CN105886443A - Bacillus licheniformis strain screening method - Google Patents
Bacillus licheniformis strain screening method Download PDFInfo
- Publication number
- CN105886443A CN105886443A CN201610433038.7A CN201610433038A CN105886443A CN 105886443 A CN105886443 A CN 105886443A CN 201610433038 A CN201610433038 A CN 201610433038A CN 105886443 A CN105886443 A CN 105886443A
- Authority
- CN
- China
- Prior art keywords
- agar
- flat board
- water
- bacillus licheniformis
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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
- 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
-
- 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
- C12N1/02—Separating microorganisms from their culture media
Abstract
Disclosed is a Bacillus licheniformis strain screening method. By using the dibbling method, screening of 200 strains can be completed by only screening one time for about 16 days. The screening process is high in objective orientation, massive screening is performed at the early stage of dibbling, and workload of screening with a shake bottle at the late stage is reduced; compared with conventional screening methods, workload is reduced by 3/4, period is shortened by 3/4, and efficiency of the method is four times of the conventional screening methods; screening is low in workload, high in objectiveness, simple to operate and few in error, and a series of problems caused by lumping after single colony culturing are avoided.
Description
Technical field
The present invention relates to microbe to screen method and technology field.
Background technology
Bacillus licheniformis has stronger broad-spectrum bactericidal action as agricultural microorganism bacterium, it is used in production manufacturing agricultural microbial agent, filter out bactericidal activity strong, and it is necessary to produce the high bacterial strain of viable count, the bacterial strain of real needs is screened in substantial amounts of cell, just as looking for a needle in a haystack, workload is the biggest.
At present, the concrete grammar that bacterial classification uses in screening process has Natural Selection, the scraps of paper to cultivate development process, variable color circle method, transparent circle method, growth circle method and inhibition zone method etc., and all there is different defects in these methods, Natural Selection workload is big, purpose is the strongest, the scraps of paper cultivate development process operation complexity, other circle methods are owing to can not can cause circle growth uneven completely by the most spreadable for single bacterium colony, producing bigger error during measuring during dilution spread.The method first cultivating single bacterium colony block hitting again that inhibition zone method uses then can cause a series of problem, such as uses unified card punch can cause between single bacterium colony and occurs polluting, and agar block cannot ultimately result in operation failure from card punch taking-up etc..
Explanation of nouns: single bacterium colony represents quantity with " individual ", passes into inclined-plane and represents inclined-plane quantity with " propping up ", represents bacterial classification quantity with " strain " after having screened.
Summary of the invention
It is an object of the invention to overcome the deficiency of existing bacillus licheniformis screening technique, it is provided that the method that a kind of dibbling method of employing carries out Bacillus licheniformis strain screening.
For achieving the above object, the technical solution used in the present invention is: a kind of Bacillus licheniformis strain screening technique: step is as follows:
Step one, preparing the fresh bacterial classification of bacillus licheniformis, the bacillus licheniformis glycerine pipe that will preserve passes No. 1 slant medium, cultivates 3-7 days for 30 DEG C-37 DEG C；
Described No. 1 slant medium formula is: glucose: 1%, yeast leaching powder: 0.5%, peptone: 0.5%, beef extract: 0.3%, NaCl:0.5%, CaCl2: 0.05%, Na2HPO4: 0.05%, soluble starch: 1%, (NH4)2SO4: 0.05%, MnSO4: 0.0005%, Na2MoO4: 0.0005%, surplus is water, supplies 100%；PH is 6.5-7.2；
Step 2, bacillus licheniformis prepared by step one fresh bacterial classification 1cm2Under aseptic condition, the single plating medium that is coated with of dilution point, cultivates 48-72h, obtains the fresh bacterial classification of plating medium bacillus licheniformis for 30 DEG C-37 DEG C；
Described plating medium formula is: fish meal protein peptone: 0.5%, NaCl:0.5%, beef extract: 0.3%, agar: 1.5%, and surplus is water, supplies 100%；PH is 6.5-7.2；
Step 3, the preparation of the alternative bacterial classification of fermentation medium agar block flat board
Step 4, prepare primary dcreening operation inclined-plane lawn
(1) identify the preparation of flat board, be divided into upper and lower two-layer, lower floor: draw 20ml
No. 1 nutrient agar is paved as lower floor in culture dish；Upper strata: wash the staphylococcus aureus taking 18 × 180 inclined-planes and smash in the sterilized water of 20ml band pearl bottle, shake up；Draw 1.5ml to 100ml
In No. 2 nutrient agars, shake up；Inhale 5ml on lower floor's culture medium, quickly pave；Prepare and identify flat board；
No. 1 described nutrient agar formula is: fish meal protein peptone: 0.5%, NaCl:0.5%, beef extract: 0.3%, agar: 1.5%, surplus is water, supplies 100%；PH is 6.5-7.2；
No. 2 described nutrient agar formulas are: fish meal protein peptone: 0.5%, NaCl:0.5%, beef extract: 0.3%, agar 0.9%, surplus is water, supplies 100%；PH is 6.5-7.2；
(2) the fermentation medium alternative bacterial classification of agar block flat board step 3 obtained, moves to identify flat board, each qualification flat board places 4 pieces, cultivates 18h for 37 DEG C, measure inhibition zone size with tweezers；
Choosing antibacterial circle diameter and pass No. 2 slant mediums 40 at single bacterium colony of 26mm-28mm, cultivate 3-5 days, obtain primary dcreening operation inclined-plane lawn for 30-37 DEG C, 4 DEG C of Refrigerator stores are standby；
Described No. 2 slant medium formulas are: glucose: 1%, yeast leaching powder: 0.5%, peptone: 0.5%, beef extract: 0.3%, NaCl:0.5%, CaCl2: 0.05%, Na2HPO4: 0.05%, soluble starch: 1%, (NH4)2SO4: 0.05%, MnSO4: 0.0005%, Na2MoO4: 0.0005%, surplus is water, supplies 100%；PH is 6.5-7.2；
Step 5, prepare Bacillus licheniformis strain
(1) preparation kind daughter bacteria
Take step 4 primary dcreening operation inclined-plane lawn, be inoculated into seed flask, inoculum concentration: 1cm2Fresh inclined-plane lawn/100ml, cultivates 18-24h, cultivation temperature: 30-37 DEG C, shaking speed: 180r/mim, obtains kind of a daughter bacteria；
Described seed flask formula is: glucose: 2%, beancake powder: 2%, K2HPO4: 0.2%, yeast leaching powder: 1%, MgSO4: 0.05%, peptone: 0.2%, sucrose: 0.8%, corn steep liquor: 2%, urea: 0.2%, Nacl: 0.1%, precipitated calcium carbonate: 0.2%, surplus is water, supplies 100%；PH is 6.5-7.2；
(2) Bacillus licheniformis strain is prepared
Daughter bacteria switching fermentation shake flask will be planted, inoculum concentration: every fermentation shake flask 5ml/50ml, cycle: 48-72h, cultivation temperature: 30-37 DEG C, shaking speed: 180r/mim, obtain the lichen bacillus ferments liquid；
Described fermentation shake flask formula is: glucose: 4%, beancake powder: 1.5%, corn flour: 2%, groundnut meal: 1%, cottonseed meal: 1%, corn steep liquor: 0.5%, yeast leaching powder: 0.5%, peptone: 0.5%, Nacl: 0.1%, precipitated calcium carbonate: 0.2%, surplus is water, supplying 100%, PH is 6.5-7.2；
Bacillus licheniformis strain detects:
1. viable count: use counting method of blood cell, draws 1ml zymotic fluid dilution 1000 times and carries out blood count；
2. bacteriostasis detection:
Identify the preparation of flat board: identify that flat board is divided into upper and lower two-layer, lower floor: draw in 20ml nutrient agar and pave as lower floor in culture dish；Upper strata: wash the staphylococcus aureus taking 18 × 180 inclined-planes and smash in 20ml pearl bottle, shake up；Draw in the nutrient agar of 1.5ml to 100ml upper strata, shake up；Inhale 5ml on lower floor's culture medium, quickly pave, prepare and identify flat board；
Detection: 4 Oxford cups of placement corresponding on each qualification flat board, numbering, draw the centrifugal the lichen bacillus ferments liquid supernatant liquor of 200 μ l, be carefully added in the cup of Oxford, after 37 DEG C of cultivation 18h, detect inhibition zone；
With viable count more than 9,000,000,000/ml and inhibition zone more than 26mm as standard, filter out Bacillus licheniformis strain；
It is characterized in that: described step 3, the preparation method of the alternative bacterial classification of fermentation medium agar block flat board be:
(1) prepared by fermentation medium agar block flat board:
1. preparation fermentation agar plate, described fermentation agar plate formula is: agar: 1.5%, glucose: 4%, beancake powder: 1.5%, corn flour: 2%, groundnut meal: 1%, cottonseed meal: 1%, corn steep liquor: 0.5%, yeast leaching powder: 0.5%, peptone: 0.5%, Nacl: 0.1%, precipitated calcium carbonate: 0.2%, surplus is water, supplies 100%；PH is 6.5-7.2；
2. preparation water agar plate, described water agar plate formula is: agar powder: 1.5%, surplus is water, supplies 100%；
3. block hitting on fermentation agar plate, is placed into block hitting on water agar plate with tweezers gripping fritter, prepares fermentation medium agar block flat board；
(2) the single bacterium colony dibbling on fermentation medium agar block flat board of plating medium bacillus licheniformis fresh bacterial classification picking 100-200 prepared from step 2,30 DEG C-37 DEG C carry out cultivating 48-72h, obtain the fermentation medium alternative bacterial classification of agar block flat board.
Compared with prior art, there is advantages that
As a example by screening 200 bacillus licheniformis bacterial classifications, conventional screening methods 1 is taken turns and has been only capable of 40 strain bacteria selection, based on 16 days used times, completes 200 strains and needs 80 days altogether；Screening technique 1 of the present invention is taken turns just can complete 200 strain bacterial classifications, 19 days used times, relatively conventional screening methods cycle time 3/4, and efficiency is 4 times of conventional screening methods.
And screening operation amount of the present invention is little, purpose is strong, simple to operate, error is little, does not exist and first cultivate single bacterium colony block hitting again and cause a series of problem.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described:
Prior art (conventional method) Bacillus licheniformis strain screening technique
Step one, preparing the fresh inclined-plane of bacillus licheniformis, 1 the bacillus licheniformis glycerine pipe that will preserve is drawn 0.1ml and is passed No. 1 slant medium, cultivates 96h for 30 DEG C；
Described No. 1 slant medium formula is: glucose: 1g, yeast leaching powder: 0.5g, peptone: 0.5g, beef extract: 0.3g, NaCl:0.5g, CaCl2: 0.05g, Na2HPO4: 0.05g, soluble starch: 1g, (NH4)2SO4: 0.05g, MnSO4: 0.0005g, Na2MoO4: 0.0005g, surplus is water, supplies 100g；PH is 7.1；
Step 2, take bacillus licheniformis fresh bacterial classification 1cm2Under aseptic condition with 0.9% physiological saline gradient dilution to 10-1～10-6Individual/ml(blood count concentration), coating separates flat board, cultivates 48h, until growing single bacterium colony for 30 DEG C；
Described plating medium formula is: fish meal protein peptone: 2.5g, NaCl:2.5g, beef extract: 1.5g, agar 7.5g, and surplus is water, and supplying 500g, PH is 7.1；
Prepared by separation flat board: in the culture medium of absorption 30ml to the culture dish of a diameter of 90mm.Solidification uses after placing 24h；
Step 3, picking grow to single bacterium colony in cycle and pass No. 2 slant mediums 40, cultivate 96h, obtain primary dcreening operation inclined-plane lawn for 30 DEG C, and 4 DEG C of Refrigerator stores are standby；
Being noted that conventional method screens 200, workload is too big, cannot realize in operating process, and sterility cannot ensure, the screening of later stage shaking flask cannot be carried out；
Described No. 2 slant medium formulas are: glucose: 12g, yeast leaching powder: 6g, peptone: 6g, beef extract: 3.6g, NaCl:6g, CaCl2: 0.6g, Na2HPO4: 0.6g, soluble starch: 12g, (NH4)2SO4: 0.6g, MnSO4: 0.006g, Na2MoO4: 0.006g, surplus is water, supplies 1200g.PH is 7.1；
Step 4, Bacillus licheniformis strain shaking flask are screened
(1) preparation kind daughter bacteria
Taking step 3 primary dcreening operation inclined-plane lawn, an inclined-plane connects a seed flask, 40 bottles altogether, inoculum concentration: 1cm2Fresh inclined-plane lawn/bottle, cultivates 24h, cultivation temperature: 30 DEG C, shaking speed: 180r/mim, obtains kind of a daughter bacteria；
Described seed flask formula is: glucose: 80g, beancake powder: 80g, K2HPO4: 8g, yeast leaching powder: 40g, MgSO4: 2g, peptone: 8g, sucrose: 32g, corn steep liquor: 80g, urea: 8g, Nacl: 4g, precipitated calcium carbonate: 8g, surplus is water, supplies 4000g；PH is 7.2；
Seed flask loading amount: 100ml culture medium is to 500ml shaking flask；
(2) Bacillus licheniformis strain is prepared
Kind of daughter bacteria is transferred one to one 40 fermentation shake flask, inoculum concentration: 5ml/ bottle, cycle: 72h, cultivation temperature: 30 DEG C, shaking speed: 180r/mim, obtain the lichen bacillus ferments liquid；
Described fermentation shake flask formula is: glucose: 80g, beancake powder: 30g, corn flour: 40g, groundnut meal: 20g, cottonseed meal: 20g, corn steep liquor: 10g, yeast leaching powder: 10g, peptone: 10g, Nacl: 2g, precipitated calcium carbonate: 4g, surplus is water, supplies 2000g；PH is 7.2
Fermentation shake flask loading amount: 50ml culture medium is to 500ml shaking flask；
Bacillus licheniformis strain detects:
1. viable count: use counting method of blood cell, draws 1ml zymotic fluid dilution 1000 times and carries out blood count；
2. bacteriostasis detection:
Identify the preparation of flat board.Identify that flat board is divided into upper and lower two-layer.Lower floor: draw in 20ml nutrient agar and pave as lower floor in culture dish.Upper strata: wash the staphylococcus aureus taking 18 × 180 inclined-planes and smash in 20ml pearl bottle, shake up.Draw in the nutrient agar of 1.5ml to 100ml upper strata, shake up.Inhale 5ml on lower floor's culture medium, quickly pave, prepare and identify flat board；
Detection: 4 Oxford cups of placement corresponding on each qualification flat board, numbering, draw the centrifugal the lichen bacillus ferments liquid supernatant liquor of 200 μ l, be carefully added in the cup of Oxford, after 37 DEG C of cultivation 18h, detect inhibition zone；
It is more than, more than 9,000,000,000/ml and inhibition zone, the Bacillus licheniformis strain that 20mm is screening with viable count；
(3) the selection result
Conclusion:
From above-mentioned the selection result it can be seen that it is the viable count the filtered out bacterial classification more than 20mm, only 1 strain more than 9,000,000,000/ml and inhibition zone that conventional method 1 takes turns only numbering 10, often the wheel cycle is about 16 days.The selection result viable count is low, and bacteriostasis is weak, and viable count and bacteriostasis fluctuation range are relatively big, there is not obvious corresponding relation, bring difficulty to screening operation.And focus concentrates on shaking flask screening, workload is big, and efficiency is low, does not has purpose guidance quality.
Embodiment
One Bacillus licheniformis strain screening technique of the present invention: step one, prepare the fresh bacterial classification of bacillus licheniformis, the bacillus licheniformis glycerine pipe that will preserve passes No. 1 slant medium, cultivates 96h for 30 DEG C；
Described No. 1 slant medium formula is: glucose: 1g, yeast leaching powder: 0.5g, peptone: 0.5g, beef extract: 0.3g, NaCl:0.5g, CaCl2: 0.05g, Na2HPO4: 0.05g, soluble starch: 1g, (NH4)2SO4: 0.05g, MnSO4: 0.0005g, Na2MoO4: 0.0005g, surplus is water, supplies 100g；PH is 7.1；
Step 2, take bacillus licheniformis fresh bacterial classification 1cm2Under aseptic condition with 0.9% physiological saline gradient dilution to 10-1～10-6Individual/ml(blood count concentration), coating separates flat board, cultivates 48h, until growing single bacterium colony for 30 DEG C；
Described plating medium formula is: fish meal protein peptone: 2.5g, NaCl:2.5g, beef extract: 1.5g, agar 7.5g, and surplus is water, and supplying 500g, PH is 7.1；
Prepared by separation flat board: in the culture medium of absorption 30ml to the culture dish of a diameter of 90mm.Solidification uses after placing 24h；
Step 3, the preparation of the alternative bacterial classification of fermentation medium agar block flat board
(1) prepared by fermentation medium agar block flat board:
1. preparing fermentation agar plate, described fermentation agar plate formula is: agar: 1.5g, glucose: 4g, beancake powder: 1.5g, corn flour: 2g, groundnut meal: 1g, cottonseed meal: 1g, corn steep liquor: 0.5g, yeast leaching powder: 0.5g, peptone: 0.5g, Nacl: 0.1g, precipitated calcium carbonate: 0.2g, surplus is water, supplies 100g；PH is 7.1.Draw the culture medium of 30ml in the plate of a diameter of 90mm, prepare fermentation agar plate；
2. preparing water agar plate, described water agar plate formula is: agar powder 1.5g, and surplus is water, supplies 100g, in the culture medium of absorption 10ml to the plate of a diameter of 90mm, prepares water agar plate；
3. with the block hitting on fermentation agar plate of the card punch of diameter 0.5mm, fritter (every piece of about 0.1cm is gripped with tweezers3) it is placed into block hitting on water agar plate, prepare fermentation medium agar block flat board；
(2) from single bacterium colony prepared by step 2,200 dibblings of picking, on fermentation medium agar block flat board, are cultivated 48h, are obtained the fermentation medium alternative bacterial classification of agar block flat board for 30 DEG C；
It is noted that the most each single bacterium colony dibbling only need to grow up to the form of single bacterium colony, and is that a single bacterium colony need to pass into an inclined-plane in above-mentioned conventional approach；
Step 4, prepare primary dcreening operation inclined-plane
(1) identify the preparation of flat board, be divided into upper and lower two-layer.Lower floor: draw 20ml
No. 1 nutrient agar is paved as lower floor in culture dish；Upper strata: wash the staphylococcus aureus taking 18 × 180 inclined-planes and smash in the sterilized water of 20ml band pearl bottle, shake up；Draw 1.5ml to 100ml
In No. 2 nutrient agars, shake up.Inhale 5ml on lower floor's culture medium, quickly pave, prepare and identify flat board；
No. 1 described nutrient agar formula is: fish meal protein peptone 5g, NaCl:5g, beef extract: 3g, agar 15g, surplus is water, supplies 1000g；PH is 7.1；
No. 2 described nutrient agar formulas are: fish meal protein peptone 1.25g, NaCl:1.25g, beef extract: 0.75g, agar 2.25g, surplus is water, supplies 250g；PH is 7.1；
(2) the fermentation medium alternative bacterial classification of agar block flat board step 3 obtained, moves to 50 with tweezers and identifies flat board, and each qualification flat board places 4 pieces, cultivates 18h for 37 DEG C, measures inhibition zone size；
The single bacterium colony choosing antibacterial circle diameter >=26.5mm passes No. 2 slant mediums 40, cultivates 96h, obtains primary dcreening operation inclined-plane lawn for 30 DEG C, and 4 DEG C of Refrigerator stores are standby；
Described No. 2 slant medium formulas are: glucose: 12g, yeast leaching powder: 6g, peptone: 6g, beef extract: 3.6g, NaCl:6g, CaCl2: 0.6g, Na2HPO4: 0.6g, soluble starch: 12g, (NH4)2SO4: 0.6g, MnSO4: 0.006g, Na2MoO4: 0.006g, surplus is water, supplies 1200g.PH is 7.1；
Step 5, prepare Bacillus licheniformis strain
(1) preparation kind daughter bacteria
Taking step 4 primary dcreening operation inclined-plane lawn, an inclined-plane connects a seed flask, 40 bottles altogether, inoculum concentration: 1cm2Fresh inclined-plane lawn/bottle, cultivates 24h, cultivation temperature: 30 DEG C, shaking speed: 180r/mim, obtains kind of a daughter bacteria；
Described seed flask formula is: glucose: 80g, beancake powder: 80g, K2HPO4: 8g, yeast leaching powder: 40g, MgSO4: 2g, peptone: 8g, sucrose: 32g, corn steep liquor: 80g, urea: 8g, Nacl: 4g, precipitated calcium carbonate: 8g, surplus is water, supplies 4000g；PH is 7.2
Seed flask loading amount: 100ml culture medium is to 500ml shaking flask；
(2) Bacillus licheniformis strain is prepared
Kind of daughter bacteria is transferred one to one 40 fermentation shake flask, inoculum concentration: 5ml/ bottle, cycle: 72h, cultivation temperature: 30 DEG C, shaking speed: 180r/mim, obtain the lichen bacillus ferments liquid；
Described fermentation shake flask formula is: glucose: 80g, beancake powder: 30g, corn flour: 40g, groundnut meal: 20g, cottonseed meal: 20g, corn steep liquor: 10g, yeast leaching powder: 10g, peptone: 10g, Nacl: 2g, precipitated calcium carbonate: 4g, surplus is water, supplies 2000g；PH is 7.2；
Fermentation shake flask loading amount: 50ml culture medium is to 500ml shaking flask；
Bacillus licheniformis strain detects:
1. viable count: use counting method of blood cell, draws 1ml zymotic fluid dilution 1000 times and carries out blood count；
2. bacteriostasis detection:
Identify the preparation of flat board: identify that flat board is divided into upper and lower two-layer, lower floor: draw in 20ml nutrient agar and pave as lower floor in culture dish；Upper strata: wash the staphylococcus aureus taking 18 × 180 inclined-planes and smash in 20ml pearl bottle, shake up；Draw in the nutrient agar of 1.5ml to 100ml upper strata, shake up.Inhale 5ml on lower floor's culture medium, quickly pave, prepare and identify flat board；
Detection: 4 Oxford cups of placement corresponding on each qualification flat board, numbering, draw the centrifugal the lichen bacillus ferments liquid supernatant liquor of 200 μ l, be carefully added in the cup of Oxford.Cultivate detection inhibition zone after 18h for 37 DEG C；
It is more than, more than 9,000,000,000/ml and inhibition zone, the Bacillus licheniformis strain that 26mm is screening with viable count；
(3) the selection result
Conclusion: can be seen that from above-mentioned the selection result, the present invention 1 takes turns viable count, and more than 9,000,000,000/ml and inhibition zone, the bacterial classification more than 26mm has 10 strains (numbered 61,19,28,84,121,56,100,86,147,140), and tradition screening 1 is taken turns and is only capable of filtering out 1 strain, the screening technique viable count of the present invention and bacteriostatic activity are apparently higher than conventional screening methods simultaneously.
In conventional screening methods screening process, focus concentrates on shaking flask screening, the only result from shaking flask screening just can find out the ability of selected single bacterium colony.In shaking flask screening, a seed bottle growth needs 24 hours, go to fermentation flask need 72 hours, this is the process of a primary dcreening operation, conventional screening methods, after inclined-plane has grown, just need to can complete through primary dcreening operation, postsearch screening in shaking flask, add inclined-plane growth time, and Counts is the hugest in shaking flask test sample, adding shaking table amount needed for Shaking culture, count number and its accuracy etc. factor, so causing once cannot be carried out large batch of screening, and the selection result is undesirable.
Screening technique of the present invention is the dibbling method used, and carries out when single bacterium colony is chosen screening, dibbling, can dibbling 200, screening quantity can be greatly improved.Carrying out antibacterial detection simultaneously and can strengthen screening purpose, can filter out the bacterial classification of high benefit accurately and rapidly, reduce later stage shaking flask screening operation amount, and the selection result viable count is high, bacteriostasis is strong, reaches ideal effect.
As a example by screening 200 strain bacterial classifications, the present invention improves efficiency situation:
Compared with embodiment one conventional screening methods, the dibbling method that the present invention uses, only take turns screening with carrying out 1, about 16 days, so that it may complete the screening of 200 strain bacterial strains.And screening process purpose guidance quality is strong, early stage dibbling is screened in a large number, reduces the workload of later stage shaking flask screening, relatively conventional screening methods, and workload reduces 3/4, and the time of cycle time 3/4, efficiency is 4 times of conventional screening methods；And screening operation amount of the present invention is little, purpose is strong, simple to operate, error is little, does not exist and first cultivate single bacterium colony block hitting again and cause a series of problem.
Claims (1)
1. a Bacillus licheniformis strain screening technique: step is as follows:
Step one, preparing the fresh bacterial classification of bacillus licheniformis, the bacillus licheniformis glycerine pipe that will preserve passes No. 1 slant medium, cultivates 3-7 days for 30 DEG C-37 DEG C；
Described No. 1 slant medium formula is: glucose: 1%, yeast leaching powder: 0.5%, peptone: 0.5%, beef extract: 0.3%, NaCl:0.5%, CaCl2: 0.05%, Na2HPO4: 0.05%, soluble starch: 1%, (NH4)2SO4: 0.05%, MnSO4: 0.0005%, Na2MoO4: 0.0005%, surplus is water, supplies 100%；PH is 6.5-7.2；
Step 2, bacillus licheniformis prepared by step one fresh bacterial classification 1cm2Under aseptic condition, the single plating medium that is coated with of dilution point, cultivates 48-72h, obtains the fresh bacterial classification of plating medium bacillus licheniformis for 30 DEG C-37 DEG C；
Described plating medium formula is: fish meal protein peptone: 0.5%, NaCl:0.5%, beef extract: 0.3%, agar: 1.5%, and surplus is water, supplies 100%；PH is 6.5-7.2；
Step 3, the preparation of the alternative bacterial classification of fermentation medium agar block flat board
Step 4, prepare primary dcreening operation inclined-plane lawn
(1) identify the preparation of flat board, be divided into upper and lower two-layer, lower floor: draw in No. 1 nutrient agar of 20ml and pave as lower floor in culture dish；Upper strata: wash the staphylococcus aureus taking 18 × 180 inclined-planes and smash in the sterilized water of 20ml band pearl bottle, shake up；Draw in No. 2 nutrient agars of 1.5ml to 100ml, shake up；Inhale 5ml on lower floor's culture medium, quickly pave；Prepare and identify flat board；
No. 1 described nutrient agar formula is: fish meal protein peptone: 0.5%, NaCl:0.5%, beef extract: 0.3%, agar: 1.5%, surplus is water, supplies 100%；PH is 6.5-7.2；
No. 2 described nutrient agar formulas are: fish meal protein peptone: 0.5%, NaCl:0.5%, beef extract: 0.3%, agar 0.9%, surplus is water, supplies 100%；PH is 6.5-7.2；
(2) the fermentation medium alternative bacterial classification of agar block flat board step 3 obtained, moves to identify flat board, each qualification flat board places 4 pieces, cultivates 18h for 37 DEG C, measure inhibition zone size with tweezers；
Choosing antibacterial circle diameter and pass No. 2 slant mediums 40 at single bacterium colony of 26mm-28mm, cultivate 3-5 days, obtain primary dcreening operation inclined-plane lawn for 30-37 DEG C, 4 DEG C of Refrigerator stores are standby；
Described No. 2 slant medium formulas are: glucose: 1%, yeast leaching powder: 0.5%, peptone: 0.5%, beef extract: 0.3%, NaCl:0.5%, CaCl2: 0.05%, Na2HPO4: 0.05%, soluble starch: 1%, (NH4)2SO4: 0.05%, MnSO4: 0.0005%, Na2MoO4: 0.0005%, surplus is water, supplies 100%；PH is 6.5-7.2；
Step 5, prepare Bacillus licheniformis strain
(1) preparation kind daughter bacteria
Take step 4 primary dcreening operation inclined-plane lawn, be inoculated into seed flask, inoculum concentration: 1cm2Fresh inclined-plane lawn/100ml, cultivates 18-24h, cultivation temperature: 30-37 DEG C, shaking speed: 180r/mim, obtains kind of a daughter bacteria；
Described seed flask formula is: glucose: 2%, beancake powder: 2%, K2HPO4: 0.2%, yeast leaching powder: 1%, MgSO4: 0.05%, peptone: 0.2%, sucrose: 0.8%, corn steep liquor: 2%, urea: 0.2%, Nacl: 0.1%, precipitated calcium carbonate: 0.2%, surplus is water, supplies 100%；PH is 6.5-7.2；
(2) Bacillus licheniformis strain is prepared
Daughter bacteria switching fermentation shake flask will be planted, inoculum concentration: every fermentation shake flask 5ml/50ml, cycle: 48-72h, cultivation temperature: 30-37 DEG C, shaking speed: 180r/mim, obtain the lichen bacillus ferments liquid；
Described fermentation shake flask formula is: glucose: 4%, beancake powder: 1.5%, corn flour: 2%, groundnut meal: 1%, cottonseed meal: 1%, corn steep liquor: 0.5%, yeast leaching powder: 0.5%, peptone: 0.5%, Nacl: 0.1%, precipitated calcium carbonate: 0.2%, surplus is water, supplying 100%, PH is 6.5-7.2；
Bacillus licheniformis strain detects:
1. viable count: use counting method of blood cell, draws 1ml zymotic fluid dilution 1000 times and carries out blood count；
2. bacteriostasis detection:
Identify the preparation of flat board: identify that flat board is divided into upper and lower two-layer, lower floor: draw in 20ml nutrient agar and pave as lower floor in culture dish；Upper strata: wash the staphylococcus aureus taking 18 × 180 inclined-planes and smash in 20ml pearl bottle, shake up；Draw in the nutrient agar of 1.5ml to 100ml upper strata, shake up；Inhale 5ml on lower floor's culture medium, quickly pave, prepare and identify flat board；
Detection: 4 Oxford cups of placement corresponding on each qualification flat board, numbering, draw the centrifugal the lichen bacillus ferments liquid supernatant liquor of 200 μ l, be carefully added in the cup of Oxford, after 37 DEG C of cultivation 18h, detect inhibition zone；
With viable count more than 9,000,000,000/ml and inhibition zone more than 26mm as standard, filter out Bacillus licheniformis strain；
It is characterized in that: described step 3, the preparation method of the alternative bacterial classification of fermentation medium agar block flat board be:
(1) prepared by fermentation medium agar block flat board:
1. preparation fermentation agar plate, described fermentation agar plate formula is: agar: 1.5%, glucose: 4%, beancake powder: 1.5%, corn flour: 2%, groundnut meal: 1%, cottonseed meal: 1%, corn steep liquor: 0.5%, yeast leaching powder: 0.5%, peptone: 0.5%, Nacl: 0.1%, precipitated calcium carbonate: 0.2%, surplus is water, supplies 100%；PH is 6.5-7.2；
2. preparation water agar plate, described water agar plate formula is: agar powder: 1.5%, surplus is water, supplies 100%；
3. block hitting on fermentation agar plate, is placed into block hitting on water agar plate with tweezers gripping fritter, prepares fermentation medium agar block flat board；
(2) the single bacterium colony dibbling on fermentation medium agar block flat board of plating medium bacillus licheniformis fresh bacterial classification picking 100-200 prepared from step 2,30 DEG C-37 DEG C carry out cultivating 48-72h, obtain the fermentation medium alternative bacterial classification of agar block flat board.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610433038.7A CN105886443A (en) | 2016-06-18 | 2016-06-18 | Bacillus licheniformis strain screening method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610433038.7A CN105886443A (en) | 2016-06-18 | 2016-06-18 | Bacillus licheniformis strain screening method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105886443A true CN105886443A (en) | 2016-08-24 |
Family
ID=56729484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610433038.7A Pending CN105886443A (en) | 2016-06-18 | 2016-06-18 | Bacillus licheniformis strain screening method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105886443A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834417A (en) * | 2017-02-16 | 2017-06-13 | 雅戈尔服装控股有限公司 | The rapid assay methods of Chinese fiber crops bast fiber extract antibacterial ability |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876614A (en) * | 2012-10-16 | 2013-01-16 | 北京龙科方舟生物工程技术有限公司 | Bacillus licheniformis and application of bacillus licheniformis |
-
2016
- 2016-06-18 CN CN201610433038.7A patent/CN105886443A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876614A (en) * | 2012-10-16 | 2013-01-16 | 北京龙科方舟生物工程技术有限公司 | Bacillus licheniformis and application of bacillus licheniformis |
Non-Patent Citations (5)
| Title |
|---|
| 孙冬雪等: "宁康霉素产生菌BAC－9912的诱变育种及培养条件的研究", 《中国优秀硕士学位论文全文数据库》 * |
| 张磊等: "利用IABS模型筛选L-异亮氨酸高产菌株", 《生物工程学报》 * |
| 彭玲等: "诱变处理增强地衣芽孢杆菌 01101107 抑菌性的研究", 《化学与生物工程》 * |
| 施巧琴、吴松刚: "《工业微生物育种学》", 31 December 1991 * |
| 李宗伟: "生长圈法应用于选育L- 异亮氨酸菌株的条件优化", 《食品工业科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834417A (en) * | 2017-02-16 | 2017-06-13 | 雅戈尔服装控股有限公司 | The rapid assay methods of Chinese fiber crops bast fiber extract antibacterial ability |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103131658B (en) | Bacillus and application thereof in control of apple ring spot | |
| CN104894035B (en) | A kind of screening technique of bacillus and its application | |
| CN101703214A (en) | Lucid Ganoderma hypra powder or Lucid Ganoderma tea and double fermentation process | |
| CN103834591B (en) | Ginseng growth-promoting bacterium FY4-2 and application thereof | |
| CN1704469A (en) | Method for producing Chinese aweto fungus | |
| CN105505836A (en) | Bacillus subtilis and methods for preparing and using bacillus subtilis microbial agent for controlling potato scab | |
| CN111690578B (en) | Salt and alkali resistant Siamese bacillus and production method and application of viable bacteria preparation thereof | |
| CN101333550B (en) | Method for preparing cyclic dipeptides compounds and use thereof | |
| CN106635884A (en) | Separation for endophytic actinomycetes of kiwifruit roots and application thereof | |
| CN101273709A (en) | Tissue culture method for rapid propagation of Dendrobium candidum | |
| CN106434490B (en) | Ginseng disease prevention growth-promoting bacterium TY15-2 and its application | |
| CN104531571A (en) | Pseudomonas fluorescens and biological preparation and application in preventing and controlling sugarcane smut | |
| CN101942403B (en) | Bacillus pumilus as well as culture method and application thereof | |
| CN103436465B (en) | Alcaligenes faecalis strain | |
| CN102766663B (en) | Preparation method of active polysaccharides from phellinus linteus | |
| CN102318545B (en) | Water culturing method for hericium erinaceus | |
| CN110396485A (en) | Generate class Brevibacillus brevis and its application of auxin | |
| CN109182151A (en) | The separating screening method of gingko endogenous fungus | |
| CN105695342B (en) | Koning trichoderma bacterium TG-72 and its application in Aspergillus flavus biological control | |
| CN106342900B (en) | A kind of Streptomyces jingyangensis wettable powder and preparation method thereof | |
| CN105400717A (en) | Bacterial strain HBRM-16 capable of promoting growth of roots of rubber tree and application of bacterial strain HBRM-16 | |
| CN105886443A (en) | Bacillus licheniformis strain screening method | |
| CN102132708B (en) | Bacteriostat from Jatropha curcas and bacteriostasis application thereof in planting edible mushrooms | |
| CN106967783A (en) | A kind of method of characterization and evaluation sesame fusarium wilt disease resistance level | |
| CN102960369A (en) | Preparation method of granular paecilomyces lilacinus biological nematocide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160824 |
|
| WD01 | Invention patent application deemed withdrawn after publication |