projection.
4, the preliminary evaluation of campylobacter jejuni: the suspicious bacterium colony of second type that grows on picking improvement Camp-BAP substratum, smear is made gram stain microscopy.According to the thalline dyeing property, get the Gram-negative bacterium colony and do oxydase and catalase test.Gram-negative bacteria, size are 0.3～0.4 μ m * 1.5～3 μ m, as little funny point-like, and when dithallic end links to each other, S-shaped, gull-shaped, spirrillum or fusiform.Oxydase and catalase test positive person can tentatively be defined as suspicious Campylobacter bacterium colony.
5, determining of campylobacter jejuni: adopt the campylobacter jejuni PCR method for detecting specificity identical to determine whether to be campylobacter jejuni with embodiment 1.
Pcr amplification product checks order through Shanghai Jierui Biology Engineering Co., Ltd, and the result proves that this PCR product is the campylobacter jejuni DNA fragment specific.Therefore, the sample that this band occurs promptly is determined campylobacter jejuni.
Following table 1 is the experimental result of pyrogallol among the present invention and anhydrous sodium carbonate consumption.
Table 1 pyrogallol and anhydrous sodium carbonate consumption experimental result
By table 1 as seen, little aerobic reagent dosage is between 2.0g/L-3.0g/L the time, and colony counts difference is not remarkable.When other culture condition was identical, the consumption of little aerobic reagent pyrogallol and anhydrous sodium carbonate was between 2.4g/L-2.6g/L, and the colony counts that campylobacter jejuni is cultivated is (25-27) at most, and culture effect is best.The consumption that is little aerobic reagent pyrogallol and anhydrous sodium carbonate is the little aerobic culture condition of optimization of campylobacter jejuni between 2.4g/L-2.6g/L.
Claims (2)
1, a kind of microaerobic isolated culture method of campylobacter jejuni is characterized in that comprising the steps:
1) aseptic get animal tissues pack into the sterilization vessel in, as sample to be checked;
2) with the test sample of gathering, directly streak inoculation places in the ground cylinder that adds pyrogallol and anhydrous sodium carbonate on the selection culture medium flat plate, puts a lighted candle again in cylinder central authorities, covers close cylinder cap; The combustion candle places incubator to cultivate for 42 ℃ in the lump together with cylinder and observes after horizontal blanking; Wherein, the consumption of pyrogallol and anhydrous sodium carbonate respectively is 2.0-3.0g/L, and both weight ratios are 1:1;
3) the suspicious bacterium colony of picking from the bacterium colony of sample cultivation gained, wherein: the suspicious bacterium colony of first type is haemolysis not, canescence, pale brown look, flat, moistening, glossy, translucent drops, protruding, smooth, the edge is not whole to be had from the tendency of inoculation alignment external diffusion; The suspicious bacterium colony of second type is haemolysis not, and is circular, glossy, translucent, often is the single bacterium colony that disperses projection, and the edge is complete, shinny;
4) with the suspicious bacterium colony of first type or the suspicious bacterium colony of the 2nd I type of picking, smear is made gram stain microscopy, get the Gram-negative bacterium colony according to the thalline dyeing property and do oxydase and catalase test, oxydase and catalase test positive person tentatively are defined as suspicious Campylobacter bacterium colony;
5) adopt the campylobacter jejuni PCR method for detecting specificity, the preliminary suspicious Campylobacter bacterium colony of determining is detected, the 358bpDNA fragment that amplifies is checked order, and sequencing result is that the bacterium colony of campylobacter jejuni specific fragment promptly is defined as campylobacter jejuni.
2, according to the microaerobic isolated culture method of the campylobacter jejuni of claim 1, it is characterized in that the consumption of described pyrogallol and anhydrous sodium carbonate respectively is 2.4-2.6g/L, both weight ratios are 1:1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462823A (en) * | 2015-12-17 | 2016-04-06 | 重庆道鑫生物科技有限公司 | Microorganism sample treatment marking system |
| CN112094328A (en) * | 2020-09-21 | 2020-12-18 | 华南农业大学 | Extraction and purification method of Campylobacter jejuni outer membrane vesicles |
-
2008
- 2008-11-06 CN CNA200810202320XA patent/CN101392228A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462823A (en) * | 2015-12-17 | 2016-04-06 | 重庆道鑫生物科技有限公司 | Microorganism sample treatment marking system |
| CN112094328A (en) * | 2020-09-21 | 2020-12-18 | 华南农业大学 | Extraction and purification method of Campylobacter jejuni outer membrane vesicles |
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