CN101270334A - Isolated culture method for anaerobic microorganism - Google Patents
Isolated culture method for anaerobic microorganism Download PDFInfo
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- CN101270334A CN101270334A CNA2007100647153A CN200710064715A CN101270334A CN 101270334 A CN101270334 A CN 101270334A CN A2007100647153 A CNA2007100647153 A CN A2007100647153A CN 200710064715 A CN200710064715 A CN 200710064715A CN 101270334 A CN101270334 A CN 101270334A
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Abstract
The invention relates to an anaerobe isolating culture method. Throughout the operational process, target microbes are prevented from contacting oxygen, and the operating cost is relatively low. The method is in particular applicable for high temperature anaerobes and strict anaerobes with the growth temperature above 50 DEG C and strict requirement for anaerobic environment difficult to be isolated. In the method, oxygen-free workstations are used only during partial operation of isolation of anaerobes. When anaerobes are cultured, common incubators can be used without anaerobic environment, thus preventing one oxygen-free workstation from being used for bacteria in just one growth temperature. The method is similarly applicable for bacteria with the culture temperature above the upper temperature limit of the oxygen-free workstations.
Description
Invention field
The present invention relates to the separation method of a kind of anaerobion, especially the research of a kind of extreme microorganism physiological ecological, natural, ecological research, genetic engineering research, life science, pathological research, indispensable novel method in Marine Geology research and the environmental pollution improvement.
Background technology
In natural ecosystems, microorganism is playing the part of very important role.In Pollution abatement, microorganism can be decomposed the organic or inorganic pollutent that a lot of additive methods can not be removed, and with low cost; In geology research, the sibship of the extreme microorganism of the particular types of different zones may indicate these zones position relation in the past.But microorganism is unit often with the flora, and multiple microorganism survives together, and therefore, separating the pure culture that obtains microorganism is the problem that must at first solve in the microorganism physiological and biochemical research.The discovery of a new microbial strains tends to cause microbiological major progress, also may cause a new subject crossing point: such as the improvement of nitrate reduction bacterium and nitrate wastewater, and the treatment of helicobacter pylori and cancer of the stomach etc.Wherein, the microorganism under the extreme environment is important role in environment, the energy, the geological research often, can provide a lot of new thinking and methods to researcher.But because the envrionment conditions of its cultivation is difficult to obtain simulation under normal condition, the microorganism under the extreme environment is difficult to purifying often to be cultivated, the situation that especially requires several extreme conditions to possess simultaneously, as require extreme anaerobic environment and hot environment to possess simultaneously.
The separation of microorganism and the method for purifying and patent have a lot, identify the substratum (patent No.: 200520003682.8) as disposable microorganism isolation diagnostic, a kind of microorganism separating and culturing method (patent No.: 94103474.7) etc., but in these separation methods, majority is at separating aerobic bacteria or facultative anaerobe.
Anerobe is divided into non-strictly anaerobic bacterium and strictly anaerobic bacterium: be exposed to that can to cause growth inhibiting bacterium in the oxygen be non-strictly anaerobic bacterium, be exposed to the strictly anaerobic bacterium that is called of causing thalline death in the oxygen.Different anerobes are to the tolerance difference of oxygen, generally with the oxytolerant time as index.In time, can not cause thalline death at oxytolerant, the oxytolerant time difference of thalline is very big, does not wait by tens of hours from several seconds.The short bacterium of compole during for oxytolerant, owing to be easy in the process of lock out operation because of contact oxygen causes thalline death, people often are difficult to be separated to, and can only remove to detect its nucleic acid fragment with the method for probe, can't obtain the pure culture bacterium.And short bacterium majority of oxytolerant time is lived in the extreme environment, as in the deep-sea because huge hydraulic pressure, bacterium can not contact with oxygen.The extreme environment microorganism is new species often, significant to scientific research; And since the sample under the extreme condition be difficult to obtain, so the microorganism that is separated under the extreme condition is very precious.But this strict demand to anaerobic condition has caused the great obstacle that separates anerobe.If do not obtain the pure culture of bacterium, people go down to study the metabolic mechanism of this anerobe with regard to being difficult in the condition of nature, anerobe are further studied be restricted.In retrieval, there are a lot of bacteriums all to be called as " not culturing bacterium " to anaerobic bacterium.This explanation is for anaerobic bacterium, and existing isolation technique also has significant limitation.
Reported at present that the method for separating anaerobic bacterium mainly contains:
1) utilize the anaerobism workstation that bacterium is carried out separation and Culture, can guarantee in the isolating whole process that purpose bacterium and oxygen are isolated, still, be limited to 50 ℃ on the general culture temperature of setting in the present anaerobism workstation, being difficult to provides enough culture temperature to thermophilc anaerobe.Even desire the bacterium of separation and Culture growing environment below 50 ℃, an anaerobism workstation only also can be set a culture temperature, and the microorganism of separation and Culture differing temps just needs a plurality of anaerobism workstations, has improved experimental cost greatly.Simultaneously, be difficult to provide the gnotobasis of strictness in the anaerobism workstation, during cultivation, microbiological contamination mutually easily between the culture dish.
2) utilize anaerobic jar, anaerobism bag and roll pipe operation and separate anaerobion, the culture temperature of bacterium more than 50 ℃ can be provided, but the entire operation process is exposed in the air, the purpose anerobe can contact with oxygen, causes the death of purpose thalline.
3) (Wan-hai is clear for document " improving one's methods of a kind of separation and Culture sulphate reducing bacteria ", use and the environmental organism journal 2003,9 (5): introduced folded ware sandwiching 561-562) and separated anaerobion, but this method is in the coating operating process, and thalline can be exposed in the air in the short period of time.If the oxytolerant time less of the bacterium of pre-separation will cause the death of purpose thalline.
4) document " to the assessment of a kind of new culture dish for anaerobe-OxyplateTM " (Zhao Hu etc., shanghai Medicine check magazine, 2002,06) introduced the method for utilizing the OxyplateTM culture dish to separate anerobe in, this method is present in the coating operating process equally, the thalline short period of time is exposed in the air, causes thalline death.
5) a kind of suitable anaerobism of patent is cultivated the culture dish that uses, (the patent No.: 200420074957.2), this patent is at discovery and the cultivation of anaerobism pathogenic bacterium in the mechanism of medical basic unit, pathogenic bacterium often survive in the environment of non-strictly anaerobic, utilize this culture dish to separate short bacterium of oxytolerant time, may cause thalline death.
Summary of the invention
The objective of the invention is: provide simply a kind of and high temperature anaerobic bacterium separation method accurately, this method can avoid purpose anerobe thalline to contact with air in operation whole process, and can separate the high temperature anaerobic bacterium of culture temperature more than 50 ℃.
Concrete solution of the present invention is:
1) will contain the nutrient agar of indicator and the Glass tubing of with closure sterilizes;
2) Glass tubing of not solidified nutrient agar, the natural material that contains the purpose anerobe, with closure is put into the sample chamber of anaerobism workstation, in the sample chamber, carried out three times draws air repeatedly, pour the routine operation of nitrogen (or hydrogen nitrogen mixed gas);
3) will contain the natural material of purpose microorganism and not solidified nutrient agar and mix after, be sub-packed in the Glass tubing screwing hermetic lid;
4) from the anaerobism workstation, Glass tubing is taken out, place incubator, set culture temperature, cultivate certain hour;
5) treat that positive reaction is arranged in the Glass tubing when (positive reaction of indicator is consistent with adding), in the anaerobic operation station, the purpose thalline is taken out.
Advantage of the present invention is as follows:
1. velocity of separation is fast, pregnant solution to the time that is separated to thalline within a week, and purity is very high;
2. the running cost that reaches same separating effect is lower than the separation method of other high temperature anaerobic bacterium;
3. simple and easy to do;
4. the separable high temperature anaerobic bacterium that is difficult to obtain to usual way.
Description of drawings
The copper wire that Fig. 1 uses when turning out the anaerobism pipe of high temperature anaerobic bacterium and digging agar
1. used copper wire when digging agar block
2. turn out the anaerobism pipe of high temperature anaerobic bacterium
Fig. 2 anaerobism workstation synoptic diagram
1. air-channel system
2. sealed chamber (being used for feeding sample)
3. sample
4. the passage that is communicated with sealed chamber and operation room
5. operation room
Embodiment
Get the natural material (hydrothermal solution mouth bed mud, end table water, anaerobic sludge) that contains the purpose anaerobion, after utilizing suitable liquid nutrient medium enrichment, in the anaerobism workstation, pregnant solution is mixed with not solidified solid medium, be sub-packed in the anaerobism pipe, from the anaerobism workstation, take out, place under the condition of different temperatures and cultivate, can be separated to the purpose bacterial classification.
1. with the liquid nutrient medium that is added with ferrous sulfate (0.0001g/L) indicator the nero deep hydrothermal solution mouth bed mud 2.5g that contains the purpose thalline is carried out enrichment.
2. under aerobic conditions, be carbon source with ethanol (concentration is 0.2mL/L), ferrous sulfate (0.006g/L) is an indicator, agar concentration is 1.8g/L, and preparation substratum 50mL places the Erlenmeyer flask of being with tampon, autoclave sterilization (121 ℃, 15 minutes).
3. before culture medium solidifying, Erlenmeyer flask and pregnant solution are placed the sample chamber of anaerobism workstation, take out the nitrogen inflated with nitrogen three times repeatedly, take the air in the substratum away.
4. in the operation box of anaerobism workstation, in not solidified substratum, insert the enrichment supernatant liquor of 3mL, be sub-packed in after shaking up in the anaerobism pipe transparent glass tube of the good screw socket plug of stopping property (or have).
5. the anaerobism pipe is taken out, put into common incubator, attemperation is 90 ℃, after 60 hours, can be observed substratum inside has pore (with the colour developing phenomenon of indicator reaction) to occur, and continues to cultivate 2 days, has different sizes, the pore that asynchronism(-nization) successively occurs is with marking pen mark on tube wall.
6. an end of copper wire is pounded the part hook flat, that this section is flat, wrap such wisp copper wire with tinfoil after, the sterilization.
7. copper wire and the anaerobism pipe with sterilization places the anaerobism workstation, with the end of bending of copper wire pore dug out, and is inoculated in new substratum and gets final product.For the purity of the bacterium that guarantees to assign to, during operation, in pipe, dig out pore successively from the mouth of pipe, whenever dig out a pore before, change a new sterilization copper wire; Whenever after digging out a pore, will dig out from this pore to agar all the mouth of pipe.
8. the purpose anerobe that separation is obtained carries out the extraction of DNA, when utilizing PCR to do 16s rDNA order-checking, can not occur bimodal, sequencing result is accurate, shows that at the ncbi database comparison result this bacterium is the most close with a strain Gram-positive mesophile, similarity 99%.
1. with the liquid nutrient medium that is added with ferrous sulfate (0.0005g/L) indicator the Atlantic Ocean deep-sea hydrothermal port bed mud 3g that contains the purpose thalline is carried out enrichment.
2. under aerobic conditions, be carbon source with ethanol (concentration is 10mL/L), ferrous sulfate (0.0005g/L) is an indicator, agar concentration is at 0.03g/L, and preparation substratum 15mL places the Erlenmeyer flask of being with tampon, autoclave sterilization (121 ℃, 15 minutes).
3. before culture medium solidifying, Erlenmeyer flask and pregnant solution are placed the sample chamber of anaerobism workstation, take out the nitrogen inflated with nitrogen three times repeatedly, take the air in the substratum away.
4. in the operation box of anaerobism workstation, in not solidified substratum, insert the enrichment supernatant liquor of 3mL, be sub-packed in after shaking up in the anaerobism pipe.
5. the anaerobism pipe is taken out, put into common incubator, attemperation is 60 ℃, after 35 hours, can be observed substratum inside has pore (with the colour developing phenomenon of indicator reaction) to occur, and continues to cultivate 4 days, has different sizes, the pore that asynchronism(-nization) successively occurs is with marking pen mark on tube wall.
6. an end of copper wire is pounded the part hook flat, that this section is flat, wrap such wisp copper wire with tinfoil after, the sterilization.
7. copper wire and the anaerobism pipe with sterilization places the anaerobism workstation, with the end of bending of copper wire pore dug out, and is inoculated in new substratum and gets final product.
8. the purpose anerobe that separation is obtained carries out the extraction of DNA, the time can not occur bimodally utilizing PCR to do 16s rDNA order-checking, and sequencing result is accurate, shows that in the ncbi database result this bacterium is the most close with a strain anaerobism mesophile, similarity 100%.
Embodiment 3
With the liquid nutrient medium that is added with ferrous sulfate (0.02g/L) indicator to the coastal waters, the Pacific Ocean that contains the purpose thalline at the bottom of table water 3-10mL carry out enrichment.
2. under aerobic conditions, be carbon source with sodium acetate (concentration is 8g/L), ferrous sulfate (0.02g/L) is an indicator, agar concentration is 0.03g/L, and preparation substratum 100mL places the Erlenmeyer flask of being with tampon, autoclave sterilization (121 ℃, 15 minutes).
3. before culture medium solidifying, Erlenmeyer flask and pregnant solution are placed the sample chamber of anaerobism workstation, take out the nitrogen inflated with nitrogen three times repeatedly, take the air in the substratum away.
4. in the operation box of anaerobism workstation, in not solidified substratum, insert the enrichment supernatant liquor of 3mL, be sub-packed in after shaking up in the anaerobism pipe.
5. the anaerobism pipe is taken out, put into common incubator, attemperation is 15 ℃, and after 24 hours, can be observed substratum inside has pore to occur, and continues to cultivate 2 days, has different sizes, the pore of asynchronism(-nization) successively occurs, with marking pen mark on tube wall.
6. an end of copper wire is pounded the part hook flat, that this section is flat, wrap such wisp copper wire with tinfoil after, the sterilization.
7. copper wire and the anaerobism pipe with sterilization places the anaerobism workstation, with the end of bending of copper wire pore dug out, and is inoculated in new substratum and gets final product.
8. the purpose anerobe that separation is obtained carries out the extraction of DNA, the time can not occur bimodally utilizing PCR to do 16s rDNA order-checking, and sequencing result is accurate, shows that in the ncbi database result this bacterium is the most close with Thioclava pacifica, similarity 100%.
Claims (5)
1. the isolation cultivation method of an anaerobion, its separating step is:
1) will contain the natural material of purpose microorganism, utilize the substratum enrichment that contains the finite concentration indicator, obtain the bacterium pregnant solution;
2) contain the sample chamber that the substratum of finite concentration agar and finite concentration carbon source, sealable container after the sterilization utilize the anaerobism workstation with pregnant solution, sterilization back are not solidified, deflate, pour nitrogen or hydrogen nitrogen mixture body, the oxygen in the substratum is removed.
3) under anaerobic, under normal temperature condition, utilize the substratum that does not solidify agar to mix, be sealed in the sterilising vessel with the bacterium pregnant solution;
4) after this need not to provide anaerobic environment, container is placed a conventional oven or incubator, its optimal temperature is provided, can isolate the bacterium colony of purpose microorganism.
2. according in the claim 1 1), described indicator is a ferrous sulfate, its concentration range is: 0.0001-0.02g/L.
3. according in the claim 1 2), but described substratum and encloses container all pass through autoclave sterilization, sterilising temp is 121 ℃, the time is 15 minutes; Described agar concentration scope of not solidifying nutrient agar is: 0.03-1.8g/L; In the described substratum, carbon source is organic compound such as ethanol, sodium acetate, Sodium.alpha.-hydroxypropionate, glucose, starch or meat soup, concentration range is respectively: ethanol: 0.2mL/L-10mL/L, sodium acetate: 0.3g/L-15g/L, Sodium.alpha.-hydroxypropionate: 0.3g/L-15g/L, glucose: 0.3g/L-20g/L, starch: 0.5g/L-20g/L, meat soup: 0.3mL/L-50mL/L.
4. according in the claim 1 3), described anaerobic condition refers to: anaerobism glove box or anaerobic operation platform need be provided during inoculation; Be meant under the described normal temperature condition that service temperature is a room temperature, promptly 15-30 ℃.
5. according in the claim 1 4), describedly need not to provide anaerobic environment to refer to that sample can place common incubator or baking oven; Optimal temperature refers to that culture temperature is 15-90 ℃.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709267B (en) * | 2009-11-27 | 2011-08-31 | 中国科学院海洋研究所 | Method for quickly separating anaerobic micro-algae |
| CN102424812A (en) * | 2011-12-30 | 2012-04-25 | 黑龙江八一农垦大学 | Method for separating cellulose-decomposing anaerobic bacteria from cellulose-decomposing composite bacterium strain |
| CN104560771A (en) * | 2013-10-29 | 2015-04-29 | 中国石油化工股份有限公司 | Separation culture method of anaerobic bacteria |
| CN106834102A (en) * | 2016-12-22 | 2017-06-13 | 中国科学院宁波城市环境观测研究站 | Cut glue formula tubulose anaerobe and be separately cultured device and the anaerobe cultural method using the device |
| CN113941305A (en) * | 2021-10-26 | 2022-01-18 | 南开大学 | Extraction method of extracellular polymer suitable for anaerobic bacteria |
| CN114252322A (en) * | 2021-12-28 | 2022-03-29 | 何武顺 | Microorganism separation method for alkaline microorganism detection |
| CN116064231A (en) * | 2023-03-06 | 2023-05-05 | 哈尔滨葵花药业有限公司 | Bifidobacterium culture equipment |
Family Cites Families (2)
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| CN1415744A (en) * | 2001-10-29 | 2003-05-07 | 大庆油田有限责任公司 | Method for screening and separating microbial edge-water encroachment fungoid |
| CN1827765A (en) * | 2006-01-23 | 2006-09-06 | 同济大学 | Screening method for anaerobe capable of degrading chlorophenol |
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2007
- 2007-03-23 CN CN2007100647153A patent/CN101270334B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709267B (en) * | 2009-11-27 | 2011-08-31 | 中国科学院海洋研究所 | Method for quickly separating anaerobic micro-algae |
| CN102424812A (en) * | 2011-12-30 | 2012-04-25 | 黑龙江八一农垦大学 | Method for separating cellulose-decomposing anaerobic bacteria from cellulose-decomposing composite bacterium strain |
| CN104560771A (en) * | 2013-10-29 | 2015-04-29 | 中国石油化工股份有限公司 | Separation culture method of anaerobic bacteria |
| CN106834102A (en) * | 2016-12-22 | 2017-06-13 | 中国科学院宁波城市环境观测研究站 | Cut glue formula tubulose anaerobe and be separately cultured device and the anaerobe cultural method using the device |
| CN106834102B (en) * | 2016-12-22 | 2019-08-02 | 中国科学院宁波城市环境观测研究站 | It cuts glue formula tubulose anaerobe and is separately cultured device and the anaerobe cultural method using the device |
| CN113941305A (en) * | 2021-10-26 | 2022-01-18 | 南开大学 | Extraction method of extracellular polymer suitable for anaerobic bacteria |
| CN114252322A (en) * | 2021-12-28 | 2022-03-29 | 何武顺 | Microorganism separation method for alkaline microorganism detection |
| CN116064231A (en) * | 2023-03-06 | 2023-05-05 | 哈尔滨葵花药业有限公司 | Bifidobacterium culture equipment |
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