CN101818189B - Detecting kit capable of simultaneously detecting 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc - Google Patents
Detecting kit capable of simultaneously detecting 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc Download PDFInfo
- Publication number
- CN101818189B CN101818189B CN 201010154457 CN201010154457A CN101818189B CN 101818189 B CN101818189 B CN 101818189B CN 201010154457 CN201010154457 CN 201010154457 CN 201010154457 A CN201010154457 A CN 201010154457A CN 101818189 B CN101818189 B CN 101818189B
- Authority
- CN
- China
- Prior art keywords
- water
- bottle
- peptone
- identify
- substratum
- 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.)
- Expired - Fee Related
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a detecting kit capable of fast, simply and simultaneously detecting 6 aquatic product pathogenic bacteria such as the pseudomonas aeruginosa and the like. The kit comprises an oxidase test paper, a hydrogen sulfide culture medium, a halophilic identifying reagent, an ortho-nitrophenol beta-D-galactoside culture medium, a pectinose culture medium, a lactobiose culture medium, saccharose culture medium, a glucose culture medium, a mannose culture medium, a cellobiose culture medium, a dextrose oxidation and fermentation identifying reagent, an arginine bi-hydrolytic enzyme identifying reagent, an ornithine identifying reagent, a lysine identifying reagent, an amino acid decarboxylation identifying reagent and an indol identifying reagent. The kit has simple reagent composition and preparation method and use method, has low cost, does not need expensive apparatuses and operators with mature technology, compared with a bacterium fast automatic identifying system and a detecting technology based on the nucleic acid. The detecting kit is suitable for initially detecting the normal pathogenic bacteria of aquatic animals, is particularly suitable for the use of aquatic product popularization staff at the production line, and immediately excludes the difficulty and the anxiety for fishing peasants.
Description
Technical field:
The present invention relates to a kind of detection kit of aquatic pathogenic bacterium, be specifically related to a kind of detection kit that can detect Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and six kinds of aquatic pathogenic bacteriums of Vibrio vulnificus simultaneously.
Background technology:
Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus are the hydrocoles common pathogenic bacteria.The existence of these pathogenic bacterias is the important measures that prevent and treat relevant aquatic animal disease in monitoring cultivated animals and the aquaculture water.The technology of identifying above-mentioned bacterium at present has: 1, the fast automatic identification systems of bacterium, this method need expensive plant and instrument and the operator that are skilled in technique; 2, based on the detection technique of nucleic acid, though have quick, succinct and sensitive characteristics, this method needs expensive plant and instrument and the operator that are skilled in technique; 3, the Physiology and biochemistry authentication method of classics; Though this method is classical, accurate; But the identification of indicator that different sections belongs to is distinguished to some extent, but all be carbohydrate fermentation basically, 10 much classes such as enzymatic determination, utilizations of biochemical measurement salt, pigment generation, power trial, the light carbohydrate fermentation just has kind more than 40; Enzymatic determination has kind more than 20, and is therefore loaded down with trivial details, consuming time and require operator that good specified quality is arranged.Because Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and six kinds of bacteriums of Vibrio vulnificus are adhered to 2 big types separately, Pseudomonas aeruginosa is non-fermented type gram negative bacillus, Rhodopseudomonas; Aeromonas hydrophila, vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus and blunt tarda are the fermented type gram negative bacillus; Wherein Aeromonas hydrophila, vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio vulnificus are the vibrionaceae bacterium, and blunt tarda is an enterobacteriaceae lactobacteriaceae.According to the Physiology and biochemistry authentication method of classics, identify above-mentioned six kinds of aquatic products pathogenetic bacterias like need, then need identify respectively, and the index difference of identifying is bigger, index is many, operates very loaded down with trivial details.Also do not see at present the relevant report that can detect Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and 6 kinds of aquatic pathogenic bacterium test kits of Vibrio vulnificus simply, simultaneously.、
Summary of the invention:
The purpose of this invention is to provide a kind of quick, simple and easy, detection kit that can detect Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and 6 kinds of aquatic pathogenic bacteriums of Vibrio vulnificus simultaneously.
The inventor is to above-mentioned 6 kinds of aquatic products encountered pathogenic bacterias; Research and analyse the different physio-biochemical characteristics of aquatic products encountered pathogenic bacteria; Through inducing classification, screening and verification experimental verification, selected 16 kinds of indexs wherein utilize these 16 kinds of indexs can be quick; Easily bacterium to be measured is classified, thereby realized the object of the invention.
The detection kit that detects 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc simultaneously of the present invention is made up of following reagent: (1) oxydase test paper, (2) hydrogen sulfide substratum, (3) halophilism indentifying substance, (4) o-nitrophenol β-D-galactoside substratum (ONPG), (5) pectinose substratum, (6) lactose medium, (7) SM, (8) dextrose culture-medium, (9) seminose substratum, (10) cellobiose substratum, (11) glucose oxidase fermentation indentifying substance, (12) arginine dihydrolase indentifying substance, (13) ornithine indentifying substance, (14) Methionin indentifying substance, (15) amino acid decarboxylase indentifying substance and (16) indoles indentifying substance.
Described oxydase test paper is to be used for checking whether bacterium contains Terminal oxidase.
Described hydrogen sulfide substratum is to be used to check that can bacterium produce hydrogen sulfide, and the screening formulation of this substratum contains peptone 2% for by massfraction 100%; Carnis Bovis seu Bubali cream powder 0.5%, sodium-chlor 0.3%, Sulfothiorine 0.02%; Ferrous sulfate 0.02%; Agar 0.6%, surplus are water, and PH 7.4.
Described halophilism indentifying substance is used to check the tolerance of bacterium to salt; This substratum preferably is made up of two substratum, is respectively salt-free peptone water (0%NaCl) substratum: by massfraction 100%, contain Tryptones 1%; Surplus is a water, PH 7.7-7.8; Salt peptone water (6%NaCl) substratum: by massfraction 100%, contain Tryptones 1%, sodium-chlor 6%, surplus is a water, PH 7.7-7.8;
Described o-nitrophenol β-D-galactoside (ONPG) substratum is to be used to check that bacterium produces the ability of beta-galactosidase enzymes; This substratum screening formulation is by massfraction 100%; Contain o-nitrophenol β-D-galactoside 0.15%, 0.01mol/LPH 7.5 phosphoric acid buffers 25%, peptone 0.75%; Surplus is a water, PH7.5.
Described pectinose substratum, lactose medium, SM, dextrose culture-medium, seminose substratum, cellobiose substratum; It is with the characteristic of bacterium to carbohydrate fermentation that conduct a survey that these carbohydrates decompose substratum, observes the acid producing ability of seized bacterium.
The screening formulation of pectinose substratum contains Carnis Bovis seu Bubali cream powder 0.5% for by massfraction 100%, peptone 1%, and sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, pectinose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH 7.4.
The screening formulation of lactose medium contains Carnis Bovis seu Bubali cream powder 0.5% for by massfraction 100%, peptone 1%, and sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, lactose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH 7.4.
The screening formulation of SM contains Carnis Bovis seu Bubali cream powder 0.5% for by massfraction 100%, peptone 1%, and sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, sucrose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH 7.4.
The screening formulation of dextrose culture-medium contains Carnis Bovis seu Bubali cream powder 0.5% for by massfraction 100%, peptone 1%, and sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, glucose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH 7.4.
The screening formulation of seminose substratum contains Carnis Bovis seu Bubali cream powder 0.5% for by massfraction 100%, peptone 1%, and sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, seminose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH 7.4.
The prescription of cellobiose substratum contains Carnis Bovis seu Bubali cream powder 0.5% for by massfraction 100%, peptone 1%, and sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, cellobiose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH 7.4.
Described glucose oxidase fermentation indentifying substance is to be used to check that bacterium is the bacterium of oxidized form or fermented type or product alkali type, and this indentifying substance preferably includes glucose oxidase fermention medium and MO, and the prescription of described glucose fermentation substratum is by massfraction 100%; Contain Carnis Bovis seu Bubali cream powder 0.5%, peptone 1%, sodium-chlor 0.3%; Potassium hydrogenphosphate 0.2%, glucose 1%, agar 0.6%; Dibromothymolsulfonphthalein 0.003%, surplus are water, and PH 7.4.For identifying that bacterium is the bacterium of oxidized form or fermented type or product alkali type, generally prepares two bottles of glucose fermentation substratum and MO.
Described arginine dihydrolase indentifying substance is the ability that is used to check the living arginine dihydrolase of Production by Bacteria, in order to identify arginine dihydrolase, preferably includes arginine dihydrolase and identifies substratum and arginine dihydrolase control medium.The prescription that described arginine dihydrolase is identified substratum contains peptone 0.1% for by massfraction 100%, sodium-chlor 0.5%, and potassium hydrogenphosphate 0.03%, phenol red 0.001%, L-arginic acid salt 1%, surplus is a water, PH 7.4.The prescription of described arginine dihydrolase control medium contains peptone 0.1% for by massfraction 100%, sodium-chlor 0.5%, and potassium hydrogenphosphate 0.03%, phenol red 0.001%, surplus is a water, PH 7.4.In order to check that Production by Bacteria gives birth to the ability of arginine dihydrolase, described arginine dihydrolase indentifying substance comprises that two bottles of arginine dihydrolases identify substratum and two bottles of arginine dihydrolase control medium and MO.
Described ornithine indentifying substance is to be used to check that bacterium produces the ability of ornithine lytic enzyme, and the screening formulation of this reagent contains peptone 0.5% for by massfraction 100%; Carnis Bovis seu Bubali cream 0.5%, D-glucose 0.05%, purpurum bromocresolis 0.001%; O-cresolsulfonphthalein 0.0005%, pyridoxal 0.0005%, ornithine 1%; Surplus is a water, PH 6.0-6.3.For identifying that this index also has MO.
Described Methionin indentifying substance is to be used to check that bacterium produces the ability of Methionin lytic enzyme, and the screening formulation of this reagent contains peptone 0.5% for by massfraction 100%; Carnis Bovis seu Bubali cream 0.5%, D-glucose 0.05%, purpurum bromocresolis 0.001%; O-cresolsulfonphthalein 0.0005%, pyridoxal 0.0005%, Methionin 1%; Surplus is a water, PH 6.0-6.3.For identifying that this index also has MO.
Described amino acid decarboxylase indentifying substance is to be used to check that the Production by Bacteria looks answers the ability of amino acid decarboxylase, and this amino acid decarboxylase indentifying substance comprises the amino acid decarboxylase control medium, and the screening formulation of this substratum is for by massfraction 100%; Contain peptone 0.5%, Carnis Bovis seu Bubali cream 0.5%, D-glucose 0.05%; Purpurum bromocresolis 0.001%, o-cresolsulfonphthalein 0.0005%, pyridoxal 0.0005%; Surplus is a water, PH 6.0-6.3.For identifying that this index also has MO.
Described indoles indentifying substance is to be used to check that can bacterium generate the indole metabolite through intracellular enzyme with the tryptophane oxygenolysis.This indentifying substance comprises indoles evaluation substratum and indoles reagent (Kovacs reagent), and this indoles identifies that the prescription of substratum for by massfraction 100%, contains peptone 1%, sodium-chlor 0.5%, and surplus is a water, PH 7.3-7.5.The prescription of indoles reagent (Kovacs reagent) is amylalcohol or primary isoamyl alcohol 150ml, to dimethylbenzaldehyde 10g, and pure concentrated hydrochloric acid 50ml.
Therefore the test kit that detects Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and 6 kinds of aquatic animal pathogenic bacterias of Vibrio vulnificus simultaneously of the present invention preferably includes:
Identify bottle 1-oxydase test paper
Identify the salt-free peptone water culture of bottle 2-: Tryptones 10g, zero(ppm) water 1000ml, PH 7.7-7.8.
Identify the salt peptone water culture of bottle 3-60g/l: Tryptones 10g, sodium-chlor 60g, zero(ppm) water 1000ml, PH 7.7-7.8.
Identify bottle 4-o-nitrophenol β-D-galactoside (ONPG) substratum: ONPG 0.6g, 0.01mol/L PH 7.5 phosphoric acid buffer 100ml, 1% peptone (PH7.5) 300ml.
Identify bottle 5-hydrogen sulfide substratum: peptone 20g, Carnis Bovis seu Bubali cream powder 5g, sodium-chlor 3g, Sulfothiorine 0.2g, ferrous sulfate 0.2g, agar 6g, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 6-pectinose substratum: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, pectinose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 7-lactose medium: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, lactose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 8-SM: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, sucrose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 9-dextrose culture-medium: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, glucose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml.
Identify bottle 10-seminose substratum: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, seminose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 11-cellobiose substratum: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, cellobiose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 12,13-glucose oxidase fermention medium: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, glucose 10g, agar 6g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 14,16-arginine dihydrolase evaluation substratum: peptone 1g, sodium-chlor 5g, potassium hydrogenphosphate 0.3g, phenol red 0.01g, L-arginic acid salt 10g, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 15,17-arginine dihydrolase control medium: peptone 1g, sodium-chlor 5g, potassium hydrogenphosphate 0.3g, phenol red 0.01g, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 18-ornithine evaluation substratum: peptone 5g, Carnis Bovis seu Bubali cream 5g, D-glucose 0.5g, 1.6% purpurum bromocresolis 0.625ml, 0.2% o-cresolsulfonphthalein 2.5ml, pyridoxal 0.005g, ornithine 10g, zero(ppm) water 1000ml, PH 6.0-6.3.
Identify bottle 19-Methionin evaluation substratum: peptone 5g, Carnis Bovis seu Bubali cream 5g, D-glucose 0.5g, 1.6% purpurum bromocresolis 0.625ml, 0.2% o-cresolsulfonphthalein 2.5ml, pyridoxal 0.005g, Methionin 10g, zero(ppm) water 1000ml, PH 6.0-6.3.
Identify bottle 20-amino acid decarboxylase control medium: peptone 5g, Carnis Bovis seu Bubali cream 5g, D-glucose 0.5g, 1.6% purpurum bromocresolis 0.625ml, 0.2% o-cresolsulfonphthalein 2.5ml, pyridoxal 0.005g, zero(ppm) water 1000ml, PH 6.0-6.3.
Identify bottle 21-indoles evaluation substratum: peptone 10g, sodium-chlor 5g, zero(ppm) water 1000ml, PH 7.3-7.5.
Reagent A-sterilization MO.
Reagent B-0.65% SPSS.
Reagent C-indoles reagent (Kovacs reagent): amylalcohol or primary isoamyl alcohol 150ml, to dimethylbenzaldehyde 10g, pure concentrated hydrochloric acid 50ml.
Reagent components can buy from currently available products among substratum in above-mentioned each evaluation bottle and reagent A, B and the C, and can prepare according to the method for known routine.
The method of use of evaluation bacterium that the present invention can detect the detection kit of Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and 6 kinds of aquatic pathogenic bacteriums of Vibrio vulnificus simultaneously is:
1, previous work
After the tested bacteria pure culture, place 30 ℃ ± 1 ℃ to cultivate 18～24h, gramstaining is selected negative bacterium to be checked.(pure bacterium directly inoculates detection behind 30 ℃ ± 1 ℃ cultivation 18～24h)
2, inoculation (under aseptic condition, operating)
Shake up to reagent B with transfering loop picking one little ring bacterium, be prepared into bacteria suspension, carry out following operation (identifying except the bottle 1):
Identify bottle 1:
Take out test paper in the bottle, smear on the test paper, observe the colour-change of smearing bacterium in 1 minute with a little tested bacteria of transfering loop picking.
Identify bottle 2-12:
Respectively draw 1～2 bacteria suspension with suction pipe and add in the evaluation bottle, cover top cement plug (jumping a queue entirely), upright, cultivate 18～24h, the colour-change of observation substratum in 30 ℃ ± 1 ℃.
Identify bottle 13-20:
Respectively draw 1～2 bacteria suspension adding with suction pipe and identify in the bottle, except that identifying bottle 14 and identify the bottle 15 that other all add 3～5 reagent A and cover media surface; Cover top cement plug (jumping a queue entirely); Uprightly, cultivate 18～24h, the colour-change of observation substratum in 30 ℃ ± 1 ℃.
Identify bottle 21:
Respectively draw 1～2 bacteria suspension adding with suction pipe and identify in the bottle, cover top cement plug (false add plug), upright, in 30 ℃ ± 1 ℃ cultivation 18～24h, add 2 reagent C, observe the colour-change of substratum.
3,,, can judge that then bacterium to be measured is corresponding bacterium, so can confirm the classification position of bacterium to be checked if consistent with bacterium result in identifying table the contrast of each reaction result of bacterium to be checked and Bacteria Identification table (table 1).
Table 1 Bacteria Identification table
It is of the present invention that to detect Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, 6 kinds of aquatic pathogenic bacteriums of Vibrio parahaemolyticus and Vibrio vulnificus simultaneously be in the different physio-biochemical characteristics of inventor's deep study and analysis aquatic products encountered pathogenic bacteria; Through inducing classification, screening and verification experimental verification; Selected 16 kinds of indexs wherein; Utilize these 16 kinds of indexs can be quick, simple Identification.Tested bacteria is after pure culture, and whether just can detect bacterium to be measured in 24 hours is 6 kinds of pathogenic bacterias of above-mentioned hydrocoles, and qualification result is sensitive, accurately.
The present invention compares with some authentication methods of prior art and has the following advantages:
1, test kit of the present invention can detect Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and 6 kinds of aquatic pathogenic bacteriums of Vibrio vulnificus simultaneously; And need be as traditional Physiology and biochemistry authentication method; Need not numerous reagent (the bacterium difference of bacterium selection of equal genus of basis; Cause also difference of a lot of reagent); Cause that workload is big, the time is long; And can not use the shortcoming that unified, simple reagent is identified above-mentioned 6 kinds of aquatic animal pathogenic bacterias simultaneously, use the indentifying substance that test kit of the present invention only needs some evaluations, just can identify Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and 6 kinds of aquatic pathogenic bacteriums of Vibrio vulnificus quick, simple and easy, accurately simultaneously.
2, test kit reagent of the present invention constitute simple, compound method is simple, method of use is also simple and cost is low, and is the same with detection technique based on nucleic acid unlike the fast automatic identification systems of bacterium, needs expensive plant and instrument and the operator that are skilled in technique.
Therefore test kit of the present invention is applicable to aquatic animal encountered pathogenic bacteria Preliminary detection, and the personnel that the spy is suitable for numerous line aquatic products centres for spreading use, and can in time remove difficulties and alleviate sufferings for the fishman.
Embodiment:
Below be to further elaboration of the present invention, rather than limitation of the present invention.
One, the preparation of test kit
According to the following component of identifying in the bottle, each identifies the reagent in the bottle ordinary method preparation:
Identify bottle 1-oxydase test paper
Identify the bottle salt-free peptone water of 2-(0%NaCl) substratum: Tryptones 10g, zero(ppm) water 1000ml, PH 7.7-7.8.
Identify salt peptone water (6%NaCl) substratum of bottle 3-60g/l: Tryptones 10g, sodium-chlor 60g, zero(ppm) water 1000ml, PH 7.7-7.8.
Identify bottle 4-o-nitrophenol β-D-galactoside (ONPG) substratum: ONPG 0.6g, 0.01mol/L PH 7.5 phosphoric acid buffer 100ml, 1% peptone (PH7.5) 300ml.
Identify bottle 5-hydrogen sulfide substratum: peptone 20g, Carnis Bovis seu Bubali cream powder 5g, sodium-chlor 3g, Sulfothiorine 0.2g, ferrous sulfate 0.2g, agar 6g, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 6-pectinose substratum: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, pectinose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 7-lactose medium: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, lactose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 8-SM: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, sucrose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 9-dextrose culture-medium: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, glucose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml.
Identify bottle 10-seminose substratum: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, seminose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 11-cellobiose substratum: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, cellobiose 10g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 12,13-glucose oxidase fermention medium: Carnis Bovis seu Bubali cream powder 5g, peptone 10g, sodium-chlor 3g, potassium hydrogenphosphate 2g, glucose 10g, agar 6g, 1% dibromothymolsulfonphthalein aqueous solution 3ml, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 14,16-arginine dihydrolase evaluation substratum: peptone 1g, sodium-chlor 5g, potassium hydrogenphosphate 0.3g, phenol red 0.01g, L-arginic acid salt 10g, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 15,17-arginine dihydrolase control medium: peptone 1g, sodium-chlor 5g, potassium hydrogenphosphate 0.3g, phenol red 0.01g, zero(ppm) water 1000ml, PH 7.4.
Identify bottle 18-ornithine evaluation substratum: peptone 5g, Carnis Bovis seu Bubali cream 5g, D-glucose 0.5g, 1.6% purpurum bromocresolis 0.625ml, 0.2% o-cresolsulfonphthalein 2.5ml, pyridoxal 0.005g, ornithine 10g, zero(ppm) water 1000ml, PH 6.0-6.3.
Identify bottle 19-Methionin evaluation substratum: peptone 5g, Carnis Bovis seu Bubali cream 5g, D-glucose 0.5g, 1.6% purpurum bromocresolis 0.625ml, 0.2% o-cresolsulfonphthalein 2.5ml, pyridoxal 0.005g, Methionin 10g, zero(ppm) water 1000ml, PH 6.0-6.3.
Identify bottle 20-amino acid decarboxylase control medium: peptone 5g, Carnis Bovis seu Bubali cream 5g, D-glucose 0.5g, 1.6% purpurum bromocresolis 0.625ml, 0.2% o-cresolsulfonphthalein 2.5ml, pyridoxal 0.005g, zero(ppm) water 1000ml, PH 6.0-6.3.
Identify bottle 21-indoles evaluation substratum: peptone 10g, sodium-chlor 5g, zero(ppm) water 1000ml, PH 7.3-7.5.
Reagent A-sterilization MO.
Reagent B-0.65% SPSS.
Reagent C-indoles reagent (Kovacs reagent): amylalcohol or primary isoamyl alcohol 150ml, to dimethylbenzaldehyde 10g, pure concentrated hydrochloric acid 50ml.
Mentioned reagent sterilization back bottling is subsequent use.
Embodiment 1:
In September, 2007, spend all tilapia kidney separation of bacterial of certain plant's morbidity from Guangzhou, the single bacterium colony of the advantage of getting carries out purifying and cultivates Gram-negative.
1, inoculation (under aseptic condition, operating)
Shake up to reagent B with transfering loop picking one little ring bacterium, be prepared into bacteria suspension, carry out following operation (identifying except the bottle 1):
Identify bottle 1:
Take out test paper in the bottle, smear on the test paper, observe the colour-change of smearing bacterium in 1 minute with a little bacterium of transfering loop picking.
Identify bottle 2-12:
Respectively draw 1～2 bacteria suspension with suction pipe and add in the evaluation bottle, cover top cement plug (jumping a queue entirely) stands in the groove of interior holder, in 30 ℃ ± 1 ℃ cultivation 18～24h, the colour-change of observing substratum.
Identify bottle 13-20:
Respectively draw 1～2 bacteria suspension adding with suction pipe and identify in the bottle, except that identifying bottle 14 and identify the bottle 15 that other all add 3～5 reagent A and cover media surface; Cover top cement plug (jumping a queue entirely); In the groove of holder, cultivate 18～24h, the colour-change of observation substratum in standing in 30 ℃ ± 1 ℃.
Identify bottle 21:
Respectively draw 1～2 bacteria suspension with suction pipe and add in the evaluation bottle, cover top cement plug (false add plug) in the groove of holder, in 30 ℃ ± 1 ℃ cultivation 18～24h, adds 2 reagent C in standing on, and observes the colour-change of substratum.
The result is following: TSA substratum color turns green; Smear on the test paper with a little bacterium of transfering loop picking, observe in 1 minute and smear bacterium and be red-purple; Identify that bottle 2 substratum become muddy, identify that bottle 3 is haze-free; Identify that bottle 4 substratum are colourless; Identify no black in the bottle 5; Identify that bottle 6-11 substratum is purple; Identify that bottle 12 is yellow, 13 nondiscolorations; Identify that bottle 14 takes on a red color, 15 is yellow; Identify that bottle 18,19 is blue-greenish colour, 20 is yellow; Identify bottle 21 redfrees (nondiscoloration).With Bacteria Identification table (table 1) contrast, the result is judged to be Pseudomonas aeruginosa.
Use the semi-automatic Bacteria Identification appearance of French Mei Liai ATB and bacterium is identified the result is a Pseudomonas aeruginosa also, this shows using test kit of the present invention judges it is feasible to Pseudomonas aeruginosa.
Embodiment 2:
Get the bacterium the table 2 from following source, after the activation culture.Carry out test according to following method:
Table 2
1, inoculation (under aseptic condition, operating)
Shake up to reagent B with transfering loop picking one little ring bacterium, be prepared into bacteria suspension, carry out following operation (identifying except the bottle 1):
Identify bottle 1:
Take out test paper in the bottle, smear on the test paper, observe the colour-change of smearing bacterium in 1 minute with a little bacterium of transfering loop picking.
Identify bottle 2-12:
Respectively draw 1～2 bacteria suspension with suction pipe and add in the evaluation bottle, cover top cement plug (jumping a queue entirely) stands in the groove of interior holder, in 30 ℃ ± 1 ℃ cultivation 18～24h, the colour-change of observing substratum.
Identify bottle 13-20:
Respectively draw 1～2 bacteria suspension adding with suction pipe and identify in the bottle, except that identifying bottle 14 and identify the bottle 15 that other all add 3～5 reagent A and cover media surface; Cover top cement plug (jumping a queue entirely); In the groove of holder, cultivate 18～24h, the colour-change of observation substratum in standing in 30 ℃ ± 1 ℃.
Identify bottle 21:
Respectively draw 1～2 bacteria suspension with suction pipe and add in the evaluation bottle, cover top cement plug (false add plug) in the groove of holder, in 30 ℃ ± 1 ℃ cultivation 18～24h, adds 2 reagent C in standing on, and observes the colour-change of substratum.
According to the result of above-mentioned reaction, contrast with identifying table (table 1), classify result such as table 3 according to results of comparison.
Table 3
Continuous table 3
Bacterial strain is used in test, comprises the reference culture of purchasing from Chinese microorganism strain preservation management committee common micro-organisms center, and all the semi-automatic Bacteria Identification appearance of application method state Mei Liai ATB is identified.
Use test kit of the present invention and the semi-automatic Bacteria Identification appearance of French Mei Liai ATB is identified bacterium, the result is consistent.Therefore using test kit of the present invention judges it is feasible to Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio vulnificus.
Claims (2)
1. detection kit that can detect Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and 6 kinds of aquatic pathogenic bacteriums of Vibrio vulnificus simultaneously; It is characterized in that, form: (1) oxydase test paper, (2) hydrogen sulfide substratum, (3) halophilism indentifying substance, (4) o-nitrophenol β-D-galactoside substratum, (5) pectinose substratum, (6) lactose medium, (7) SM, (8) dextrose culture-medium, (9) seminose substratum, (10) cellobiose substratum, (11) glucose oxidase fermentation indentifying substance, (12) arginine dihydrolase indentifying substance, (13) ornithine indentifying substance, (14) Methionin indentifying substance, (15) amino acid decarboxylase indentifying substance and (16) indoles indentifying substance by following reagent.
2. the detection kit that can detect Pseudomonas aeruginosa, Aeromonas hydrophila, blunt tarda, vibrio alginolyticus, Vibrio parahaemolyticus and 6 kinds of aquatic pathogenic bacteriums of Vibrio vulnificus simultaneously according to claim 1; It is characterized in that; This can detect the detection kit of 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc simultaneously, specifically is made up of following evaluation bottle:
Identify bottle 1-oxydase test paper;
Identify the salt-free peptone water culture of bottle 2-: by massfraction 100%, contain Tryptones 1%, surplus is a water, PH7.7-7.8;
Identify the salt peptone water culture of bottle 3-60g/l: by massfraction 100%, contain Tryptones 1%, sodium-chlor 6%, surplus is a water, PH7.7-7.8;
Identify bottle 4-o-nitrophenol β-D-galactoside substratum: by massfraction 100%, contain o-nitrophenol β-D-galactoside 0.15%, 0.01mol/L PH7.5 phosphoric acid buffer 25%, peptone 0.75%, surplus is a water, PH7.5;
Identify bottle 5-hydrogen sulfide substratum: by massfraction 100%, contain peptone 2%, Carnis Bovis seu Bubali cream powder 0.5%, sodium-chlor 0.3%, Sulfothiorine 0.02%, ferrous sulfate 0.02%, agar 0.6%, surplus is a water, PH7.4;
Identify bottle 6-pectinose substratum: by massfraction 100%, contain Carnis Bovis seu Bubali cream powder 0.5%, peptone 1%, sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, pectinose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH7.4;
Identify bottle 7-lactose medium: by massfraction 100%, contain Carnis Bovis seu Bubali cream powder 0.5%, peptone 1%, sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, lactose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH7.4;
Identify bottle 8-SM: by massfraction 100%, contain Carnis Bovis seu Bubali cream powder 0.5%, peptone 1%, sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, sucrose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH7.4;
Identify bottle 9-dextrose culture-medium: by massfraction 100%, contain Carnis Bovis seu Bubali cream powder 0.5%, peptone 1%, sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, glucose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH7.4;
Identify bottle 10-seminose substratum: by massfraction 100%, contain Carnis Bovis seu Bubali cream powder 0.5%, peptone 1%, sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, seminose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH7.4;
Identify bottle 11-cellobiose substratum: by massfraction 100%, contain Carnis Bovis seu Bubali cream powder 0.5%, peptone 1%, sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, cellobiose 1%, dibromothymolsulfonphthalein 0.003%, surplus is a water, PH7.4;
Identify bottle 12,13-glucose oxidase fermention medium: by massfraction 100%, contain Carnis Bovis seu Bubali cream powder 0.5%, peptone 1%; Sodium-chlor 0.3%, potassium hydrogenphosphate 0.2%, glucose 1%, agar 0.6%; Dibromothymolsulfonphthalein 0.003%, surplus are water, PH7.4;
Identify that bottle 14,16-arginine dihydrolase identify substratum: by massfraction 100%, contain peptone 0.1%, sodium-chlor 0.5%, potassium hydrogenphosphate 0.03%, phenol red 0.001%, L-arginic acid salt 1%, surplus is a water, PH7.4;
Identify bottle 15,17-arginine dihydrolase control medium: by massfraction 100%, contain peptone 0.1%, sodium-chlor 0.5%, potassium hydrogenphosphate 0.03%, phenol red 0.001%, surplus is a water, PH7.4;
Identify bottle 18-ornithine evaluation substratum: by massfraction 100%, contain peptone 0.5%, Carnis Bovis seu Bubali cream 0.5%, D-glucose 0.05%; Purpurum bromocresolis 0.001%, o-cresolsulfonphthalein 0.0005%, pyridoxal 0.0005%; Ornithine 1%, surplus are water, PH6.0-6.3;
Identify bottle 19-Methionin evaluation substratum: by massfraction 100%, contain peptone 0.5%, Carnis Bovis seu Bubali cream 0.5%, D-glucose 0.05%; Purpurum bromocresolis 0.001%, o-cresolsulfonphthalein 0.0005%, pyridoxal 0.0005%; Methionin 1%, surplus are water, PH6.0-6.3;
Identify bottle 20-amino acid decarboxylase control medium: by massfraction 100%, contain peptone 0.5%, Carnis Bovis seu Bubali cream 0.5%, D-glucose 0.05%, purpurum bromocresolis 0.001%, o-cresolsulfonphthalein 0.0005%, pyridoxal 0.0005%, surplus is a water, PH6.0-6.3;
Identify bottle 21-indoles evaluation substratum: by massfraction 100%, contain peptone 1%, sodium-chlor 0.5%, surplus is a water, PH7.3-7.5;
Reagent A-sterilization MO;
Reagent B-0.65% SPSS;
Reagent C-indoles reagent: Kovacs reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010154457 CN101818189B (en) | 2010-04-16 | 2010-04-16 | Detecting kit capable of simultaneously detecting 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010154457 CN101818189B (en) | 2010-04-16 | 2010-04-16 | Detecting kit capable of simultaneously detecting 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101818189A CN101818189A (en) | 2010-09-01 |
| CN101818189B true CN101818189B (en) | 2012-01-25 |
Family
ID=42653463
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010154457 Expired - Fee Related CN101818189B (en) | 2010-04-16 | 2010-04-16 | Detecting kit capable of simultaneously detecting 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101818189B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732599A (en) * | 2011-04-12 | 2012-10-17 | 南开大学 | Method for detecting Vibrio vulnificus and Vibrio parahaemolyticus in seawater sample by duplex polymerase chain reaction |
| CN103194404A (en) * | 2013-03-07 | 2013-07-10 | 中国水产科学研究院珠江水产研究所 | Simple method for screening aquatic source aeromonas |
| CN106148477A (en) * | 2016-08-29 | 2016-11-23 | 郑州点石生物技术有限公司 | A kind of Pseudomonas aeruginosa chromogenic culture medium and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101631874A (en) * | 2007-02-08 | 2010-01-20 | 生物梅里埃公司 | Be used to detect and/or differentiate the medium of bacterium |
| CN101914613A (en) * | 2010-08-12 | 2010-12-15 | 上海市疾病预防控制中心 | Kit for screening four enteric pathogenic bacteria by using biochemical and enzyme reaction test sieve and screening method |
-
2010
- 2010-04-16 CN CN 201010154457 patent/CN101818189B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101631874A (en) * | 2007-02-08 | 2010-01-20 | 生物梅里埃公司 | Be used to detect and/or differentiate the medium of bacterium |
| CN101914613A (en) * | 2010-08-12 | 2010-12-15 | 上海市疾病预防控制中心 | Kit for screening four enteric pathogenic bacteria by using biochemical and enzyme reaction test sieve and screening method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101818189A (en) | 2010-09-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8778629B2 (en) | Sterility test method and totally enclosed bacterial ampoule incubator used by it | |
| CN102424832B (en) | Chromogenic medium used for detecting esherichia coli O157:H7 | |
| KR20000048673A (en) | Method and apparatus for detecting bacteria | |
| CN104198357B (en) | Flow Cytometry methods application in production of vaccine is monitored in real time | |
| He et al. | Label-free detection of bacteria in fruit juice by nano-flow cytometry | |
| CN101818189B (en) | Detecting kit capable of simultaneously detecting 6 aquatic product pathogenic bacteria such as pseudomonas aeruginosa, etc | |
| CN101497915B (en) | Reagent and method for fast detecting Staphylococcus aureus | |
| CN103290094A (en) | Staphylococcus aureus chromogenic medium and test piece thereof | |
| CN101186891A (en) | Salmonella color culture medium, detection kit an detection method | |
| CN106433631A (en) | Fluorescent carbon dots as well as preparation method and applications thereof | |
| Mesa et al. | Use of the direct epifluorescent filter technique for the enumeration of viable and total acetic acid bacteria from vinegar fermentation | |
| CN101402989A (en) | Syringe-shaped microorganism culture device | |
| CN101970682A (en) | Method for detecting and/or identifying clostridium difficile | |
| CN101631875A (en) | Be used to detect and/or differentiate the medium of bacterium | |
| CN102146429B (en) | Vibrio alginolyticus selective differential medium | |
| CN105779564A (en) | Rapid detection method of escherichia coli in food | |
| Reiter et al. | Comparative study of alternative methods for food safety control in poultry slaughterhouses | |
| CN102304585A (en) | Immunocapture PCR (polymerase chain reaction) detection kit of staphylococcus aureus and using method of kit | |
| CN109439725A (en) | Chromogenic culture medium and its application for Escherichia coli identification | |
| CN104178416A (en) | Detection kit for distinguishing three aquatic source aeromonas composite groups | |
| Tebaldi et al. | Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting | |
| Fröhling et al. | A method for viability testing of Pectobacterium carotovorum in postharvest processing by means of flow cytometry | |
| Assunção et al. | Quantification of mycoplasmas in broth medium with sybr green-I and flow cytometry | |
| CN102816715B (en) | Kit for separating and identifying yersinia enterocolitica as well as preparation and application thereof | |
| CN203117162U (en) | Integrated membrane biosensor for quickly detecting escherichia coli |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120125 Termination date: 20130416 |