CN105861451A - Method for fast screening of virulent phage - Google Patents
Method for fast screening of virulent phage Download PDFInfo
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- CN105861451A CN105861451A CN201610377461.XA CN201610377461A CN105861451A CN 105861451 A CN105861451 A CN 105861451A CN 201610377461 A CN201610377461 A CN 201610377461A CN 105861451 A CN105861451 A CN 105861451A
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Abstract
The invention discloses a method for fast screening of virulent phages, comprising the steps of: collection of samples, acquisition of phages, activation of test bacteria, preparation of single colony, decussation streak inoculation of the bacterial single colony and phages, and screening of phages with lysis test strains. The method provided by the invention can conveniently, quickly and accurately screen out test bacteria specific phages, avoids influences of bacterial mutation and other adverse factors on screening of the virulent phages, and is favorable for sufficient research and excavation of phage resources in nature.
Description
Technical field
The present invention relates to a kind of method screening phage from nature, the side of a kind of rapid screening virulent phage
Method.
Background technology
Phage is a kind of bacterial virus, as all viruses, does not has cellularity and complete enzyme system and energy synthesis
System, can only colonize in cell.The most often there is corresponding phage in every antibacterial.Phage exists
Abundance in soil can reach 107pfu/ml；In aquatic environment, phage abundance is from 104Pfu/ml to 108More than pfu/ml,
It it is 3-10 times of number of bacteria.Phage affects biogeochemical cycle, red tide phenomenon etc. in nature.
During Phage Infection antibacterial, it is divided into lysogeny and cracking type.Lysogenic phage is otherwise known as mild phage, invades
Bacteria lysis is not caused after entering host bacteria；On the contrary, cracking type phage can crack antibacterial and make it dead, and has height
The specificity of degree, can kill specific bacterial type.In view of phage is to the specificity of bacteria lysis and high efficiency, people are again
Pay close attention to virulent phage important function in controlling bacteria growth process, including soil bacteria and the water body in ecological environment field
Antibacterial, and the pathogenetic bacteria etc. of pesticide, veterinary drug and field of medicaments.Particularly pathogenetic bacteria drug resistance phenomenon is increasingly severe
In the present age, screen fragility phage, study Phage therapy, have become as antibacterial research focus.And on the other hand, some grinds
Study carefully and commercial production, in order to avoid phage is disturbed, need specifically to screen phage-resistant bacteria variants.As Yoghourt is sent out
During ferment, when lactic acid bacteria is by Phage Infection, its fermentation activity is destroyed, and whole sweat slows down or terminates,
Thus had a strong impact on the quality of product, and bringing huge economic loss to Producer, solution is maximally effective to be obtained exactly
Obtain phage-resistant bacterial strain.Screened by virulent phage, utilize virulent phage to distinguish phage-sensitive strain and phage
Resistant strain.
The most conventional virulent phage screening technique has two kinds, and 1) coating culture dish after antibacterial mixes will be tested with phage,
See whether after cultivation that plaque occurs；2) test antibacterial is first coated on culture dish, then not same district on culture dish
Territory dropping phage, sees whether plaque occur after cultivation.But, owing to antibacterial spontaneous mutation is very frequent, can produce
Raw phage resistant strains.During according to above two method screening virulent phage, when the test inoculum used contains
During more resistant strain, often lead to phage selection failure, although containing corresponding phage in extracting solution；And bite
Bacterial plaque method there will be the plaque that some are comparatively fine in screening process, or plaque can along with incubation time extend by
Diminish greatly, cause observation fault.So both approaches reduces phage selection efficiency.
Summary of the invention
The technical problem to be solved is to provide a kind of method of rapid screening virulent phage, can convenient, fast,
Screen the virulent phage of test antibacterial exactly.
For solving above-mentioned technical problem, the technical solution adopted in the present invention is: a kind of method of rapid screening virulent phage,
Concretely comprise the following steps:
1) collecting sample from natural environment, directly obtains water body sample or is prepared as suspension, then filter or from
The heart, obtains phage；
2) test microbionation is activated on solid medium, be transferred to fluid medium and cultivate；Then carry out thin
Bacterium list bacterium colony is cultivated, and obtains single bacterium colony of different saltant；
3) solid medium is poured culture dish into, at a plurality of long line of culture dish back labelling after solidification, then with square crossing side
Formula is a plurality of short-term of labelling on every long line；Along long wire tag, inoculation step 1) phage that obtains, along short-term mark
Note inoculation step 2) the middle single bacterium colony obtained so that antibacterial list bacterium colony forms square crossing with phage；
4) to step 3) culture dish of gained cultivates 12-36 hour under conditions of being placed in test antibacterial suitable growth, observes thin
Whether bacterium and phage infall have the cleaved phenomenon of antibacterial；
5) would indicate that phage and the bacterial strain of lytic activity are marked, more corresponding phage and correspondence list bacterium colony are total to
Be inoculated in fluid medium amplification culture obtain phage, repeat step 2) to step 4) in operation carry out multiple sieve；
Carry out the phage extracting solution still after multiple sieve with cracking test strain preserving the screening that can complete virulent phage.
Further, step 1) in collecting sample time, collecting sample from different environment, collection for non-water body sample
Time, add sterilized water and make suspension.
Further, step 1) use the direct filtration sterilization of microporous filter membrane to obtain phage；Or it is broken to extract reagent by phage
Broken bacterial cell, is then centrifuged for or is filtrated to get phage.
Further, the aperture of described microporous filter membrane is 0.22 micron；It is chloroform that described phage extracts reagent.
Further, the mass concentration of described chloroform is 10%-15%.
Further, described step 2) in carry out antibacterial list bacterium colony and cultivate and will be coated with after the antibacterial gradient dilution of liquid culture
Or streak inoculation is on solid medium.Avoid reducing phage selection efficiency because mutant bacteria produces resistance.Gradient dilution
Time concentration be 10-1、10-2...10-6, select culture dish during cultivation, then choose a cultivation containing 20-50 single bacterium colony
Ware, is numbered each single bacterium colony, for phage-antibacterial antagonism test.
Further, described step 3) the acceptance of the bid employing marking pen that clocks carries out, a plurality of long line is parallel to each other, between distance
It is spaced apart 2-3cm；A plurality of short-term is parallel to each other, between distance be spaced apart 0.7-1.2cm.The culture dish of 9-10cm diameter
Back labelling 2-3 bar parallel long line, labelling 7-12 bar short-term on the long line of every, culture dish of a diameter of 9-10cm；Inoculation is thin
During bacterium list bacterium colony, according to number order along short-term labelling streak inoculation；Observation is facilitated to process.Solid culture ware back is entered
Line flag, reduces streak inoculation error, it is to avoid different bacteriophages and difference list bacterium colony are overlapping.Phage and antibacterial is used to carry out
Decussation antagonism detects, and confirms phage virulence and bacterial resistance more rapidly.
If the phage of same sample source inoculated by 3 long lines of a culture dish, 30 can be inoculated in a culture dish
Individual antibacterial difference list bacterium colony of the same race, generally comprises phage sensitive and resistant mutant in these single bacterium colonies；It is perpendicular to long line
The antibacterial of up to 30 kinds of different generas can also be inoculated, compare the most each culture dish and only inoculate a kind of antibacterial efficiency
Improve a lot.Phage sensitive bacterial strain and resistant mutant strains are often mixed by existing method, pass through plaque
Observe cracking performance；Owing to some mutant bacteria type can crack with phage resistance, a considerable amount of when inoculum is mixed with
After resistant strain, plaque is hardly formed, and causes the discovery of sample pnagus medius to be omitted with screening, and the present invention is by single bacterium
Fall to separating different genotype (including resistant mutant bacterial strain and sensitive strain), it is ensured that resistant mutant strains and sensitive organism
Strain expose bacteriophage independently so that the cracking phenomenon of sensitive strain shows and do not affected by resistant mutant strains, increases
Screening obtains phage probability.
Further, described step 3) in every long line inoculate identical phage, every short-term one single bacterium colony of inoculation, and
The different single bacterium colony of different short-term inoculations.
Further, described step 3) in inoculation time all take the mode of streak inoculation to carry out.
Further, described step 5) in phage extracting solution refer to the extracting solution that obtains during amplification culture.
The list that the different genotype (including resistant mutant) of same antibacterial is carried out separating and formed by the method that the present invention provides
Bacterium colony is directly and phage carries out antagonism test, and the phage selection that because of bacterial resistance sudden change cause has been reduced to a great extent
Failure, improves phage selection efficiency；The present invention can also inoculate in same culture dish up to 30 the most of the same race
Antibacterial, the simultaneously phage of one sample source of the screening cracking character to 30 different bacterium kinds, with an existing training
Foster ware can only be inoculated the screening technique of a kind of antibacterial and compare, and has higher screening efficiency.The decussation method energy simultaneously used
The fine melt being enough clearly observed phage virulence and bacterial resistance situation and Changing Pattern, such as phage (intersects
Place without bacterial growth), middle cracking performance (several sparse bacterium colony only occurs in infall), slack melt (infall have antibacterial give birth to
Long, but between bacterium colony, have visible blank), without cracking performance (infall bacterial growth does not has visible blank).The i.e. present invention
By using single bacterium colony, decussation method antagonism means, test bacteriophage can be screened convenient, fast, exactly,
Avoid the unfavorable factor interference such as mutant bacteria, be conducive to fully research and excavate nature pnagus medius resource, be a kind of fast
The method of speed screening virulent phage, is worthy to be popularized.
Accompanying drawing explanation
1 one the primary dcreening operation postvaccinal state diagrams of culture dish labelling of Fig. 1 embodiment.
Detailed description of the invention
Below in conjunction with embodiment, it is further elucidated with the present invention.These embodiments be interpreted as being merely to illustrate the present invention rather than
For limiting the scope of the invention.After having read the content that the present invention records, those skilled in the art can be to this
Invention makes various changes or modifications, and these equivalence changes and modification fall into claims of the present invention limited range equally.
Embodiment 1:
1) 9, small stream piecewise acquisition water body sample is seeked from SanXia University；
2) adding 10% chloroform in water sample, acutely concussion 5 minutes, centrifugal (4 DEG C, 10 000rpm, 5min) obtain
Phage；
3) by the escherichia coli streak inoculation of preservation on Nutrient agar solid medium, 36h, picking edge light are cultivated for 37 DEG C
Sliding single bacterium colony is transferred in the test tube equipped with nutrient broth fluid medium, cultivates 18h for 37 DEG C；
4) by step 3) the escherichia coli sterilized water gradient dilution of liquid culture, take 10-5With 10-6Coat Nutrient agar
On solid medium, cultivating 24h for 37 DEG C, single bacterium colony is numbered after being formed；
5) culture dish labelling, as shown in Figure 1: solid medium is poured into culture dish, uses mark below culture dish after solidification
3 long lines of stroke (diameter 9cm culture dish), then use mark stroke 7-9 bar short on every long line in square crossing mode
Line；
6) along long wire tag, the phage (identical phage inoculated by every long line) obtained with inoculating loop streak inoculation, edge
Short-term labelling streak inoculation and derive from step 4) different single bacterium colonies (according to number order picking list bacterium colony, when single bacterium colony
When quantity is many, it is possible to use multiple culture dishs, these single bacterium colonies comprise saltant), every short-term one list of inoculation
Bacterium colony so that the bacterial strain of streak inoculation forms square crossing with the phage of streak inoculation in advance；
7) by step 6) culture dish inoculated is placed in 37 DEG C when cultivating 12h, 18h, 24h, 36h and observes respectively,
Then record antagonistic effect is the cleaved situations of escherichia coli: fine melt (infall is without bacterial growth), middle cracking (intersect
Only there is several sparse bacterium colony in place), slack melt (infall has bacterial growth, but has visible blank between bacterium colony), without splitting
Solve (infall bacterial growth does not has visible blank)；
8) cultivating 36 hours, SanXia University seeks the phage of small stream administrative building section acquisition to escherichia coli list bacterium colony cracking situation:
Cracking in 2 single bacterium colony fine melts, 7 single bacterium colonies, 19 single bacterium colony slack melts, 8 single bacterium colonies are without cracking；
9) by step 8) in demonstrate that single bacterium colony of fine melt and the phage of middle cracking performance and correspondence is marked, will
The escherichia coli list bacterium colony co-inoculation of corresponding phage and corresponding activation is in fluid medium amplification culture 16h, according to step
Rapid 2) method extracts phage, repeats step 3) to step 7)；
10) by step 9) obtain after multiple sieve and derive from SanXia University and seek the phage strong inhibition of small stream administrative building section for examination
Escherichia coli are up to 90% single bacterium colony (including fine melt and middle cracking, cultivate 36h), then with 25% Freezing Glycerine preservation.
Embodiment 2:
1) from Yiling District dragon's fountain breeding water body collecting sample；
2) water sample is degerming with 0.22 μm filtering with microporous membrane, it is thus achieved that phage stock solution；
3) by the bacillus subtilis streak inoculation of preservation on Nutrient agar solid medium, 36h, picking limit are cultivated for 37 DEG C
It is transferred in the test tube equipped with nutrient broth fluid medium along smooth single bacterium colony, cultivates 18h for 37 DEG C；
4) by step 3) the bacillus subtilis sterilized water gradient dilution of liquid culture, take 10-5With 10-6Coat nutrition
On Solid agar culture, cultivating 24h for 37 DEG C, single bacterium colony is numbered after being formed；
5) culture dish labelling: solid medium is poured into culture dish, with 3 long lines of mark stroke below culture dish after solidification
(diameter 9cm culture dish), then on every long line, use mark stroke 8-10 bar short-term in square crossing mode；
6) along long wire tag, the phage (phage of every long line one sample of inoculation) extracted with inoculating loop streak inoculation,
Derive from step 4 along short-term labelling streak inoculation) single bacterium colony (according to number order picking list bacterium colony, when single clump count
When measuring many, it is possible to use multiple culture dishs, these single bacterium colonies comprise saltant), every short-term one single bacterium of inoculation
Fall so that the bacterial strain of streak inoculation forms square crossing with the phage of streak inoculation in advance；
7) by step 6) culture dish inoculated is placed in 37 DEG C when cultivating 12h, 18h, 24h, 36h and observes respectively,
Then record antagonistic effect is the cleaved situation of bacillus subtilis: fine melt (infall is without bacterial growth), middle cracking (are handed over
Several sparse bacterium colony only occurs at fork), slack melt (infall has bacterial growth, but has visible blank between bacterium colony), nothing
Cracking (infall bacterial growth does not has visible blank)；
8) cultivating 36 hours, the phage that certain breeding water body obtains is to bacillus subtilis list bacterium colony cracking situation: 3 lists
Cracking in bacterium colony fine melt, 8 single bacterium colonies, 15 single bacterium colony slack melts, 5 single bacterium colonies are without cracking；
9) by step 8) in demonstrate that single bacterium colony of fine melt and the phage of middle cracking performance and correspondence is marked, will
The bacillus subtilis list bacterium colony co-inoculation of corresponding phage and corresponding activation, in fluid medium amplification culture 16h, is pressed
According to step 2) method extraction phage, repeat step 3) to step 7)；
10) by step 9) obtain the phage strong inhibition deriving from dragon's fountain pond for examination bacillus subtilis after multiple sieve
Up to 85% single bacterium colony (includes fine melt and middle cracking, cultivate 36h), then with 25% Freezing Glycerine preservation.
Claims (10)
1. the method for a rapid screening virulent phage, it is characterised in that concretely comprise the following steps:
1) collecting sample from natural environment, directly obtains water body sample or is prepared as suspension, then filters or centrifugal, obtains phage；
2) test microbionation is activated on solid medium, be transferred to fluid medium and cultivate；Then carry out antibacterial list bacterium colony to cultivate, obtain single bacterium colony of different saltant；
3) solid medium is poured culture dish into, at a plurality of long line of culture dish back labelling after solidification, then with square crossing mode a plurality of short-term of labelling on every long line；Along long wire tag, inoculation step 1) phage that obtains, along short-term labelling inoculation step 2) in single bacterium colony of obtaining so that antibacterial list bacterium colony forms square crossing with phage；
4) culture dish to step 3) gained is cultivated 12-36 hour under conditions of being placed in test antibacterial suitable growth, observes whether antibacterial has the cleaved phenomenon of antibacterial with phage infall；
5) would indicate that phage and the bacterial strain of lytic activity are marked, more corresponding phage and correspondence list bacterium colony co-inoculation obtained phage in fluid medium amplification culture, repeat step 2) operation to step 4) carries out multiple sieve；Carry out the phage extracting solution still after multiple sieve with cracking test strain preserving the screening that can complete virulent phage.
Method the most according to claim 1, it is characterised in that: in step 1) during collecting sample, collecting sample from different environment, collection for non-water body sample time, add sterilized water make suspension.
Method the most according to claim 1, it is characterised in that: step 1) uses the direct filtration sterilization of microporous filter membrane to obtain phage；Or crush bacterial cell with phage extraction reagent, it is then centrifuged for or is filtrated to get phage.
Method the most according to claim 3, it is characterised in that: the aperture of described microporous filter membrane is 0.22 μm；It is chloroform that described phage extracts reagent.
Method the most according to claim 4, it is characterised in that: the mass concentration of described chloroform is 10%-15%.
Method the most according to claim 1, it is characterised in that: described step 2) in carry out antibacterial list bacterium colony cultivate will after the antibacterial gradient dilution of liquid culture coating or streak inoculation on solid medium.
Method the most according to claim 1, it is characterised in that: described step 3) acceptance of the bid clock employing marking pen carry out, a plurality of long line is parallel to each other, between distance be spaced apart 2-3cm；A plurality of short-term is parallel to each other, between distance be spaced apart 0.7-1.2cm.
Method the most according to claim 1, it is characterised in that: in described step 3), every long line inoculates identical phage, every short-term one single bacterium colony of inoculation, and the different single bacterium colony of different short-term inoculation.
Method the most according to claim 1, it is characterised in that: described step 3) all take the mode of streak inoculation to carry out during inoculation.
Method the most according to claim 1, it is characterised in that: the phage extracting solution in step 5) refers to the extracting solution obtained during amplification culture.
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| CN201610377461.XA CN105861451A (en) | 2016-05-31 | 2016-05-31 | Method for fast screening of virulent phage |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108651522A (en) * | 2018-04-09 | 2018-10-16 | 广州诺晶生物技术有限公司 | A kind of vibrio alginolyticus phage preparation, preparation method and applications |
-
2016
- 2016-05-31 CN CN201610377461.XA patent/CN105861451A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 何觅之: "大肠杆菌K88、K99广谱噬菌体的分离与生物学特性鉴定", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 张晓冬: "鸡大肠杆菌宽谱噬菌体的分离及裂解性研究", 《中国兽医杂志》 * |
| 李冰等: "鸡大肠杆菌噬菌体分离及裂解性试验", 《中国家禽》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108651522A (en) * | 2018-04-09 | 2018-10-16 | 广州诺晶生物技术有限公司 | A kind of vibrio alginolyticus phage preparation, preparation method and applications |
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Application publication date: 20160817 |