CN106754623A - A kind of anthrax bacteria for efficiently avoiding polluting is conidial to prepare cultural method - Google Patents
A kind of anthrax bacteria for efficiently avoiding polluting is conidial to prepare cultural method Download PDFInfo
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- CN106754623A CN106754623A CN201710033999.3A CN201710033999A CN106754623A CN 106754623 A CN106754623 A CN 106754623A CN 201710033999 A CN201710033999 A CN 201710033999A CN 106754623 A CN106754623 A CN 106754623A
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- spore
- culture medium
- culture
- triangular flask
- anthrax bacteria
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- 241000193738 Bacillus anthracis Species 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 42
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 9
- 244000061456 Solanum tuberosum Species 0.000 claims abstract description 9
- 235000002595 Solanum tuberosum Nutrition 0.000 claims abstract description 9
- 239000002609 medium Substances 0.000 claims abstract description 9
- 239000008121 dextrose Substances 0.000 claims abstract description 5
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- 238000010828 elution Methods 0.000 claims abstract description 4
- 229920001817 Agar Polymers 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 239000000356 contaminant Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 abstract description 5
- 241001411320 Eriogonum inflatum Species 0.000 abstract description 4
- 238000011109 contamination Methods 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 3
- 238000005273 aeration Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 240000001131 Nostoc commune Species 0.000 description 3
- 235000013817 Nostoc commune Nutrition 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 235000004936 Bromus mango Nutrition 0.000 description 2
- 241000222199 Colletotrichum Species 0.000 description 2
- 241001529387 Colletotrichum gloeosporioides Species 0.000 description 2
- 240000007228 Mangifera indica Species 0.000 description 2
- 235000014826 Mangifera indica Nutrition 0.000 description 2
- 235000009184 Spondias indica Nutrition 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002073 mitogenetic effect Effects 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 241000233866 Fungi Species 0.000 description 1
- 241000228150 Penicillium chrysogenum Species 0.000 description 1
- 241001529246 Platymiscium Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
Abstract
Conidial cultural method is prepared the invention discloses a kind of anthrax bacteria for efficiently avoiding pollution, the characteristic of common micro-organisms can be effectively intercepted using triangular flask, make to prepare the utensil of culture spore using common triangular flask, the high-quality anthrax bacteria conidium of culture is prepared, cultural method step is prepared as follows：1) it is common triangular flask cleaning is standby；2) make product spore culture medium with potato dextrose medium, prepare triangle vial-type culture medium；3) anthrax bacteria is implanted into vial-type culture medium；4) spore culture is produced under the conditions of suitable temperature；5) conidium is collected with aseptic water elution.It is an advantage of the invention that：1) after triangular flask mixes conventional bottle stopper, passing through for common micro-organisms can be intercepted, protects be not bacterial contamination in triangular flask well, obtain high-quality spore finished product；2) operation of the spore than collecting spore in culture dish is collected in triangular flask more convenient；3) protection of triangular flask may be such that product spore bacterium colony can arbitrarily place a period of time without contaminated, and keep producing spore state, be easy to be connected with the working procedure of spore is needed.
Description
Technical field
The present invention relates to Plant Pathology technology, specifically a kind of conidial system of anthrax bacteria for efficiently avoiding polluting
Standby cultural method.
Background technology
Anthracnose is the important disease on many crops, and heavy economic losses is often resulted on many crops.Cause anthrax
The pathogen of disease is the fungi of colletotrichum (Colletotrichum), it can be common that colletotrichum gloeosporioides Penz (Colletotrichum
gloeosporioides).Conidium is the vegetative propagule of anthrax bacteria, in the Disease Cycle and plant disease epidemic of anthracnose
Middle performance significant role.In laboratory, conidium is also the indispensable material of anthracnose research.About the formation of spore
The researchs such as the identification interaction reaction of physiology of development, adaptability of existence or repellence to poor environment, spore and host, are required for
Use the material of spore shape；Relevant research in terms of new protective agents research and development, such as medicament to the Toxic efficiency of spore,
Need spore；The mutagenesis of the relevant research of biology field, such as pathogenic related gene, positioning and clone；Also it be unable to do without
Spore；It can be seen that, conidium is the important thalli morphology of the daily research of anthracnose.And in many work, often require that
The conidium of research institute will reach the state without living contaminants.
Anthrax bacteria can form conidium in conventional medium, and laboratory prepares the conidial conventional method of culture
It is, by the use of Nostoc commune Vanch ware as culture utensil, culture medium to be poured into culture dish and is made culture plate, anthrax bacteria is flat in culture
Conidium is grown and formed on plate.
The culture dish that common laboratory is used is made up of ware bottom and ware lid, is limited to the Manufacturing Techniques of current culture dish,
The degree uniformly fitted is extremely difficult between ware bottom and ware lid, is existed between the ware bottom of most of culture dishes and ware lid compared with big gap, training
The air flow supported inside and outside ware can be unblocked.Culture dish this smoothly aeration, the spore shape to needing good aeration condition
It is highly beneficial into developing, but exactly this smoothly aeration, cause the biomaterial in culture dish easily to receive outside contamination air
Interference, culture obtain with living contaminants spore finished product probability it is larger.Especially general Plant Pathology laboratory,
Current to prefer to incubator of the configuration with cooling/liter temperature function, such incubator is quick in operating room generally for realizing
Constant temperature and temperature uniform distribution, operating room is often designed as the pattern of circulation air.Carried out using this kind of incubator
Spore culture is produced, culture dish surrounding air is in normal flow regime, causes the probability of material contamination in culture dish at a relatively high, generally training
Supporting can cause more than 50% culture plate for 7 days by living contaminants, even if can't see obvious Penicillium notatum on some culture plates
Deng the bacterium colony of laboratory pollution bacterium, but the pollution condition being invisible to the naked eye is still present.Therefore, charcoal is prepared using culture dish culture
Subcutaneous ulcer germ conidium, is technically difficult the possibility for decontaminating.
The content of the invention
Conidial cultural method is prepared it is an object of the invention to provide a kind of anthrax bacteria for efficiently avoiding pollution.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
It is a kind of efficiently to avoid that the anthrax bacteria of pollution is conidial to prepare cultural method, the step of prepare cultural method such as
Under：
1. make to prepare the utensil of culture spore using common triangular flask, triangular flask cleaning is standby.
2. the preparation of triangle vial-type culture medium：Make product spore culture medium with potato dextrose medium, the culture medium is matched somebody with somebody
Than for：Potato 200g, glucose 20g, agar 20g, water 1000mL；Culture medium is sub-packed in the triangular flask that step 1 prepares, often
Turn into triangle vial-type culture medium after rule high pressure-temperature sterilizing, it is standby.
3. the transplanting of anthrax bacteria：With transplanting tool by the standing anthrax bacteria mycelium in laboratory, it is implanted into step 2 and makes
On standby triangle vial-type culture medium.
4. spore is produced in culture：By step 3 operate it is complete after triangle vial-type culture medium be transferred to the Nostoc commune Vanch that temperature is 28 DEG C
Culture in case；After being generally incubated 6 days, triangle vial-type culture medium has formed the intensive bacterium colony of mycelia on face, forms a large amount of on bacterium colony
Conidium.
5. conidial collection：The spore on bacterium colony is obtained with sterilized water elution step 4, and focuses on sterilization container
It is interior, that is, obtain the anthrax bacteria conidium liquid of the better quality without living contaminants.
Characteristic of the invention is with advantage：
1) after triangular flask mixes conventional bottle stopper, passing through for common micro-organisms can be intercepted, protects do not received in triangular flask well
Living contaminants, obtains high-quality spore finished product.
2) operation of the spore than collecting spore in culture dish is collected in triangular flask more convenient.
3) protection of triangular flask may be such that product spore bacterium colony can arbitrarily place a period of time without contaminated, and keep producing spore shape
State, is easy to be connected with the working procedure of spore is needed.
Specific embodiment
With reference to embodiment, the invention will be further described.
Key technology of the invention is the combination and application of triangular flask and agar class culture medium.
Although the everyday devices of triangular flask platymiscium PAL, its normal usage is mainly and contains culture medium and go out
Bacterium；Also being commonly used for containing fluid nutrient medium or plant tissue class culture medium (such as seed, stalk) carries out Bacteria culturing.
The application of agar class culture medium, existing technical specification is all to make culture utensil using culture dish, by agar culture
After base heating fusing, pour into culture dish and be made culture medium flat plate, and pathogen is cultivated on the culture plate.
Preparation culture of the anthrax bacteria conidium on agar medium, is using conventional technique specification, i.e. profit
Culture is prepared with the culture technique of culture dish combination agar class culture medium；So far yet there are no and make incubator using triangular flask
Tool, carries out the spore technology of preparing that anthrax bacteria produces spore culture on agar class culture medium.Possible cause is the length to culture dish
Phase is relied on and custom is continued to use, and being generally considered pathogen product spore needs the good environmental condition of aeration, and culture dish is lucky
Good aeration can be met.
After common triangular flask puts bottle stopper, although form the poor enclosed environment of aeration, but inventor's repetition test is sent out
It is existing, form conidium on the agar medium in environment that anthrax bacteria can more be closed at this；And the application of triangular flask, then
Just solve the key technical problem that applied culture ware is easily caused pollution.
Anthrax bacteria can grow on many agar class culture mediums and form conidium, product spore culture of the present invention
Base is the conventional potato dextrose medium in laboratory, and its component is：Potato 200g, glucose 20g, agar 20g, water
1000mL；Boil according to a conventional method and match somebody with somebody.Culture medium is boiled with after finishing, can direct packaging in triangular flask；Because triangular flask bottom is more
Category curved surface state, culture base unit weight is very few to cause bottom of bottle local excessively thin without culture medium or culture medium, and culture medium is crossed and at most easily made
Into waste；Usual every bottle (250mL specifications) loads 20~30mL culture mediums and is advisable, and the triangular flask of other specifications takes the circumstances into consideration to increase and decrease liquid
Amount.
After packaging bottle stopper, conventional high-pressure high-temperature sterilization takes out triangular flask and is flat on straight desktop, after cooling after sterilizing
Form triangle vial-type culture medium.
Vial-type culture medium can also be dispensed alternatively, first with larger triangular flask load in the usual way liquid measure compared with
Many culture mediums are sterilized；For another example it is the same with the flat board of falling culture dish, after being melted using preceding heating, it is dispensed into the blank three of sterilizing
In the bottle of angle.
The transplanting of anthrax bacteria can be transplanted in the usual way, and the parent bacterium of transplanting is used as with the mycelium on common bacterium colony
Body；To accelerate culture process, polylith mycelia block can be implanted on a vial-type culture medium.
Producing spore culture can implement in standard incubator, the preference temperature that temperature control grows in anthrax bacteria, the present invention
Cultivation temperature uses 28 DEG C；Incubation is to illumination condition and damp condition without special demands.
After generally product spore prepares culture 6 days, dense aerial hyphae has been formed with a large amount of points on culture basal plane in triangular flask
Raw spore.
The conidium for obtaining is collected with sterilized water, practical operation can scrub bacterium colony, make spores release to washing with T-shaped glass rod
In spore aqueous.
Embodiment 1
Cultural method is prepared using a kind of anthrax bacteria for efficiently avoiding polluting of the present invention is conidial, Pei Yang Mango is prepared
The conidium of fruit anthrax bacteria bacterial strain Cg-1, implements operation as follows：
1. make to prepare the utensil of culture spore using common triangular flask, triangular flask cleaning is standby.
2. the preparation of triangle vial-type culture medium：Make product spore culture medium with potato dextrose medium, the culture medium is matched somebody with somebody
Than for：Potato 200g, glucose 20g, agar 20g, water 1000mL；Culture medium is sub-packed in the triangular flask that step 1 prepares, often
Bottled 30mL, turns into triangle vial-type culture medium after conventional high-pressure high-temperature sterilization, standby.
3. the transplanting of anthrax bacteria：With transplanting pin by the standing Mango anthrax bacteria bacterial strain Cg-1 mycelium in laboratory, move
On triangle vial-type culture medium prepared by implantation step 2.
4. spore is produced in culture：By step 3 operate it is complete after triangle vial-type culture medium be transferred to the Nostoc commune Vanch that temperature is 28 DEG C
Culture in case；After culture 6 days, triangle vial-type culture medium has formed the intensive bacterium colony of mycelia on face, and substantial amounts of point is formed on bacterium colony
Raw spore.
5. conidial collection：The spore on bacterium colony obtained with sterilized water elution step 4, and focuses on sterilization container
It is interior, that is, obtain the better quality Mango anthrax bacteria conidium liquid without living contaminants.
The spore on one bottle of bacterium colony is eluted with sterilized water 15mL, it is 12.3 × 10 that can obtain spore concentration6Individual/mL's is mitogenetic
Spore liquid.
Embodiment 2
Cultural method is prepared using a kind of anthrax bacteria for efficiently avoiding polluting of the present invention is conidial, Pei Yang Mango is prepared
The conidium of fruit anthrax bacteria bacterial strain Cg-2,1 operates to step 5 and implements the step of by embodiment 1, different simply steps 3
The bacterial strain for using is Cg-2.
The spore on one bottle of bacterium colony is eluted with sterilized water 15mL, it is 1.6 × 10 that can obtain spore concentration6Individual/mL's is mitogenetic
Spore liquid.
Claims (1)
1. a kind of anthrax bacteria for efficiently avoiding polluting is conidial prepares cultural method, it is characterised in that prepare culture side
The step of method, is as follows：
1) make to prepare the utensil of culture spore using common triangular flask, triangular flask cleaning is standby；
2) preparation of triangle vial-type culture medium：Make product spore culture medium with potato dextrose medium, the proportioning of the culture medium is：
Potato 200g, glucose 20g, agar 20g, water 1000mL；Culture medium is sub-packed in step 1) triangular flask for preparing, it is conventional high
Turn into triangle vial-type culture medium after super pressure-high temperature sterilizing, it is standby；
3) transplanting of anthrax bacteria：With transplanting tool by the standing anthrax bacteria mycelium in laboratory, step 2 is implanted into) prepare
Triangle vial-type culture medium on；
4) spore is produced in culture：By step 3) operation it is complete after triangle vial-type culture medium be transferred in the standard incubator that temperature is 28 DEG C
Culture；After being generally incubated 6 days, triangle vial-type culture medium has formed the intensive bacterium colony of mycelia on face, and substantial amounts of point is formed on bacterium colony
Raw spore；
5) conidial collection：With sterilized water elution step 4) spore on bacterium colony is obtained, and focus in sterilization container, i.e.,
Obtain the anthrax bacteria conidium liquid of the better quality without living contaminants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710033999.3A CN106754623A (en) | 2017-01-10 | 2017-01-10 | A kind of anthrax bacteria for efficiently avoiding polluting is conidial to prepare cultural method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710033999.3A CN106754623A (en) | 2017-01-10 | 2017-01-10 | A kind of anthrax bacteria for efficiently avoiding polluting is conidial to prepare cultural method |
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| Publication Number | Publication Date |
|---|---|
| CN106754623A true CN106754623A (en) | 2017-05-31 |
Family
ID=58946170
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|---|---|---|---|
| CN201710033999.3A Pending CN106754623A (en) | 2017-01-10 | 2017-01-10 | A kind of anthrax bacteria for efficiently avoiding polluting is conidial to prepare cultural method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4017697B2 (en) * | 1996-12-06 | 2007-12-05 | 大日本除蟲菊株式会社 | Novel antibacterial active substance and plant disease control agent containing the same |
| CN104480060A (en) * | 2014-12-02 | 2015-04-01 | 福建省农业科学院果树研究所 | Rapid spore-producing method of colletotrichum gloeosporioides |
| CN105420115A (en) * | 2015-12-10 | 2016-03-23 | 三峡旅游职业技术学院 | Culture medium for armillaria mellea spore separation and culture, method and application |
-
2017
- 2017-01-10 CN CN201710033999.3A patent/CN106754623A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4017697B2 (en) * | 1996-12-06 | 2007-12-05 | 大日本除蟲菊株式会社 | Novel antibacterial active substance and plant disease control agent containing the same |
| CN104480060A (en) * | 2014-12-02 | 2015-04-01 | 福建省农业科学院果树研究所 | Rapid spore-producing method of colletotrichum gloeosporioides |
| CN105420115A (en) * | 2015-12-10 | 2016-03-23 | 三峡旅游职业技术学院 | Culture medium for armillaria mellea spore separation and culture, method and application |
Non-Patent Citations (4)
| Title |
|---|
| 刘威: "茶树炭疽病的病原鉴定及其遗传多样性分析", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 胡文浪: "《黄酒工艺学》", 31 August 1998, 中国轻工业出版社 * |
| 范巴陵: "（三）AS.3.4309的种曲三角瓶", 《实用酒精工艺基础》 * |
| 陈聃: "葡萄炭疽病菌的抗药性检测和治理研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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