CN105803041A - Acinetobacter baumannii capsule staining kit and detection method - Google Patents
Acinetobacter baumannii capsule staining kit and detection method Download PDFInfo
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- CN105803041A CN105803041A CN201610207220.0A CN201610207220A CN105803041A CN 105803041 A CN105803041 A CN 105803041A CN 201610207220 A CN201610207220 A CN 201610207220A CN 105803041 A CN105803041 A CN 105803041A
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Abstract
The invention discloses an Acinetobacter baumannii capsule staining kit and a detection method and belongs to the field of clinical laboratory diagnostics. The detection method comprises the following steps: Acinetobacter baumannii is mixed with a Congo red dye and an adhesive, then flakes are prepared through pushing, and Acinetobacter baumannii capsules are stained with a Congo red dyeing method. One simple, convenient, rapid and feasible evaluation method for the virulence of the Acinetobacter baumannii is established, the capsule generation condition of the clinically separated Acinetobacter baumannii can be detected, the strain virulence and treatment difficulty level can be evaluated, and reference is provided for selection of clinic treatment schemes.
Description
Technical field
The invention belongs to Clinical Laboratory Diagnostics field, relate to a kind of Acinetobacter bauamnnii pod membrane quick detection kit and detection method.
Background technology
Acinetobacter bauamnnii (Acinetobacterbaumannii, Ab) is a modal strain in acinetobacter, is a kind of azymic saccharide, Gram's staining coccobacillus negative, motorless, it is common to be present in nature and human body.This bacterium Distribution in hospital extensively and can long-term surviving, be one of important conditioned pathogen of nosocomial infection.
Acinetobacter bauamnnii infects can cause the multiple diseases such as patient's septicemia, pneumonia and meningitis, even causes the death of patient.Existing research is disclosed in Acinetobacter bauamnnii attack to host cell processes, and antibacterial can cause the various immunocyte apoptosis of host or death and monocytes/macrophages respiratory burst, causes each organ Normal cell death of patient.But clinical Acinetobacter bauamnnii research focuses mostly in Plasmid at present, its pathogenicity or virulence factor are studied relatively fewer, and treatment clinical course finding, Acinetobacter bauamnnii drug-resistant intensity and antibacterial pathogenicity are not perfectly correlated, cause bigger puzzlement to this bacterium treatment of infection.In recent years by genome sequencing, gene Knockout and in conjunction with several animal models, the Major Virulence Factors determining Acinetobacter bauamnnii has outer membrane protein A(outermembraneproteinA, OmpA), capsular polysaccharide (capsularpolysaccharide, CPS), lipopolysaccharide (lipopolysaccharide, LPS) etc..
Pod membrane is an important virulence factor of antibacterial, one layer of polysaccharose substance that the most appearance of many antibacterials covers, by antibacterial and external environment isolation, it is prevented that the killing of various antibiotic and antibody on bacterial.Simultaneously pod membrane may also help in antibacterial and resists poor environment, and antibacterial self is not swallowed by human leukocytes in protection, and the property of can select that be attached on the surface of specific cells (such as lymphocyte or macrophage etc.), show the single-minded attacking ability to target cell.Researcher finds very early, has encapsulated virulence of Streptococcus pneumoniae apparently higher than not having encapsulated streptococcus pneumoniae, encapsulated for tool streptococcus pneumoniae is injected in Mice Body, mice will soon be dead, and not having after encapsulated streptococcus pneumoniae is injected in Mice Body, mice can survive the long period.Acinetobacter bauamnnii also has pod membrane, and the pod membrane growing amount of different strains is widely different.Russo etc. study discovery, and acapsular Acinetobacter bauamnnii is injected into mouse peritoneal, in 24h mice can fully erased intraperitoneal antibacterial, if but infect have encapsulated Acinetobacter bauamnnii, in 24h ascites, bacterial number is more than 107/ml(Russo,TA,LukeNR,BeananJM,etal.TheK1capsularpolysaccharideofAcinetobacterbaumanniistrain307-0294isamajorvirulencefactor[J].InfectImmun.2010,78(9):p.3993-4000.).The studies above result shows, Acinetobacter bauamnnii pod membrane is closely related with its virulence, and Acinetobacter bauamnnii drug resistance with virulence and is not exclusively equal to, clinical treatment weighs Acinetobacter bauamnnii virulence with greater need for a kind of method, and pod membrane is a kind of potential desirable Testing index.
Summary of the invention
The invention process provides a kind of Acinetobacter bauamnnii pod membrane method for quick, is used for solving clinical rapid evaluation Acinetobacter bauamnnii virulence, and guiding clinical treatment works.
The present invention can be specific binding with bacterial eapsular polysaccharide composition according to congo red characteristic, set up Acinetobacter bauamnnii pod membrane method for quick.
A kind of Acinetobacter bauamnnii capsule stain detection method, carries out as steps described below:
(1) Congo red dye liquor is taken with Ovum Gallus domesticus album by 4:1(v/v) mix, on inoculating loop picking blood plate, Acinetobacter bauamnnii is abundant with aforesaid liquid, makes push jack, natural drying by the method for pushing blood sheet.
(2) dropping destaining solution decolouring 1min, running water.
(3) dropping crystal violet dye liquor dyeing 1min, running water, microscopy after natural drying；Under microscope, visible Acinetobacter bauamnnii surrounding pod membrane is not painted by dye liquor, and Acinetobacter bauamnnii thalline purple, whole background presents salmon pink.
The wherein Congo red dye liquor described in step (1): Congo red (congored) 0.8g is completely dissolved in 20ml distilled water, saves backup under room temperature.
The wherein destaining solution described in step (2): in fume hood, 5ml concentrated hydrochloric acid is added in 95ml distilled water, save backup under room temperature.
The wherein crystal violet dye liquor described in step (3): first add in 25ml95% ethanol (v/v) after finely ground for 2.5g crystal violet, fully dissolve and be made into A liquid.1g ammonium oxalate is completely dissolved in 100ml distilled water, is made into B liquid.Adopt after A liquid and B liquid are fully mixed and stands 24h Medium speed filter paper filter crystal violet dye liquor, be placed in room temperature and preserve.
Beneficial effect
Acinetobacter bauamnnii is current clinical most important many/general fastbacteria, but its Resistance detection result can not be used for assessing clinical treatment complexity and the rear situation of patient, and a kind of more efficiently detection guidance of urgent clinical needs is to evaluate bacterial virulence.A kind of method that the present invention provides quick detection bacterial capsule to clinic, it is possible to be used for evaluating bacterial virulence, the selection for clinical treatment provides reference information.
Accompanying drawing explanation
Few pod membrane Acinetobacter bauamnnii coloration result (1000 ×) of Fig. 1, Acinetobacter bauamnnii is after congo red staining method dyes, visible Acinetobacter bauamnnii thalline purple under microscope, whole background presents salmon pink, and few pod membrane Acinetobacter bauamnnii surrounding has no the pod membrane not being colored；
Fig. 2 many pod membranes Acinetobacter bauamnnii coloration result (1000 ×), Acinetobacter bauamnnii is after congo red staining method dyes, visible Acinetobacter bauamnnii thalline purple under microscope, whole background presents salmon pink, many pod membranes Acinetobacter bauamnnii surrounding can be thicker the pod membrane not being colored, be wrapped in whole thalline.
Detailed description of the invention
Embodiment 1: Acinetobacter bauamnnii capsule stain
Randomly select Acinetobacter bauamnnii 10 strain (deriving from Affiliated Hospital of Jiangsu University), including multi-drug resistant bacteria 5 strain (to cephalosporins, carbapenem antibiotic, fluoroquinolone antibiotics, aminoglycoside antibiotics and beta-lactamase inhibitor class antibiotic drug resistance simultaneously), sensitive organism 5 strain (non-concurrent to cephalosporins, fluoroquinolone antibiotics, aminoglycoside antibiotics and beta-lactamase inhibitor class antibiotics resistance, and to carbapenem antibiotic not drug resistance).The kind of all bacterial strains and drug resistance all adopt France bioMerieux's VITEK2-Compact automatic bacterial to identify and susceptibility instrument is identified again, qualification result shows that all 10 strain antibacterials are Acinetobacter bauamnnii (Acinetobacterbaumannii), wherein 5 strain multi-drug resistant bacteria, and another 5 strains are sensitive organism.
All Acinetobacter bauamnniis are inoculated in blood agar plate, cultivate 16h for 35 DEG C and are formed after single bacterium colony for capsule stain.
Concrete staining procedure:
(1) Congo red dye liquor is taken with Ovum Gallus domesticus album by 4:1(v/v) mix, on inoculating loop picking blood plate, Acinetobacter bauamnnii is abundant with aforesaid liquid, makes push jack, natural drying by the method for pushing blood sheet.
(2) dropping destaining solution decolouring 1min, running water.
(3) dropping crystal violet dye liquor dyeing 1min, running water, microscopy after natural drying.
Under microscope, visible Acinetobacter bauamnnii can be divided into many Capsular strains and few Capsular strains.Only 1 strain obvious pod membrane as seen in 5 strain Multi-drug resistant Acinetobacter baumannii, another 4 strains have no obvious pod membrane；3 strains obvious pod membrane as seen in 5 strain sensitive strain Acinetobacter bauamnniis.Pod membrane is not painted by dye liquor, and Acinetobacter bauamnnii thalline purple, whole background presents salmon pink.See Fig. 1 and 2.
The wherein Congo red dye liquor described in step (1): Congo red (congored) 0.8g is completely dissolved in 20ml distilled water, saves backup under room temperature.
The wherein destaining solution described in step (2): in fume hood, 5ml concentrated hydrochloric acid is added in 95ml distilled water, save backup under room temperature.
The wherein crystal violet dye liquor described in step (3): first add in 25ml95% ethanol (v/v) after finely ground for 2.5g crystal violet, fully dissolve and be made into A liquid.1g ammonium oxalate is completely dissolved in 100ml distilled water, is made into B liquid.Adopt after A liquid and B liquid are fully mixed and stands 24h Medium speed filter paper filter crystal violet dye liquor, be placed in room temperature and preserve.
Claims (4)
1. an Acinetobacter bauamnnii capsule stain detection method, it is characterised in that carry out as steps described below:
(1) Congo red dye liquor is taken with Ovum Gallus domesticus album by 4:1(v/v) mix, on inoculating loop picking blood plate, Acinetobacter bauamnnii is abundant with aforesaid liquid, makes push jack, natural drying by the method for pushing blood sheet；
(2) dropping destaining solution decolouring 1min, running water；
(3) dropping crystal violet dye liquor dyeing 1min, running water, microscopy after natural drying；Under microscope, visible Acinetobacter bauamnnii surrounding pod membrane is not painted by dye liquor, and Acinetobacter bauamnnii thalline purple, whole background presents salmon pink.
2. according to a kind of Acinetobacter bauamnnii capsule stain detection method, it is characterised in that the wherein Congo red dye liquor described in step (1): Congo red 0.8g is completely dissolved in 20ml distilled water, saves backup under room temperature.
3. according to a kind of Acinetobacter bauamnnii capsule stain detection method, it is characterised in that the wherein destaining solution described in step (2): in fume hood, 5ml concentrated hydrochloric acid is added in 95ml distilled water, save backup under room temperature.
4. according to a kind of Acinetobacter bauamnnii capsule stain detection method, it is characterised in that the wherein crystal violet dye liquor described in step (3): first add in 25ml95% ethanol (v/v) after finely ground for 2.5g crystal violet, fully dissolve and be made into A liquid；1g ammonium oxalate is completely dissolved in 100ml distilled water, is made into B liquid；Adopt after A liquid and B liquid are fully mixed and stands 24h Medium speed filter paper filter crystal violet dye liquor, be placed in room temperature and preserve.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815486A (en) * | 2017-11-10 | 2018-03-20 | 西北农林科技大学 | The method that quick screening produces Salmonella organisms film |
| 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 |
|---|---|---|---|---|
| WO2008003114A2 (en) * | 2006-07-05 | 2008-01-10 | Austrian Research Centers Gmbh - Arc | Identification of pathogens |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008003114A2 (en) * | 2006-07-05 | 2008-01-10 | Austrian Research Centers Gmbh - Arc | Identification of pathogens |
Non-Patent Citations (2)
| Title |
|---|
| YKERNV79: "荚膜染色法", 《丁香园论坛》 * |
| 王怀兵等: "细菌荚膜染色的改良", 《黔南民族医专学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815486A (en) * | 2017-11-10 | 2018-03-20 | 西北农林科技大学 | The method that quick screening produces Salmonella organisms film |
| 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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Application publication date: 20160727 |