CN112322543A - Flavobacterium columnare isolated culture medium - Google Patents
Flavobacterium columnare isolated culture medium Download PDFInfo
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- CN112322543A CN112322543A CN202011300034.4A CN202011300034A CN112322543A CN 112322543 A CN112322543 A CN 112322543A CN 202011300034 A CN202011300034 A CN 202011300034A CN 112322543 A CN112322543 A CN 112322543A
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- flavobacterium columnare
- culture medium
- sulfate heptahydrate
- chloride dihydrate
- flavobacterium
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- 241000604777 Flavobacterium columnare Species 0.000 title claims abstract description 55
- 239000001963 growth medium Substances 0.000 title claims abstract description 42
- 241000894006 Bacteria Species 0.000 claims abstract description 15
- 229960000707 tobramycin Drugs 0.000 claims abstract description 14
- NLVFBUXFDBBNBW-PBSUHMDJSA-N tobramycin Chemical compound N[C@@H]1C[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N NLVFBUXFDBBNBW-PBSUHMDJSA-N 0.000 claims abstract description 14
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 13
- 239000001632 sodium acetate Substances 0.000 claims abstract description 13
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 13
- 108010013198 Daptomycin Proteins 0.000 claims abstract description 12
- 108010040201 Polymyxins Proteins 0.000 claims abstract description 12
- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 claims abstract description 12
- 229960005484 daptomycin Drugs 0.000 claims abstract description 12
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 12
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 12
- 210000002966 serum Anatomy 0.000 claims abstract description 12
- PWHCIQQGOQTFAE-UHFFFAOYSA-L barium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ba+2] PWHCIQQGOQTFAE-UHFFFAOYSA-L 0.000 claims abstract description 11
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims abstract description 11
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims abstract description 11
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 11
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims abstract description 11
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims abstract description 11
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims abstract description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 11
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000000843 powder Substances 0.000 claims abstract description 11
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims abstract description 7
- 239000001888 Peptone Substances 0.000 claims abstract description 6
- 108010080698 Peptones Proteins 0.000 claims abstract description 6
- 235000019319 peptone Nutrition 0.000 claims abstract description 6
- MYPYJXKWCTUITO-KIIOPKALSA-N chembl3301825 Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)C(O)[C@H](C)O1 MYPYJXKWCTUITO-KIIOPKALSA-N 0.000 claims abstract description 5
- 238000000926 separation method Methods 0.000 claims description 12
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 10
- 229920001817 Agar Polymers 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 108010059993 Vancomycin Proteins 0.000 claims description 6
- 238000002955 isolation Methods 0.000 claims description 6
- 239000012137 tryptone Substances 0.000 claims description 6
- 229960003165 vancomycin Drugs 0.000 claims description 6
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 claims description 6
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 5
- 239000000499 gel Substances 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 claims 1
- 241000251468 Actinopterygii Species 0.000 abstract description 7
- 201000010099 disease Diseases 0.000 abstract description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 7
- 238000011841 epidemiological investigation Methods 0.000 abstract description 3
- 208000024891 symptom Diseases 0.000 abstract description 3
- 230000000844 anti-bacterial effect Effects 0.000 abstract 1
- 239000003899 bactericide agent Substances 0.000 abstract 1
- 239000007787 solid Substances 0.000 description 11
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 5
- 238000009630 liquid culture Methods 0.000 description 5
- 241000252230 Ctenopharyngodon idella Species 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 235000019797 dipotassium phosphate Nutrition 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 108010093965 Polymyxin B Proteins 0.000 description 3
- 238000009360 aquaculture Methods 0.000 description 3
- 244000144974 aquaculture Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000013505 freshwater Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229920000024 polymyxin B Polymers 0.000 description 3
- 229960005266 polymyxin b Drugs 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000252228 Ctenopharyngodon Species 0.000 description 2
- 241000589565 Flavobacterium Species 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000606125 Bacteroides Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241001609213 Carassius carassius Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241000404975 Synchiropus splendidus Species 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229960001212 bacterial vaccine Drugs 0.000 description 1
- 229910001626 barium chloride Inorganic materials 0.000 description 1
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 241001233037 catfish Species 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
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- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention provides a flavobacterium columnare isolated culture medium, which comprises the following components: 0.5-1g/L of inorganic salt, 500 mu g/mL of 200-one bacteria remover, 4-5g/L of peptone, 0.1-1% of serum and 15-18g/L of gel powder, wherein the inorganic salt comprises one or more of sodium acetate, barium chloride dihydrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, ferrous sulfate heptahydrate and sodium bicarbonate, and the bacteria remover comprises one or more of polymyxin, tobramycin, daptomycin and vancomycin. Compared with the prior art, the invention can rapidly separate the flavobacterium columnare from the gill-rot disease fish gill and the body surface of the flavobacterium columnare by matching inorganic salt with a bactericide, form a single colony, has a small quantity of mixed bacteria, completes the epidemiological investigation of 11 provinces of the flavobacterium columnare in China by practical application, and separates 45 strains of the flavobacterium columnare in 46 typical symptom focus positions.
Description
Technical Field
The invention relates to a separation culture medium, in particular to a flavobacterium columnare separation culture medium.
Background
The grass carp is a main variety for freshwater aquaculture in China, the fresh water aquaculture yield is over 25 percent, the aquaculture yield of the grass carp in China in 2018 is about 534 million tons, and the loss caused by diseases is nearly 21 hundred million yuan each year. The gram-negative bacteria of the genus flavobacterium columniformis infect about 20 families of freshwater fishes such as grass carp, clear fish, crucian carp, mandarin fish, longsnout catfish, weever and the like. The gill rot disease of the grass carps caused by the yellow-dry fungus is outbreak in recent years nationwide, and is one of the most serious 'three old diseases' in grass carp culture. As the national administration and supervision of aquatic antibiotics tend to be strict, the aquatic bacterial vaccine with preventive and safety characteristics is increasingly paid more attention by the nation and scientific research units in recent years.
The separation of the flavobacterium columnare and epidemiological investigation are taken as one of key factors for researching and developing vaccines of the flavobacterium columnare, the traditional flavobacterium columnare separation method has the defects of poor specificity and low separation rate, the epidemiological investigation of 11 provinces of the flavobacterium columnare in China is completed through practical application, and 45 strains of the flavobacterium columnare separated from 46 focus parts with typical symptoms.
Disclosure of Invention
The invention provides a flavobacterium columnare separation culture medium, which is used for improving the separation effect of the flavobacterium columnare.
The invention provides a flavobacterium columnare isolated culture medium, which comprises the following components: 0.5-1g/L of inorganic salt, 500 mu g/mL of 100-one bacteria remover, 4-5g/L of peptone, 0.1-1% of serum and 15-18g/L of gel powder, wherein the inorganic salt comprises one or more of sodium acetate, barium chloride dihydrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, ferrous sulfate heptahydrate and sodium bicarbonate, and the bacteria remover comprises one or more of polymyxin, tobramycin, daptomycin and vancomycin.
Further, the inorganic salt is composed of sodium acetate, barium chloride dihydrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, ferrous sulfate heptahydrate, and sodium bicarbonate.
Still further, the inorganic salt comprises: 0.02g/L of sodium acetate, 0.01g/L of barium chloride dihydrate, 0.1g/L of dipotassium hydrogen phosphate, 0.05g/L of potassium dihydrogen phosphate, 0.6g/L of magnesium sulfate heptahydrate, 0.0067g/L of calcium chloride dihydrate, 0.002g/L of ferrous sulfate heptahydrate and 0.05g/L of sodium bicarbonate.
Further, the flavobacterium columnare separation culture medium also comprises 0.4-0.5g/L of yeast extract.
Further, the serum content is 0.1% -0.6%.
Still further, the serum content is 0.6%.
Further, the degerming agent consists of polymyxin, tobramycin, daptomycin and vancomycin.
Further, the degerming agent comprises polymyxin 140-200U/mL, tobramycin 2-5 μ g/mL, daptomycin 100-200 μ g/mL and vancomycin 2-10 μ g/mL.
Further, the gel powder is agar powder.
Further, the peptone is tryptone.
Compared with the prior art, the method has the advantages that the inorganic salt is matched with the degerming agent, the flavobacterium columnare can be rapidly separated from the gill and the body surface of the gill rot disease fish of the flavobacterium columnare, a single colony is formed, the quantity of mixed bacteria is small, compared with the traditional separation method, the problem that the quantity of the mixed bacteria is large is solved, the separation efficiency is greatly improved, the bottleneck that the flavobacterium columnare difficult to separate is broken through, the epidemiological survey of 11 provincial flavobacterium columnare of 11 grass carps in China is completed through practical application, and 45 strains of the flavobacterium columnare are separated from 46 focus parts with typical symptoms.
Drawings
FIG. 1 is a colony cultivation diagram of an isolation plate according to example 1 of the present invention;
FIG. 2 is a colony cultivation diagram of an isolation plate in example 2 of the present invention;
FIG. 3 is a colony culture diagram of an isolation plate according to a control example of the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1
0.02g/L of sodium acetate, 0.01g/L of barium chloride dihydrate, 0.1g/L of dipotassium phosphate, 0.05g/L of potassium dihydrogen phosphate, 0.6g/L of magnesium sulfate heptahydrate, 0.0067g/L of calcium chloride dihydrate, 0.002g/L of ferrous sulfate heptahydrate, 0.05g/L of sodium bicarbonate, 5g/L of tryptone, 0.5g/L of yeast extract, 0.6% of serum, 200U/mL of polymyxin, 5 mu g/mL of tobramycin, 150 mu g/mL of daptomycin, 10 mu g/mL of vancomycin and 15g/L of agar powder.
Example 2
0.02g/L of sodium acetate, 0.01g/L of barium chloride dihydrate, 0.1g/L of dipotassium phosphate, 0.05g/L of potassium dihydrogen phosphate, 0.6g/L of magnesium sulfate heptahydrate, 0.0067g/L of calcium chloride dihydrate, 0.002g/L of ferrous sulfate heptahydrate, 0.05g/L of sodium bicarbonate, 5g/L of tryptone, 0.5g/L of yeast extract, 0.1% of serum, 200U/mL of polymyxin, 200 mu g/mL of daptomycin and 15g/L of agar powder (solid culture medium).
Example 3
0.01g/L of sodium acetate, 0.01g/L of barium chloride dihydrate, 0.1g/L of dipotassium phosphate, 0.05g/L of potassium dihydrogen phosphate, 0.6g/L of magnesium sulfate heptahydrate, 0.0067g/L of calcium chloride dihydrate, 0.002g/L of ferrous sulfate heptahydrate, 0.05g/L of sodium bicarbonate, 5g/L of tryptone, 0.5g/L of yeast extract, 0.6% of serum, 200U/mL of polymyxin, 5 mu g/mL of tobramycin, 150 mu g/mL of daptomycin, 10 mu g/mL of vancomycin and 15g/L of agar powder.
Example 4
0.02g/L of sodium acetate, 0.01g/L of barium chloride dihydrate, 0.1g/L of dipotassium phosphate, 0.05g/L of potassium dihydrogen phosphate, 0.6g/L of magnesium sulfate heptahydrate, 0.0067g/L of calcium chloride dihydrate, 0.002g/L of ferrous sulfate heptahydrate, 0.05g/L of sodium bicarbonate, 5g/L of tryptone, 0.5g/L of yeast extract, 0.1% of serum, 200U/mL of polymyxin, 5 mu g/mL of tobramycin, 150 mu g/mL of daptomycin, 10 mu g/mL of vancomycin and 15g/L of agar powder.
Comparative example
The culture medium of the comparative example of the present invention includes conventional culture medium of Flavobacterium columnare, selective solid culture medium of Flavobacterium columnare, and selective liquid culture medium of Flavobacterium columnare.
The conventional culture medium of flavobacterium columnare conventional Shieh solid culture medium, and the preparation method comprises the following steps: collecting peptone 5g, yeast extract 0.5g, Noble agar 10g, and K2HPO4 0.1g、KH2PO40.05g, sodium acetate 0.01g, NaHCO3 0.05g、MgSO4·7H2O0.3g、BaCl2·H2O 0.01g、FeSO4·7H2O 0.001g、CaCl2·2H2O0.0067 g is added into 1000mL of distilled water, the pH is adjusted to 7.2, and the mixture is sterilized to obtain the product.
Flavobacterium columnare selective solid culture medium: sterilizing and cooling the conventional culture medium of Flavobacterium columnare, adding tobramycin to make the final concentration of the tobramycin to be 2 mug/mL and polymyxin B to make the final concentration of the polymyxin B to be 20U/mL, and pouring the mixture into a flat plate to obtain the flavobacterium columnare culture medium;
flavobacterium columnare selective liquid culture medium: removing agar from the above conventional culture medium of Flavobacterium columnare, sterilizing, cooling, adding tobramycin to give a final concentration of 2 μ g/mL, and adding polymyxin B to give a final concentration of 20U/mL.
The method for using the culture medium of the comparative example of the invention is as follows:
1. taking the prepared conventional solid culture medium, selective solid culture medium and selective liquid culture medium of the flavobacterium columnare for later use;
2. the disease fish gill part focus is picked, streaked and inoculated to a conventional solid culture medium of flavobacterium columnare, and cultured for 48 hours at 28 ℃, and a typical yellow colony of the flavobacterium columnare can be observed on the culture medium.
3. The single yellow colony obtained by the above culture was selected, streaked and inoculated on a Flavobacterium columnare selective solid medium, and cultured at 28 ℃ for 48 hours.
4. Selecting the single yellow colony obtained by the culture, inoculating the single yellow colony to a flavobacterium columnare selective liquid culture medium, and culturing for 24 hours at 28 ℃;
5. diluting the yellow bacterial liquid according to a 10-fold series (the dilution after dilution is 10 < -5 > to 10 < -8 >), uniformly coating the yellow bacterial liquid on a flavobacterium bacteroid selective solid culture medium, and culturing for 48 hours at 28 ℃;
6. selecting single yellow colony on 10-8 dilution plate, inoculating to Flavobacterium columnare selective liquid culture medium, and culturing at 28 deg.C for 24 hr to obtain relatively pure culture;
7. and (3) strain confirmation:
and (3) performing gram staining on the purified bacterial liquid obtained in the step 6), wherein the bacteria are red and have single shape.
To compare the separation effects of examples 1 to 4 according to the invention and the comparative example, tests were carried out by the following method:
1. taking the isolated culture medium of the flavobacterium columnare of the embodiments 1-4 and the conventional solid culture medium of the flavobacterium columnare of the comparison example for later use;
2. the diseased fish gill lesions are picked and streaked on the isolated culture medium of flavobacterium columnare in examples 1-4 and the conventional solid culture medium of flavobacterium columnare in a control example respectively, the separated culture medium is cultured at 28 ℃ for 48 hours, and the colony growth situation on the culture medium is observed, wherein the colony growth situation is shown in figure 1 of example 1 of the invention, figure 2 of example 2 of the invention, and figure 3 of the control example of the invention.
As shown in FIGS. 1 to 3, in examples 1 to 4 of the present invention, Flavobacterium columnare rapidly isolated from the gill and body surface of a gill rot disease fish with Flavobacterium columnare, a single colony is formed, and the number of mixed bacteria is small, while the control example has a large number of mixed bacteria and grows faster, and is difficult to isolate even if purified for many times.
Meanwhile, the method disclosed by the embodiment 1-4 is simple to operate, and the flat plate does not need to be worried about pollution by the mixed bacteria in the air.
In addition, as shown in fig. 1 to 3, in example 1 of the present invention, compared to example 2, by using a combination of polymyxin, tobramycin, daptomycin and vancomycin, the inhibition of infectious microbes is effectively improved; compared with the embodiment 3, the growth rate of the flavobacterium columnare improved by adopting 0.02g/L sodium acetate, 0.002g/L ferrous sulfate and 0.6g/L magnesium sulfate; compared with the embodiment 4, after 12 hours of cultivation under the conventional condition, the OD value of the embodiment 1 can reach 0.680, but the OD value of the embodiment 4 is only 0.455, and the embodiment 1 of the invention effectively improves the growth speed of the flavobacterium columnare by adopting 0.6% of serum.
Finally, it should be noted that the above-mentioned embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the modifications and equivalents of the specific embodiments of the present invention can be made by those skilled in the art after reading the present specification, but these modifications and variations do not depart from the scope of the claims of the present application.
Claims (10)
1. The flavobacterium columnare separation culture medium is characterized by comprising the following components: 0.5-1g/L of inorganic salt, 500 mu g/mL of 100-one bacteria remover, 4-5g/L of peptone, 0.1-1% of serum and 15-18g/L of gel powder, wherein the inorganic salt comprises one or more of sodium acetate, barium chloride dihydrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, ferrous sulfate heptahydrate and sodium bicarbonate, and the bacteria remover comprises one or more of polymyxin, tobramycin, daptomycin and vancomycin.
2. The isolated medium of Flavobacterium columnare as claimed in claim 1, wherein said inorganic salt is composed of sodium acetate, barium chloride dihydrate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate, ferrous sulfate heptahydrate, sodium bicarbonate.
3. The isolated culture medium of Flavobacterium columnare as claimed in claim 3, wherein said inorganic salts comprise: 0.02g/L of sodium acetate, 0.01g/L of barium chloride dihydrate, 0.1g/L of dipotassium hydrogen phosphate, 0.05g/L of potassium dihydrogen phosphate, 0.6g/L of magnesium sulfate heptahydrate, 0.0067g/L of calcium chloride dihydrate, 0.002g/L of ferrous sulfate heptahydrate and 0.05g/L of sodium bicarbonate.
4. The isolated culture medium of Flavobacterium columnare as claimed in claim 1, wherein the isolated culture medium further comprises yeast extract 0.4-0.5 g/L.
5. The isolated culture medium of Flavobacterium columnare as claimed in claim 1, wherein said serum content is 0.1% -0.6%.
6. The isolated culture medium of Flavobacterium columnare as claimed in claim 5, wherein said serum content is 0.6%.
7. The isolated culture medium for flavobacterium columnare as claimed in claim 1, wherein the degerming agent is composed of polymyxin, tobramycin, daptomycin and vancomycin.
8. The Flavobacterium columnare isolation medium as claimed in claim 7, wherein the bacteria removing agent comprises polymyxin 140-200U/mL, tobramycin 2-5 μ g/mL, daptomycin 100-200 μ g/mL, vancomycin 2-10 μ g/mL.
9. The flavobacterium columnare isolation medium according to claim 1, wherein the gel powder is agar powder.
10. The Flavobacterium columnare isolation medium of claim 1, wherein the peptone is tryptone.
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