CN108018251A - One species impoverishment strain returns the rejuvenation cultural method of natural environment - Google Patents
One species impoverishment strain returns the rejuvenation cultural method of natural environment Download PDFInfo
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- CN108018251A CN108018251A CN201810095654.5A CN201810095654A CN108018251A CN 108018251 A CN108018251 A CN 108018251A CN 201810095654 A CN201810095654 A CN 201810095654A CN 108018251 A CN108018251 A CN 108018251A
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- rejuvenation
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- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses the rejuvenation cultural method that a species impoverishment strain returns natural environment, it includes following steps：1. the activation of strain；2. decline strain returns natural environment rejuvenation；3. rejuvenation strain expands numerous.Wherein, rejuvenation method is to complete under field conditions (factors).Not only exclude pollution of the non-targeted microorganism to target pure culture in natural environment, it can also make to have occurred and that the individual of decline is eliminated due to not adapting to original natural conditions in the pure culture of multiple secondary culture, so as to which only survival adapts to the individual of the condition in device when making the rejuvenation phase complete, complete the rejuvenation of specific degradation strain, the also interference without human factor.It is an advantage of the invention that it can realize decline strain simply and effectively rejuvenation.
Description
Technical field
The present invention relates to microbial culture technique, particularly a species impoverishment strain to return the rejuvenation cultural method of natural environment.
Background technology
In the basic research and application field of microorganism, one desirable strain of selection and breeding is an arduous job, and is wanted
The genetic stability for keeping bacterial strain is even more difficulty.Long-term application, cell division propagation passage repeatedly, the degeneration of strain can not
Avoid, this often causes bacterial strain performance degradation to cause productivity effect decline or particular studies result to deviate reality.
At present, the method for being usually used in decline rejuvenation of spawn mainly has：
（1）Pure strain separation：Using plate streaking or dilution spread flat band method, still keeping the unicellular of original typical merit
Separate, recover the typical merit of original bacterial strain through expanding culture.
（2）Rejuvenation is carried out by host's tumor growth：The mainly bacterial strain of some parasitics, can connect the bacterial strain of decline
Kind is arrived in corresponding host, improves its parasitic performance and other performances.
（3）Handled using fiercer chemical factors, stayed with killing the poor impaired individual of vitality
The individual of robust growth in lower colony.
（4）Using effective culture collection process.
Above-mentioned microbial decay rejuvenation of spawn method is rule repeatedly in laboratory conditions according to relative physiologic index
Separated or separated one by one further according to relative physiologic index by meeting former host repeatedly one by one, taken, take a lot of work, inefficiency.
The content of the invention
In view of the above problems, it is an object of the invention to provide the rejuvenation culture side that a species impoverishment strain returns natural environment
The problem of method, this method can be used for rejuvenation of the decline strain under the conditions of nature, can effectively solve the problem that strain decline.
To achieve these goals, the present invention is achieved through the following technical solutions：
One species impoverishment strain returns the rejuvenation cultural method of natural environment, includes the following steps：
（1）Actication of culture：Culture medium is prepared, is sterilized, cooling, under sterile working, with sterile micro suction pipe, from the bacterium thawed
A certain amount of bacterium solution is drawn in kind preservation pipe, instills in culture medium, the culture medium being inoculated with is placed in the shaking table of design temperature, is pressed
The revolution culture of setting is to logarithmic phase；
（2）Decline strain returns natural environment rejuvenation：
1）On superclean bench, prepare the device to interact under analysis natural conditions between particular types microorganism, take 2 layers of circle
Shape miillpore filter is placed on the wide orle concave surface of device reaction device main body, makes miillpore filter that rubber seal to be completely covered；
2）By step（1）Treat that the appropriate bacterium mud or bacterium solution of the particular types microorganism of rejuvenation are placed in miillpore filter center；
3）2 layers of miillpore filter separately are taken, by step 2）Bacterium mud or bacterium solution be clipped among 4 layers of miillpore filter；
4）Reactor is placed in nature environment, makes its rejuvenation under field conditions (factors)；
（3）The expansion of rejuvenation strain is numerous：
1）After rejuvenation process, reactor is taken out and is opened, will contact that 2 layers of external environment filter are cured to be discarded, centre is pressed from both sides
There is what 2 layers of inner membrance of bacterium mud or bacterium solution were placed in sterilizing to take diaphragm base central, and compressed film edge completely by fixator；
2）By lower handle, part of the film not in contact with external environment microorganism is cut by blade, is placed in sterile saline
Rinse；
3）To be coated on target solids culture medium, be inverted after the dilution of physiological saline bacteria suspension, thermophilic culture to growing bacterium colony, from
And obtain returning the bacterial strain of natural environment rejuvenation.
Step（1）The strain is the strain of decline.
Step（1）Solid medium or Liquid Culture of the culture medium needed for growth used in the actication of culture
Base.
Step（2）The device to interact under the analysis natural conditions between particular types microorganism is the prior art,
Had been described in Chinese patent CN104745458A.
Step（2）Described in miillpore filter be diameter slightly larger than the rubber seal that cross section is T-shaped diameter, it is but small
The circular miillpore filter of circular diameter where the wide orle of reactor body.
Compared with prior art, it is an advantage of the invention that：The device of application is simple, easy to operate, without using any reagent,
Without interference from human factor rejuvenation process, significant effect, can easily solve the problems, such as spawn degeneration, be plant produced and scientific research
Reliable and stable strain source is provided.
Brief description of the drawings
Fig. 1 is the strain schematic diagram before 1 citric acid acid-producing bacteria mutant strain TR-M30-1 of embodiment declines；
Fig. 2 is the strain schematic diagram after 1 citric acid acid-producing bacteria mutant strain TR-M30-1 of embodiment declines；
Fig. 3 is the strain schematic diagram after 1 citric acid acid-producing bacteria mutant strain TR-M30-1 rejuvenation of embodiment；
Fig. 4 is the strain schematic diagram before the decline of embodiment 2G4 bacterium；
Fig. 5 is the strain schematic diagram after the decline of embodiment 2G4 bacterium；
Fig. 6 returns the strain schematic diagram after natural environment rejuvenation for embodiment 2G4 bacterium.
Embodiment
The present invention is further described in detail with reference to embodiments, so that those skilled in the art can be preferably
Understand the present invention and be practiced, but illustrated embodiment is not as a limitation of the invention, and any those skilled in the art exists
In the field of the invention, the change or modification made all are covered among the scope of the claims of the present invention.
The rejuvenation method of one species impoverishment strain, including following steps：
A, the activation of strain
B, natural environment rejuvenation of spawn is returned
C, rejuvenation strain expands numerous
Embodiment 1
The citric acid acid-producing bacteria mutant strain TR-M30-1 that the preservation of this laboratory has been carried out with the present invention is [blue in dawn space Jiang Shaofeng
Fortune China, etc. to the screening Guangxi Normal Universitys journal of the sour kirschner bacterium mutant of mycorhiza secretion strong taxis production:Natural science edition,
2014,32 (2):130-136] decline strain rejuvenation, the method and result of rejuvenation be as follows：
1. experiment material
（1）The decline bacterial strain of rejuvenation：The sour kirschner bacterium mutant strain TR-M30-1, Kan of productionr（Kalamycin resistance）, by transposonsmTn5gusA-pgfp21Genetic marker, bacterium colony shows green fluorescence under ultraviolet light；Growing nitrogen-fixing, can be on nitrogen-free agar
Growth.
（2）Strain activation and culture base, LB culture mediums：Tryptone 10g, yeast extract 5g, NaCl 10g, are used
1mol/LNaOH tune pH to 7.0,1L is settled to deionized water, the steam sterilizing 20min at 121 DEG C.
（3）The device that rejuvenation uses under strain natural conditions：Using published entitled " a kind of to analyze spy under natural conditions
Surely the device of the device to interact between kind quasi-microorganism " patent（CN104745458A）.
（4）The culture medium of rejuvenation Spawn incubation：Ah 's shellfish nitrogen-free solid medium：Glucose 10g, MgSO4.7H2O
0.2g, KH2PO40.2g, K2SO40.2g, NaCl 0.2g, CaCO35g, agar 1.5-2%, deionized water are settled to 1000ml,
PH7.0-7.2, the steam sterilizing 20min at 121 DEG C.Ah 's shellfish nitrogen-free fluid nutrient medium is then added without agar.
（5）Kanamycin mother liquid（50mg/mL）：Weigh 0.5g kanamycins powder to be dissolved in 10mL distilled water, use
0.22 μm of syringe filter disk sterilizing is spare.
2. method
A, the activation of strain
（1）In superclean bench, protected using pipettor from the citric acid acid-producing bacteria mutant strain TR-M30-1 strains to have thawed
The bacterium solution that 20 μ L are drawn in pipe is hidden, is added in the LB fluid nutrient mediums of 5mL；
（2）The good culture medium of above-mentioned inoculation is placed in 180r/min in 28 DEG C of shaking table and cultivates 3-6h to logarithmic phase；
B, decline strain returns natural environment rejuvenation
（1）In superclean bench, it is ready to " a kind of to analyze the dress to interact under natural conditions between particular types microorganism
Put " device of patent（ZL201520168825.4）, the rubber seal loop diameter by 2 layers of diameter slightly larger than cross section for T-shaped,
But the circular miillpore filter less than circular diameter where the wide orle of reactor body is placed on the wide orle concave surface of reactor body, is made
Rubber seal is completely covered miillpore filter；
（2）The bacterium solution room temperature 3000r/min that citric acid acid-producing bacteria mutant strain TR-M30-1 is activated to logarithmic phase centrifuges 2min
After take appropriate bacterium mud be placed in miillpore filter center；
（3）It is another to take 2 layers of miillpore filter for being same as above diameter, by step（2）Bacterium mud and step（1）Miillpore filter be completely covered；
（4）Reactor lid is tightened by screw socket and reactor body, bacterium mud is clipped among 4 layers of miillpore filter；
（5）Reactor is placed in nature environment（Humidity is 70% or so vegetable soil）, make its rejuvenation under field conditions (factors)
Culture（About 15 days by 1 month）；
C, rejuvenation strain expands numerous
（1）Terminate when the above-mentioned rejuvenation stage, reactor is taken out and is rinsed well and is placed in super-clean bench with clear water；
（2）It is careful to open reactor, 2 layers of filter membrane of external environment will be contacted（Outmost 2 tunic）Discard, bacterium will be sandwiched between
The base for taking diaphragm that the tunic of mud is placed in sterilizing is central, and is compressed at the edge of film completely by fixator；
（3）By lower handle, part of the film not in contact with external environment microorganism is cut by blade, is placed in sterile saline
Middle flushing；
（4）100 μ L physiological saline bacteria suspension dilution spreads are drawn in being 50 μ g/mL kanamycins added with concentration with pipettor
Ah 's shellfish nitrogen-free solid medium on, inversion is incubated in 28 DEG C of constant incubator, until grow bacterium colony, so as to be returned
The citric acid acid-producing bacteria mutant strain TR-M30-1 of natural environment rejuvenation.
Citric acid acid-producing bacteria mutant strain TR-M30-1 has nitrogen fixing capacity can be added with raw on kanamycins nitrogen-free agar
Long, the nitrogen compound from external world's suction or nitrogen can be converted into the protein, nucleic acid and other nitrogens of itself and closed by bacterial strain
Thing.Bibliography（Tan Zewen, Tan Zhiyuan, Huang Huiling, wait azotobacter separation identification and system in Chinese parasol trees county oryza officinalis
Developmental analysis [J] is applied and environmental organism journal, 2017,23 (4):622-627.）, bacterial strain is detected by acetylene reduction method
Nitrogenase activity size before and after rejuvenation.By culture in LB culture mediums to logarithmic phase（OD600Equal to 0.7）Citric acid production acid it is wild
Before raw bacterial strain, decline, after decline, the TR-M30-1 after rejuvenation, bacterium mud is collected by centrifugation, washs 5 times with sterile saline, finally
It is diluted to same concentrations（OD600Equal to 0.5）；Draw 10 μ L and be inoculated in the band for filling 5 mL Ah 's shellfish nitrogen-free fluid nutrient mediums
In caulking gum lid finger-type, when culture is to logarithmic phase in 28 DEG C of constant incubators of sealing（OD600Equal to 0.7）, second is injected into pipe
Alkynes gas（Final concentration of 1%）, 24 h are further cultured under the same conditions, use gas chromatograph（Beijing Tian Pu analytical instrument factory SP-
2100）Before measure ethylene emanation is to reflect the sour wild strain of citric acid production, decline（As shown in Figure 1）, after decline（Such as Fig. 2
It is shown）, after rejuvenation（As shown in Figure 3）TR-M30-1 nitrogen fixing capacity.Negative blank control is to be trained in Ah 's shellfish nitrogen-free liquid
Support and sterile deionized water is added in base, positive control is to add citric acid acid-producing bacteria not in Ah 's shellfish nitrogen-free fluid nutrient medium
Fail wild strain；External standard method calculates measurement result, with a μm olC2H4/ mL bacterium solutions/h represents nitrogenase activity.
The size of nitrogenase activity is calculated according to following equation：ρx= Ax ×ρs × V / (24.9 ×As× t),
Wherein,
AxFor sample peak area value,
AsFor standard C2H4Peak area value,
ρsFor standard C2H4Concentration（μmol/L）,
V is culture vessel volume（m L）,
T is the sample culturing time（h）,
ρxFor the C of generation2H4Concentration（μmol m L-1 h-1）
It is positive control through above method measurement result（The sour wild strain of citric acid production）Nitrogenase activity for 33.457 ±
1.245nmol m L-1 h-1, TR-M30-1 do not fail bacterial strain for 32.523 ± 2.352 nmol m L-1 h-1, negative blank
Compare as 0.003 ± 0.001 nmol m L-1 h-1（As shown in Figure 1）, the TR-M30-12 nitrogenase activities of decline are 18.827
±1.637nmol m L-1 h-1（As shown in Figure 2）, the TR-M30-1 nitrogenase activities after rejuvenation are 31.619 ± 3.169nmol
m L-1 h-1（As shown in Figure 3）, bacterial strain nitrogenase activity changes greatly before and after rejuvenation, illustrates the bacterial strain fixed nitrogen after rejuvenation that fails
Enzymatic activity is restored raising.
Embodiment 2：
Using the present invention to from hazard prevention Yan Shan towns root-knot nematode seriously occur to separate in vegetable soil can be with several
Fourth matter carries out rejuvenation as the decline pure culture of the G4 bacterium of only nitrogen source, and the method and result of rejuvenation are as follows：
1. experiment material
（1）Fail bacterial strain：What from hazard prevention Yan Shan towns, root-knot nematode seriously occurred to separate in vegetable soil can be with several
G4 bacterium of the fourth matter as only nitrogen source；Transparent circle is presented in its periphery of bacterial colonies when being grown on chitin colloid culture medium.
（2）Strain activation and culture base, LB culture mediums：Tryptone 10g, yeast extract 5g, NaCl 10g, are used
1mol/LNaOH tune pH to 7.0,1L is settled to deionized water, the steam sterilizing 20min at 121 DEG C.
（3）The device that rejuvenation uses under strain natural conditions：Using published entitled " a kind of to analyze spy under natural conditions
Surely the device to interact between quasi-microorganism is planted " patent（CN104745458A）Device.
（4）The preparation of 3% tobacco brown spot pathogen：30g chitin powder is weighed, is slowly added into the concentrated hydrochloric acid of 300mL precoolings
In, in 4 DEG C of vigorous stirring overnights；Said mixture is added to the sterile of 95% ice-cold ethanol of 2000mL or 2000mL precoolings
In distilled water, quickly stir after room temperature（25℃）Stand overnight；In 4 DEG C, 5000g centrifugations 20min collects precipitation；Using sterile
Distilled water rinses precipitation repeatedly, until pH7.0；1000ml sterile distilled waters are added, 3% tobacco brown spot pathogen solution are prepared, in 4
DEG C save backup.
（5）Rejuvenation strain expands breeding culture medium（Chitin colloid culture medium）：K2HPO40.7g, MgSO4.7H2O 0.5g,
KH2PO40.3g, FeSO4..7H2O 0.01g, agar 15g, 3% tobacco brown spot pathogen 80mL, deionized water are settled to 1000ml,
PH7.0-7.2, the steam sterilizing 20min at 121 DEG C.
2. method
Specific method is similar to Example 1, after its difference is that decline bacterial strain returns natural environment rejuvenation culture, uses pipettor
100 μ L physiological saline bacteria suspension dilution spreads are drawn on chitin colloid culture medium, inversion is incubated at 28 DEG C of constant temperature incubation
In case, until bacterium colony is grown, so as to obtain returning the G4 bacterium of natural environment rejuvenation.
Using transparent circle method, by the inoculation before rejuvenation and after rejuvenation into solid chitin culture medium, in 28 DEG C
Transparent loop diameter R2 and colony diameter R1 are measured respectively after cultivating 3d in constant incubator, compare its chitin using R2/R1 ratios
Matter enzymatic activity.Fig. 4,5,6 for G4 bacterium decline before, decline after, the upgrowth situation after rejuvenation on solid chitin culture medium；Fig. 4,
6 transparent loop diameter and colony diameter ratio are apparently higher than Fig. 5 person.
The average value of R2/R1 before the decline of G4 bacterium is 2.18 ± 0.07（Fig. 4）, bacterial strain produces chitinase after passing on repeatedly
Function reduction, the average value of R2/R1 ratios are reduced to 1.32 ± 0.04（Fig. 5）, after the method rejuvenation by the present invention, G4 bacterium chitins
Matter enzyme function substantially returns to the state before decline, and the average value of R2/R1 ratios is 2.15 ± 0.06（Fig. 6）, illustrate this patent
Rejuvenation method there is preferable effect of rejuvenation.
Claims (4)
1. a species impoverishment strain returns the rejuvenation cultural method of natural environment, it is characterized in that：Include the following steps：
（1）Actication of culture：Culture medium is prepared, is sterilized, cooling, under sterile working, with sterile micro suction pipe, from the bacterium thawed
A certain amount of bacterium solution is drawn in kind preservation pipe, instills in culture medium, the culture medium being inoculated with is placed in the shaking table of design temperature, is pressed
The revolution culture of setting is to logarithmic phase；
（2）Decline strain returns natural environment rejuvenation：
1）On superclean bench, prepare the device to interact under analysis natural conditions between particular types microorganism, take 2 layers of circle
Shape miillpore filter is placed on the wide orle concave surface of device reaction device main body, makes miillpore filter that rubber seal to be completely covered；
2）By step（1）Treat that the appropriate bacterium mud or bacterium solution of the particular types microorganism of rejuvenation are placed in miillpore filter center；
3）2 layers of miillpore filter separately are taken, by step 2）Bacterium mud or bacterium solution be clipped among 4 layers of miillpore filter；
4）Reactor is placed in nature environment, makes its rejuvenation under field conditions (factors)；
（3）The expansion of rejuvenation strain is numerous：
1）After rejuvenation process, reactor is taken out and is opened, will contact that 2 layers of external environment filter are cured to be discarded, centre is pressed from both sides
There is what 2 layers of inner membrance of bacterium mud or bacterium solution were placed in sterilizing to take diaphragm base central, and compressed film edge completely by fixator；
2）By lower handle, part of the film not in contact with external environment microorganism is cut by blade, is placed in sterile saline
Rinse；
3）To be coated on target solids culture medium, be inverted after the dilution of physiological saline bacteria suspension, thermophilic culture to growing bacterium colony, from
And obtain returning the bacterial strain of natural environment rejuvenation.
2. rejuvenation cultural method according to claim 1, it is characterized in that：Step（1）The strain is the strain of decline.
3. rejuvenation cultural method according to claim 1, it is characterized in that：Step（1）Training used in the actication of culture
Support solid medium or fluid nutrient medium of the base needed for growth.
4. rejuvenation cultural method according to claim 1, it is characterized in that：Step（2）Described in miillpore filter be diameter omit
Diameter more than cross section for the rubber seal of T-shaped, but it is circular micro- less than circular diameter where the wide orle of reactor body
Hole filter membrane.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110337986A (en) * | 2019-07-10 | 2019-10-18 | 杭州丝绸之路文化艺术有限公司 | A kind of Spawn incubation method of Phellinus |
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| CN103404368A (en) * | 2013-07-12 | 2013-11-27 | 重庆市中药研究院 | Rejuvenation culture method for artificial breeding of cordyceps militaris strain |
| CN104745458A (en) * | 2015-03-25 | 2015-07-01 | 广西师范大学 | Device for analyzing interaction between microorganisms of specific categories under natural conditions |
| CN105724049A (en) * | 2016-01-27 | 2016-07-06 | 浙江省农业科学院 | Mulberry parasitical phellinus strain wild environment returning rejuvenation culture method |
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2018
- 2018-01-31 CN CN201810095654.5A patent/CN108018251A/en active Pending
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| CN103404368A (en) * | 2013-07-12 | 2013-11-27 | 重庆市中药研究院 | Rejuvenation culture method for artificial breeding of cordyceps militaris strain |
| CN104745458A (en) * | 2015-03-25 | 2015-07-01 | 广西师范大学 | Device for analyzing interaction between microorganisms of specific categories under natural conditions |
| CN105724049A (en) * | 2016-01-27 | 2016-07-06 | 浙江省农业科学院 | Mulberry parasitical phellinus strain wild environment returning rejuvenation culture method |
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| ANHUI CHEN等: "A Novel Technique for Rejuvenation of Degenerated Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), a Valued Traditional Chinese Medicine", 《INTERNATIONAL JOURNAL OF MEDICINAL MUSHROOMS》 * |
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Application publication date: 20180511 |