CN112646786A - Rapid preliminary separation method for vibrio kammaticus phage - Google Patents
Rapid preliminary separation method for vibrio kammaticus phage Download PDFInfo
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- 241000607598 Vibrio Species 0.000 title claims abstract description 30
- 238000000926 separation method Methods 0.000 title abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 58
- 239000001963 growth medium Substances 0.000 claims abstract description 31
- 230000001580 bacterial effect Effects 0.000 claims abstract description 16
- 238000012258 culturing Methods 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 241001515965 unidentified phage Species 0.000 claims abstract description 14
- 238000005336 cracking Methods 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 10
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 6
- 238000002474 experimental method Methods 0.000 claims abstract description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 27
- 229910052760 oxygen Inorganic materials 0.000 claims description 27
- 239000001301 oxygen Substances 0.000 claims description 27
- 239000007787 solid Substances 0.000 claims description 25
- 239000012880 LB liquid culture medium Substances 0.000 claims description 20
- 238000009395 breeding Methods 0.000 claims description 10
- 230000001488 breeding effect Effects 0.000 claims description 10
- 230000009089 cytolysis Effects 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 10
- 230000002101 lytic effect Effects 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 239000000706 filtrate Substances 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000010865 sewage Substances 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 239000012982 microporous membrane Substances 0.000 claims description 4
- 244000005700 microbiome Species 0.000 abstract description 6
- 238000005273 aeration Methods 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 238000009360 aquaculture Methods 0.000 description 3
- 244000144974 aquaculture Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 241000607323 Vibrio campbellii Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 241000143060 Americamysis bahia Species 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000604931 Bdellovibrio bacteriovorus Species 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- 241000607618 Vibrio harveyi Species 0.000 description 1
- 206010047400 Vibrio infections Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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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
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/00051—Methods of production or purification of viral material
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Abstract
The invention belongs to the technical field of microorganisms, and relates to a quick preliminary separation method of vibrio kammaticus phage, which specifically comprises the following steps: step 1: preparing a culture medium and a reagent; step 2: culturing a bacterial liquid; and step 3: collecting a water sample; and 4, step 4: treating a water sample; and 5: a cracking experiment; step 6: judging the transparency of the cracking ring; the method for preliminarily separating the vibrio kammii bacteriophages quickly can preliminarily separate the vibrio kammii bacteriophages quickly and efficiently at low cost, and solves the problems of few samples and difficulty in preliminarily separating the bacteriophages.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a quick preliminary separation method of vibrio kammaticus phage.
Background
Vibrio campbellii is an important member of vibrio harveyi flora (v. harveyigaroup), is widely distributed in marine environment, can cause various aquatic animal diseases, is one of pathogenic bacteria of aquaculture animals, can cause mass death of fishes, shrimps and shellfishes, and causes huge economic loss to aquaculture industry, so people pay attention to the vibrio campbellii.
Bacteriophages (phase) are a class of viruses that parasitize spirochetes, bacteria, actinomycetes, cyanobacteria, and the like, and are beneficial and harmless to living organisms, and are widely present in nature. The phage product has been used in aquaculture due to its non-pollution and non-residue properties, and its high specificity makes it have strong erosion-cracking ability for pathogenic microorganisms. It is reported that the bacteriophage product has better therapeutic effect on cultured animals than antibiotics, especially on vibriosis. Therefore, a method for preliminarily separating the vibrio kammaticus phage with high speed, high efficiency and low cost is needed, and the problems of less sampling and difficulty in preliminarily separating the phage are solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention mainly aims to provide a method for preliminarily separating the vibrio kammaticus phage with high speed, high efficiency and low cost, solves the problems of few samples and difficult preliminary separation of the phage, and provides a new idea for optimizing and separating the phage in the future on the basis of the phenomenon.
The technical scheme of the invention is as follows: the method for quickly and preliminarily separating the vibrio kammaticus phage specifically comprises the following steps:
step 1: preparation of culture medium and reagents: LB liquid culture medium, 0.75% LB solid culture medium, 1.5% LB solid culture medium, 1% LB solid culture medium, 0.85% -0.9% normal saline;
step 2: culturing a bacterial liquid: inoculating 1% of Vibrio kamtschaticus into 500mL of LB liquid culture medium and 100mL of LB liquid culture medium respectively, and culturing for 24h at 30 ℃ in a constant temperature shaking table at 180 r/min;
and step 3: collecting a water sample: 10L of water is collected by three groups of disinfected buckets near the seaside of a breeding plant area, a breeding workshop focus pool and a sewage discharge port of a workshop seedling collecting pool, and each group of collected water samples is divided into 3 same groups serving as parallel test groups, so that experimental errors or missing conditions are reduced;
and 4, step 4: treating a water sample: respectively pouring the cultured vibrio kangii bacterial liquid into three groups of water buckets containing water samples, simultaneously pouring LB liquid culture medium with the same amount as the bacterial liquid, and exposing oxygen indoors for 24 hours, wherein the oxygen is just about the oxygen bubbles just emerging out of the water surface; after the culture is finished, collecting 10mL of water sample in each barrel by using a 10mL syringe, filtering through a 0.22-micron microporous membrane, collecting filtrate, and repeatedly filtering for 3 times, so that the phage can be preliminarily filtered and collected; the oxygen aeration quantity is standard, on one hand, oxygen required for ensuring the normal growth and metabolism of microorganisms is ensured, on the other hand, the oxygen quantity is prevented from being too large, and the water solution is easy to spill out of the barrel;
and 5: lysis experiments: taking out 100mL of cultured culture solution of the fluorescent vibrio, placing the culture solution on a super clean workbench, taking out 100 mu L of the culture solution, uniformly coating the culture solution on a 1% LB solid culture medium, drying the culture solution, dropping 10 mu L of filter liquor into the LB solid culture medium, drying filter liquor water drops by opening a fan, and placing the filter liquor water drops in a 30 ℃ constant temperature incubator for culturing for 16-18 h;
step 6: judging the transparency of the cracking ring: the obvious transparent circle is ++, which shows that the bacteriophage has strong cracking effect; a fuzzy transparent circle is + so as to indicate that the phage has the lysis capability; no clearing circle is-indicating that the phage is very weak or non-lytic.
The invention has the beneficial effects that: the method for preliminarily separating the vibrio kammii bacteriophages quickly can preliminarily separate the vibrio kammii bacteriophages quickly and efficiently at low cost, and solves the problems of few samples and difficulty in preliminarily separating the bacteriophages.
Detailed Description
The following further illustrates embodiments of the invention:
example 1
The method for quickly and preliminarily separating the vibrio kammaticus phage specifically comprises the following steps:
step 1: preparation of culture medium and reagents: LB liquid culture medium, 0.75% LB solid culture medium, 1.5% LB solid culture medium, 1% LB solid culture medium, 0.85% normal saline;
step 2: culturing a bacterial liquid: inoculating 1% of Vibrio kamtschaticus into 500mL of LB liquid culture medium and 100mL of LB liquid culture medium respectively, and culturing for 24h at 30 ℃ in a constant temperature shaking table at 180 r/min;
and step 3: collecting a water sample: 10L of water is collected by three groups of disinfected buckets near the seaside of a breeding plant area, a breeding workshop focus pool and a sewage discharge port of a workshop seedling collecting pool, and each group of collected water samples is divided into 3 same groups serving as parallel test groups, so that experimental errors or missing conditions are reduced;
and 4, step 4: treating a water sample: respectively pouring the cultured vibrio kangii bacterial liquid into three groups of water buckets containing water samples, simultaneously pouring LB liquid culture medium with the same amount as the bacterial liquid, and exposing oxygen indoors for 24 hours, wherein the oxygen is just about the oxygen bubbles just emerging out of the water surface; after the culture is finished, collecting 10mL of water sample in each barrel by using a 10mL syringe, filtering the water sample through a 0.22-micron microporous filter membrane, collecting filtrate, and repeatedly filtering for 3 times, so that phage can be preliminarily filtered and collected, and most bdellovibrio bacteriovorus have the diameter larger than 0.22 micron; the oxygen aeration quantity is standard, on one hand, oxygen required for ensuring the normal growth and metabolism of microorganisms is ensured, on the other hand, the oxygen quantity is prevented from being too large, and the water solution is easy to spill out of the barrel;
and 5: lysis experiments: taking out 100mL of cultured culture solution of the fluorescent vibrio, placing the culture solution on a super clean workbench, taking out 100 mu L of the culture solution, uniformly coating the culture solution on a 1% LB solid culture medium, drying the culture solution, dropping 10 mu L of filter liquor into the LB solid culture medium, drying filter liquor water drops by opening a fan, and placing the filter liquor water drops in a 30 ℃ constant temperature incubator for culturing for 16 hours; the plate coating and filtrate dripping can effectively and intuitively verify the lytic property of the phage, determine whether the subsequent purification step is necessary or not, and is superior to the verification of the lytic property by adopting a complicated double-layer plate culture method;
step 6: judging the transparency of the cracking ring: the obvious transparent circle is ++, which shows that the bacteriophage has strong cracking effect; a fuzzy transparent circle is + so as to indicate that the phage has the lysis capability; no clearing circle is-indicating that the phage is very weak or non-lytic.
Example 2
The method for quickly and preliminarily separating the vibrio kammaticus phage specifically comprises the following steps:
step 1: preparation of culture medium and reagents: LB liquid culture medium, 0.75% LB solid culture medium, 1.5% LB solid culture medium, 1% LB solid culture medium, 0.88% normal saline;
step 2: culturing a bacterial liquid: inoculating 1% of Vibrio kamtschaticus into 500mL of LB liquid culture medium and 100mL of LB liquid culture medium respectively, and culturing for 24h at 30 ℃ in a constant temperature shaking table at 180 r/min;
and step 3: collecting a water sample: 10L of water is collected by three groups of disinfected buckets near the seaside of a breeding plant area, a breeding workshop focus pool and a sewage discharge port of a workshop seedling collecting pool, and each group of collected water samples is divided into 3 same groups serving as parallel test groups, so that experimental errors or missing conditions are reduced;
and 4, step 4: treating a water sample: respectively pouring the cultured vibrio kangii bacterial liquid into three groups of water buckets containing water samples, simultaneously pouring LB liquid culture medium with the same amount as the bacterial liquid, and exposing oxygen indoors for 24 hours, wherein the oxygen is just about the oxygen bubbles just emerging out of the water surface; after the culture is finished, collecting 10mL of water sample in each barrel by using a 10mL syringe, filtering through a 0.22-micron microporous membrane, collecting filtrate, and repeatedly filtering for 3 times, so that the phage can be preliminarily filtered and collected; the oxygen aeration quantity is standard, on one hand, oxygen required for ensuring the normal growth and metabolism of microorganisms is ensured, on the other hand, the oxygen quantity is prevented from being too large, and the water solution is easy to spill out of the barrel;
and 5: lysis experiments: taking out 100mL of cultured culture solution of the fluorescent vibrio, placing the culture solution on a super clean workbench, taking out 100 mu L of the culture solution, uniformly coating the culture solution on a 1% LB solid culture medium, drying the culture solution, dropping 10 mu L of filter liquor into the LB solid culture medium, drying filter liquor water drops by opening a fan, and placing the filter liquor water drops in a 30 ℃ constant temperature incubator for culturing for 17 hours;
step 6: judging the transparency of the cracking ring: the obvious transparent circle is ++, which shows that the bacteriophage has strong cracking effect; a fuzzy transparent circle is + so as to indicate that the phage has the lysis capability; no clearing circle is-indicating that the phage is very weak or non-lytic.
Example 3
The method for quickly and preliminarily separating the vibrio kammaticus phage specifically comprises the following steps:
step 1: preparation of culture medium and reagents: LB liquid culture medium, 0.75% LB solid culture medium, 1.5% LB solid culture medium, 1% LB solid culture medium, 0.9% normal saline;
step 2: culturing a bacterial liquid: inoculating 1% of Vibrio kamtschaticus into 500mL of LB liquid culture medium and 100mL of LB liquid culture medium respectively, and culturing for 24h at 30 ℃ in a constant temperature shaking table at 180 r/min;
and step 3: collecting a water sample: 10L of water is collected by three groups of disinfected buckets near the seaside of a breeding plant area, a breeding workshop focus pool and a sewage discharge port of a workshop seedling collecting pool, and each group of collected water samples is divided into 3 same groups serving as parallel test groups, so that experimental errors or missing conditions are reduced;
and 4, step 4: treating a water sample: respectively pouring the cultured vibrio kangii bacterial liquid into three groups of water buckets containing water samples, simultaneously pouring LB liquid culture medium with the same amount as the bacterial liquid, and exposing oxygen indoors for 24 hours, wherein the oxygen is just about the oxygen bubbles just emerging out of the water surface; after the culture is finished, collecting 10mL of water sample in each barrel by using a 10mL syringe, filtering through a 0.22-micron microporous membrane, collecting filtrate, and repeatedly filtering for 3 times, so that the phage can be preliminarily filtered and collected; the oxygen aeration quantity is standard, on one hand, oxygen required for ensuring the normal growth and metabolism of microorganisms is ensured, on the other hand, the oxygen quantity is prevented from being too large, and the water solution is easy to spill out of the barrel;
and 5: lysis experiments: taking out 100mL of cultured culture solution of the fluorescent vibrio, placing the culture solution on a super clean workbench, taking out 100 mu L of the culture solution, uniformly coating the culture solution on a 1% LB solid culture medium, drying the culture solution, dropping 10 mu L of filter liquor into the LB solid culture medium, drying filter liquor water drops by opening a fan, and placing the filter liquor water drops in a 30 ℃ constant temperature incubator for culturing for 18 hours;
step 6: judging the transparency of the cracking ring: the obvious transparent circle is ++, which shows that the bacteriophage has strong cracking effect; a fuzzy transparent circle is + so as to indicate that the phage has the lysis capability; no clearing circle is-indicating that the phage is very weak or non-lytic.
The foregoing embodiments and description have been presented only to illustrate the principles and preferred embodiments of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention as hereinafter claimed.
Claims (1)
1. A method for quickly and preliminarily separating a vibrio kammaticus bacteriophage is characterized by comprising the following steps: the method specifically comprises the following steps:
step 1: preparation of culture medium and reagents: LB liquid culture medium, 0.75% LB solid culture medium, 1.5% LB solid culture medium, 1% LB solid culture medium, 0.85% -0.9% normal saline;
step 2: culturing a bacterial liquid: inoculating 1% of Vibrio kamtschaticus into 500mL of LB liquid culture medium and 100mL of LB liquid culture medium respectively, and culturing for 24h at 30 ℃ in a constant temperature shaking table at 180 r/min;
and step 3: collecting a water sample: collecting 10L of water by using three groups of disinfected water buckets near a breeding plant area, a breeding workshop focus pool and a sewage discharge port of a workshop seedling collecting pool, wherein each group of collected water samples is divided into 3 same groups serving as parallel test groups;
and 4, step 4: treating a water sample: respectively pouring the cultured vibrio kangii bacterial liquid into three groups of water buckets containing water samples, simultaneously pouring LB liquid culture medium with the same amount as the bacterial liquid, and exposing oxygen indoors for 24 hours, wherein the oxygen is just about the oxygen bubbles just emerging out of the water surface; after the culture is finished, collecting 10mL of water sample in each barrel by using a 10mL syringe, filtering through a 0.22-micron microporous membrane, collecting filtrate, and repeatedly filtering for 3 times, so that the phage can be preliminarily filtered and collected;
and 5: lysis experiments: taking out 100mL of cultured culture solution of the fluorescent vibrio, placing the culture solution on a super clean workbench, taking out 100 mu L of the culture solution, uniformly coating the culture solution on a 1% LB solid culture medium, drying the culture solution, dropping 10 mu L of filter liquor into the LB solid culture medium, drying filter liquor water drops by opening a fan, and placing the filter liquor water drops in a 30 ℃ constant temperature incubator for culturing for 16-18 h;
step 6: judging the transparency of the cracking ring: the obvious transparent circle is ++, which shows that the bacteriophage has strong cracking effect; a fuzzy transparent circle is + so as to indicate that the phage has the lysis capability; no clearing circle is-indicating that the phage is very weak or non-lytic.
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| 朱秋华等: "溶藻弧菌噬菌体Va2001 的分离鉴定及其应用", 食品工业科技, vol. 1988, pages 102 - 109 * |
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