CN105861372A - Preparation method of gardnerella vaginalis culture medium - Google Patents
Preparation method of gardnerella vaginalis culture medium Download PDFInfo
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- CN105861372A CN105861372A CN201610271076.7A CN201610271076A CN105861372A CN 105861372 A CN105861372 A CN 105861372A CN 201610271076 A CN201610271076 A CN 201610271076A CN 105861372 A CN105861372 A CN 105861372A
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- 241000207201 Gardnerella vaginalis Species 0.000 title claims abstract description 30
- 239000001963 growth medium Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 238000004264 monolayer culture Methods 0.000 claims abstract description 12
- 239000006781 columbia blood agar Substances 0.000 claims abstract description 9
- 239000012153 distilled water Substances 0.000 claims abstract description 4
- 239000011521 glass Substances 0.000 claims abstract description 4
- 238000010438 heat treatment Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 19
- 238000011177 media preparation Methods 0.000 claims description 18
- 239000006166 lysate Substances 0.000 claims description 9
- 229920001817 Agar Polymers 0.000 claims description 8
- 239000008272 agar Substances 0.000 claims description 8
- 206010018910 Haemolysis Diseases 0.000 claims description 6
- 239000000835 fiber Substances 0.000 claims description 6
- 230000008588 hemolysis Effects 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 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
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 claims description 3
- 229930182566 Gentamicin Natural products 0.000 claims description 3
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 claims description 3
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 3
- 229960003942 amphotericin b Drugs 0.000 claims description 3
- 230000012447 hatching Effects 0.000 claims description 3
- MHWLWQUZZRMNGJ-UHFFFAOYSA-N nalidixic acid Chemical compound C1=C(C)N=C2N(CC)C=C(C(O)=O)C(=O)C2=C1 MHWLWQUZZRMNGJ-UHFFFAOYSA-N 0.000 claims description 3
- 229960000210 nalidixic acid Drugs 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 230000003245 working effect Effects 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 abstract description 9
- 239000008280 blood Substances 0.000 abstract description 9
- 235000015097 nutrients Nutrition 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 abstract 4
- 239000007382 columbia agar Substances 0.000 abstract 2
- 238000001816 cooling Methods 0.000 abstract 2
- 239000000022 bacteriostatic agent Substances 0.000 abstract 1
- 239000006161 blood agar Substances 0.000 abstract 1
- 230000002349 favourable effect Effects 0.000 abstract 1
- 238000011534 incubation Methods 0.000 abstract 1
- 238000011081 inoculation Methods 0.000 abstract 1
- 238000005303 weighing Methods 0.000 abstract 1
- 230000012010 growth Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 3
- 208000004926 Bacterial Vaginosis Diseases 0.000 description 2
- 208000037009 Vaginitis bacterial Diseases 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 206010060937 Amniotic cavity infection Diseases 0.000 description 1
- 208000008158 Chorioamnionitis Diseases 0.000 description 1
- 208000004145 Endometritis Diseases 0.000 description 1
- 206010053636 Obstetric infection Diseases 0.000 description 1
- 241000283898 Ovis Species 0.000 description 1
- 208000006399 Premature Obstetric Labor Diseases 0.000 description 1
- 208000003107 Premature Rupture Fetal Membranes Diseases 0.000 description 1
- 206010036600 Premature labour Diseases 0.000 description 1
- HVYLDJKDVOOTHV-UHFFFAOYSA-N acetic acid;2-iminoethanethiol Chemical compound CC(O)=O.CC(O)=O.SCC=N HVYLDJKDVOOTHV-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 208000026440 premature labor Diseases 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 208000006685 tubal pregnancy Diseases 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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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)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a preparation method of a gardnerella vaginalis culture medium, comprising the steps of: step 1, accurately weighing 4.1g of Columbia blood agar base, and adding 95ml of distilled water for dissolving with heating; step 2: autoclaving the dissolved solution of the step 1; step 3: cooling the dissolved solution to about 50 DEG C, adding 5ml of defibered rabbit blood and combined bacteriostatic agent under the condition of sterile operation; step 4, pouring the solution into a sterilized glass plate, regarding the solution as a rabbit blood Columbia agar monolayer culture medium, cooling the solution for later use; step 5: inoculating the prepared rabbit blood Columbia agar monolayer culture medium with gardnerella vaginalis, carrying out partitioned streak inoculation; and step 6: carrying out incubation at 35 DEG C with 5% CO2 for 48 hours. The preparation method only uses two components namely the rabbit blood and Colombia blood agar as nutrient substances to prepare the monolayer culture medium, can achieve the effect of separating the gardnerella vaginalis, realizes the characteristics of low cost, convenient operation, easy availability of raw materials and the like in preparation of culture media, and is favorable for wide development in different levels of hospitals.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of gardnerella vaginalis culture medium preparation method.
Background technology
Gardnerella vaginalis (gardnerella vaginalis
GV) in the microorganism species of bacterial vaginosis, content, apparently higher than other normal flora, is always thought one of important pathogenic bacteria of bacterial vaginosis by Chinese scholars.GV infects also can cause the Averse pregnancy outcomes such as fallopian tube ectopic pregnancy, premature rupture of fetal membrane, chorioamnionitis, obstetric infection, endometritis and neonate premature labor.
The isolated culture of GV remains the internationally recognized goldstandard of GV detection.GV growth conditions is the harshest, needs multiple nutrients material to participate in growth course.Common Sanguis caprae seu ovis flat board, through 35 ~ 37 DEG C, 5%CO2,48 ~ 72h cultivation can form the bacterium colony of needle point size, and is difficult to identification, lacks specificity colony morphology characteristic, easily causes the missing inspection in routine work.Domestic scholars select cultivation in add multiple nutrients composition (brother promote the growth of GV than agar, people's whole blood and blood plasma, brain-heart-infusion, yeast extract, nutrient broth etc., improves the positive rate of detection.GV is containing producing β haemolysis on people's blood meida, the appearance of β haemolysis acts not only as the specific colony growth feature of GV, is simultaneously also beneficial to the raising of positive rate.
The most most document has been adopted the Double-Medium containing human blood and has been added multiple nutrition separation gardnerella vaginalis, and in the case of national centre's blood station blood supply wretched insufficiency, human blood is expensive and is difficult to obtain.Double-Medium preparation is complicated, increase opportunities for contamination.The interpolation of the nutrition such as brain-heart-infusion, yeast extract causes cost to increase.Therefore, research can with low cost, preparation procedure simple, ensure stable bacterial separating effect culture medium simultaneously, be wide variety of precondition.
Summary of the invention
The purpose of the present invention: provide a kind of gardnerella vaginalis culture medium preparation method, material is easy to get, prepare simplicity, with low cost so that it is all can extensively carry out at situation of all-level hospitals, improve gardnerella vaginalis separation rate.
To achieve these goals, the technical scheme is that
A kind of gardnerella vaginalis culture medium preparation method, the method at least comprises the steps:
Step 1: precise columbia blood agar base 4.1g, and add 95ml distilled water heating for dissolving.
Step 2: to the lysate autoclaving in step 1.
Step 3: lysate is cooled to about 50 DEG C sterile workings addition 5ml and takes off fiber Sanguis Leporis seu oryctolagi and combined bacteriostat.
Step 4: sterile working is poured into sterilizing glass dish, is prepared as Sanguis Leporis seu oryctolagi brother than agar monolayer culture base, cold the most standby.
Step 5: gardnerella vaginalis is inoculated in the Sanguis Leporis seu oryctolagi brother prepared and inoculates than agar monolayer culture base, sectional streak.
Step 6: be placed in 35 DEG C, 5%CO2 hatches 48 hours.
Above-mentioned gardnerella vaginalis culture medium preparation method, wherein, in described step 1, the pH value after described columbia blood agar base 4.1g dissolves is 7.2-7.4.
Above-mentioned gardnerella vaginalis culture medium preparation method, wherein, in described step 2, described autoclaved temperature is 121 DEG C, and the autoclaved time is 15 minutes.
Above-mentioned gardnerella vaginalis culture medium preparation method, wherein, in described step 3, it is final concentration of that described lysate adds after de-fiber Sanguis Leporis seu oryctolagi and combined bacteriostat: amphotericin B 2 g/ml；Gentamycin 5 g/ml；Nalidixic acid 30 g/ml.
Above-mentioned gardnerella vaginalis culture medium preparation method, wherein, in described step 6, after hatching 48 hours, bacterium colony is rounded, smooth, moistening, dew drips sample form, and maximum thalline diameter 0.985mm, β zone of hemolysis maximum gauge reaches 2.273mm.
The present invention only uses Sanguis Leporis seu oryctolagi and two kinds of compositions of Columbia Blood Agar as nutrient substance, only it is configured to monolayer culture base, just can reach gardnerella vaginalis separating effect, achieve the features such as culture medium preparation cost is cheap, easy to operate, raw material is easy to get, be conducive to extensively carrying out in different grades of hospital.
Accompanying drawing explanation
Fig. 1 is the flow chart of the present invention a kind of gardnerella vaginalis culture medium preparation method.
Detailed description of the invention
Embodiments of the invention are further illustrated below in conjunction with accompanying drawing.
Referring to shown in accompanying drawing 1, a kind of gardnerella vaginalis culture medium preparation method, the method at least comprises the steps:
Step 1: precise columbia blood agar base 4.1g, and add 95ml distilled water heating for dissolving.
Step 2: to the lysate autoclaving in step 1.
Step 3: lysate is cooled to about 50 DEG C sterile workings addition 5ml and takes off fiber Sanguis Leporis seu oryctolagi and combined bacteriostat.
Step 4: sterile working is poured into sterilizing glass dish, is prepared as Sanguis Leporis seu oryctolagi brother than agar monolayer culture base, cold the most standby.
Step 5: gardnerella vaginalis is inoculated in the Sanguis Leporis seu oryctolagi brother prepared and inoculates than agar monolayer culture base, sectional streak.
Step 6: be placed in 35 DEG C, 5%CO2 hatches 48 hours.
In described step 1, the pH value after described columbia blood agar base 4.1g dissolves is 7.2-7.4.
In described step 2, described autoclaved temperature is 121 DEG C, and the autoclaved time is 15 minutes.
In described step 3, it is final concentration of that described lysate adds after de-fiber Sanguis Leporis seu oryctolagi and combined bacteriostat: amphotericin B 2 g/ml；Gentamycin 5 g/ml；Nalidixic acid 30 g/ml.
In described step 6, after hatching 48 hours, bacterium colony is rounded, smooth, moistening, dew drips sample form, records maximum thalline diameter up to 0.985mm with slide gauge, obvious because adding the β zone of hemolysis size of Sanguis Leporis seu oryctolagi generation, β zone of hemolysis maximum gauge is up to 2.273mm.
In the present invention, bacterium colony people naked eyes are clear and legible, it is possible to reach gardnerella vaginalis separating effect.Sanguis Leporis seu oryctolagi brother is that one is cost-effective, preparation is convenient than agar monolayer culture base, has the gardnerella vaginalis isolation medium of important clinical value.
In sum, the present invention only uses Sanguis Leporis seu oryctolagi and two kinds of compositions of Columbia Blood Agar as nutrient substance, only it is configured to monolayer culture base, just can reach gardnerella vaginalis separating effect, achieve the features such as culture medium preparation cost is cheap, easy to operate, raw material is easy to get, be conducive to extensively carrying out in different grades of hospital.
The foregoing is only the preferred embodiments of the present invention; not thereby the scope of the claims of the present invention is limited; every equivalent structure transformation utilizing description of the invention content to be made; or directly or indirectly use the technical field being attached to other Related products, the most in like manner it is included in the scope of patent protection of the present invention.
Claims (5)
1. a gardnerella vaginalis culture medium preparation method, it is characterised in that: the method at least comprises the steps:
Step 1: precise columbia blood agar base 4.1g, and add 95ml distilled water heating for dissolving；
Step 2: to the lysate autoclaving in step 1；
Step 3: lysate is cooled to about 50 DEG C sterile workings addition 5ml and takes off fiber Sanguis Leporis seu oryctolagi and combined bacteriostat；
Step 4: sterile working is poured into sterilizing glass dish, is prepared as Sanguis Leporis seu oryctolagi brother than agar monolayer culture base, cold the most standby；
Step 5: gardnerella vaginalis is inoculated in the Sanguis Leporis seu oryctolagi brother prepared and inoculates than agar monolayer culture base, sectional streak；
Step 6: be placed in 35 DEG C, 5%CO2 hatches 48 hours.
Gardnerella vaginalis culture medium preparation method the most according to claim 1, it is characterised in that: in described step 1, the pH value after described columbia blood agar base 4.1g dissolves is 7.2-7.4.
Gardnerella vaginalis culture medium preparation method the most according to claim 1, it is characterised in that: in described step 2, described autoclaved temperature is 121 DEG C, and the autoclaved time is 15 minutes.
Gardnerella vaginalis culture medium preparation method the most according to claim 1, it is characterised in that: in described step 3, it is final concentration of that described lysate adds after de-fiber Sanguis Leporis seu oryctolagi and combined bacteriostat: amphotericin B 2 g/ml；Gentamycin 5 g/ml；Nalidixic acid 30 g/ml.
Gardnerella vaginalis culture medium preparation method the most according to claim 1, it is characterized in that: in described step 6, after hatching 48 hours, bacterium colony is rounded, smooth, moistening, dew drips sample form, maximum thalline diameter 0.985mm, β zone of hemolysis maximum gauge reaches 2.273mm.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610271076.7A CN105861372A (en) | 2016-04-27 | 2016-04-27 | Preparation method of gardnerella vaginalis culture medium |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610271076.7A CN105861372A (en) | 2016-04-27 | 2016-04-27 | Preparation method of gardnerella vaginalis culture medium |
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| Publication Number | Publication Date |
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| CN105861372A true CN105861372A (en) | 2016-08-17 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201610271076.7A Pending CN105861372A (en) | 2016-04-27 | 2016-04-27 | Preparation method of gardnerella vaginalis culture medium |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173236A (en) * | 2007-11-22 | 2008-05-07 | 中国医科大学绍兴华宇医院 | Selective culture medium for gardnerella vaginalis and method for producing the same |
-
2016
- 2016-04-27 CN CN201610271076.7A patent/CN105861372A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101173236A (en) * | 2007-11-22 | 2008-05-07 | 中国医科大学绍兴华宇医院 | Selective culture medium for gardnerella vaginalis and method for producing the same |
Non-Patent Citations (2)
| Title |
|---|
| B. WESLEY CATLIN: "Gardnerella vaginalis: Characteristics, Clinical Considerations, and Controversies", 《CLINICAL MICROBIOLOGY REVIEWS》 * |
| 陈远翔 等: "三种培养基对阴道加德纳菌分离效果评价", 《现代预防医学》 * |
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