CN110055198A - 一种快速培养结核病致病菌的培养方法 - Google Patents
一种快速培养结核病致病菌的培养方法 Download PDFInfo
- Publication number
- CN110055198A CN110055198A CN201910415433.6A CN201910415433A CN110055198A CN 110055198 A CN110055198 A CN 110055198A CN 201910415433 A CN201910415433 A CN 201910415433A CN 110055198 A CN110055198 A CN 110055198A
- Authority
- CN
- China
- Prior art keywords
- added
- culture medium
- culture
- tuberculosis
- mixes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 57
- 201000008827 tuberculosis Diseases 0.000 title claims abstract description 26
- 244000052616 bacterial pathogen Species 0.000 title claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims abstract description 66
- 241000894006 Bacteria Species 0.000 claims abstract description 32
- 238000002360 preparation method Methods 0.000 claims abstract description 20
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 7
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 42
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 34
- 239000002253 acid Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 229940054269 sodium pyruvate Drugs 0.000 claims description 21
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- 239000003513 alkali Substances 0.000 claims description 20
- 229920001817 Agar Polymers 0.000 claims description 18
- 239000008272 agar Substances 0.000 claims description 18
- 239000002504 physiological saline solution Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 14
- 102000002322 Egg Proteins Human genes 0.000 claims description 13
- 108010000912 Egg Proteins Proteins 0.000 claims description 13
- 210000004681 ovum Anatomy 0.000 claims description 13
- 229920002472 Starch Polymers 0.000 claims description 11
- 230000001376 precipitating effect Effects 0.000 claims description 11
- 235000019698 starch Nutrition 0.000 claims description 11
- 239000008107 starch Substances 0.000 claims description 11
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 10
- 238000004090 dissolution Methods 0.000 claims description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 10
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 10
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 10
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 8
- 239000000839 emulsion Substances 0.000 claims description 8
- 238000012545 processing Methods 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 6
- 229960001230 asparagine Drugs 0.000 claims description 6
- 235000009582 asparagine Nutrition 0.000 claims description 6
- 210000004072 lung Anatomy 0.000 claims description 6
- 229960005336 magnesium citrate Drugs 0.000 claims description 6
- 235000002538 magnesium citrate Nutrition 0.000 claims description 6
- 239000004337 magnesium citrate Substances 0.000 claims description 6
- FDZZZRQASAIRJF-UHFFFAOYSA-M malachite green Chemical compound [Cl-].C1=CC(N(C)C)=CC=C1C(C=1C=CC=CC=1)=C1C=CC(=[N+](C)C)C=C1 FDZZZRQASAIRJF-UHFFFAOYSA-M 0.000 claims description 6
- 229940107698 malachite green Drugs 0.000 claims description 6
- 238000012856 packing Methods 0.000 claims description 6
- PLSARIKBYIPYPF-UHFFFAOYSA-H trimagnesium dicitrate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O PLSARIKBYIPYPF-UHFFFAOYSA-H 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 230000035479 physiological effects, processes and functions Effects 0.000 claims description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 4
- 238000005299 abrasion Methods 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 229960002413 ferric citrate Drugs 0.000 claims description 4
- -1 ferric citrate amine Chemical class 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 4
- 210000003097 mucus Anatomy 0.000 claims description 4
- 235000011837 pasties Nutrition 0.000 claims description 4
- 229920001592 potato starch Polymers 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000003860 storage Methods 0.000 claims description 4
- 230000001360 synchronised effect Effects 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 208000008771 Lymphadenopathy Diseases 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 208000018555 lymphatic system disease Diseases 0.000 claims description 3
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims 2
- 235000005979 Citrus limon Nutrition 0.000 claims 1
- 244000131522 Citrus pyriformis Species 0.000 claims 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- 240000003273 Passiflora laurifolia Species 0.000 claims 1
- 235000013762 Passiflora laurifolia Nutrition 0.000 claims 1
- 244000144977 poultry Species 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 241000367152 Mycobacterium tuberculosis complex bacterium Species 0.000 abstract description 4
- 238000013461 design Methods 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 230000007774 longterm Effects 0.000 abstract description 2
- 238000003239 susceptibility assay Methods 0.000 abstract description 2
- 238000010998 test method Methods 0.000 abstract description 2
- 235000013601 eggs Nutrition 0.000 description 7
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 5
- 238000011534 incubation Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 238000002255 vaccination Methods 0.000 description 4
- LRZSAGKIMYFLHY-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;dihydrate Chemical compound O.O.OC(=O)CC(O)(C(O)=O)CC(O)=O LRZSAGKIMYFLHY-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012092 media component Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- 235000011083 sodium citrates Nutrition 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 244000191482 Cantharellus cibarius Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000692870 Inachis io Species 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000186366 Mycobacterium bovis Species 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- 240000001462 Pleurotus ostreatus Species 0.000 description 1
- 235000001603 Pleurotus ostreatus Nutrition 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical compound ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000001458 anti-acid effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000003771 laboratory diagnosis Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
Classifications
-
- 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
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Toxicology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于生物实验方法的设计技术领域,具体公开一种快速培养结核病致病菌的培养方法,包括下述步骤:(一)培养基的制备与保存;(二)样本处理;(三)结核病致病原快速培养。同时使用该培养基和培养方法可快速进行结核病致病菌培养、鉴定;获得的纯菌落可进行多种药物敏感实验,并可长期保存结核分枝杆菌复合群细菌。该培养基和培养方法培养结果容易观察,应用范围广泛。
Description
技术领域
本发明属于生物实验方法的设计技术领域,具体公开一种快速培养结核病致病菌的培养方法。
背景技术
结核病是由结核分枝杆菌复合群细菌所引起的人畜共患的慢性消耗性传染病。要控制结核病的发生和流行,必须加强对该病的检测。结核分枝杆菌复合群细菌是结核病的致病菌,为抗酸的杆菌,主要通过呼吸道传播。及时、有效的诊断是控制结核病关键,其中其致病菌培养是实验室诊断的主要方法之一,但结核分枝杆菌复合群细菌属缓慢生长分枝杆菌,其自身分裂周期长、生长速度慢,且在样本处理过程中易受损伤,导致分离培养耗时长。当前,实验室的常用培养基有改良罗氏培养基(L-J培养基)和丙酮酸钠培养基,虽然这两种培养基已使用有百年的历史,但是这两种培养基培养时间长,已分离的纯培养物需25天以上,临床病料中的病原分离需要60天以上才可见到典型菌落,达不到快速诊断的目的。
近年来,用于培养结核病致病菌的新型改良培养基越来越多,在国内相关实验中,添加烟酸的新型改良罗氏培养基,添加维生素E及猪肺汤的改良培养基,以及在原有培养基成分中加入土豆、鸡蛋或平菇等营养物质的培养基均有出现。以上这些培养基和培养方法尽管缩短了结核致病菌的培养时间,仍需要20天以上,且所需药品或培养基成分要求高,受设施和条件限制,不利于广泛使用。
发明内容
本发明的目的在于:设计一种能快速分离结核病致病菌的培养方法,在结核病致病菌损伤修复培养基(DRM培养基)中进行预培养后,可提高细菌增殖率,并降低了在生长过程中的高营养需求,从而加速了结核病致病菌的生长,缩短了培养时间。
本发明的技术方案:一种快速培养结核病致病菌的培养方法,该培养方法包括下述步骤:
一、培养基的制备与保存
(一)0.3%琼脂高糖DRM培养基的制备
(1)将L-天冬酰胺1g,磷酸二氢钾1.5g,磷酸氢二钠2.5g,七水硫酸镁0.5g,二水柠檬酸纳1.3g,柠檬酸铁胺0.05g,丙酮酸钠2.4g,琼脂粉3g,水1000mL溶解后,在115-121℃,高压条件下,20min,制备成0.3%琼脂快速培养基,备用;
(2)称取100g白糖,加入50mL水,在45-50℃充分溶解,110-115℃,高压10-15min灭菌后制备成200%高糖液,备用;
(3)无菌条件下,在加高压除菌后处于60-80℃的143mL的200%高糖液加到同等条件下1000mL 0.3%琼脂快速培养基中,充分混匀,制备成0.3%琼脂高糖DRM培养基分装,18mm×150mm试管,每管7mL,4℃保存备用;
(二)Lowenstein-Jensen培养基的制备
(1)将天门冬素3.6g,磷酸二氢钾2.4g,七水硫酸镁0.24g,柠檬酸镁0.6g,丙三醇12mL,蒸馏水600mL,充分溶解后备用;
(2)向步骤(1)中加入可溶性淀粉30g,混匀,煮沸35-45min,成透明糊状溶液,冷却后备用;
(3)向步骤(2)中加入经过滤后的新鲜鸡卵液1000mL,混匀,备用;
(4)最后,向步骤(3)中加入2%孔雀绿水溶液20mL,混匀,分装,18mm×150mm试管,每管7mL,放置斜面高度占试管三分之二,在温度条件85℃,45min间歇灭菌两次,37℃无菌测定24h后,4℃贮存备用;
(三)丙酮酸钠培养基的制备
(1)先将天门冬素3.6g,磷酸二氢钾2.4g,七水硫酸镁0.24g,柠檬酸镁0.6g,丙酮酸钠1.6g,蒸馏水600mL,充分溶解后,用4%NaOH校正至pH7.2后,加入葡萄糖4.0g混匀备用;
(2)向步骤(1)中加入可溶性淀粉30g,混匀,煮沸35-45min成透明糊状溶液,冷却后备用;
(3)向步骤(2)中加入经过滤后的新鲜鸡卵液1000mL,混匀,备用;
(4)最后,向步骤(3)中加入2%孔雀绿水溶液20mL,混匀,分装,18mm×150mm试管,每管7mL,放置斜面高度占试管三分之二,在温度条件85℃,45min间歇灭菌两次,37℃无菌测定24h后,4℃贮存备用;
二、样本处理
(一)组织样本处理
(1)研磨组织:将采集到的肺脏、淋巴病变组织,用剪刀剪成小块,然后加入生理盐水,用研磨棒研磨,制成乳剂;
(2)碱处理:取2mL~4mL乳剂液,加人等量的4%NaOH溶液,充分振摇5~10min;
(3)酸平衡:碱处理10min后,用2mol/L的HCL调pH值至6.5-7.0,在10min之内完成;
(4)离心:酸碱处理后的菌液,离心20min,4000rpm,弃上清,沉淀加入生理盐水,充分混匀;
(二)鼻拭子样品的处理
(1)将含鼻拭子样品的生理盐水用4000rpm离心20min,收集沉淀,加入适量的生理盐水;
(2)碱处理:同步加入等量4%NaOH和2.94%枸橼酸钠溶液中,其中枸橼酸钠溶液中含1%N-乙酰-L半胱胺酸,室温静置20分钟,使粘液液化至清亮;
(3)酸平衡:碱处理20min后,用2mol/L的HCL调pH值至6.5-7.0,在10min之内完成;(4)离心:酸碱处理后的菌液,离心20min,4000rpm,弃上清,沉淀加入生理盐水,充分混匀;
(三)结核病致病原快速培养:酸、碱处理后的菌液,用无菌的一次性滴管取0.2mL菌液垂直穿刺至含0.3%琼脂高糖DRM培养基,置37℃恒温培养箱中预培养3天;3天后用无菌的一次性滴管取0.2mL菌液转接至丙酮酸钠培养基和/或L-J培养基斜面,置37℃恒温培养箱中培养,从第三天开始进行观察培养基中细菌的生长情况并做记录。
优化设计,所述新鲜鸡卵液的制备过程为:先将新鲜鸡蛋清洗干净,晾干,再浸泡在75%酒精中10-15min;再挑拣消毒后的无裂缝鸡蛋取鸡卵。
优化设计,所述的可溶性淀粉为马铃薯淀粉。
本发明的有益效果:该DRM培养基具有使抗酸染色阳性菌培养时间较短,生长速度较快且能快速形成典型菌落等特点,检测效果好及成本低。结核杆菌培养前在DRM培养基中进行预培养能够缩短培养基时间,对结核病的科研研究和早期诊断有一定的意义。同时使用该培养基和培养方法可快速进行结核与非结核分支杆菌培养、鉴定;获得的纯菌落可进行多种药物敏感实验,并可长期保存结核与非结核分支杆菌。该培养基和培养方法培养结果容易观察,应用范围广泛。
具体实施方式
实施例1、快速培养结核病致病菌的培养方法
一、试剂及制备
样品处理溶液的制备:(1)生理盐水:氯化钠0.85g,加蒸馏水至100mL,15磅高压20min,4℃保存备用;(2)4%NaOH;(3)2.94%枸橼酸钠;(4)1%N-乙酰基-L-半胱氨酸
1、0.1%琼脂DRM培养基:(1)培养基组成:L-天冬酰胺1g,磷酸二氢钾1.5g,磷酸氢二钠2.5g,七水硫酸镁0.5g,二水柠檬酸纳1.3g,柠檬酸铁胺0.05g,丙酮酸钠2.4g,琼脂粉1g,水1000mL;(2)制备和保存:上述成分溶解后充分混匀,115-121℃,高压20min,分装,18mm×150mm试管,每管7mL,置4℃保存备用。
2、0.3%琼脂高糖DRM培养基
(1)培养基组分:L-天冬酰胺1g,磷酸二氢钾1.5g,磷酸氢二钠2.5g,七水硫酸镁0.5g,二水柠檬酸纳1.3g,柠檬酸铁胺0.05g,丙酮酸钠2.4g,琼脂粉3g,水1000mL。上述成分溶解后充分混匀,115-121℃,高压20min备用。(2)200%高糖的制备:称取100g白糖,加入50mL的水,在45-50℃水中充分溶解。再置110-115℃高压10-15min灭菌。(3)制备和保存:无菌条件下,加高压除菌后处于60-80℃的143mL的200%高糖到同等条件下1000mL 0.3%琼脂快速培养基中,充分混匀,分装,18mm×150mm试管,每管7mL,待凝固后置4℃保存备用。
3、Lowenstein-Jensen培养基的制备
(1)培养基组分:天门冬素3.6g,磷酸二氢钾2.4g,七水硫酸镁0.24g,柠檬酸镁0.6g,丙三醇12mL,蒸馏水600mL,可溶性淀粉(马铃薯淀粉)30g,新鲜鸡卵液1000mL,2%孔雀绿水溶液20mL。(2)制备与保存:各盐类成份充分溶解后,加入马铃薯淀粉,混匀,沸水浴煮沸35-45min至淀粉成透明糊状。待冷却后加入经消毒纱布过滤的新鲜鸡卵液,混匀;最后加入2%孔雀绿水溶液,混匀,分装,18mm×150mm试管,每管7mL,放置斜面高度占试管2/3。85℃,45min间歇灭菌两次。37℃无菌测定24h后贮存4℃冰箱备用。(3)注意事项a.配试剂用的灭菌水要预先加热。b.鸡蛋清洗干净,先晾干,再浸泡在75%酒精中约10-15min。c.挑拣消毒后的无裂缝鸡蛋取鸡卵,鸡蛋要一个一个的先在无菌的50mL的烧杯中磕破,检查蛋黄和蛋清的新鲜状况,以免有不新鲜的鸡卵液污染,影响培养基的营养成份。
4、丙酮酸钠培养基的制备(1)培养基组分:天门冬素3.6g,磷酸二氢钾2.4g,七水硫酸镁0.24g,柠檬酸镁0.6g,丙酮酸钠1.6g,蒸馏水600mL,调PH值至7.2,葡萄糖4.0g,可溶性淀粉30g,新鲜鸡卵液1000mL,2%孔雀绿水溶液20mL。(2)制备与保存:各盐类成份充分溶解后,用4%NaOH校正至PH7.2后,加入葡萄糖混匀。再加入可溶性淀粉,混匀,沸水浴煮沸(约40min)至淀粉成透明糊状。以下步骤同“Lowenstein-Jensen培养基的制备”中的“(2)制备与保存”。(3)注意事项同“Lowenstein-Jensen培养基的制备”中的“(3)注意事项”。
二、样本处理
1、组织样本处理
(1)研磨组织:将采集到的肺脏、淋巴病变组织,用剪刀剪成小块,然后加入少量生理盐水,用研磨棒研磨,制成乳剂。(2)碱处理:取2mL~4mL乳剂液,加人等量的4%NaOH溶液,充分振摇5min~10min,或摇至发生液化为止。(3)酸平衡:碱处理10min后,用2mol/L的HCL调PH值至6.5-7.0,在10min之内完成。(4)离心:酸碱处理后的菌液,离心20min,4000rpm。弃上清,沉淀加入适量的生理盐水,充分混匀。
2、鼻拭子样品的处理
(1)将含鼻拭子样品的生理盐水用4000rpm离心20min,收集沉淀,加入适量的生理盐水。
(2)碱处理:同步加入等量4%NaOH和2.94%枸橼酸钠中(含1%N-乙酰-L半胱胺酸),室温静置20分钟,使粘液液化至清亮。(3)酸平衡:碱处理20min后,用2mol/L的HCL调PH值至6.5-7.0,在10min之内完成。(4)离心:酸碱处理后的菌液,离心20min,4000rpm。弃上清,沉淀加入适量的生理盐水,充分混匀。
三、结核病致病原快速培养
酸、碱处理后的菌液,用无菌的一次性滴管取0.2mL菌液垂直穿刺至含0.3%琼脂高糖DRM培养基,置37℃恒温培养箱中预培养3天,3天后用无菌的一次性滴管取0.2mL菌液分别转接至丙酮酸钠培养基和L-J培养基斜面,置37℃恒温培养箱中培养,从第三天开始进行观察培养基中细菌的生长情况并做记录。
实施例2、牛结核分枝杆菌参考菌株培养方法比较
1.材料方法
1.1菌株:牛结核分枝杆菌参考菌株。
1.2预处理:将单个牛结核分枝杆菌参考菌株菌落稀释到1mL生理盐水中,再混入30mL痰液中,充分混匀,使用鼻拭子蘸取后放入10mL生理盐水中,按照以下进行步骤处理:
(1)将含鼻拭子样品的生理盐水用4000rpm离心20min,收集沉淀,加入适量的生理盐水;
(2)碱处理:同步加入等量4%NaOH和2.94%枸橼酸钠中(含1%N-乙酰-L半胱胺酸),室温静置20分钟,使粘液液化至清亮;
(3)酸平衡:碱处理20min后,用2mol/L的HCL调PH值至6.5-7.0,在10min之内完成;
(4)离心:酸碱处理后的菌液,离心20min,4000rpm。弃上清,沉淀加入适量的生理盐水,充分混匀。
2、培养
按照实施例1中的“结核病致病原快速培养方法”进行,对照组将处理的样本直接接种到丙酮酸钠培养基或L-J培养基。
3、结果:直接接种于丙酮酸钠和L-J培养基中,在丙酮酸钠培养基上出现可见菌落需要13d,26d后长出典型的菌落;在L-J培养基上出现可见菌落需要15d,31d后长出典型的菌落(表1)。
牛型结核分枝杆菌参考菌株按照上述处理后,使用三步法培养,预培养后接种于丙酮酸钠培养基,样本处理后初次接种至出现可见菌落平均需要9天,第18天出现典型菌落;预培养后接种于L-J培养基,样本处理后初次接种至出现可见菌落平均需要9天,第18天出现典型菌落(表1)。
表1:牛结核分枝杆菌参考菌株用直接法和间接法培养比较结果
实施例3、六份牛结核病病理组织样本的培养比较
1.材料方法
1.1菌株:从屠宰场采集的六份牛结核病肺脏病理组织样本。
1.2预处理:按照实施例1中的组织样本处理方式进行组织样本的处理,具体如下:
(1)研磨组织:将采集到的肺脏剪刀剪成小块,然后加入少量生理盐水,用研磨棒研磨,制成乳剂。(2)碱处理:取2mL~4mL乳剂液,加人等量的4%NaOH溶液,充分振摇5min~10min,或摇至发生液化为止。(3)酸平衡:碱处理10min后,用2mol/L的HCL调PH值至6.5-7.0,在10min之内完成。(4)离心:酸碱处理后的菌液,离心20min,4000rpm。弃上清,沉淀加入适量的生理盐水,充分混匀。
2、培养
按照实施例1中的“结核病致病原快速培养方法”进行,对照组将处理的样本直接接种到丙酮酸钠培养基或L-J培养基。
3、结果:酸、碱处理后的菌液直接接种在丙酮酸钠培养基和L-J培养基上,其中,接种于丙酮酸钠培养基中,平均在第64天出现可见菌落,继续培养平均在第86天出现典型菌落;接种于L-J培养基的细菌平均第66天出现可见菌落,继续培养平均在第87天出现典型菌落(表2)。
使用结核病致病原快速培养方法,预培养后接种于丙酮酸钠培养基,样本处理后初次接种至出现可见菌落平均需要19天,第31天出现典型菌落;预培养后接种于L-J培养基,样本处理后初次接种至出现可见菌落平均需要24天,第35天出现典型菌落(表2)。
表2结核分枝杆菌病理组织经不同方法培养结果的比较
Claims (3)
1.一种快速培养结核病致病菌的培养方法,其特征在于:该培养方法包括下述步骤:
一、培养基的制备与保存
(一)0.3%琼脂高糖DRM培养基的制备
(1)将L-天冬酰胺 1g,磷酸二氢钾 1.5g,磷酸氢二钠 2.5g,七水硫酸镁 0.5g,二水柠檬酸纳 1.3g,柠檬酸铁胺 0.05g,丙酮酸钠 2.4g,琼脂粉 3g,水 1000mL溶解后,在115-121℃,高压条件下,20min,制备成0.3%琼脂快速培养基,备用;
(2)称取100g白糖,加入50mL水,在45-50℃充分溶解,110-115℃,高压10-15 min灭菌后制备成200%高糖液,备用;
(3)无菌条件下,在加高压除菌后处于60-80℃的143mL的200%高糖液加到同等条件下1000mL 0.3%琼脂快速培养基中,充分混匀,制备成0.3%琼脂高糖DRM培养基分装,18mm×150mm试管,每管7mL, 4℃保存备用;
(二)Lowenstein-Jensen培养基的制备
(1)将天门冬素 3.6g,磷酸二氢钾 2.4g,七水硫酸镁 0.24g,柠檬酸镁0.6g,丙三醇12mL,蒸馏水600mL,充分溶解后备用;
(2)向步骤(1)中加入可溶性淀粉30g,混匀,煮沸35-45min,成透明糊状溶液,冷却后备用;
(3)向步骤(2)中加入经过滤后的新鲜鸡卵液1000mL,混匀,备用;
(4)最后,向步骤(3)中加入2%孔雀绿水溶液20mL,混匀,分装,18mm×150mm试管,每管7mL,放置斜面高度占试管三分之二,在温度条件85℃,45min间歇灭菌两次,37℃无菌测定24h后,4℃贮存备用;
(三)丙酮酸钠培养基的制备
(1)先将天门冬素 3.6g,磷酸二氢钾 2.4g,七水硫酸镁 0.24g,柠檬酸镁 0.6g,丙酮酸钠 1.6g,蒸馏水 600mL,充分溶解后,用4%NaOH校正至pH7.2后,加入葡萄糖4.0g混匀备用;
(2)向步骤(1)中加入可溶性淀粉30g,混匀,煮沸35-45min成透明糊状溶液,冷却后备用;
(3)向步骤(2)中加入经过滤后的新鲜鸡卵液1000mL,混匀,备用;
(4)最后,向步骤(3)中加入2%孔雀绿水溶液20mL,混匀,分装,18mm×150mm试管,每管7mL,放置斜面高度占试管三分之二,在温度条件85℃,45min间歇灭菌两次,37℃无菌测定24h后,4℃贮存备用;
二、样本处理
(一)组织样本处理
(1)研磨组织:将采集到的肺脏、淋巴病变组织,用剪刀剪成小块,然后加入生理盐水,用研磨棒研磨,制成乳剂;
(2)碱处理:取2mL~4mL乳剂液,加人等量的4%NaOH溶液,充分振摇5~10min;
(3)酸平衡:碱处理10min后,用2mol/L的HCL调pH值至6.5-7.0,在10min之内完成;
(4)离心:酸碱处理后的菌液,离心20min,4000rpm,弃上清,沉淀加入生理盐水,充分混匀;
(二)鼻拭子样品的处理
(1)将含鼻拭子样品的生理盐水用4000 rpm离心20 min,收集沉淀,加入适量的生理盐水;
(2)碱处理:同步加入等量4% NaOH和2.94%枸橼酸钠溶液中,其中枸橼酸钠溶液中含1%N-乙酰-L半胱胺酸,室温静置20分钟,使粘液液化至清亮;
(3)酸平衡:碱处理20min后,用2mol/L的HCL调pH值至6.5-7.0,在10min之内完成;
(4)离心:酸碱处理后的菌液,离心20min,4000rpm,弃上清,沉淀加入生理盐水,充分混匀;
(三)结核病致病原快速培养:酸、碱处理后的菌液,用无菌的一次性滴管取0.2mL菌液垂直穿刺至含0.3%琼脂高糖DRM培养基,置37℃恒温培养箱中预培养3天;3天后用无菌的一次性滴管取0.2mL菌液转接至丙酮酸钠培养基和/或L-J培养基斜面,置37℃恒温培养箱中培养,从第三天开始进行观察培养基中细菌的生长情况并做记录。
2.根据权利要求1所述的一种快速培养结核病致病菌的培养方法,其特征在于:所述新鲜鸡卵液的制备过程为:先将新鲜鸡蛋清洗干净,晾干,再浸泡在75%酒精中10-15min;再挑拣消毒后的无裂缝鸡蛋取鸡卵。
3.根据权利要求1所述的一种快速培养结核病致病菌的培养方法,其特征在于:所述的可溶性淀粉为马铃薯淀粉。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910415433.6A CN110055198A (zh) | 2019-05-17 | 2019-05-17 | 一种快速培养结核病致病菌的培养方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910415433.6A CN110055198A (zh) | 2019-05-17 | 2019-05-17 | 一种快速培养结核病致病菌的培养方法 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110055198A true CN110055198A (zh) | 2019-07-26 |
Family
ID=67323536
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910415433.6A Pending CN110055198A (zh) | 2019-05-17 | 2019-05-17 | 一种快速培养结核病致病菌的培养方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110055198A (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101560490A (zh) * | 2009-04-28 | 2009-10-21 | 熊礼宽 | 分支杆菌培养基及制备方法及培养、鉴定和药敏测定方法 |
-
2019
- 2019-05-17 CN CN201910415433.6A patent/CN110055198A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101560490A (zh) * | 2009-04-28 | 2009-10-21 | 熊礼宽 | 分支杆菌培养基及制备方法及培养、鉴定和药敏测定方法 |
Non-Patent Citations (4)
Title |
---|
古努尔·吐尔逊等: "牛结核分枝杆菌株快速培养方法的对比", 《中国动物检疫》 * |
彭丽等: "结核分枝杆菌噬菌体试剂盒的研制与初步评价", 《重庆医科大学学报》 * |
王存利等: "CT检查在糖尿病并发肺结核病中的优势(附260例报告)", 《当代医学》 * |
陆恩词等: "肺结核合并糖尿病患者T淋巴细胞亚群及红细胞免疫功能的变化及意义", 《中国老年学杂志》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN102154167B (zh) | 一种猪肺炎支原体培养基及其制备方法 | |
CN101412977B (zh) | 用于沙门氏菌、副溶血弧菌和霍乱弧菌复合增菌的培养基及制备方法 | |
CN103409375A (zh) | 一种接种鸡胚用病毒稀释液及其制备方法 | |
CN115710566B (zh) | 一种用于稻田综合种养的菌株及其应用 | |
CN101485300B (zh) | 一种拟丽突线虫的培养方法 | |
CN104403952B (zh) | 一种菌核侧耳新菌种及其栽培方法和应用 | |
CN109182151A (zh) | 银杏内生真菌的分离筛选方法 | |
CN106244498B (zh) | 一种果蔬农药残留净化菌剂的制备方法 | |
CN110760456B (zh) | 一种用于降解胆固醇的植物乳杆菌La1及其应用 | |
CN110055198A (zh) | 一种快速培养结核病致病菌的培养方法 | |
CN105779373A (zh) | 一种适宜稻曲病菌产孢的核糖培养基及其应用方法 | |
CN114196580B (zh) | 一种淡紫灰链霉菌海南变种菌株及利用其制备中生菌素制品的方法 | |
RU2681285C1 (ru) | ПИТАТЕЛЬНАЯ СРЕДА ПЛОТНАЯ ДЛЯ КУЛЬТИВИРОВАНИЯ БРУЦЕЛЛ ВИДА Brucella neotomae | |
CN106635865B (zh) | 一种用于牛鼻支原体分离纯化的培养基及其制备方法和应用 | |
CN1325630C (zh) | 一种无菌松材线虫的培养方法 | |
JP6404072B2 (ja) | アカントアメーバの消毒評価方法 | |
CN110904007B (zh) | 兽用诺维氏梭菌外毒素及其制备方法、产毒培养基和应用 | |
CN109400444A (zh) | 抑制植物病源真菌的倍半萜类化合物及其制备方法 | |
CN114317670A (zh) | 一种筛选培养基及其制备方法和应用 | |
CN103952460B (zh) | 一种鉴别天然蜂蜜和人造蜂蜜的方法 | |
Al-Azzauy et al. | The beetroot juice as a bacterial growth and maintenance medium for many pathogenic bacteria | |
CN108676737A (zh) | 大豆内生菌及其应用 | |
RU2111245C1 (ru) | Питательная среда для культивирования дрожжевидных грибов рода candida | |
CN1162549C (zh) | 结核菌双相快速培养基及其制备方法 | |
RU2484141C1 (ru) | Элективно-дифференциальная питательная среда для выделения холерных вибрионов (варианты) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190726 |