CN117903997A - Rhodococcus rhodochrous selective separation culture medium and preparation method and application thereof - Google Patents
Rhodococcus rhodochrous selective separation culture medium and preparation method and application thereof Download PDFInfo
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- CN117903997A CN117903997A CN202410206856.8A CN202410206856A CN117903997A CN 117903997 A CN117903997 A CN 117903997A CN 202410206856 A CN202410206856 A CN 202410206856A CN 117903997 A CN117903997 A CN 117903997A
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- rhodococcus equi
- culture medium
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- selective
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- 239000001963 growth medium Substances 0.000 title claims abstract description 81
- 238000000926 separation method Methods 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 241000187693 Rhodococcus rhodochrous Species 0.000 title description 7
- 241000158504 Rhodococcus hoagii Species 0.000 claims abstract description 99
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims abstract description 34
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims abstract description 32
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 claims abstract description 26
- 229920002472 Starch Polymers 0.000 claims abstract description 17
- 235000015278 beef Nutrition 0.000 claims abstract description 17
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 17
- 239000005018 casein Substances 0.000 claims abstract description 17
- 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 abstract description 17
- 235000021240 caseins Nutrition 0.000 claims abstract description 17
- 239000000284 extract Substances 0.000 claims abstract description 17
- 229940107700 pyruvic acid Drugs 0.000 claims abstract description 17
- 235000019698 starch Nutrition 0.000 claims abstract description 17
- 239000008107 starch Substances 0.000 claims abstract description 17
- 239000012138 yeast extract Substances 0.000 claims abstract description 17
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 16
- FRHBOQMZUOWXQL-UHFFFAOYSA-L ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 claims abstract description 16
- 239000001110 calcium chloride Substances 0.000 claims abstract description 16
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 16
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 16
- 229960004642 ferric ammonium citrate Drugs 0.000 claims abstract description 16
- 239000004313 iron ammonium citrate Substances 0.000 claims abstract description 16
- 235000000011 iron ammonium citrate Nutrition 0.000 claims abstract description 16
- 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 16
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims abstract description 16
- 229940099596 manganese sulfate Drugs 0.000 claims abstract description 16
- 239000011702 manganese sulphate Substances 0.000 claims abstract description 16
- 235000007079 manganese sulphate Nutrition 0.000 claims abstract description 16
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims abstract description 16
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 16
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 16
- 239000004323 potassium nitrate Substances 0.000 claims abstract description 16
- 235000010333 potassium nitrate Nutrition 0.000 claims abstract description 16
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229960001082 trimethoprim Drugs 0.000 claims abstract description 16
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 16
- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 16
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 16
- 229920001817 Agar Polymers 0.000 claims abstract description 15
- 239000008272 agar Substances 0.000 claims abstract description 15
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims abstract description 15
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims abstract description 15
- BFPJYWDBBLZXOM-UHFFFAOYSA-L potassium tellurite Chemical compound [K+].[K+].[O-][Te]([O-])=O BFPJYWDBBLZXOM-UHFFFAOYSA-L 0.000 claims abstract description 15
- GCFBRXLSHGKWDP-XCGNWRKASA-N cefoperazone Chemical compound O=C1C(=O)N(CC)CCN1C(=O)N[C@H](C=1C=CC(O)=CC=1)C(=O)N[C@@H]1C(=O)N2C(C(O)=O)=C(CSC=3N(N=NN=3)C)CS[C@@H]21 GCFBRXLSHGKWDP-XCGNWRKASA-N 0.000 claims abstract description 14
- 229960004682 cefoperazone Drugs 0.000 claims abstract description 14
- 108010093965 Polymyxin B Proteins 0.000 claims abstract description 13
- 229920000024 polymyxin B Polymers 0.000 claims abstract description 13
- 229960005266 polymyxin b Drugs 0.000 claims abstract description 13
- 239000002689 soil Substances 0.000 claims description 46
- 239000002609 medium Substances 0.000 claims description 44
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 36
- 239000000843 powder Substances 0.000 claims description 25
- 238000012258 culturing Methods 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 239000000203 mixture Substances 0.000 claims description 19
- 238000002955 isolation Methods 0.000 claims description 18
- 239000002253 acid Substances 0.000 claims description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 16
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 16
- 239000001632 sodium acetate Substances 0.000 claims description 16
- 235000017281 sodium acetate Nutrition 0.000 claims description 16
- 235000006408 oxalic acid Nutrition 0.000 claims description 12
- 239000003242 anti bacterial agent Substances 0.000 claims description 11
- 229940088710 antibiotic agent Drugs 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 229910052799 carbon Inorganic materials 0.000 claims description 8
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 8
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 238000010790 dilution Methods 0.000 claims description 7
- 239000012895 dilution Substances 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 239000013049 sediment Substances 0.000 claims description 7
- 239000008223 sterile water Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 241001465754 Metazoa Species 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- JNKJTXHDWHQVDL-UHFFFAOYSA-N potassiotellanylpotassium Chemical compound [K][Te][K] JNKJTXHDWHQVDL-UHFFFAOYSA-N 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 230000002906 microbiologic effect Effects 0.000 abstract description 2
- 229940023476 agar Drugs 0.000 abstract 1
- 229940093928 potassium nitrate Drugs 0.000 abstract 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 abstract 1
- 230000012010 growth Effects 0.000 description 42
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- 238000011084 recovery Methods 0.000 description 18
- 239000000243 solution Substances 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 11
- 241000283073 Equus caballus Species 0.000 description 11
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 10
- 239000011248 coating agent Substances 0.000 description 10
- 238000000576 coating method Methods 0.000 description 10
- 238000007865 diluting Methods 0.000 description 10
- 244000005700 microbiome Species 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 241000191967 Staphylococcus aureus Species 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 230000005764 inhibitory process Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 244000063299 Bacillus subtilis Species 0.000 description 7
- 235000014469 Bacillus subtilis Nutrition 0.000 description 7
- 241000194032 Enterococcus faecalis Species 0.000 description 7
- 241000228150 Penicillium chrysogenum Species 0.000 description 7
- 241000233639 Pythium Species 0.000 description 7
- 241000194048 Streptococcus equi Species 0.000 description 7
- 241000187759 Streptomyces albus Species 0.000 description 7
- 229940032049 enterococcus faecalis Drugs 0.000 description 7
- 241000159512 Geotrichum Species 0.000 description 6
- 238000009630 liquid culture Methods 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 239000006152 selective media Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000000654 additive Substances 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 241000233866 Fungi Species 0.000 description 3
- 108010040201 Polymyxins Proteins 0.000 description 3
- 206010065041 Rhodococcus infection Diseases 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003864 humus Substances 0.000 description 3
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- 241001655308 Nocardiaceae Species 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- HVFLCNVBZFFHBT-ZKDACBOMSA-N cefepime Chemical compound S([C@@H]1[C@@H](C(N1C=1C([O-])=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1C[N+]1(C)CCCC1 HVFLCNVBZFFHBT-ZKDACBOMSA-N 0.000 description 2
- 229960002100 cefepime Drugs 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
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- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910021645 metal ion Inorganic materials 0.000 description 2
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241001465318 Aspergillus terreus Species 0.000 description 1
- 201000004813 Bronchopneumonia Diseases 0.000 description 1
- 238000009641 CAMP test Methods 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241000187562 Rhodococcus sp. Species 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000191963 Staphylococcus epidermidis Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000003782 beta lactam antibiotic agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000006161 blood agar Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000006781 columbia blood agar Substances 0.000 description 1
- -1 cyclic imide Chemical class 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 244000038280 herbivores Species 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229950003442 methoprene Drugs 0.000 description 1
- 229930002897 methoprene Natural products 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
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- 235000013343 vitamin Nutrition 0.000 description 1
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- 229930003231 vitamin Natural products 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics 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
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a rhodococcus equi selective separation culture medium, a preparation method and application thereof, belonging to the fields of microbiological technology and environmental microbiology. The culture medium comprises the following components: soluble starch, pyruvic acid, sodium acetate, acid hydrolyzed casein, beef extract, yeast extract, calcium chloride, monopotassium phosphate, disodium hydrogen phosphate, potassium nitrate, zinc sulfate, manganese sulfate, magnesium sulfate heptahydrate, ferric ammonium citrate, potassium tellurite, agar, trimethoprim, cefoperazone, polymyxin B and cycloheximide.
Description
Technical Field
The invention belongs to the fields of microbiological technology and environmental microbiology, and particularly relates to a rhodococcus equi selective separation culture medium, a preparation method and application thereof.
Background
Rhodococcus equi is a veterinary accepted bacterial pathogen, a gram-positive obligate aerobic bacterium of the genus Rhodococcus (Rhodococcus sp.) of the family nocardiaceae (Nocardiaceae), the growth morphology of which varies with the growth conditions and growth cycle. It is widely distributed in nature, especially in soil and herbivore feces, and there are a large number of rhodococcus equi strains in the horse cultivation environment. The foal infected by the colts can generate severe chronic suppurative bronchopneumonia, and the mortality rate is high after the disease, so that the loss of the horse farming industry is huge. Although rare, it is also sometimes observed in other mammalian infections, and is common in immunocompromised individuals such as human immunodeficiency virus infected patients, and the main clinical symptoms are fever, headache, and weakness.
Rhodococcus equi is cultured on a blood plate at 37 ℃ to slowly proliferate, and the strain has small colony, is moist, smooth, semitransparent and mucilaginous, and can produce characteristic orange-red pigment. Rhodococcus equi cultured on sheep blood agar plates can undergo synergistic hemolysis with staphylococcus aureus and listeria monocytogenes (positive in CAMP test), and antagonism between iminothiolycin and other beta-lactam antibiotics widely exists in rhodococcus equi isolates.
Rhodococcus rhodochrous is generally deposited in soil and intestinal tracts of foal, how to accurately screen rhodococcus rhodochrous from the soil is an important means for effectively monitoring rhodochrous infection in a horse yard, a Columbia blood agar plate is generally used for culturing rhodochrous and observing colony morphology of rhodochrous at present, however, rhodochrous grows slowly on the culture medium, the colony is smaller, a large number of microorganisms such as bacteria and fungi and humus exist in the soil of an animal culture area, no selective separation culture medium for rhodococcus rhodochrous exists in domestic market at present, and rhodococcus rhodochrous cannot be accurately separated from detection samples with a large number of other microorganisms such as bacteria. In order to further strengthen the understanding of the bacteria in the laboratory and reduce rhodococcus equi infection in the horse field, a culture medium which can effectively meet the growth, metabolism and propagation of rhodococcus equi needs to be developed, and the culture medium is beneficial to timely monitoring the change of rhodococcus equi in the environment of the horse field and is beneficial to discovering new therapeutic drugs, so that the ecological research of rhodococcus equi is enhanced.
Disclosure of Invention
The invention aims to provide a rhodococcus equi selective separation medium for improving the separation rate of rhodococcus equi in the soil of an animal cultivation area, and a preparation method and application thereof, wherein the medium is prepared by optimizing the nutrient components such as a carbon source, a nitrogen source, antibiotics and the like in the rhodococcus equi selective separation medium, so that the medium suitable for screening and separating rhodococcus equi from the soil is prepared, and the technical scheme adopted by the invention is as follows: a rhodococcus equi selective isolation medium, the rhodococcus equi selective isolation medium comprising: carbon source, nitrogen source, antibiotics, 0.005-0.015 g/L of calcium chloride, 0.3-0.5 g/L of monopotassium phosphate, 0.5-1.0 g/L of disodium hydrogen phosphate, 0.05-0.15 g/L of potassium nitrate, 0.01-0.02 g/L of zinc sulfate, 0.01-0.03 g/L of manganese sulfate, 0.1-0.2 g/L of magnesium sulfate heptahydrate, 0.03-0.07 g/L of ferric ammonium citrate, 0.01-0.02 g/L of potassium tellurite and 16-18 g/L of agar;
The carbon source comprises the following components: 1-5 g/L of soluble starch, 1-5 g/L of pyruvic acid and 1-5 g/L of sodium acetate;
the nitrogen source comprises the following components: 10-25 g/L of acid hydrolyzed casein, 1-10 g/L of beef extract powder and 1-5 g/L of yeast extract powder;
The composition of the antibiotics is as follows: 1 to 10mg/L of trimethoprim, 1 to 10mg/L of cefoperazone, 1 to 10mg/L of polymyxin B and 50 to 200mg/L of cycloheximide.
A preparation method of a rhodococcus equi selective separation culture medium is characterized by weighing 1-5 g of soluble starch, 1-5 g of pyruvic acid, 1-5 g of sodium acetate, 10-25 g of acid hydrolyzed casein, 1-10 g of beef extract, 1-5 g of yeast extract, 0.005-0.015 g of calcium chloride, 0.3-0.5 g of monopotassium phosphate, 0.5-1.0 g of disodium hydrogen phosphate, 0.05-0.15 g of potassium nitrate, 0.01-0.02 g of zinc sulfate, 0.01-0.03 g of manganese sulfate, 0.1-0.2 g of magnesium sulfate heptahydrate, 0.03-0.07 g of ferric ammonium citrate, 0.01-0.02 g of potassium tellurite and 16-18 g of agar, dissolving in deionized water, fixing volume to 1000 mL, adjusting pH to 7.5-7.6 with sodium carbonate and citric acid, sterilizing at 121 ℃ for 15 minutes under high pressure, cooling to 50 ℃, adding 1-10 mg of methoprene, 1-10 mg of cefepime, 1-10 mg of perzine B and 200mg of perzine B, and carrying out selective perfusion culture medium to obtain sterile rhodococcus equi.
The Rhodococcus equi selective separation medium is applied to the separation of Rhodococcus equi in the soil of an animal cultivation area, namely the microorganism is Rhodococcus equi (Rhodococcus equi).
The culture temperature was 30 ℃.
The culture time is 48-72 h.
The application method of the rhodococcus equi selective separation culture medium comprises the following steps:
(1) Placing the collected soil sample in a sterile plate, naturally drying, and grinding into powder by a sterile pestle;
(2) Weighing 1g of soil sample, adding the soil sample into 10 mL of 2% oxalic acid, oscillating 20 min in a shaking table of 180 r.min -1, and naturally precipitating 5 min;
(3) The supernatant was poured off and centrifuged at 3000 rpm at 5 min;
(4) Suspending the sediment in 5 mL of 1% sterile peptone water, sucking 1 mL soil suspension into 9 mL sterile water, and carrying out 10-time gradient dilution to the concentration of 1 g.L -1;
(5) 200. Mu.L of the culture medium was spread on a plate of the isolation medium and cultured in a constant temperature incubator at 30 ℃.
The rhodococcus equi selective separation culture medium provided by the invention is used for enrichment culture, separation and screening of rhodococcus equi for non-disease diagnosis.
The beneficial effects of the invention are as follows:
(1) The culture medium provided by the invention has the advantages that the raw materials adopted by the culture medium are cheap and easy to obtain, the economic cost is low, the nutrition components of the culture medium are optimized through multiple tests, and a proper amount of microelements required by normal growth of rhodococcus equi are added, so that not only is the nutrition requirement of rhodococcus equi met, but also the colony morphology of rhodococcus equi is more characteristic, the growth speed of a specific strain in the strain separation process can be increased, the separation time is shortened, the separation efficiency is improved, the enrichment culture of rhodococcus equi can be realized, the growth, metabolism and reproduction of rhodococcus equi in the equine environment are met, the detection and monitoring capability of rhodococcus equi in the culture environment is improved, and the prevention and control of rhodococcus equi infection in animal culture areas such as a horse farm are facilitated;
(2) According to the method, by utilizing the difference of the sensitivity degree of different microorganisms in soil to different medicaments, the rhodococcus equi inhibiting effect is weak, and the antibiotics with strong inhibiting effect on various bacteria such as enterobacter, staphylococcus, streptococcus, salmonella and some fungi such as penicillium and aspergillus are added into the culture medium, so that rhodococcus equi grows slowly or does not grow in the environment, the success rate of separating rhodococcus equi from the soil can be effectively improved, and the operation time is greatly saved;
(3) When the rhodococcus equi selective separation culture medium is prepared, the components except antibiotics are mixed, heated, sterilized and cooled, and then antibiotics such as trimethoprim and the like are added, so that the basic structure and the properties of the antibiotics are not damaged, and the culture medium can well inhibit other miscellaneous bacteria;
(4) The preparation process of the culture medium is simple and convenient to operate, the time required for manufacturing is short, complex supporting equipment is not needed, manpower and material resources in the actual production process can be reduced, the practical operation feasibility is strong, and the use convenience is high;
(5) When the culture medium is applied to separating rhodococcus equi from a soil sample, the sample to be detected is primarily treated with oxalic acid with the concentration of 2%, and by influencing the acid-base balance of the soil, the mixed bacteria, metal ions and humus in the soil can be primarily eliminated, and the rhodococcus equi has stronger tolerance to oxalic acid environment, so that the biomass of rhodococcus equi in the sample is improved.
Drawings
FIG. 1 is a graph showing comparison of recovery rates of rhodococcus equi from the selective isolation medium of examples 1-4;
FIG. 2 is a graph comparing growth curves of rhodococcus equi in selective media in examples 1-4;
FIG. 3 is a graph of the growth inhibition capacity of the selective media of examples 1-4 versus rhodococcus equi;
FIG. 4 is a graph showing the comparison of the effect of selective media on screening of rhodococcus equi from soil in examples 1-4.
Detailed Description
The following three embodiments will be described to more clearly show the technical solutions and advantages of the present invention. By adjusting the nutrition composition of the culture medium, the selective separation effect of the rhodococcus equi is evaluated from the aspects of the growth promotion capability of the culture medium and the inhibition capability of the rhodococcus equi by using methods such as count recovery, growth curve test and the like.
Soil samples in the following examples were all from the same horse farm locally, surface soil samples were collected in the region where the horse survived in the farm, and after soil sample collection were kept in ice bags and boxes and immediately returned to the laboratory for processing.
The standard strains used in the following examples were purchased from China general microbiological culture Collection center, and the instruments and reagents used were commercially available as conventional products.
Table 1 reference strains for experiments
| Bacterial name | Bacterial number |
| Rhodococcus rhodochrous | CGMCC1.6322 |
| Rhodococcus faecalis | CGMCC4.1813 |
| Streptococcus equi | CGMCC1.10838 |
| Enterococcus faecalis | CGMCC1.125 |
| Staphylococcus aureus | CGMCC1.4519 |
| Pseudomonas aeruginosa | CGMCC1.1785 |
| Bacillus subtilis | CGMCC1.439 |
| Streptomyces albus | CGMCC4.7658 |
| Aspergillus terreus | CGMCC3.3934 |
| Penicillium chrysogenum | CGMCC3.15539 |
| Pythium isox | CGMCC3.17815 |
1. The formula of the culture medium comprises:
Per liter of medium contained: carbon source, nitrogen source, antibiotics, 0.005-0.015 g of calcium chloride, 0.3-0.5 g of monopotassium phosphate, 0.5-1.0 g of disodium hydrogen phosphate, 0.05-0.15 g of potassium nitrate, 0.01-0.02 g of zinc sulfate, 0.01-0.03 g of manganese sulfate, 0.1-0.2 g of magnesium sulfate heptahydrate, 0.03-0.07 g of ferric ammonium citrate, 0.01-0.02 g of potassium tellurite and 16-18 g of agar;
Wherein the carbon source composition is: 1-5 g/L of soluble starch, 1-5 g/L of pyruvic acid and 1-5 g/L of sodium acetate; soluble starch, pyruvic acid and sodium acetate provide the carbon source required for rhodococcus equi growth;
the nitrogen source comprises the following components: 10-25 g/L of acid hydrolyzed casein, 1-10 g/L of beef extract powder and 1-5 g/L of yeast extract powder; acid hydrolyzed casein, beef extract and yeast extract provide nitrogen sources, vitamins and amino acids for the growth of rhodococcus equi;
The composition of the antibiotics is as follows: 1 to 10mg/L of trimethoprim, 1 to 10mg/L of cefoperazone, 1 to 10mg/L of polymyxin B and 50 to 200mg/L of cycloheximide. The trimethoprim can interfere folic acid metabolism of bacteria, has inhibition effect on various gram-positive bacteria and gram-negative bacteria, the cefoperazone belongs to a broad-spectrum antibiotic, has better antibacterial activity on common staphylococcus aureus, staphylococcus epidermidis, beta-hemolytic streptococcus, escherichia coli and the like, and the polymyxin B has obvious inhibition effect on gram-negative bacteria such as pseudomonas aeruginosa, klebsiella and the like, and the cycloheximide has obvious inhibition effect on fungi.
2. Preparing culture medium
The preparation of the solid culture medium comprises the following steps: 1 to 5g of soluble starch, 1 to 5g of pyruvic acid, 1 to 5g of sodium acetate, 10 to 25g of acid hydrolyzed casein, 1 to 10g of beef extract, 1 to 5g of yeast extract, 0.005 to 0.015g of calcium chloride, 0.3 to 0.5g of monopotassium phosphate, 0.5 to 1.0g of disodium hydrogen phosphate, 0.05 to 0.15g of potassium nitrate, 0.01 to 0.02g of zinc sulfate, 0.01 to 0.03g of manganese sulfate, 0.1 to 0.2g of magnesium sulfate heptahydrate, 0.03 to 0.07g of ferric ammonium citrate, 0.01 to 0.02g of potassium tellurite and 16 to 18g of agar are weighed, dissolved in deionized water, the volume is fixed to 1000 mL, the pH is adjusted to 7.5 to 7.6 by sodium carbonate and citric acid, the pH is adjusted to be 15 minutes, the temperature is adjusted to 121 ℃, 105 to be sterilized for 15 minutes, 1 to 10mg of trimethoprim, 1 to 10mg of cefepime, 1 to 10mg of polymyxin B and 50 mg of polymyxin B are added to be subjected to sterile and the solid is selectively mixed to prepare a sterile medium of a solid culture vessel, and the sterile medium is filled in a pot, and the sterile medium is subjected to 53 to filtration.
The preparation of the liquid culture medium comprises the following steps: 1 to 5g of soluble starch, 1 to 5g of pyruvic acid, 1 to 5g of sodium acetate, 10 to 25g of acid hydrolyzed casein, 1 to 10g of beef extract, 1 to 5g of yeast extract, 0.005 to 0.015g of calcium chloride, 0.3 to 0.5g of monopotassium phosphate, 0.5 to 1.0g of disodium hydrogen phosphate, 0.05 to 0.15g of potassium nitrate, 0.01 to 0.02g of zinc sulfate, 0.01 to 0.03g of manganese sulfate, 0.1 to 0.2g of magnesium sulfate heptahydrate, 0.03 to 0.07g of ferric ammonium citrate and 0.01 to 0.02g of potassium tellurite are weighed, dissolved in deionized water, the volume is fixed to 1000 mL, the pH is adjusted to 7.5 to 7.6 by sodium carbonate and citric acid, the temperature is adjusted to 121 ℃, the temperature is 105 to kPa minutes, 1 to 10mg of trimethoprim, 1 to 10mg of cefoperazone, 1 to 10mg of polymyxin B and 50mg of cycloheximide are added, and the solution is subjected to selective uniform mixing and culturing to prepare a liquid medium.
3. Inoculating and culturing
(1) Standard strain recovery experiments: gradually diluting the activated rhodococcus equi strain to be not more than 100 cfu.mL -1 by using a sterile NaCl solution, coating the strain on TSA and rhodococcus equi selective separation culture mediums, respectively coating 3 plates on the two culture mediums, culturing at 30 ℃ for 48-72 h, observing the growth condition of the strain, counting colonies, and calculating the recovery rate;
Recovery = (number of selectively isolated media colonies/number of TSA colonies) ×100%
(2) Gradually diluting the activated rhodococcus equi strain to about 10 5 cfu·mL-1 by using a 0.9% sterile NaCl solution, mixing 20 mu L of bacterial liquid with 180 mu L of selective liquid culture medium, placing the mixture in a growth curve instrument, culturing the mixture at 30 ℃ to 72 h, reading OD 600 value of a culture hole, and drawing a growth curve;
(3) Inhibition ability of the selective media: taking common microorganisms including rhodococcus faecalis, streptococcus equi, enterococcus faecalis, pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, streptomyces albus, geotrichum, penicillium chrysogenum and Pythium isox in soil as test strains for detecting the growth ability of other microorganisms, inoculating about 10 4 cfu of each activated strain on a selective separation culture medium, placing the selective separation culture medium in a constant-temperature incubator at 30 ℃ for culturing 72h, and observing whether colonies appear;
(4) The practical application method of the rhodococcus equi selective separation culture medium comprises the following steps: the collected soil sample was placed in a sterile plate for natural drying, ground into powder with a sterile pestle, 1g of soil sample was weighed, added to 10mL of 2% oxalic acid, shaken in a shaker of 180r min -1 for 20min, naturally precipitated for 5min, poured out the supernatant, centrifuged at 3000 rpm for 5min, the sediment was suspended in 5mL of 1% sterile peptone water, 1mL of the soil suspension was sucked into 9 mL sterile water, 10-fold gradient dilution was performed to a concentration of 1g L -1, 200. Mu.L was spread on a separation medium plate, and cultured in a constant temperature incubator at 30 ℃.
When the rhodococcus equi selective separation culture medium is practically applied, the collected soil sample is ground by using a sterile pestle, so that a soil sample can be fully contacted with oxalic acid, the oxalic acid can primarily eliminate mixed bacteria, metal ions and humus in the soil by influencing the acid-base balance of the soil in the subsequent oscillation process, rhodococcus equi has stronger tolerance to oxalic acid environment, the biomass of rhodococcus equi in the sample can be improved, the concentration of rhodococcus equi is moderate after the soil suspension is centrifuged and diluted to a certain concentration, the growth quantity coated on the selective separation culture medium is convenient to count, and bacterial colonies are easy to observe.
The rhodococcus equi selective isolation medium can be either solid or liquid, and is described in the examples below in terms of the manner in which the solids are produced.
Example 1
This example provides a rhodococcus equi selective isolation medium.
1. The formula of the culture medium is as follows:
Per liter of medium contained: 1.5g of soluble starch, 2g of pyruvic acid, 1.85g of sodium acetate, 17.5g of acid hydrolyzed casein, 6g of beef extract powder, 4g of yeast extract powder, 0.01g of calcium chloride, 0.46g of monopotassium phosphate, 0.73g of disodium hydrogen phosphate, 0.1g of potassium nitrate, 0.016g of zinc sulfate, 0.022g of manganese sulfate, 0.12g of magnesium sulfate heptahydrate, 0.054g of ferric ammonium citrate, 0.015g of potassium tellurite and 17g of agar; the additive is trimethoprim 5.8mg, cefoperazone 6.75mg, polymyxin B6.45 mg and cycloheximide 90mg.
2. Preparing culture medium
1.5G of soluble starch, 2g of pyruvic acid, 1.85g of sodium acetate, 17.5g of acid hydrolyzed casein, 6g of beef extract powder, 4g of yeast extract powder, 0.01g of calcium chloride, 0.46g of monopotassium phosphate, 0.73g of disodium hydrogen phosphate, 0.1g of potassium nitrate, 0.016g of zinc sulfate, 0.022g of manganese sulfate, 0.12g of magnesium sulfate heptahydrate, 0.054g of ferric ammonium citrate, 0.015g of potassium tellurite and 17g of agar are weighed, dissolved in deionized water, the volume is fixed to 1000 mL, the pH is adjusted to 7.5-7.6 by sodium carbonate and citric acid, the mixture is sterilized for 15 minutes at 121 ℃ and 105-kPa, cooled to 50 ℃, 5.8mg of trimethoprim, 6.75mg of cefoperazone, 6.45mg of polymyxin B, 90mg of cycloheximide are added and mixed, and the mixture is aseptically filled into 90-mm sterile dishes to prepare a rhodococcus equi selective separation culture medium.
3. Inoculating and culturing
(1) Gradually diluting the activated rhodococcus equi strain to not more than 100 cfu mL -1 by using a sterile NaCl solution, coating the strain on a TSA and a separation culture medium, respectively coating 3 plates on the two culture mediums, culturing at 30 ℃ for 48-72 h, observing the growth condition of the strain, counting colonies, and taking an average value to calculate the recovery rate;
Recovery = (number of selectively isolated media colonies/number of TSA colonies) ×100%
(2) Gradually diluting the activated rhodococcus equi strain to about 10 5 cfu·mL-1 by using a 0.9% sterile NaCl solution, mixing 20 mu L of bacterial liquid with 180 mu L of selective liquid culture medium, placing the mixture in a growth curve instrument, culturing the mixture at 30 ℃ to 72 h, reading OD 600 value of a culture hole, and drawing a growth curve;
(3) Taking rhodococcus faecalis, streptococcus equi, enterococcus faecalis, pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, streptomyces albus, geotrichum, penicillium chrysogenum and Pythium isosilk as test strains for detecting the growth ability of other microorganisms of the culture medium, inoculating about 10 4 cfu of each activated strain on a selective separation culture medium, placing the culture medium in a constant-temperature incubator at 30 ℃ for culturing 72 h, and observing whether colonies appear;
(4) The practical application method of the rhodococcus equi selective separation culture medium comprises the following steps: the collected soil sample was placed in a sterile plate for natural drying, ground into powder with a sterile pestle, 1g of soil sample was weighed, added to 10mL of 2% oxalic acid, shaken in a shaker of 180r min -1 for 20min, naturally precipitated for 5min, poured out the supernatant, centrifuged at 3000 rpm for 5min, the sediment was suspended in 5mL of 1% sterile peptone water, 1mL of the soil suspension was sucked into 9 mL sterile water, 10-fold gradient dilution was performed to a concentration of 1g L -1, 200. Mu.L was spread on a separation medium plate, and cultured in a constant temperature incubator at 30 ℃.
Example 2
This example provides a rhodococcus equi selective isolation medium.
1. The formula of the culture medium is as follows:
Per liter of medium contained: 4.5g of soluble starch, 4.46g of pyruvic acid, 4.85g of sodium acetate, 24g of acid hydrolyzed casein, 9.2g of beef extract powder, 4.9g of yeast extract powder, 0.015g of calcium chloride, 0.46g of monopotassium phosphate, 0.95g of disodium hydrogen phosphate, 0.148g of potassium nitrate, 0.019g of zinc sulfate, 0.028g of manganese sulfate, 0.195g of magnesium sulfate heptahydrate, 0.06g of ferric ammonium citrate, 0.018g of potassium tellurite and 17g of agar; the additives are trimethoprim 10mg, cefoperazone 9.25mg, polymyxin B9.5 mg and cycloheximide 190mg.
2. Preparing culture medium
4.5G of soluble starch, 4.46g of pyruvic acid, 4.85g of sodium acetate, 24g of acid hydrolyzed casein, 9.2g of beef extract, 4.9g of yeast extract, 0.015g of calcium chloride, 0.46g of monopotassium phosphate, 0.95g of disodium hydrogen phosphate, 0.148g of potassium nitrate, 0.019g of zinc sulfate, 0.028g of manganese sulfate, 0.195g of magnesium sulfate heptahydrate, 0.06g of ferric ammonium citrate, 0.018g of potassium tellurite and 17g of agar are weighed, dissolved in deionized water, fixed to 1000 mL, and adjusted to pH 7.5-7.6 by sodium carbonate and citric acid, sterilized at 121 ℃ for 15 minutes, cooled to 50 ℃,10 mg of trimethoprim, 9.25mg of cefoperazone, 9.5mg of polymyxin B, 190mg of cyclic imide and mixed evenly, and aseptically filled into a 90 mm dish to prepare a rhodococcus equisimilis selective isolation medium.
3. Inoculating and culturing
(1) Gradually diluting the activated rhodococcus equi strain to not more than 100 cfu mL -1 by using a sterile NaCl solution, coating the strain on a TSA and a separation culture medium, respectively coating 3 plates on the two culture mediums, culturing at 30 ℃ for 48-72 h, observing the growth condition of the strain, counting colonies, and taking an average value to calculate the recovery rate;
Recovery = (number of selectively isolated media colonies/number of TSA colonies) ×100%
(2) Gradually diluting the activated rhodococcus equi strain to about 10 5 cfu·mL-1 by using a 0.9% sterile NaCl solution, mixing 20 mu L of bacterial liquid with 180 mu L of selective liquid culture medium, placing the mixture in a growth curve instrument, culturing the mixture at 30 ℃ to 72 h, reading OD 600 value of a culture hole, and drawing a growth curve;
(3) Taking rhodococcus faecalis, streptococcus equi, enterococcus faecalis, pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, streptomyces albus, geotrichum, penicillium chrysogenum and Pythium isosilk as test strains for detecting the growth ability of other microorganisms of the culture medium, inoculating about 10 4 cfu of each activated strain on a selective separation culture medium, placing the culture medium in a constant-temperature incubator at 30 ℃ for culturing 72 h, and observing whether colonies appear;
(4) The practical application method of the rhodococcus equi selective separation culture medium comprises the following steps: the collected soil sample was placed in a sterile plate for natural drying, ground into powder with a sterile pestle, 1g of soil sample was weighed, added to 10mL of 2% oxalic acid, shaken in a shaker of 180r min -1 for 20min, naturally precipitated for 5min, poured out the supernatant, centrifuged at 3000 rpm for 5min, the sediment was suspended in 5mL of 1% sterile peptone water, 1mL of the soil suspension was sucked into 9 mL sterile water, 10-fold gradient dilution was performed to a concentration of 1g L -1, 200. Mu.L was spread on a separation medium plate, and cultured in a constant temperature incubator at 30 ℃.
Example 3
This example provides a rhodococcus equi selective isolation medium.
1. The formula of the culture medium is as follows:
Per liter of medium contained: 3g of soluble starch, 4g of pyruvic acid, 3.15g of sodium acetate, 11.5g of acid hydrolyzed casein, 9g of beef extract powder, 2.5g of yeast extract powder, 0.01g of calcium chloride, 0.4g of potassium dihydrogen phosphate, 0.65g of disodium hydrogen phosphate, 0.1g of potassium nitrate, 0.015g of zinc sulfate, 0.02g of manganese sulfate, 0.14g of magnesium sulfate heptahydrate, 0.05g of ferric ammonium citrate, 0.014g of potassium tellurite and 17g of agar; the additives are trimethoprim 2.15mg, cefoperazone 9.45mg, polymyxin B8.75 mg and cycloheximide 60 mg mg.
2. Preparing culture medium
3G of soluble starch, 4g of pyruvic acid, 3.15g of sodium acetate, 11.5g of acid hydrolyzed casein, 9g of beef extract powder, 2.5g of yeast extract powder, 0.01g of calcium chloride, 0.4g of monopotassium phosphate, 0.65g of disodium hydrogen phosphate, 0.1g of potassium nitrate, 0.015g of zinc sulfate, 0.02g of manganese sulfate, 0.14g of magnesium sulfate heptahydrate, 0.05g of ferric ammonium citrate, 0.014g of potassium tellurite and 17g of agar are weighed, dissolved in deionized water, fixed to 1000mL, and adjusted to pH 7.5-7.6 by sodium carbonate and citric acid, sterilized for 15 minutes at 121 ℃ and 105: 105 kPa, cooled to 50 ℃, added with 2.15mg of trimethoprim, 9.45mg of cefoperazone, 8.75mg of polymyxin B, 60mg of cycloheximide and mixed evenly, and aseptically filled into 90 mm sterile culture dishes to prepare rhodococcus equi selective separation culture media.
3. Inoculating and culturing
(1) Gradually diluting the activated rhodococcus equi strain to not more than 100 cfu mL -1 by using a sterile NaCl solution, coating the strain on a TSA and a separation culture medium, respectively coating 3 plates on the two culture mediums, culturing at 30 ℃ for 48-72 h, observing the growth condition of the strain, counting colonies, and taking an average value to calculate the recovery rate;
Recovery = (number of selectively isolated media colonies/number of TSA colonies) ×100%
(2) Gradually diluting the activated rhodococcus equi strain to about 10 5cfu·mL-1 by using a 0.9% sterile NaCl solution, mixing 20 mu L of bacterial liquid with 180 mu L of selective liquid culture medium, placing the mixture in a growth curve instrument, culturing the mixture at 30 ℃ to 72 h, reading OD 600 value of a culture hole, and drawing a growth curve;
(3) Taking rhodococcus faecalis, streptococcus equi, enterococcus faecalis, pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, streptomyces albus, geotrichum, penicillium chrysogenum and Pythium isosilk as test strains for detecting the growth ability of other microorganisms of the culture medium, inoculating about 10 4 cfu of each activated strain on a selective separation culture medium, placing the culture medium in a constant-temperature incubator at 30 ℃ for culturing 72 h, and observing whether colonies appear;
(4) The practical application method of the rhodococcus equi selective separation culture medium comprises the following steps: the collected soil sample was placed in a sterile plate for natural drying, ground into powder with a sterile pestle, 1g of soil sample was weighed, added to 10mL of 2% oxalic acid, shaken in a shaker of 180r min -1 for 20min, naturally precipitated for 5min, poured out the supernatant, centrifuged at 3000 rpm for 5min, the sediment was suspended in 5mL of 1% sterile peptone water, 1mL of the soil suspension was sucked into 9 mL sterile water, 10-fold gradient dilution was performed to a concentration of 1g L -1, 200. Mu.L was spread on a separation medium plate, and cultured in a constant temperature incubator at 30 ℃.
Example 4
This example provides a rhodococcus equi selective isolation medium.
1. The formula of the culture medium is as follows:
Per liter of medium contained: 1.25g of soluble starch, 1.2g of pyruvic acid, 1.1g of sodium acetate, 11.8g of acid hydrolyzed casein, 1.75g of beef extract powder, 1.5g of yeast extract powder, 0.006g of calcium chloride, 0.32g of monopotassium phosphate, 0.55g of disodium hydrogen phosphate, 0.06g of potassium nitrate, 0.011g of zinc sulfate, 0.012g of manganese sulfate, 0.114g of magnesium sulfate heptahydrate, 0.035g of ferric ammonium citrate, 0.012g of potassium tellurite and 17g of agar; the additive comprises 1.5mg of trimethoprim, 1.8mg of cefoperazone, 2mg of polymyxin B and 70mg of cycloheximide.
2. Preparing culture medium
1.25G of soluble starch, 1.2g of pyruvic acid, 1.1g of sodium acetate, 11.8g of acid hydrolyzed casein, 1.75g of beef extract, 1.5g of yeast extract, 0.006g of calcium chloride, 0.32g of monopotassium phosphate, 0.55g of disodium hydrogen phosphate, 0.06g of potassium nitrate, 0.011g of zinc sulfate, 0.012g of manganese sulfate, 0.114g of magnesium sulfate heptahydrate, 0.035g of ferric ammonium citrate, 0.012g of potassium tellurite and 17g of agar are weighed, dissolved in deionized water, fixed volume is adjusted to 1000 mL, pH is adjusted to 7.5-7.6 by sodium carbonate and citric acid, sterilization is carried out for 15 minutes at 121 ℃, cooling is carried out to 50 ℃, 1.5mg of trimethoprim, 1.8mg of cefoperazone, 2mg of polymyxin B, 70mg of cycloheximide are added and mixed, and the mixture is aseptically poured into a 90 mm aseptic culture dish to prepare a rhodococcus equisimilis selective separation culture medium.
3. Inoculating and culturing
(1) Gradually diluting the activated rhodococcus equi strain to not more than 100 cfu mL -1 by using a sterile NaCl solution, coating the strain on a TSA and a separation culture medium, respectively coating 3 plates on the two culture mediums, culturing at 30 ℃ for 48-72 h, observing the growth condition of the strain, counting colonies, and taking an average value to calculate the recovery rate;
Recovery = (number of selectively isolated media colonies/number of TSA colonies) ×100%
(2) Gradually diluting the activated rhodococcus equi strain to about 10 5cfu·mL-1 by using a 0.9% sterile NaCl solution, mixing 20 mu L of bacterial liquid with 180 mu L of selective liquid culture medium, placing the mixture in a growth curve instrument, culturing the mixture at 30 ℃ to 72 h, reading OD 600 value of a culture hole, and drawing a growth curve;
(3) Taking rhodococcus faecalis, streptococcus equi, enterococcus faecalis, pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, streptomyces albus, geotrichum, penicillium chrysogenum and Pythium isosilk as test strains for detecting the growth ability of other microorganisms of the culture medium, inoculating about 10 4 cfu of each activated strain on a selective separation culture medium, placing the culture medium in a constant-temperature incubator at 30 ℃ for culturing 72 h, and observing whether colonies appear;
(4) The practical application method of the rhodococcus equi selective separation culture medium comprises the following steps: the collected soil sample was placed in a sterile plate for natural drying, ground into powder with a sterile pestle, 1g of soil sample was weighed, added to 10mL of 2% oxalic acid, shaken in a shaker of 180r min -1 for 20min, naturally precipitated for 5min, poured out the supernatant, centrifuged at 3000 rpm for 5min, the sediment was suspended in 5mL of 1% sterile peptone water, 1mL of the soil suspension was sucked into 9 mL sterile water, 10-fold gradient dilution was performed to a concentration of 1g L -1, 200. Mu.L was spread on a separation medium plate, and cultured in a constant temperature incubator at 30 ℃.
As shown in FIG. 1, the average recovery rates of the selective separation media of examples 1-4 were 91%, 66%, 81%, 62%, respectively, and the above results indicate that the recovery rates of the selective separation media of examples 1-4 were higher than 50% for rhodococcus equi, but the recovery rates of the media of example 1 were significantly higher than those of examples 2-4. Rhodococcus equi grows well in the media of examples 1-4, and the slope of the growth curve of the liquid medium of example 1 is slightly greater than that of examples 2-4, suggesting that Rhodococcus equi grows faster in the liquid medium of example 1 and reaches stationary phase in a shorter period. The test strains used for detecting the inhibition ability of the culture medium, namely rhodococcus faecalis, streptococcus equi, enterococcus faecalis, pseudomonas aeruginosa, staphylococcus aureus, bacillus subtilis, streptomyces albus, geotrichum, penicillium chrysogenum and Pythium isosilk, do not grow on the selective culture medium, so that the selective culture medium of the examples 1-4 can meet the inhibition ability requirement to a certain extent. The use of a selective medium is important when rhodococcus equi is isolated from a highly contaminated source, as shown in figure 4, on the selective medium of examples 1-4, the medium of example 1 is very well able to inhibit the contaminating flora and the colony morphology of rhodococcus equi on this medium is more characteristic (glossy, smooth, moist), rhodococcus equi has a greater number of growths, whereas only weaker growths of rhodococcus equi are observed on the medium of example 3. The media of example 2 and example 4 had a smaller number of rhodococcus equi growths than the media of example 3, and the recovery of rhodococcus equi on the media of this example was lower.
In summary, the invention optimizes the formulation of the culture medium according to the growth requirement and the characteristics of rhodococcus equi to form a rhodococcus equi selective separation culture medium, and the selective culture medium in the given examples 1-4 is compared with the selective culture medium in the given examples 1-4 by the recovery, the growth curve and the growth colony phenotype characteristics of rhodococcus equi on the selective culture medium and the growth condition of non-rhodococcus equi, so that the selective culture medium in the example 1 has better rhodococcus equi growth promoting capability and can achieve corresponding inhibition capability, and therefore, the example 1 is the best example of the invention. The rhodococcus equi selective separation culture medium can effectively detect the existence of rhodococcus equi in a equine farm cultivation environment, can be applied to rhodococcus equi enrichment culture, separation screening, quantity detection and the like, and has important value for preventing and controlling rhodococcus equi infection problems in animal cultivation areas of equine farms and the like.
Finally, it should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the spirit and scope of the invention.
Claims (6)
1. The rhodococcus equi selective separation medium is characterized by comprising the following components: carbon source, nitrogen source, antibiotics, 0.005-0.015 g/L of calcium chloride, 0.3-0.5 g/L of monopotassium phosphate, 0.5-1.0 g/L of disodium hydrogen phosphate, 0.05-0.15 g/L of potassium nitrate, 0.01-0.02 g/L of zinc sulfate, 0.01-0.03 g/L of manganese sulfate, 0.1-0.2 g/L of magnesium sulfate heptahydrate, 0.03-0.07 g/L of ferric ammonium citrate, 0.01-0.02 g/L of potassium tellurite and 16-18 g/L of agar;
The carbon source comprises the following components: 1-5 g/L of soluble starch, 1-5 g/L of pyruvic acid and 1-5 g/L of sodium acetate;
the nitrogen source comprises the following components: 10-25 g/L of acid hydrolyzed casein, 1-10 g/L of beef extract powder and 1-5 g/L of yeast extract powder;
The composition of the antibiotics is as follows: 1 to 10mg/L of trimethoprim, 1 to 10mg/L of cefoperazone, 1 to 10mg/L of polymyxin B and 50 to 200mg/L of cycloheximide.
2. A method for preparing a rhodococcus equi selective separation culture medium according to claim 1, which is characterized by weighing 1-5 g of soluble starch, 1-5 g of pyruvic acid, 1-5 g of sodium acetate, 10-25 g of acid hydrolyzed casein, 1-10 g of beef extract, 1-5 g of yeast extract, 0.005-0.015 g/L of calcium chloride, 0.3-0.5 g/L of monopotassium phosphate, 0.5-1.0 g/L of disodium hydrogen phosphate, 0.05-0.15 g/L of potassium nitrate, 0.01-0.02 g/L of zinc sulfate, 0.01-0.03 g/L of manganese sulfate, 0.1-0.2 g/L of magnesium sulfate heptahydrate, 0.03-0.07 g/L of ferric ammonium citrate, 0.01-0.02 g/L of potassium telluride, 16-18 g/L of agar, dissolving in deionized water, setting to 1000.26, adjusting the pH to 7.5 ℃ by sodium carbonate, sterilizing to be 1-10 mg of hyperbaric, preparing 10mg of hyperbaric acid, filling 10mg of rhodochrom, sterilizing to obtain the rhodochrous, and preparing the sterile rhodochrous medium by filling 1-50 mg of the medium.
3. The use of rhodococcus equi selective isolation medium according to claim 1, characterized in that it is applied for the isolation of rhodococcus equi in the soil of animal cultivation areas.
4. The use of a rhodococcus equi selective isolation medium according to claim 3, characterized in that the culture temperature of rhodococcus equi in the isolation medium is 30 ℃.
5. The use of a rhodococcus equi selective isolation medium according to claim 3, wherein the isolation medium is used for culturing rhodococcus equi for a period of 48-72 h.
6. A method for using the rhodococcus equi selective isolation medium of claim 1, characterized in that,
(1) Placing the collected soil sample in a sterile plate, naturally drying, and grinding into powder by a sterile pestle;
(2) Weighing 1g of soil sample, adding the soil sample into 10 mL of 2% oxalic acid, oscillating 20 min in a shaking table of 180 r.min -1, and naturally precipitating 5 min;
(3) The supernatant was poured off and centrifuged at 3000 rpm at 5 min;
(4) Suspending the sediment in 5 mL of 1% sterile peptone water, sucking 1 mL soil suspension into 9 mL sterile water, and carrying out 10-time gradient dilution to the concentration of 1 g.L -1;
(5) 200. Mu.L of the culture medium was spread on a plate of the isolation medium and cultured in a constant temperature incubator at 30 ℃.
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| EP0244912A1 (en) * | 1986-05-08 | 1987-11-11 | Gist-Brocades N.V. | A process for the preparation of R and S-2,2-R1,R2-1,3-dioxolane-4-methanol |
| US6254777B1 (en) * | 1998-12-28 | 2001-07-03 | Institut Francais Du Petrole | Process for bacterial treatment of effluents that contain at least one ether |
| US20110274660A1 (en) * | 2009-01-12 | 2011-11-10 | Antonius Arnoldus Christiaan Jacobs | Pharmaceutical composition to protect an animal against a disorder arising from an infection with a bacterium that belongs to the group of nocardioform actinomycetes |
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|---|---|---|---|---|
| EP0244912A1 (en) * | 1986-05-08 | 1987-11-11 | Gist-Brocades N.V. | A process for the preparation of R and S-2,2-R1,R2-1,3-dioxolane-4-methanol |
| US6254777B1 (en) * | 1998-12-28 | 2001-07-03 | Institut Francais Du Petrole | Process for bacterial treatment of effluents that contain at least one ether |
| US20110274660A1 (en) * | 2009-01-12 | 2011-11-10 | Antonius Arnoldus Christiaan Jacobs | Pharmaceutical composition to protect an animal against a disorder arising from an infection with a bacterium that belongs to the group of nocardioform actinomycetes |
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| L. MAKRAI 等: "COMPARISON OF SELECTIVE MEDIA FOR THE ISOLATION OF RHODOCOCCUS EQUI AND DESCRIPTION OF A NEW SELECTIVE PLATING MEDIUM", 《ACTA VETERINARIA HUNGARICA 》, vol. 53, no. 3, 31 December 2005 (2005-12-31), pages 275 * |
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