CN102618464A - Resuscitation medium capable of promoting growth of VBNC (viable but non-culture) bacteria and improving separation abundance, and preparation method and application thereof - Google Patents
Resuscitation medium capable of promoting growth of VBNC (viable but non-culture) bacteria and improving separation abundance, and preparation method and application thereof Download PDFInfo
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- CN102618464A CN102618464A CN2012100811267A CN201210081126A CN102618464A CN 102618464 A CN102618464 A CN 102618464A CN 2012100811267 A CN2012100811267 A CN 2012100811267A CN 201210081126 A CN201210081126 A CN 201210081126A CN 102618464 A CN102618464 A CN 102618464A
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- 241000894006 Bacteria Species 0.000 title claims abstract description 98
- 238000000926 separation method Methods 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title abstract description 11
- 230000001737 promoting effect Effects 0.000 title abstract description 6
- 239000002689 soil Substances 0.000 claims abstract description 48
- 230000001954 sterilising effect Effects 0.000 claims abstract description 45
- 239000012530 fluid Substances 0.000 claims abstract description 43
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000012535 impurity Substances 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 244000005700 microbiome Species 0.000 claims abstract description 11
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 11
- 241000191938 Micrococcus luteus Species 0.000 claims abstract description 9
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 238000011084 recovery Methods 0.000 claims description 58
- 230000001580 bacterial effect Effects 0.000 claims description 57
- 239000007788 liquid Substances 0.000 claims description 54
- 238000000034 method Methods 0.000 claims description 35
- 238000004659 sterilization and disinfection Methods 0.000 claims description 28
- 239000000203 mixture Substances 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 22
- 235000015097 nutrients Nutrition 0.000 claims description 19
- 229940041514 candida albicans extract Drugs 0.000 claims description 16
- 239000012138 yeast extract Substances 0.000 claims description 16
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- 239000000284 extract Substances 0.000 claims description 14
- 239000008103 glucose Substances 0.000 claims description 13
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- 239000007787 solid Substances 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 10
- 239000008272 agar Substances 0.000 claims description 10
- 235000018102 proteins Nutrition 0.000 claims description 10
- 241001478240 Coccus Species 0.000 claims description 9
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- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- 229940117709 gamboge Drugs 0.000 claims description 9
- 239000008399 tap water Substances 0.000 claims description 9
- 235000020679 tap water Nutrition 0.000 claims description 9
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 8
- 235000010755 mineral Nutrition 0.000 claims description 8
- 239000011707 mineral Substances 0.000 claims description 8
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- 238000012360 testing method Methods 0.000 claims description 8
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- 150000007513 acids Chemical class 0.000 claims description 7
- 241000186361 Actinobacteria <class> Species 0.000 claims description 6
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 6
- 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 description 6
- 238000004321 preservation Methods 0.000 claims description 6
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 5
- 239000012137 tryptone Substances 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- 229930010555 Inosine Natural products 0.000 claims description 4
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 claims description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 4
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- 241000589516 Pseudomonas Species 0.000 claims description 4
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- 235000016709 nutrition Nutrition 0.000 claims description 4
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- 235000019319 peptone Nutrition 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 4
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- 239000002699 waste material Substances 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 241000186063 Arthrobacter Species 0.000 claims description 3
- 244000063299 Bacillus subtilis Species 0.000 claims description 3
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- 244000046052 Phaseolus vulgaris Species 0.000 claims description 3
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- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims description 3
- 241000187747 Streptomyces Species 0.000 claims description 3
- 229930003756 Vitamin B7 Natural products 0.000 claims description 3
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- 238000004458 analytical method Methods 0.000 claims description 3
- 238000012790 confirmation Methods 0.000 claims description 3
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 3
- 239000006071 cream Substances 0.000 claims description 3
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 claims description 3
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 3
- 239000011790 ferrous sulphate Substances 0.000 claims description 3
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- 235000012204 lemonade/lime carbonate Nutrition 0.000 claims description 3
- CNFDGXZLMLFIJV-UHFFFAOYSA-L manganese(II) chloride tetrahydrate Chemical compound O.O.O.O.[Cl-].[Cl-].[Mn+2] CNFDGXZLMLFIJV-UHFFFAOYSA-L 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims description 3
- 229960004452 methionine Drugs 0.000 claims description 3
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 235000003784 poor nutrition Nutrition 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 3
- 239000011735 vitamin B7 Substances 0.000 claims description 3
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- 238000005303 weighing Methods 0.000 claims description 3
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 3
- 229960001763 zinc sulfate Drugs 0.000 claims description 3
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- 239000005018 casein Substances 0.000 claims description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 2
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- 238000011160 research Methods 0.000 description 4
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- 241000186367 Mycobacterium avium Species 0.000 description 2
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Abstract
The invention relates to the field of microorganism screening and culturing, in particular to a resuscitation medium capable of promoting growth of VBNC (viable but non-culture) bacteria and improving separation abundance, and a preparation method and application thereof. The resuscitation medium capable of promoting growth of VBNC bacteria and improving separation abundance is composed of medium base fluid, bacterium fluid and soil fluid, the bacterium fluid is prepared by culturing micrococcus luteus on culturing fluid medium to the later period of log phase, contains resuscitation promoting factors Rpf after culturing fluid medium is removed by centrifuging to extract protein and is 0.5-30% of the medium base fluid according to the volume ratio, and the soil fluid is prepared by adding water to the soil which is dried, screened and subjected to impurity removal, sterilizing multiple times and filtering to obtain filtrate or centrifuging to obtain supernatant, and is 0.1-20% of the medium base fluid according to the volume ratio. By means of the resuscitation promoting factors Rpf and by adding the soil fluid to prepare the resuscitation medium, resuscitation of the VBNC bacteria can be promoted further, and abundance of separating the VBNC bacteria from the ecological environment is improved. The resuscitation medium and the preparation method thereof have the advantages of simplicity in operation and fast resuscitation.
Description
Technical field
The present invention relates to microbe to screen and cultivation field, relate in particular to a kind of recovery substratum of non-cultivation the (VBNC) bacterial classification of work.
Background technology
VBNC (viable but non-culture) refers to some mikrobe can be detected existence in their habitat, but can not carry out artificial culture in the laboratory.Nineteen eighty-two Xu Huai pardon to wait through to the research of V.cholerae with E.coli survival rule, find first and proposed bacterium " the non-cultivation alive " state (Viable but non-culturable state, VBNC); Be that bacterium is under the unsuitable environmental condition, its cell shortens sphere (finding in nearest many researchs that also some bacterium shows as volume increase, cell elongation) usually into; Can not make its growth and breeding with the ordinary method cultivation, but still have metabolic activity, under the effect of DNA synthetic inhibitor; When adding a certain amount of nutrition and cultivating; Though these cells can not divide, but still can elongation growth, prove it and still survive; Be not death. be in the bacterium of VBNC state, can recover under certain conditions.
Now the bacterium in natural ecological environment can only have a 0.01-10% through what traditional partition method was obtained, and the overwhelming majority is in non-cultivation the (VBNC) alive, is similar to the state of dormancy.With regard to the environmental microorganism resource, the resource bacterial classification that how to obtain occurring in nature 90-99.99% needs the innovation of new approaches, novel method, could excavate, develop, utilize these unknown Microbial resources.
Chinese invention patent application (application number: 200810051555.3 applyings date: 2008-12-09) disclose resuscitation fluid and preparation method thereof and the method for resuscitation of a kind of VBNC Salmonellas; Resuscitation fluid is made up of for 1～3 part 1 part of serum, sterilization pure water; Through mixing stirring and evenly mixing, make with the millipore filter filtration sterilization; The Salmonellas that gets into VBNC is placed resuscitation fluid, in the program control circulation appearance of heat hole, take to increase progressively heating mode, hot program control circulation appearance recovery heats up and the incubation time-program(me) is selected from 5 ℃ to 37 ℃ intervals, and the incubation time is 2MIN～1.5H; Be inoculated in the SS liquid nutrient medium then, and in gas bath vibration shaking table, 200～220R/MIN cultivates.
(Ding Linxian; Su Xiaomei; Horizontal field is bright. live but the progress of non-cultivation the (VBNC) state bacterium and use prospect. " mikrobe journal " .2011,51 (7): formation mechanism, transformation and kind, recovery, Research Significance and the application thereof of 858-862.) having set forth VBNC state bacterium are looked forward to.And reported in the period of Ding Linxian etc. is surplus ten to the recovery that is in VBNC state bacterium in the ecotope, can cultivation, some achievements in research of aspects such as phyletic evolution relation and potential function, the exploitation of intending to Microbial resources provides new scientific basis with application.And disclose bring back to life to promote the factor (Rpf, resuscitation promotingfactor) be by the gamboge coccus (
M. luteus) a kind of protein that can make this bacterium that is in the VBNC state recover its growth and breeding ability again of excretory, molecular weight is about 16-17 kDa.Mukamolova has reported that Rpf can recover and has been in the VBNC gram-positive microorganism in period
MycobacteriumThe mycobacterium tuberculosis that belongs to (
M. tuberculosis),
Mycobacterium bovis (
M. bovis), mycobacterium kansasii (
M.kansasii), M. smegmatics (
M. smegmatis) and mycobacterium avium (
M. avium) etc.And the gene similar with Rpf also comes to light in the gram-positive microorganism of GC such as height such as streptomycete, tubercule bacillus, coryneform bacteria grade.In addition, people such as Mukamolova have reported that also Rpf adopts the mode secretion signal of autocrine or paracrine, and the mikrobe of dormancy is brought back to life, the growth that can also stimulate normal bacterium, and the reproductive process of ability regulating cell.But in the document, openly do not adopt the recovery substratum of which kind of prescription, and in practical application, simple resurrection promotes the factor promoting the recovery growth of non-cultivation the (VBNC) state bacterium and separating on the abundance also highly significant not of effect.
Summary of the invention
For recovery growth that solves non-cultivation the (VBNC) bacterial classification alive and the technical problem of separating abundance.First purpose of the present invention provides a kind of recovery substratum that can promote the VBNC bacterial growth and improve the separation abundance; This substratum utilizes a kind of specific proteins signaling molecule to make up can promote the growth of VBNC bacteria resuscitation, improves and from ecotope, separates the VBNC bacteria abundance.Second purpose provides the preparation method of above-mentioned recovery substratum.The 3rd purpose provides the application of above-mentioned recovery substratum.
In order to realize first above-mentioned purpose, the technical scheme below the present invention has adopted:
A kind of recovery substratum that can promote the VBNC bacterial growth and improve the separation abundance, this recovery substratum is made up of substratum base fluid, bacterium liquid and liquid soil; Described bacterium liquid is cultured to the logarithmic phase later stage by the micrococcus luteus bacterial classification on the bacterial classification liquid nutrient medium, extract through centrifugal removal bacterial classification liquid nutrient medium and obtain protein, contains to bring back to life to promote factor R pf, and bacterium liquid is the 0.5-30% of substratum base fluid by volume; Described liquid soil adds water by through soil air-dry, that sieve, remove impurity, and leaching filtrating or centrifuging and taking supernatant are crossed in repeatedly sterilization, and liquid soil is the 0.1-20% of substratum base fluid by volume.
As preferably, above-mentioned bacterium liquid is the 1-10% of substratum base fluid by volume, and liquid soil is the 1-5% of substratum base fluid by volume.
As preferably, above-mentioned substratum base fluid is selected a kind of in the following prescription for use:
A, general bacteria resuscitation are cultivated the prescription that uses:
Bacto-tryptone 8.0 ~ 15.0g,
Yeast extract 3.0 ~ 8.0g,
Malt extract 3.0 ~ 8.0g,
Carnis Bovis seu Bubali cream 1.0 ~ 3.0g,
Casamino acids 3.0 ~ 8.0g,
Glycerine 1.0 ~ 3.0g,
Tween-80 0.03 ~ 0.1g,
MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.5 ~ 2.0g,
Add deionized water 1000mL, regulate pH7.0;
B, be fit to gram-positive microorganism do not have the mycolic acids Pseudomonas (
AmycolatopsisGenus) prescription:
Glucose 2.0 ~ 6.0g,
Yeast extract 2.0 ~ 6.0g,
Fructus Hordei Germinatus extract 0.6 ~ 2.0 g,
Zero(ppm) water 1000.0 ml,
PH 7.2, add during the configuration nutrient agar and use lime carbonate CaCO
3,1.0 ~ 3.0g;
C, be fit to the gram-positive microorganism genus arthrobacter (
ArthrobacterBelong to), rhodococcus (
RhodococcusBelong to), Lay Fu Shi belong to (
LeifsoniaBelong to), promise Ka Shi belong to (
NocardiaBelong to), in the north spore belong to (
KitasatosporaBelong to), streptomyces (
StreptomycesBelong to), Bacillus subtilus belong to (
BacillusBelong to), series bacillus belong to (
PaenibacillusGenus) prescription:
Peptone 3.0 ~ 8.0 g
Yeast extract 1.0 ~ 5.5 g,
Glucose 0.5 ~ 2.0 g,
Bata-phenethyl alcohol 2.0 ~ 4.0ml,
Kabicidin 0.03 ~ 0.08 g,
Cycloheximide 0.02 ~ 0.08 g,
Deionized water 1000 ml, pH 7.0;
The prescription of bacterium in D, the suitable poor nutrient soil ecology:
Nutrient broth 1.0 ~ 2.50 g,
Yeast extract 0.20 ~ 1.0g,
Zero(ppm) water 1000 ml, pH 7.0;
The prescription of bacterium in E, the suitable poor nutrition deep water ecology:
Casein 1.0 ~ 3.0 g,
Dregs of beans 0.10 ~ 0.50 g,
Sodium-chlor 0.20 ~ 1.0 g,
Potassium hydrogenphosphate 0.1 ~ 0.5 g,
Glucose 0.1 ~ 0.5 g,
Zero(ppm) water 1000 ml, pH 7.3.
When needs preparation solid medium, in each assembly side of described A, B, C, D or E, add agar 15 ~ 20g, through 110 ~ 130 ℃, the use of 10 ~ 30min autoclaving postcooling.
In order to realize second above-mentioned purpose, the technical scheme below the present invention has adopted:
A kind of method for preparing above-mentioned recovery substratum, this method comprises the steps:
1) take a morsel gamboge coccus bacterial classification inoculation in the bacterial classification liquid nutrient medium, 25 ~ 35 C, 100 ~ 150rpm vibrate cultivation; In bacterial growth to the logarithmic phase later stage, nutrient solution is with 3000 ~ 8000rpm, and thalline is removed in the centrifugal extracting of 10 ~ 30min; After the sterilization of 0.20 ~ 0.25 μ m membrane filtration ,-30 ~-10 ℃ of preservations are subsequent use;
2) the garden mould 1kg air-dry, that sieve, remove impurity that learns from else's experience adds tap water 1 ~ 2L, 110 ~ 130 ℃, the sterilization of 10 ~ 40min high-pressure sterilizing pot; After room temperature is treated an evening, use once again with method and sterilize; 1500 ~ 2500r/min low-speed centrifugal is got supernatant or is obtained filtrating with the filter paper filtering waste, and through 110 ~ 130 ℃, 10 ~ 30min high-pressure sterilizing pot sterilization postcooling ,-30 ~-10 ℃ of preservations are subsequent use;
3) accurately take by weighing each component of substratum base fluid, it is for use that pH transfers to 7.0,110 ~ 130 ℃, 10 ~ 30min high-pressure sterilizing pot sterilization postcooling to room temperature; The separation of the suitable most bacteriums of substratum base fluid is for particular requirement being arranged and selectively bacterium can corresponding plus-minus nutrition composition;
4) before the use, under aseptic condition, add the bacterium liquid of respective amount in the substratum base fluid, add the liquid soil of respective amount again, use behind the mixing.
As preferably, the prescription of above-mentioned bacterial classification liquid nutrient medium is following:
Ammonium chloride 3.0 ~ 5.0g,
Potassium primary phosphate 1.2 ~ 1.6g,
Vitamin H 0.003 ~ 0.008g,
L-methionine(Met) 0.01 ~ 0.04g,
VitB1 0.02 ~ 0.05g,
Inosine 0.8 ~ 1.5g,
MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.05 ~ 0.1g,
Mineral solution 0.8 ~ 1.5ml,
L-lithium lactate 5.0 ~ 20.0g,
Zero(ppm) water 1000ml, pH7.5.
Preferred as again, the prescription of above-mentioned mineral solution is following:
Copper sulfate 0.20 ~ 0.30g,
Manganous chloride tetrahydrate 0.30 ~ 0.80g,
Ferrous sulfate 0.08 ~ 0.20g,
Sodium orthomolybdate 0.01 ~ 0.03g,
Zinc sulfate 0.02 ~ 0.08g,
Zero(ppm) water 1000ml.
In order to realize the 3rd above-mentioned purpose, the invention discloses the scheme of three kinds of application.
1, a kind of method that can promote the VBNC bacterial growth and improve the separation abundance, this method comprises the steps:
1) get 1 part of separation source and add 7 ~ 12 parts of above-mentioned recovery substratum, fully mixing is made mother liquor, arrives to a certain degree through 10 times of gradient dilutions;
2) be coated with the identical component solid plate substratum that adds 2% agar, the dull and stereotyped inversion cultivated under the conventional culture temperature of bacterium, general bacterium 24-48h, and actinomycetes 5-14d, picking list bacterium colony, enlarged culturing obtains pure strain.
2, a kind of method that can promote VBNC bacteria resuscitation growth, this method will tests the preceding cultured strain of not growing on the substratum or be kept at the bacterial classification of very low temperature-80 ℃, be inoculated on the above-mentioned recovery substratum, and the part bacterial classification recovers to grow.
3, a kind of confirmation method of VBNC bacterial classification; Whether this method belongs to the bacterium that is in VBNC in the separation source to using above-mentioned recovery substratum to separate the bacterial classification that obtains; The recovery substratum that adopts above-mentioned recovery substratum and do not add bacterium liquid is as contrast, adopts conventional MPN counting process, DGGE or the analysis of FISH molecular biology method to demonstrate,prove.
The present invention is owing to adopted above-mentioned technical scheme; Utilize a kind of specific proteins signaling molecule to bring back to life and promote factor R pf; Add liquid soil simultaneously, be built into the recovery substratum, owing to comprise multiple special trace element in the liquid soil; Can further promote the growth of VBNC bacteria resuscitation, improve and from ecotope, separated the VBNC bacteria abundance.Simple to operate, the fast characteristics of recovering that the present invention has.
Description of drawings
Fig. 1 separates comparing result figure for the park soil sample.
Fig. 2 is that island sand ground soil sample separates comparing result figure.
Embodiment
Embodiment 1
A kind of recovery substratum that can promote the VBNC bacterial growth and improve separate abundance, substratum is formed: by A, B, constitute with the C composition and (be called for short: ABC recovery substratum).
The A composition:
Bacterium liquid: gamboge coccus Micrococcus luteus IAM14879 bacterial classification is cultured to the logarithmic phase later stage on the LMMD substratum, extracts through centrifugal substratum and obtains protein, contains to bring back to life to promote that factor R pf, consumption are 5% of C one-tenth partial volume.
The B composition:
Liquid soil: the garden mould 1kg air-dry, that sieve, remove impurity such as stone grass roots that learns from else's experience adds tap water 1L, 121 ℃, the sterilization of 30min high-pressure sterilizing pot; After room temperature is treated an evening, once again the sterilization, triplicate; The 2000r/min low-speed centrifugal is got supernatant (or with filter paper filtering acquisition filtrating), and consumption is 2% of a C one-tenth partial volume.
The C composition:
Bacto-tryptone (Difco company) (Bacto peptone (Difco)) 10.0 g,
Yeast extract (Yeast extract (Difco)) 5.0 g,
Malt extract (Malt extract (Difco)) 5.0 g,
(hydrolysis) casamino acids (Casamino acids (Difco)) 5.0g,
Carnis Bovis seu Bubali cream (Beef extract (Difco)) 2.0g,
Glycerine (Glycerol) 2.0g,
Tween 80 (Tween 80) 0.05g,
MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 (MgSO
47H
2O) 1.0 g,
Add deionized water 1000mL, pH7.0.
Above substratum is the liquid culture based component, during like the preparation solid medium, need add agar 15-20g, and through 121 ℃, 15min autoclaving postcooling uses.
The LMMD substratum is formed in the A composition:
Ammonium chloride (NH
4Cl) 4.0 g,
Potassium primary phosphate (KH
2PO
4) 1.4 g,
Vitamin H (Biotin) 0.005 g,
L-methionine(Met) (L-Methionine) 0.02 g,
VitB1 (Thiamine) 0.04 g,
Inosine (Inosine) 1.0 g,
MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 (MgSO
47H
2O) 0.07 g,
Mineral solution (Mineral solution) 1.0 ml,
L-lithium lactate (Lithium L-lactate) 10.0 g,
Zero(ppm) water (Distilled water) 1000 ml, pH 7.5.
Wherein, above-mentioned mineral solution (Mineral solution):
Copper sulfate (CuSO
4) 0.24 g,
Manganous chloride tetrahydrate (MnCl
2) 0.50 g,
Ferrous sulfate (FeSO
4) 0.10 g,
Sodium orthomolybdate (Na
2MoO
4) 0.025 g,
Zinc sulfate (ZnSO
4) 0.05 g,
Zero(ppm) water (distilled water) 1000 ml.
More than be that general bacteria resuscitation is cultivated the prescription that uses.According to the different ecological environment requirement, the C composition can be done corresponding adjustment.
Above-mentioned recovery culture medium preparation method:
1. take a morsel gamboge coccus bacterial classification inoculation in the LMMD liquid nutrient medium, 30 C, 120 rpm vibrate cultivation; In bacterial growth to the logarithmic phase later stage, nutrient solution is used 5000 rpm, and thalline is removed in the centrifugal extracting of 20 min; After the sterilization of 0.22 μ m membrane filtration ,-20 ℃ of preservations are subsequent use;
2. the garden mould 1kg air-dry, that sieve, remove impurity such as stone grass roots that learns from else's experience adds tap water 1L, 121 ℃, the sterilization of 30min high-pressure sterilizing pot; After room temperature is treated an evening, use once again with method and sterilize; The 2000r/min low-speed centrifugal is got supernatant (or with filter paper filtering waste acquisition filtrating), and through 121 ℃, 15min high-pressure sterilizing pot sterilization postcooling ,-20 ℃ of preservations are subsequent use;
3. accurately take by weighing each component of C composition, it is for use that pH transfers to 7.0,121 ℃, 15min high-pressure sterilizing pot sterilization postcooling to room temperature.The separation of the suitable most bacteriums of C component is for particular requirement being arranged and selectively bacterium can corresponding plus-minus nutrition composition;
4. before using, should under aseptic condition, add 5% (A/C) A composition in the C composition, add 3% (B/C) B composition again, use behind the mixing.
Embodiment 2
A kind of recovery substratum that can promote the VBNC bacterial growth and improve separate abundance, substratum is formed: by A, B, constitute with the C composition (be called for short: ABC recovery substratum), suitable gram-positive microorganism do not have the mycolic acids Pseudomonas (
AmycolatopsisBelong to).
The A composition:
Bacterium liquid: gamboge coccus Micrococcus luteus IAM14879 bacterial classification is cultured to the logarithmic phase later stage on the LMMD substratum, extracts through centrifugal substratum and obtains protein, contains to bring back to life to promote that factor R pf, consumption are 1% of C one-tenth partial volume.
The B composition:
Liquid soil: the garden mould 1kg air-dry, that sieve, remove impurity such as stone grass roots that learns from else's experience adds tap water 1L, 121 ℃, the sterilization of 30min high-pressure sterilizing pot; After room temperature is treated an evening, once again the sterilization, triplicate; The 2000r/min low-speed centrifugal is got supernatant (or with filter paper filtering acquisition filtrating), and consumption is 10% of a C one-tenth partial volume.
The C composition---prescription:
Glucose Glucose, 4.0 g
Yeast extract Yeast extract, 4.0 g
Fructus Hordei Germinatus extract Malt extract, 10.0 g
Zero(ppm) water Distilled water, 1000.0 ml;
PH 7.2, add during the configuration nutrient agar and use lime carbonate CaCO
3,2.0 g.
Above substratum is the liquid culture based component, during like the preparation solid medium, need add agar 15-20g, and through 121 ℃, 15min autoclaving postcooling uses.
Embodiment 3
A kind of recovery substratum that can promote the VBNC bacterial growth and improve separate abundance, substratum is formed: by A, B, constitute with the C composition (be called for short: ABC recovery substratum), be fit to the gram-positive microorganism genus arthrobacter (
ArthrobacterBelong to), rhodococcus (
RhodococcusBelong to), Lay Fu Shi belong to (
LeifsoniaBelong to), promise Ka Shi belong to (
NocardiaBelong to), in the north spore belong to (
KitasatosporaBelong to), streptomyces (
StreptomycesBelong to), Bacillus subtilus belong to (
BacillusBelong to), series bacillus belong to (
PaenibacillusBelong to).
The A composition:
Bacterium liquid: gamboge coccus Micrococcus luteus IAM14879 bacterial classification is cultured to the logarithmic phase later stage on the LMMD substratum, extracts through centrifugal substratum and obtains protein, contains to bring back to life to promote that factor R pf, consumption are 8% of C one-tenth partial volume.
The B composition:
Liquid soil: the garden mould 1kg air-dry, that sieve, remove impurity such as stone grass roots that learns from else's experience adds tap water 1L, 121 ℃, the sterilization of 30min high-pressure sterilizing pot; After room temperature is treated an evening, once again the sterilization, triplicate; The 2000r/min low-speed centrifugal is got supernatant (or with filter paper filtering acquisition filtrating), and consumption is 15% of a C one-tenth partial volume.
The C composition---prescription:
Peptone Tryptone, 5.0 g
Yeast extract Yeast extract, 2.5 g
Glucose Glucose, 1.0 g
Bata-phenethyl alcohol β-Phenethyl alcohol, 3.0 ml
Kabicidin Kabicidin, 0.05 g
Cycloheximide Cycloheximide, 0.05 g
Deionized water Deionized water, 1000 ml, pH 7.0.
Above substratum is the liquid culture based component, during like the preparation solid medium, need add agar 15-20g, and through 121 ℃, 15min autoclaving postcooling uses.
Embodiment 4
A kind of recovery substratum that can promote the VBNC bacterial growth and improve separate abundance, substratum is formed: by A, B, constitute with the C composition and (be called for short: ABC recovery substratum), be fit to the bacterium in the poor nutrient soil ecology.
The A composition:
Bacterium liquid: gamboge coccus Micrococcus luteus IAM14879 bacterial classification is cultured to the logarithmic phase later stage on the LMMD substratum, extracts through centrifugal substratum and obtains protein, contains to bring back to life to promote that factor R pf, consumption are 15% of C one-tenth partial volume.
The B composition:
Liquid soil: the garden mould 1kg air-dry, that sieve, remove impurity such as stone grass roots that learns from else's experience adds tap water 1L, 121 ℃, the sterilization of 30min high-pressure sterilizing pot; After room temperature is treated an evening, once again the sterilization, triplicate; The 2000r/min low-speed centrifugal is got supernatant (or with filter paper filtering acquisition filtrating), and consumption is 18% of a C one-tenth partial volume.
The C composition---prescription:
Peptone Tryptone, 5.0 g
Yeast extract Yeast extract, 2.5 g
Glucose Glucose, 1.0 g
Bata-phenethyl alcohol β-Phenethyl alcohol, 3.0 ml
Kabicidin Kabicidin, 0.05 g
Cycloheximide Cycloheximide, 0.05 g
Deionized water Deionized water, 1000 ml, pH 7.0.
Above substratum is the liquid culture based component, during like the preparation solid medium, need add agar 15-20g, and through 121 ℃, 15min autoclaving postcooling uses.
Embodiment 4
A kind of recovery substratum that can promote the VBNC bacterial growth and improve separate abundance, substratum is formed: by A, B, constitute with the C composition and (be called for short: ABC recovery substratum), be fit to the bacterium in the poor nutrition deep water ecology.
The A composition:
Bacterium liquid: gamboge coccus Micrococcus luteus IAM14879 bacterial classification is cultured to the logarithmic phase later stage on the LMMD substratum, extracts through centrifugal substratum and obtains protein, contains to bring back to life to promote that factor R pf, consumption are 25% of C one-tenth partial volume.
The B composition:
Liquid soil: the garden mould 1kg air-dry, that sieve, remove impurity such as stone grass roots that learns from else's experience adds tap water 1L, 121 ℃, the sterilization of 30min high-pressure sterilizing pot; After room temperature is treated an evening, once again the sterilization, triplicate; The 2000r/min low-speed centrifugal is got supernatant (or with filter paper filtering acquisition filtrating), and consumption is 1% of a C one-tenth partial volume.
The C composition---prescription:
Casein Casein, 1.70 g
Dregs of beans Soybean meal, 0.30 g
Sodium chloride nacl, 0.50 g
Potassium hydrogenphosphate K
2HPO
4,0.25 g
Glucose Dextrose, 0.25 g
Zero(ppm) water Distilled water, 1000 ml; PH 7.3.
Above substratum is the liquid culture based component, during like the preparation solid medium, need add agar 15-20g, and through 121 ℃, 15min autoclaving postcooling uses.
Application examples 1
Liquid nutrient medium separates the VBNC bacterium.Get 1 part of separation source (water or soil) and add 9 parts of embodiment 3 described ABC recovery substratum, fully mixing is made mother liquor, through 10 times of gradient dilutions to a certain degree (water sample is generally 10-6, and soil sample generally should be 10-8); Identical component solid plate substratum with adding 2% agar is coated with, and dull and stereotyped the inversion cultivated (general bacterium 24-48h, actinomycetes 5-14d) under the conventional culture temperature of bacterium, picking list bacterium colony, and enlarged culturing obtains pure strain.The pure strain that obtains can carry out various tests; As 16S rRNA gene amplification also being checked order through PCR; GenBank does BLAST retrieval contrast in the NCBI website, can roughly identify the level that this pure strain belongs to, and helps to analyze this pure strain and whether is derived from the VBNC state.
Application examples 2
Solid medium is to being derived from the activation culture that VBNC can the cultivation bacterial classification.Through embodiment 1 described ABC recovery screening of medium to some bacterial strains in culture of continuous cultivation; Under the situation that particularly leaves standstill for a long time; Possibly get back to the VBNC state, at this moment, can be with certain bacterial classification of testing the preceding cultured strain of not growing on the substratum or being kept at very low temperature-80 ℃; Through being inoculated on the ABC recovery substratum, the part bacterial classification can recover growth.
Application examples 3
The confirmation method of VBNC bacterial classification.Above use embodiment 1 described ABC recovery substratum is separated the bacterial classification that obtains whether belong to the bacterium that is in VBNC in the separation source; Can be with ABC recovery substratum and the BC substratum that does not add A as contrast, adopt conventional MPN counting process and DGGE or the analysis of FISH equimolecular biological method to demonstrate,prove.
Test Example 1
The separation of VBNC bacterium in the soil.To 68 pedotheques from park, island and forest, adopt embodiment 3 described ABC recovery substratum to be separated to belong to the VBNC state through recovery become can cultivation 40 bacterial strains, improve 5-490 separation abundance doubly.Wherein high GC Gram positive actinomycetes accounts for 63%, and low GC gram-positive microorganism accounts for 37%, finds a novel species bacterium at least.
Test Example 2
The separation of VBNC bacterium in the Sewage treatment systems.In the city sewage deep treatment system, adopt A embodiment 3 described BC recovery substratum to be separated to belong to the VBNC state through recovery become can cultivation 10 bacterial strains, be high GC Gram positive actinomycetes, find 6 novel species bacteriums at least.
Test Example 3
The difficult growth-promoting effect of cultivating gram negative bacterium.Belong to (spiral Pseudomonas), Curvibacter and belong to the difficult Gram-negative bacterial classification of cultivating of the part of (crooked bacterium belongs to) and the A composition in the embodiment 4 described ABC recovery substratum is added to the result finds the obvious growth promoter action in the former substratum cultivate Aquaspirillum from the deep-well shipwreck; Cell concentration increases more than 100 times, makes 3 difficult cultivation novel species bacteriums obtain to identify.
Test Example 4
The recovery that is derived from the VBNC bacterium promotes to keep cultivating.To belonging to the actinomycetes of VBNC state from soil through embodiment 1 described ABC recovery substratum, this bacterium is returned to the VBNC state again after leaving standstill for a long time.Adopt the ABC recovery to cultivate the back and recover nearly 100 times growth vigor.
Comparative Examples 1
When in utilizing resuscitation fluid separation soil, being in VBNC state bacterium, observing having contrasted and add soil extraction liquid separating the effect of soil bacteria.The making method that soil extracts liquid is: the garden mould 1kg air-dry, that sieve, remove impurity that learns from else's experience adds tap water 1L, 121 ℃, the sterilization of 30min high-pressure sterilizing pot; After room temperature is treated an evening, use once again with method and sterilize; The 2000r/min low-speed centrifugal is got supernatant or is obtained filtrating with the filter paper filtering waste, and through 121 ℃, 15min high-pressure sterilizing pot sterilization postcooling, addition is 1% (v/v); Maximum MPN method (MPN) is adopted in experimental observation, and looks into MPN numerical tabular counting, and the result of triplicate is following.
Fig. 1 is the park soil sample, and the bacterium number that basic medium is separated to is that visible bacterium number was 9.3x10 in the 2nd day
5Cfu/g (bacterium in every gram soil sample forms colony count, down together), adding liquid soil is 9.3x10
6Cfu/g, both bacterium numbers all are stabilized in 9.3x10 after the 3rd day
6Cfu/g; Adding the 2nd day bacterium number of resuscitation fluid is 9.3x10
6The bacterium number that cfu/g, resuscitation fluid add liquid soil is 1.5x10
7Cfu/g; It is 4.3x10 that the 4th day resuscitation fluid adds the bacterium number of liquid soil
8Cfu/g, and list reached identical bacterium number on the 5th day with resuscitation fluid, was stabilized in 4.3x10 later on
8Cfu/g shows that soil extracts the growth that fluid power promotes bacterium.Arrived the highest separable bacterial count in one day approximately ahead of time with regard to seeing on its time than adding, see effect and not obvious on the bacteria total amount but just obtain.
Fig. 2 is an island sand ground soil sample, and the bacterium number that basic medium is separated to is that visible bacterium number was 2.1x10 in the 2nd day
5Cfu/g, adding liquid soil is 9.3x10
6Cfu/g, both bacterium numbers all are stabilized in 1.5x10 after the 3rd day
6Cfu/g; Adding the 2nd day bacterium number of resuscitation fluid is 2.4x10
6The bacterium number that cfu/g, resuscitation fluid add liquid soil is 4.6x10
6Cfu/g; It is 1.5x10 that the 4th day resuscitation fluid adds the bacterium number of liquid soil
7Cfu/g reaches the highest number of cultivating, and single reaches identical bacterium number on the 5th day with resuscitation fluid, has shown that equally soil extracts the growth that fluid power promotes bacterium, and arrival in one day approximately ahead of time is the effect of high separable bacterial count.
Experimental observation is the result show, adding soil extraction liquid has the obvious growth promoter action to separating soil bacteria.Add the raising of recovery fluid power and obtain VBNC state bacterium in the soil, separate abundance and can reach 10-46 doubly.
Claims (10)
1. one kind can promote the VBNC bacterial growth and improve the recovery substratum that separates abundance, and it is characterized in that: this recovery substratum is made up of substratum base fluid, bacterium liquid and liquid soil; Described bacterium liquid is cultured to the logarithmic phase later stage by the micrococcus luteus bacterial classification on the bacterial classification liquid nutrient medium, extract through centrifugal removal bacterial classification liquid nutrient medium and obtain protein, contains to bring back to life to promote factor R pf, and bacterium liquid is the 0.5-30% of substratum base fluid by volume; Described liquid soil adds water by through soil air-dry, that sieve, remove impurity, and leaching filtrating or centrifuging and taking supernatant are crossed in repeatedly sterilization, and liquid soil is the 0.1-20% of substratum base fluid by volume.
2. a kind of recovery substratum that can promote the VBNC bacterial growth and improve the separation abundance according to claim 1 is characterized in that bacterium liquid is the 1-10% of substratum base fluid by volume, and liquid soil is the 1-5% of substratum base fluid by volume.
3. a kind of recovery substratum that can promote the VBNC bacterial growth and improve separate abundance according to claim 1 and 2 is characterized in that the substratum base fluid selects a kind of in the following prescription for use:
A, general bacteria resuscitation are cultivated the prescription that uses:
Bacto-tryptone 8.0 ~ 15.0g,
Yeast extract 3.0 ~ 8.0g,
Malt extract 3.0 ~ 8.0g,
Carnis Bovis seu Bubali cream 1.0 ~ 3.0g,
Casamino acids 3.0 ~ 8.0g,
Glycerine 1.0 ~ 3.0g,
Tween-80 0.03 ~ 0.1g,
MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.5 ~ 2.0g,
Add deionized water 1000mL, regulate pH7.0;
B, suitable gram-positive microorganism do not have the prescription of mycolic acids Pseudomonas:
Glucose 2.0 ~ 6.0g,
Yeast extract 2.0 ~ 6.0g,
Fructus Hordei Germinatus extract 0.6 ~ 2.0 g,
Zero(ppm) water 1000.0 ml, pH 7.2, add with lime carbonate 1.0 ~ 3.0g during the configuration nutrient agar;
The prescription that spore genus, streptomyces, Bacillus subtilus genus, series bacillus belong in C, suitable gram-positive microorganism genus arthrobacter, rhodococcus, Lay Fu Shi genus, promise Ka Shi genus, the north:
Peptone 3.0 ~ 8.0 g
Yeast extract 1.0 ~ 5.5 g,
Glucose 0.5 ~ 2.0 g,
Bata-phenethyl alcohol 2.0 ~ 4.0ml,
Kabicidin 0.03 ~ 0.08 g,
Cycloheximide 0.02 ~ 0.08 g,
Deionized water 1000 ml, pH 7.0;
The prescription of bacterium in D, the suitable poor nutrient soil ecology:
Nutrient broth 1.0 ~ 2.50 g,
Yeast extract 0.20 ~ 1.0g,
Zero(ppm) water 1000 ml, pH 7.0;
The prescription of bacterium in E, the suitable poor nutrition deep water ecology:
Casein 1.0 ~ 3.0 g,
Dregs of beans 0.10 ~ 0.50 g,
Sodium-chlor 0.20 ~ 1.0 g,
Potassium hydrogenphosphate 0.1 ~ 0.5 g,
Glucose 0.1 ~ 0.5 g,
Zero(ppm) water 1000 ml, pH 7.3.
4. a kind of recovery substratum that can promote the VBNC bacterial growth and improve the separation abundance according to claim 3; When it is characterized in that preparing solid medium; In each assembly side of described A, B, C, D or E, add agar 15 ~ 20g, through 110 ~ 130 ℃, the use of 10 ~ 30min autoclaving postcooling.
5. a method for preparing claim 1 or 2 described recovery substratum is characterized in that this method comprises the steps:
1) take a morsel gamboge coccus bacterial classification inoculation in the bacterial classification liquid nutrient medium, 25 ~ 35 C, 100 ~ 150rpm vibrate cultivation; In bacterial growth to the logarithmic phase later stage, nutrient solution is with 3000 ~ 8000rpm, and thalline is removed in the centrifugal extracting of 10 ~ 30min; After the sterilization of 0.20 ~ 0.25 μ m membrane filtration ,-30 ~-10 ℃ of preservations are subsequent use;
2) the garden mould 1kg air-dry, that sieve, remove impurity that learns from else's experience adds tap water 1 ~ 2L, 110 ~ 130 ℃, the sterilization of 10 ~ 40min high-pressure sterilizing pot; After room temperature is treated an evening, use once again with method and sterilize; 1500 ~ 2500r/min low-speed centrifugal is got supernatant or is obtained filtrating with the filter paper filtering waste, and through 110 ~ 130 ℃, 10 ~ 30min high-pressure sterilizing pot sterilization postcooling ,-30 ~-10 ℃ of preservations are subsequent use;
3) accurately take by weighing each component of substratum base fluid, it is for use that pH transfers to 7.0,110 ~ 130 ℃, 10 ~ 30min high-pressure sterilizing pot sterilization postcooling to room temperature; The separation of the suitable most bacteriums of substratum base fluid is for particular requirement being arranged and selectively bacterium can corresponding plus-minus nutrition composition;
4) before the use, under aseptic condition, add the bacterium liquid of respective amount in the substratum base fluid, add the liquid soil of respective amount again, use behind the mixing.
6. a kind of method for preparing the recovery substratum according to claim 5 is characterized in that the prescription of bacterial classification liquid nutrient medium is following:
Ammonium chloride 3.0 ~ 5.0g,
Potassium primary phosphate 1.2 ~ 1.6g,
Vitamin H 0.003 ~ 0.008g,
L-methionine(Met) 0.01 ~ 0.04g,
VitB1 0.02 ~ 0.05g,
Inosine 0.8 ~ 1.5g,
MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.05 ~ 0.1g,
Mineral solution 0.8 ~ 1.5ml,
L-lithium lactate 5.0 ~ 20.0g,
Zero(ppm) water 1000ml, pH7.5.
7. a kind of method for preparing the recovery substratum according to claim 6 is characterized in that the prescription of mineral solution is following:
Copper sulfate 0.20 ~ 0.30g,
Manganous chloride tetrahydrate 0.30 ~ 0.80g,
Ferrous sulfate 0.08 ~ 0.20g,
Sodium orthomolybdate 0.01 ~ 0.03g,
Zinc sulfate 0.02 ~ 0.08g,
Zero(ppm) water 1000ml.
8. one kind can promote the VBNC bacterial growth and improve the method for separating abundance, it is characterized in that this method comprises the steps:
1) get 1 part of separation source and add 7 ~ 12 parts of right requirement 1 or 2 described recovery substratum, fully mixing is made mother liquor, arrives to a certain degree through 10 times of gradient dilutions;
2) be coated with the identical component solid plate substratum that adds 2% agar, the dull and stereotyped inversion cultivated under the conventional culture temperature of bacterium, general bacterium 24-48h, and actinomycetes 5-14d, picking list bacterium colony, enlarged culturing obtains pure strain.
9. method that can promote VBNC bacteria resuscitation growth; It is characterized in that this method will test the preceding cultured strain of not growing on the substratum or be kept at the bacterial classification of very low temperature-80 ℃; Be inoculated on claim 1 or the 2 described recovery substratum, the part bacterial classification recovers growth.
10. the confirmation method of a VBNC bacterial classification; It is characterized in that whether this method belongs to the bacterium that is in VBNC in the separation source to using claim 1 or 2 described recovery substratum to separate the bacterial classification that obtains; The recovery substratum that adopts claim 1 or 2 described recovery substratum and do not add bacterium liquid is as contrast, adopts conventional MPN counting process, DGGE or the analysis of FISH molecular biology method to demonstrate,prove.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103436460A (en) * | 2013-06-24 | 2013-12-11 | 浙江大学 | Method for enriching efficient biphenyl degradation flora by using accelerator SRpf |
| CN109536431A (en) * | 2018-12-04 | 2019-03-29 | 江苏省农业科学院 | Composite bio-chemical Resuscitation promoting factor composition and its application |
| CN109626595A (en) * | 2019-01-14 | 2019-04-16 | 浙江师范大学 | Purification of water quality microbial ecological unit, purification of water quality microbial ecosystem and its method |
| CN109626595B (en) * | 2019-01-14 | 2024-06-11 | 浙江师范大学 | Water quality purifying microorganism ecological unit, water quality purifying microorganism ecological system and method thereof |
| CN110484465A (en) * | 2019-08-01 | 2019-11-22 | 浙江浩岚投资有限公司 | A kind of recovery medium and the preparation method and application thereof of VBNC bacterium |
| CN110484465B (en) * | 2019-08-01 | 2021-06-04 | 金华康扬环境科技有限公司 | Resuscitation medium of VBNC bacteria and preparation method and application thereof |
| CN116286364A (en) * | 2022-09-08 | 2023-06-23 | 杭州秀川科技有限公司 | Compound for promoting anaerobic microorganism separation and culture and application thereof |
| CN116286364B (en) * | 2022-09-08 | 2023-11-10 | 杭州秀川科技有限公司 | Compound for promoting anaerobic microorganism separation and culture and application thereof |
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