CN105861622B - A kind of viable count method of bacteria cellulose production bacterial strain - Google Patents
A kind of viable count method of bacteria cellulose production bacterial strain Download PDFInfo
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- CN105861622B CN105861622B CN201610355676.1A CN201610355676A CN105861622B CN 105861622 B CN105861622 B CN 105861622B CN 201610355676 A CN201610355676 A CN 201610355676A CN 105861622 B CN105861622 B CN 105861622B
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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Abstract
The present invention relates to a kind of viable count methods of bacteria cellulose production bacterial strain, (1) prepare the thick suspension of thallus；(2) the thick suspension of gained obtains thallus after being centrifuged in method three, with containing a certain amount of cellulase physiological saline or phosphate buffer thallus is resuspended, until initial volume；(3) gradient dilution: a certain amount of cellulase is first added into gradient dilution liquid, then carries out gradient dilution experiment；(4) after mixing well, coating counts or pours into counting.This method is discharged thallus with cellulase from bacteria cellulose, and uniform bacteria suspension is made.And by adding cellulase into gradient dilution liquid, achieve the purpose that the cellulose silk connected between fully degraded thallus, it avoids in counting process, since the presence of cellulose distinguish thallus can not completely, bacterium colony is caused to assemble or can not count, when this method is applied to coating counting and pours into counting method, thallus separating effect is fabulous.
Description
Technical field
The present invention relates to microbial culture technique, in particular to a kind of count plate side of bacteria cellulose production bacterial strain
Method.
Background technique
Bacteria cellulose (Bacterial Cellulose, BC) refers to by acetic acid Pseudomonas (Acetobacter), rhizobium
The Microbe synthesis such as category (Rhizobium), Agrobacterium (Agrobacterium) and Sarcina (Sarcina), be
It is distinguished with plant fiber, is named as " bacteria cellulose " or " micro organism cellulose ".Currently, research range is most wide, it is thin to produce
Fungin property advantageously acetobacter relatively.
Czaja W etc. and Tang WH etc. are studies have shown that acetobacter produces bacterial fibers in stationary culture and shaken cultivation
In the process of element, thallus is wrapped in mostly in membranaceous and spherical bacteria cellulose.The researchs such as Brown report shows the wooden vinegar bar
The cell membrane surface of bacterium is dispersed with intensive cellulose secretion site.Fiber filament is constantly extended with the speed of 2um/min, and is pushed away
Dynamic cell movement.It is connected with each other between fiber filament further through hydrogen bond, forms the unique tridimensional network of bacteria cellulose.Due to
Demand in scientific research or application, is often required to thallus to separate from the reticular structure of bacteria cellulose.Currently, domestic surgery
Scholar and bacteria cellulose manufacturer are ground, mostly acid-alkali treatment method ruptures somatic cells, to reach thallus and bacterium
The purpose of cellulose separation.If then the method is infeasible however, to carry out count plate to thallus.Usually applied using dilution
Cloth method Dichlorodiphenyl Acetate bacillus carries out bacterium colony counting, however, bacterium colony can not separate, it can only be with bacterium colony area coverage, rough comparative sample
Bacteria reducing falls the difference of concentration.Zhao Qiong et al. obtains uniform bacteria suspension and adding cellulase into culture medium.However, this
When method is applied to bacterium colony counting, it is still unable to get single bacterium colony, thus can not effectively be counted.
The reason of we have found during experiment, and the method fails mainly has ignored thallus institute during the experiment
The cellulose of generation.Therefore, the present invention proposes a kind of count plate side of bacteria cellulose production bacterial strain as starting point
Method.
Summary of the invention
The present invention in order to overcome the deficiencies of the prior art, provides a kind of viable count method of bacteria cellulose production bacterial strain,
This method is up to and carries out the purpose of effective count plate to bacteria cellulose production bacterial strain using the methods of pouring into and be coated with.
To realize goal of the invention, steps are as follows for specific experiment of the present invention:
A kind of viable count method of bacteria cellulose production bacterial strain, steps are as follows:
(1) the thick suspension of thallus is prepared；By thallus culture, the thick suspension of thallus is formed, cellulose in the thick suspension of thallus thallus is dropped
Solution is to being visible by naked eyes fiber filament；
(2) the thick suspension of thallus is after cellulose is completely degraded, and centrifugation obtains thallus, with the physiological saline of cellulase
Or thallus is resuspended phosphate buffer, until initial volume；
(3) gradient dilution: cellulase is first added into gradient dilution liquid, then carries out gradient dilution experiment；
(4) after mixing well, coating counts or pours into counting.
Moreover, there are three types of different methods for the preparation of the thick suspension of thallus: 1. adding cellulose into fermentation medium
Enzyme, inoculation production bacterial strain are cultivated；2. or to culture a period of time fermentation liquid in add cellulase, continue culture one section
After time, cellulose is degraded；3. or cultured fermentation liquid is centrifuged, the whole thallus of acquirement and bacteria cellulose, then be dipped in
In cellulase solution, degradation is to being visible by naked eyes fiber filament.
Moreover, the preparation method of the thick suspension of thallus is suitable for stationary culture, shaken cultivation, is bubbled culture and turntable training
It supports.
Moreover, gradient dilution liquid cellulase content 0.5U-10U.
Moreover, the dilution gradient of gradient dilution experiment is 10-4、10-5With 10-6。
The advantages and positive effects of the present invention are as follows:
The present invention provides the viable count method of the bacterial strain of bacteria cellulose production always, and this method follows typical viable bacteria meter
Number method, experimental result are highly reliable.
The viable count method that the present invention provides bacteria cellulose production bacterial strain solves perplexs correlative study all the time
The technical problem of field scientific research personnel, the technological progress for having pushed related scientific research to work.
Detailed description of the invention
Fig. 1 is the plate photo using the thick suspension dilution spread acetobacter of thallus；
Fig. 2 is the plate photo using method dilution spread acetobacter of the invention；
Fig. 3 is that the bacterium colony of embodiment 1 is coated with photo；
Fig. 4 is that the bacterium colony of embodiment 2 is coated with photo；
Fig. 5 is that the bacterium colony of embodiment 4 is coated with photo.
Case is embodied
Below with reference to case study on implementation, the present invention is further described, and it is not limited that following case study on implementation, which are illustrative,
, it cannot be limited the scope of protection of the present invention with following case study on implementation.
Bacterial strain used by following case study on implementation is wooden gluconic acid acetobacter (the Gluconacetobacter xylinus
CGMCC No.2955), the ribbon-like fibre element of synthesis has many advantages, such as that purity is high, the degree of polymerization are high, crystallinity is high, is thin at present
Fungin first closes one of most commonly used bacterial strain of area research.
Embodiment 1
A kind of viable count method of bacteria cellulose production bacterial strain, steps are as follows:
(1) a ring bacterium colony is chosen, is seeded in 150mL fermentation medium.Culture medium prescription: glucose 25g/L, yeast powder
7.5g/L, peptone 10g/L, Na2HPO410g/L, the cellulase (1000U) of 3% (v/v) filtration sterilization.28 DEG C of shaken cultivations
(180rpm)48h。
(2) in the test tube for taking cellulase (1000U) of the 1mL bacterium solution injection containing 8.9mL sterile saline and 0.1mL,
Test tube is shaken, solution in pipe is mixed, the dilution of 1:10 is made.
By aforesaid operations take turns doing 10 times be incremented by be diluted to 10-6, 10-4、10-5With 10-6Divide in the corresponding test tube of gradient
100 μ L dilutions are not taken, are spread evenly across on solid plate, after 28 DEG C of inversions are cultivated 3-4 days, are carried out bacterium colony counting, are seen Fig. 3.
Embodiment 2
A kind of viable count method of bacteria cellulose production bacterial strain, steps are as follows:
(1) embodiment chooses a ring bacterium colony, is seeded in 50mL fermentation medium.Culture medium prescription: glucose 25g/L, yeast
Powder 7.5g/L, peptone 10g/L, Na2HPO410g/L.30 DEG C of stationary culture 72h.
(2) fermentation liquid and produced BC are all poured into 50mL centrifuge tube, 5000rpm is centrifuged 5min.Abandon supernatant, Xiang Chen
The cellulase (1000U) of 5mL film filtering is added in shallow lake, 30 DEG C of standing 30min are degradable to cellulose.5000rpm centrifugation
5min abandons supernatant.
(3) precipitating is resuspended with 50mL sterile saline, after mixing well, takes the injection of 1mL bacterium solution sterile containing 8.8mL
In the test tube of phosphate buffer and 0.2mL cellulase (1000U).Shaking test tube mixes solution in pipe, is made 1:10's
Dilution.10 times of incremental dilutions are taken turns doing by aforesaid operations, until 10-7。
(4) 10-5、10-6With 10-7100 μ L dilutions are taken in the corresponding test tube of gradient respectively, are spread evenly across solid plate
On.After 30 DEG C of inversions are cultivated 3-4 days, bacterium colony counting is carried out, sees Fig. 4.
Embodiment 3
A kind of viable count method of bacteria cellulose production bacterial strain, steps are as follows:
(1) a ring bacterium colony is chosen, is seeded in 150mL fermentation medium.Culture medium prescription is the same as case 2.30 DEG C of shaken cultivations
It after (160rpm) 20h, adds 5mL cellulase (1000U), continues shaken cultivation 4h.
(2), according to the method gradient dilution of case 1,150 μ L dilutions is taken to be spread evenly across on solid plate.30 DEG C of inversions
After culture 3-4 days, bacterium colony counting is carried out.
Embodiment 4
A kind of viable count method of bacteria cellulose production bacterial strain, steps are as follows:
(1) a ring bacterium colony is chosen, is seeded in 5L bubbling fermentation tank.Culture medium prescription is the same as case 2.After 30 DEG C of cultures for 24 hours,
Take fermentation liquid of the 100mL containing cotton-shaped bacteria cellulose into sterile triangular flask, sufficiently oscillation mixes.
(2) according to the method degraded cellulose of case 2 and gradient dilution is to 10-5Afterwards, 10-5、10-6With 10-7Gradient is corresponding
Test tube in take 100 μ L dilutions respectively, counted using tilt-pour process.After 30 DEG C of inversions are cultivated 3-4 days, bacterium colony counting is carried out, is seen
Fig. 5.
Claims (3)
1. a kind of viable count method of bacteria cellulose production bacterial strain, it is characterised in that: steps are as follows:
(1) the thick suspension of thallus is prepared；Thallus is cultivated, by the cellulose degradation in fermentation liquid to being visible by naked eyes fiber filament；
(2) the thick suspension of thallus is after cellulose is completely degraded, and centrifugation obtains thallus, with the physiological saline or phosphorus of cellulase
Thallus is resuspended phthalate buffer, until initial volume；
(3) gradient dilution: cellulase is first added into gradient dilution liquid, then carries out gradient dilution experiment；It is fine in gradient dilution liquid
Tie up plain 0.5 U-10 U of enzyme content；
(4) after mixing well, coating counts or pours into counting.
2. the viable count method of bacteria cellulose production bacterial strain according to claim 1, it is characterised in that: the thallus
There are three types of different methods for the preparation of thick suspension: cellulase is 1. added into fermentation medium, inoculation production bacterial strain is trained
It supports；2. or to culture a period of time fermentation liquid in add cellulase, continue cultivate a period of time after, cellulose is degraded；
3. or cultured fermentation liquid is centrifuged, the whole thallus of acquirement and bacteria cellulose, then be dipped in cellulase solution, degradation to nothing
Visually visible fiber filament.
3. the viable count method of bacteria cellulose production bacterial strain according to claim 2, it is characterised in that: the thallus
The preparation method of thick suspension is suitable for stationary culture, shaken cultivation, is bubbled culture and turntable culture.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101314788A (en) * | 2008-07-11 | 2008-12-03 | 天津实发中科百奥工业生物技术有限公司 | Method for bacteria cellulose high yield bacterial strain cultivation sifting motion |
| CN102212589A (en) * | 2011-04-29 | 2011-10-12 | 钟春燕 | Method for preparing bacterial cellulose |
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2016
- 2016-05-26 CN CN201610355676.1A patent/CN105861622B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101314788A (en) * | 2008-07-11 | 2008-12-03 | 天津实发中科百奥工业生物技术有限公司 | Method for bacteria cellulose high yield bacterial strain cultivation sifting motion |
| CN102212589A (en) * | 2011-04-29 | 2011-10-12 | 钟春燕 | Method for preparing bacterial cellulose |
Non-Patent Citations (2)
| Title |
|---|
| Bacterial Cellulose Production by Acetobacter xylinum Strains from Agricultural Waste Products;Sasithorn Kongruang;《Appl Biochem Biotechnol》;20080103;第245-256页 |
| 基于固态培养基的醋酸菌扩大培养研究;郇阿梅等;《中国调味品》;20140430;第39卷(第4期);第13-17页 |
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