CN108593395A - The preparation method and application method of acid-fast stain Quality Control piece - Google Patents
The preparation method and application method of acid-fast stain Quality Control piece Download PDFInfo
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- CN108593395A CN108593395A CN201810418417.8A CN201810418417A CN108593395A CN 108593395 A CN108593395 A CN 108593395A CN 201810418417 A CN201810418417 A CN 201810418417A CN 108593395 A CN108593395 A CN 108593395A
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- 238000003908 quality control method Methods 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims abstract description 44
- 238000004043 dyeing Methods 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000002253 acid Substances 0.000 claims abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000007037 hydroformylation reaction Methods 0.000 claims abstract description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000001035 drying Methods 0.000 claims abstract description 6
- HZLHRDBTVSZCBS-UVJJDBRNSA-N 4-[(e)-(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline;hydrochloride Chemical compound Cl.C1=CC(=N)C(C)=C\C1=C(C=1C=C(C)C(N)=CC=1)/C1=CC=C(N)C=C1 HZLHRDBTVSZCBS-UVJJDBRNSA-N 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims abstract description 5
- 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 abstract description 4
- 229940107698 malachite green Drugs 0.000 claims abstract description 4
- 238000000386 microscopy Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 28
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 11
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 8
- 102000002322 Egg Proteins Human genes 0.000 claims description 8
- 108010000912 Egg Proteins Proteins 0.000 claims description 8
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 8
- 235000014103 egg white Nutrition 0.000 claims description 8
- 210000000969 egg white Anatomy 0.000 claims description 8
- 210000002969 egg yolk Anatomy 0.000 claims description 8
- 235000011187 glycerol Nutrition 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 239000000047 product Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000000975 dye Substances 0.000 claims description 5
- 239000008399 tap water Substances 0.000 claims description 5
- 235000020679 tap water Nutrition 0.000 claims description 5
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 claims description 3
- 241000186000 Bifidobacterium Species 0.000 claims description 3
- 230000001458 anti-acid effect Effects 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 3
- 229940031551 inactivated vaccine Drugs 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000011156 evaluation Methods 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 108010077544 Chromatin Proteins 0.000 claims 1
- 210000003483 chromatin Anatomy 0.000 claims 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003245 coal Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229960000907 methylthioninium chloride Drugs 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of preparation method and application method of acid-fast stain Quality Control piece, the preparation of Quality Control piece includes that bacterium solution preparation and hydroformylation are fixed.In use, first adding carbolfuchsin solution dyeing, reagent is completely covered sample and stops 5 minutes, and circulating water is rinsed until not having the appearance of cloud stain, drains remaining water；Then plus ethanol solution hydrochloride decoloration, reagent are completely covered sample and stop, rinse and drain remaining water；Finally plus malachite green solution dyeing, reagent are completely covered sample and stop 1 minute, rinse and drain remaining water；Microscopy after drying.Acid fast bacteria takes on a red color after dyeing, and sharp contrast is formed with environment, can clearly be observed that form, arrangement and the certain structure features of acid fast bacteria, and the substance in other bacteriums and background is blue-green.Be carried out at the same time the process for just contaminating, decolourizing, redying by Quality Control piece and clinical samples smear, can detect dyeing liquor product quality, whether the quality of expired and operating personnel progress acid-fast stain.
Description
Technical field
The present invention relates to the preparation of technical field of biological more particularly to a kind of acid-fast stain Quality Control piece and users
Method.
Background technology
Ziehi-Neelsen stain acid-fast staining method are initiated simultaneously for 1882 by Paul Ehrlich (F.Ehrlich)
Through F.Ehrlich) Qi Er (Ziehl) is improved and the bacterial stain that creates.It is wherein most representative for tulase
Qi Er-Nelson (Ziehl-Neelsen) decoration methods and Qi Er-Jia Beite (Ziehl-Gabbet) decoration method.
Together-Nissl Ziehi-Neelsen stain is to make mycolic acid with carbolfuchsin firm connection at compound in a heated condition
Object handles also nondiscoloration with hydrochloride alcohol.When again plus after alkaline methylene blue is redyed, mycobacteria remains as red, and other bacteriums
And the substance in background is blue.
Acid fast bacteria cell wall causes staining efficiency low rich in wax with lipid, needs to heat coal acid solution acceleration cell in the past
Wall forms compound, and the modern times are prepared using special process, need not carry out heating and dyeing can be completed.Acid fast bacteria cell wall is by product
It is hardly possible after red dye dyeing to be decolourized, it can not decolourize using ethanolic HCl destainer.Utilize acid fast bacteria
This feature can be distinguished with non-acid fast bacteria under the microscope.
Acid-fast stain Quality Control piece can simultaneously be dyed with sample smear, carry out comparing determining positive bacteria and negative bacterium, be examined anti-
The quality and reviewer's qualification of sour dyeing liquor.Existing market does not have a prepared domestic acid-fast stain Quality Control in advance
Piece is compareed for acid-fast stain and is used.
Based on the above present situation, biotechnology urgent need want one kind ensure bio-safety, it is easy to operate, can detect it is antiacid
The quality of dyeing liquor, the acid-fast stain liquid quality-control product that training on operation, technical ability supervision are carried out for reviewer.
Invention content
The object of the present invention is to provide a kind of acid-fast stain liquid quality-control products, it can be ensured that bio-safety, it is easy to operate, can
It detects the quality of acid-fast stain liquid, carry out training on operation and technical ability supervision for reviewer.
Technical solution is used by the present invention solves the above problems：A kind of preparation method of acid-fast stain Quality Control piece, packet
It includes bacterium solution preparation and hydroformylation is fixed, concrete operations are as follows：
(1) prepared by bacterium solution
A liquid：Egg white is drawn respectively and the slide adherency system being made containing egg white, yolk and glycerine is added in glycerine in yolk；
B liquid：Bifidobacterium live triple capsule is taken, with normal saline dilution, placement makes medicated premix precipitate, takes supernatant
Liquid, Aspirate supernatant add A liquid mixing smears, carry out acid-fast stain, and micro- sem observation negative bacterium is blue-green, is used as and matches after qualified
Bacterium solution processed is spare；
C liquid：Inactivated vaccine freeze-dried powder is taken, is mixed with normal saline dilution fully shaking, adds A liquid mixing smears, is carried out
Acid-fast stain, micro- sem observation positive bacteria are red, spare as bacterium solution is prepared after qualified.
(2) hydroformylation is fixed
A liquid is added after taking formalin, B liquid that C liquid mixings are added forms joint bacterium solution mixing or oscillator oscillation repeatedly,
It is used for acid-fast stain Quality Control piece smear；
Mixed bacteria liquid is drawn with sample injector, the mycoderm piece of 2cm diameters is painted with microbionation ring, after natural drying
It heats and fixes on alcolhol burner, the acid-fast stain Quality Control piece labeling that will be made is stored in spare in 2-8 DEG C of environment.
Preferably, the mass content of egg white, yolk and glycerine is respectively 40%, 10%, 50% in A liquid.
Preferably, in hydroformylation fixing step formalin a concentration of 40wt%.
A kind of application method of acid-fast stain Quality Control piece first carries out acid-fast stain and then evaluates dyeing liquor.
The step of acid-fast stain is
(1), plus carbolfuchsin solution dyes, and reagent is completely covered sample and stops 5 minutes or more, and circulating water rinses straight
Occur to no cloud stain, drains remaining water on sample；
(2), plus ethanol solution hydrochloride decoloration, reagent are completely covered sample and stop 15-30 seconds, residence time sighting target sheet
Thickness determines that flowing tap water rinses immediately, drains remaining water on sample；
(3), plus malachite green solution dyes, and reagent is completely covered sample and stops 1 minute or more, and circulating water rushes immediately
It washes, drains remaining water on sample；Blotted with filter paper or air-dry in air or 50 DEG C drying after microscopy；
Acid-fast stain result is as follows：
Acid fast bacteria takes on a red color after dyeing, and sharp contrast is formed with environment, can clearly be observed that form, the row of acid fast bacteria
Row and certain structure features, and the substance in other bacteriums and background is blue-green；
Acid-fast stain Quality Control piece is as follows to the evaluation of dyeing liquor：
It is carried out at the same time the process for just contaminating, decolourizing, redying by the acid-fast stain Quality Control piece and clinical samples smear that prepare,
It can detect the product quality of dyeing liquor, whether expired and operating personnel carry out the quality of acid-fast stain.
Compared with the prior art, the advantages of the present invention are as follows：This application provides a kind of acid-fast stain liquid quality-control products, are used for
Dyeing liquor is evaluated, it is ensured that bio-safety, quality easy to operate, that acid-fast stain liquid can be detected, for reviewer
Carry out training on operation and technical ability supervision.
Specific implementation mode
Present invention is further described in detail with reference to embodiments.
The present embodiment is related to a kind of acid-fast stain Quality Control piece, for evaluating the antibacterial dyeing of dyeing liquor.The Quality Control
The preparation of piece includes that bacterium solution preparation and hydroformylation are fixed, and the specific method is as follows
(1) prepared by bacterium solution
A liquid：Egg white is drawn respectively and yolk is added in glycerine and the glass containing 40% egg white, 10% yolk and 50% glycerine is made
Piece adherency system；
B liquid：Bifidobacterium live triple capsule (specification 210mg/) 1 is taken, with 0.5ml normal saline dilutions, is put
Setting 15 minutes makes medicated premix precipitate, and takes out supernatant, draws 7ul supernatants and adds A liquid 7ul mixing smears, carries out antiacid dye
Color, micro- sem observation negative bacterium are blue-green, and it is spare to make preparation bacterium solution after qualified；
C liquid：Inactivated vaccine freeze-dried powder 50mg/ branch is mixed with 1ml normal saline dilution fully shakings, is drawn 7ul and is added A
Liquid 7ul mixing smears carry out acid-fast stain, and micro- sem observation positive bacteria is red, and it is spare to prepare bacterium solution for work after qualification.
(2) hydroformylation is fixed
It is anti-that A liquid 1.2ml formation joint bacterium solution is added after taking 40% formalin 150ul, B liquid 0.2ml that C liquid mixings are added
Compound is even or oscillator vibrates, and is used for acid-fast stain Quality Control piece smear.
Mixed bacteria liquid 15ul is drawn with sample injector, the mycoderm piece of 2cm diameters is painted with microbionation ring, through naturally dry
It heats and fixes on alcolhol burner after dry, the acid-fast stain Quality Control piece labeling that will be made is stored in spare in 2-8 DEG C of refrigerator.
The method and step of acid-fast stain is as follows：
1, plus carbolfuchsin solution dyeing, reagent be completely covered sample and stop 5 minutes, flowing tap water rinse until
There is no the appearance of cloud stain, drains remaining water on sample；
2, plus ethanol solution hydrochloride decoloration, reagent are completely covered sample and stop 15~30 seconds (being determined depending on Sample thickness),
Flowing tap water rinses immediately, drains remaining water on sample；
3, plus malachite green solution dyeing, reagent are completely covered sample and stop 1 minute, and flowing tap water rinses immediately,
Drain remaining water on sample；Blotted with filter paper or air-dry in air or 50 DEG C drying after microscopy；
Acid-fast stain result is as follows：
Acid fast bacteria takes on a red color after dyeing, and sharp contrast is formed with environment, can clearly be observed that form, the row of acid fast bacteria
Row and certain structure features, and the substance in other bacteriums and background is blue-green.
It is carried out at the same time the process for just contaminating, decolourizing, redying by the acid-fast stain Quality Control piece and clinical samples smear that prepare,
It can detect the product quality of dyeing liquor, whether expired and operating personnel carry out the quality of acid-fast stain.
In addition to the implementation, all to use equivalent transformation or equivalent replacement the invention also includes there is an other embodiment
The technical solution that mode is formed should all be fallen within the scope of the hereto appended claims.
Claims (4)
1. a kind of preparation method of acid-fast stain Quality Control piece, it is characterised in that：It is prepared including bacterium solution and hydroformylation is fixed, concrete operations
It is as follows：
(1) prepared by bacterium solution
A liquid：Egg white is drawn respectively and the slide adherency system being made containing egg white, yolk and glycerine is added in glycerine in yolk；
B liquid：Bifidobacterium live triple capsule is taken, with normal saline dilution, placement makes medicated premix precipitate, takes supernatant,
Aspirate supernatant adds A liquid mixing smears, carries out acid-fast stain, and micro- sem observation negative bacterium is blue-green, is used as and prepares after qualified
Bacterium solution is spare；
C liquid：Inactivated vaccine freeze-dried powder is taken, is mixed with normal saline dilution fully shaking, A liquid mixing smears are added, is carried out antiacid
Dyeing, micro- sem observation positive bacteria are red, spare as bacterium solution is prepared after qualified；
(2) hydroformylation is fixed
A liquid is added after taking formalin, B liquid that C liquid mixings are added and forms joint bacterium solution mixing or oscillator oscillation repeatedly, for anti-
Sour chromatin control wafer smear uses；
Mixed bacteria liquid is drawn with sample injector, the mycoderm piece of 2cm diameters is painted with microbionation ring, in wine after natural drying
It heats and fixes on smart lamp, the acid-fast stain Quality Control piece labeling that will be made is stored in spare in 2-8 DEG C of environment.
2. the preparation method of acid-fast stain Quality Control piece according to claim 1, it is characterised in that：Egg white in A liquid, yolk and
The mass content of glycerine is respectively 40%, 10%, 50%.
3. the preparation method of acid-fast stain Quality Control piece according to claim 1, it is characterised in that：First in hydroformylation fixing step
A concentration of 40wt% of aldehyde solution.
4. a kind of application method of acid-fast stain Quality Control piece, it is characterised in that：Acid-fast stain is first carried out then to evaluate dyeing liquor,
It is specific as follows
The step of acid-fast stain is
(1), plus carbolfuchsin solution dyes, and reagent is completely covered sample and stops 5 minutes or more, and circulating water is rinsed until not having
There is the appearance of cloud stain, drains remaining water on sample；
(2), plus ethanol solution hydrochloride decoloration, reagent are completely covered sample and stop 15-30 seconds, and the residence time determines depending on Sample thickness
Fixed, flowing tap water rinses immediately, drains remaining water on sample；
(3), plus malachite green solution dyes, and reagent is completely covered sample and stops 1 minute or more, and circulating water rinses immediately, drip
Remove remaining water on sample；Blotted with filter paper or air-dry in air or 50 DEG C drying after microscopy；
Acid-fast stain result is as follows：
Acid fast bacteria takes on a red color after dyeing, with environment formed sharp contrast, can clearly be observed that acid fast bacteria form, arrangement and
Certain structure features, and the substance in other bacteriums and background is blue-green；
Acid-fast stain Quality Control piece is as follows to the evaluation of dyeing liquor：
It is carried out at the same time the process for just contaminating, decolourizing, redying by the acid-fast stain Quality Control piece and clinical samples smear that prepare, it can be with
Detect the product quality of dyeing liquor, whether expired and operating personnel carry out the quality of acid-fast stain.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810418417.8A CN108593395A (en) | 2018-05-04 | 2018-05-04 | The preparation method and application method of acid-fast stain Quality Control piece |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810418417.8A CN108593395A (en) | 2018-05-04 | 2018-05-04 | The preparation method and application method of acid-fast stain Quality Control piece |
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| CN108593395A true CN108593395A (en) | 2018-09-28 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129610A (en) * | 2020-09-22 | 2020-12-25 | 无锡市第二人民医院 | Bacterial capsule staining method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451260A (en) * | 2013-09-09 | 2013-12-18 | 上海兰卫临床检验有限公司 | Preparation method of anti-acid staining solution quality control product |
-
2018
- 2018-05-04 CN CN201810418417.8A patent/CN108593395A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451260A (en) * | 2013-09-09 | 2013-12-18 | 上海兰卫临床检验有限公司 | Preparation method of anti-acid staining solution quality control product |
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| 上海市立医学化验所: "《实用临床检验》", 31 March 1965, 上海科学技术出版社 * |
| 董彬 等: "痰标本抗酸杆菌涂片检查染色方法研究", 《医学动物防制》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112129610A (en) * | 2020-09-22 | 2020-12-25 | 无锡市第二人民医院 | Bacterial capsule staining method and application thereof |
| CN112129610B (en) * | 2020-09-22 | 2021-07-16 | 无锡市第二人民医院 | Bacterial capsule staining method and application thereof |
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