CN102199530A - Preparation method for drug sensitivity kit and drug sensitivity detection method - Google Patents
Preparation method for drug sensitivity kit and drug sensitivity detection method Download PDFInfo
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- CN102199530A CN102199530A CN2011100720511A CN201110072051A CN102199530A CN 102199530 A CN102199530 A CN 102199530A CN 2011100720511 A CN2011100720511 A CN 2011100720511A CN 201110072051 A CN201110072051 A CN 201110072051A CN 102199530 A CN102199530 A CN 102199530A
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Abstract
The invention relates to a preparation method for a drug sensitivity kit and a drug sensitivity detection method, which are applied in the field of aquiculture. The drug sensitivity kit comprises a centrifuge tube with enriched bacterial culture solution, a culture dish with solid culture medium and a drug sensitivity drug storage ring. According to the invention, drug sensitivity detection is carried out on suscepts and water samples and the drug sensitivity drug storage rings are observed after the detection; drugs in drug sensitivity plasters where a transparent area appears can all be used as alternative drugs, and the drug in a drug sensitivity plaster where a big clear area appears can be used as a preferable drug; therefore a most appropriate antibiotic is selected out. Using a drug based on a correct diagnosis can avoid abuse of fish drugs and particularly abuse of antibiotics, and prevent medicinal residues from remaining in aquatic products. It only needs 12 hours to complete the preparation of the drug sensitivity kit; thus selection of drugs and corresponding prevention and treatment can be carried out in a same day as the preparation. The invention has the advantages of simple and convenient operation and low cost, is applicable to selection of drugs for controlling bacterial diseases in basic level of aquiculture, and plays a positive role in protecting ecological environment for aquiculture.
Description
Technical field
The present invention relates to a kind of susceptibility test kit and susceptibility detection method that is used for aquaculture, a kind of specifically preparation method and susceptibility detection method that detects the susceptibility test kit of sick body or water sample when being used for freshwater aquiculture or sea farming.
Background technology
, environmental degradation excessive owing to cultivation density and other multiple reason cause in recent years that the aquaculture bacteriosis takes place frequently, and cause enormous economic loss.Because the shortage of culturist's scientific knowledge and ordering about of market economy interests, the particularly antibiotic phenomenon ubiquity of abuse fishing medicine causes that the resistance of aquatic products pathogenic bacterium, fishery products medicine are residual, problems such as environmental pollution and ecological damage increasingly sharpen.Will solve microbiotic abuse problem from the source, key is to carry out drug sensitive test before treatment, thereby suits the remedy to the case on the basis of correct diagnosis.Chang Yong drug sensitive test method is the K-B paper disk method in the world.Adopt this method to carry out the drug sensitive test requirement, need spend the time about a week, and must under Microbiological Lab's condition of specialty, finish, therefore be not suitable in numerous culturists, promoting by the personnel that possess expertise with the pathogenic bacterium separation and purification.Patent Office of the People's Republic of China is disclosed on February 28th, 2007, Granted publication number is " the susceptibility detection box " of " CN2874498Y ", it is used for the animal epidemic diagnostic detection, comprises being with to be stamped on the detection box 2 that detects hole 1 detecting lid 3, is carved with on detection box 2 bottom surfaces and measures scale 4.It directly adds to detect in the hole according to test objective and can carry out drug sensitive test by various medicines.This detection box is not owing to virus, bacterium are cultivated, and the virus, the bacterium that are obtained are limited, can't effectively detect at all.
Summary of the invention
The objective of the invention is to defective and deficiency at above-mentioned prior art, for people provide a kind of easy and simple to handle, moderate cost is applicable to that aquatic products basic unit is as the preparation method who selects the susceptibility test kit that the pharmaceutical worker does of control fresh water bacteria property disease and use this susceptibility test kit to carry out the method that susceptibility detects.
The technical solution used in the present invention is for achieving the above object: a kind of preparation method of susceptibility test kit, described susceptibility examination box comprises centrifuge tube that enrichment culture medium is housed, be equipped with solid medium culture dish and susceptibility storage medicine circle.
The preparation of the centrifuge tube of enrichment culture medium is housed:
A, be that the peptone of 5-15 part, the yeast extract of 3-8 part, the sodium chloride of 5-15 part are poured in the beaker that 600-1000 part distilled water is housed, stir weight proportion;
B, in beaker, pour hydrochloric acid or sodium hydroxid into, the solution pH value in the beaker is adjusted to 7.3 ± 0.1, be mixed with enrichment culture medium;
C, pour the solution in the beaker into adjustable skimmer, enrichment culture medium is injected in the centrifuge tube, lid is screwed on but do not screw, stay half-turn to a circle by adjustable skimmer;
D, the centrifuge tube that enrichment culture medium will be housed are placed on the test-tube stand, put into the automatic high pressure Autoclave again, autoclaving 10-20 minute;
After e, sterilization finish centrifuge tube is taken out, screw the pipe lid of centrifuge tube then, put into true packing bag and vacuumize.
Be equipped with solid medium the preparation of culture dish:
A, be that the peptone of 5-15 part, the yeast extract of 3-8 part, the sodium chloride of 5-15 part, the agar of 10-15 part are poured in the Erlenmeyer flask that 600-1000 part distilled water is housed, stir weight proportion;
B, in Erlenmeyer flask, pour hydrochloric acid or sodium hydroxid into, the solution pH value in the beaker is adjusted to 7.3 ± 0.1, make nutrient solution;
C, Erlenmeyer flask is put into the automatic high pressure Autoclave, autoclaving 10-20 minute;
D, the liquid storage funnel to automatic skimmer in super clean bench are sterilized, and the nutrient solution that will be cooled to the 55-65 degree is then poured in the liquid storage funnel of automatic skimmer, divides to install in the culture dish;
E, culture dish is lain against on the super clean bench, and get to the super clean bench ultraviolet lamp, make the nutrient solution cooled and solidified become solid medium;
F, the lid of culture dish is covered, the vacuum packaging bag of packing into then is evacuated.
The preparation of susceptibility storage medicine circle:
Make the drug sensitive test paper of 15-20 kind medicine, drug sensitive test paper is pasted on the front of storing the medicine circle, the vacuum packaging bag of packing into then is evacuated.
After in the step b of the preparation of the centrifuge tube that enrichment culture medium is housed, adding sea crystal or concentrated seawater, adjust salinity, between the pH6.8-7.2, be mixed with enrichment culture medium between the 24-28; Be equipped with solid medium the step b of preparation of culture dish in add sea crystal or concentrated seawater after, adjust salinity between the 24-28, between the pH6.8-7.2, be mixed with nutrient solution.
A kind of according to using the susceptibility test kit to carry out the susceptibility detection method, it is characterized in that
A, preparation: hand, scissors and dissection container are cleaned up, and with forms such as 75% medical alcohol, poach or baked wheaten cakes to scissors with dissect sterilizing containers;
B, sampling: select individualities such as the typical dying fishes and shrimps of illness, clear water is cleaned the region of anatomy; Dissect with scissors, observe pathology, sterilize once more behind the scissors, the clip typical focus of soya bean size is approximately directly put into the centrifuge tube that enrichment culture medium is housed, and screw centrifuge tube pipe lid, if detect water sample, then directly get the 5-10 sample that drips and splash in the centrifuge tube that enrichment culture medium is housed, and screw centrifuge tube pipe lid;
C, liquid culture: place the back for several times with the forced oscillation centrifuge tube, and should avoid the sunlight direct projection, afterwards every half hour to 1 hour for several times with the forced oscillation centrifuge tube, up to the liquid muddiness;
D, coating: centrifugal liquid in pipe is poured into the culture dish centre that solid medium is housed, should avoid pouring into the throw out at the bottom of the centrifuge tube, on solid medium, liquid is smeared evenly lightly with spreading rod then, and should be avoided the solid culture primary surface is scratched;
E, subsides susceptibility are store the medicine circle: the spacious lid of culture dish is kept flat dry, to surperficial substantially dry, take out drug allergy ring then, drug sensitive test paper is put into the cultivation box down, the light pressure, the scraps of paper are fully contacted with media surface, cover back-off behind the lid of culture dish, be statically placed in temperature local 8-15 hour more than 20 degree;
F, result judge:
1), the medicine that occurs in the susceptibility paster of clear area all can be used as drug candidate, medicine in the bigger susceptibility paster in clear area can be used as preferred agents, take all factors into consideration pharmacopoeia class commonly used, medication history, medicine valency, the administering mode of this class disease, the factor of import-export regulations, select only microbiotic;
2), be mottled distribution, do not show that then disease may not be to be caused by infectation of bacteria as growing bacterium or bacterium.
3), be covered with whole cultivation box as bacterium, but the clear area do not occur, it is invalid to pathogenic bacterium then to show when medicine in all the susceptibility pasters on susceptibility storage medicine circle is at the standard using dosage, advises that other products select medicine again.
The present invention is by carrying out the susceptibility detection to sick body and water sample, after detection, observe susceptibility storage medicine circle, medicine in the susceptibility paster of appearance clear area all can be used as drug candidate, medicine in the bigger susceptibility paster in clear area can be used as preferred agents, take all factors into consideration pharmacopoeia class commonly used, medication history, medicine valency, the administering mode of this class disease, the factor of import-export regulations, select only microbiotic, on the basis of correct diagnosis, suit the remedy to the case, avoided abuse fishing medicine, particularly the phenomenon of abuse of antibiotics prevents that the fishery products medicine is residual.The present invention is the fastest only to need to finish in 12 hours; can select medicine and corresponding prevention and treatment the same day, easy and simple to handle, cost is low; be applicable to that aquaculture basic unit does as the pharmaceutical worker that selects of control bacteriosis, and the ecotope of protection aquaculture is played active effect.
Embodiment
The present invention relates to a kind of aquaculture susceptibility test kit preparation method, its susceptibility test kit comprises centrifuge tube that enrichment culture medium is housed, be equipped with solid medium culture dish and susceptibility storage medicine circle.Described susceptibility test kit is divided into fresh water type and sea-water type again, and wherein the susceptibility test kit of fresh water type is used for the susceptibility of freshwater aquiculture is detected, and the susceptibility test kit of sea-water type is used for the susceptibility of sea farming is detected.
The preparation method of the susceptibility test kit of fresh water type.
The preparation of the centrifuge tube of enrichment culture medium is housed:
A, be that the peptone of 5-15 part, the yeast extract of 3-8 part, the sodium chloride of 5-15 part are poured in the beaker that 600-1000 part distilled water is housed, stir weight proportion.
B, in beaker, pour hydrochloric acid or sodium hydroxid into, the solution pH value in the beaker is adjusted to 7.3 ± 0.1, be mixed with enrichment culture medium.
C, pour the solution in the beaker into adjustable skimmer, enrichment culture medium is injected in the centrifuge tube, lid is screwed on but do not screw, stay half-turn to a circle by adjustable skimmer.
D, the centrifuge tube that enrichment culture medium will be housed are placed on the test-tube stand, put into the automatic high pressure Autoclave again, autoclaving 10-20 minute.
After e, sterilization finish centrifuge tube is taken out, screw the pipe lid of centrifuge tube then, put into true packing bag and vacuumize.
Be equipped with solid medium the preparation of culture dish:
A, be that the peptone of 5-15 part, the yeast extract of 3-8 part, the sodium chloride of 5-15 part, the agar of 10-15 part are poured in the Erlenmeyer flask that 600-1000 part distilled water is housed, stir weight proportion.
B, in Erlenmeyer flask, pour hydrochloric acid or sodium hydroxid into, the solution pH value in the beaker is adjusted to 7.3 ± 0.1, make nutrient solution.
C, Erlenmeyer flask is put into the automatic high pressure Autoclave, autoclaving 10-20 minute.
D, the liquid storage funnel to automatic skimmer in super clean bench are sterilized, and the nutrient solution that will be cooled to the 55-65 degree is then poured in the liquid storage funnel of automatic skimmer, divides to install in the culture dish.Wherein can be by in super clean bench, 2.5% sodium hypochlorite being poured in the liquid storage funnel of the automatic skimmer that has cleaned up to the sterilization of the liquid storage funnel of automatic skimmer, separatory is 10 times continuously.Pour nutrient solution when being cooled to 70 ℃ of left and right sides again into, separatory is 4 times continuously.
E, culture dish is lain against on the super clean bench, and get to the super clean bench ultraviolet lamp, make the nutrient solution cooled and solidified become solid medium.
F, the lid of culture dish is covered, the vacuum packaging bag of packing into then is evacuated.
The preparation of susceptibility storage medicine circle:
Make the drug sensitive test paper of 15-20 kind medicine, drug sensitive test paper is pasted on the front of storing the medicine circle, the vacuum packaging bag of packing into then is evacuated.According to the bacteriosis that freshwater aquiculture occurs, can select the drug sensitive test paper of totally 16 kinds of medicines such as amoxycilline Trihydrate bp, kantlex, gentamicin, Xin Meisu, paraxin, erythromycin, PXB, lincomycin, trimethoprim-sulfamethoxazole SMZ/TMP, doxycycline, Norxin, Rifampin, Cephradine, Ofloxacine USP 23, Enrofloxacin and sulphafurazole usually for use.
The preparation method of the susceptibility test kit of sea-water type.
After the susceptibility test kit of sea-water type is to add sea crystal or concentrated seawater in the step b of the preparation of the centrifuge tube that enrichment culture medium is housed with the difference of fresh water type susceptibility test kit in the preparation, adjust salinity between the 24-28, between the pH6.8-7.2, be mixed with enrichment culture medium.Be equipped with solid medium the step b of preparation of culture dish in add sea crystal or concentrated seawater after, adjust salinity between the 24-28, between the pH6.8-7.2, be mixed with nutrient solution, the bacterium that produces in the sea farming is cultivated being suitable for.And centrifuge tube preferably charges into a certain amount of carbon dioxide when encapsulation.
According to the bacteriosis that sea farming occurs, can select the drug sensitive test paper of totally 19 kinds of medicines such as penicillin G, Streptomycin sulphate, Rifampin, kantlex, gentamicin, Xin Meisu, paraxin, erythromycin, PXB, lincomycin, trimethoprim-sulfamethoxazole SMZ/TMP, doxycycline, Norxin, Cephradine, Ofloxacine USP 23, amoxycilline Trihydrate bp, levofloxacin, Enrofloxacin and sulphafurazole usually for use.
Embodiment:
1) take by weighing peptone 10g yeast extract 5g, sodium-chlor 10g pours in the beaker that 800ml left and right sides distilled water is housed, stirs.Adjust pH to 7.3 ± 0.1 with hydrochloric acid or sodium hydroxide, be settled to 1000ml with distilled water again, be mixed with enrichment culture medium.
Above enrichment culture medium is packed in the adjustable skimmer, in each centrifuge tube, add the 0.5ml enrichment culture medium, lid is screwed on gently but do not screw, stay a half-turn or a circle approximately.
The centrifuge tube that enrichment culture medium will be housed is at last put into respectively on the stainless test-tube stand, puts into the automatic high pressure Autoclave again, 0.102MPa (121 ℃), autoclaving 15 minutes.
After sterilization finishes centrifuge tube is taken out, screw centrifuge tube pipe lid, put into vacuum packaging bag and vacuumize.Each vacuum-packed band is adorned 1 centrifuge tube.
2) take by weighing peptone 10g yeast extract 5g sodium-chlor 10g, agar 12g pours in the Erlenmeyer flask that 800ml left and right sides distilled water is housed, stirs.Adjust pH to 7.3 ± 0.1 with hydrochloric acid or sodium hydroxide, be settled to 1000ml with distilled water again, be mixed with nutrient solution.
The Erlenmeyer flask that nutrient solution is housed is put into the automatic high pressure Autoclave, 0.102MPa (121 ℃), autoclaving 15 minutes.
After sterilization finishes, in super clean bench be that 2.5% sodium hypochlorite is poured in the liquid storage funnel of the automatic skimmer that has cleaned up with 250ml content, adjusting the separatory range is 25ml, operate continuously 10 times.Nutrient solution when pouring 100ml again into and being cooled to 70 ℃ of left and right sides, operate continuously 4 times.Be cooled to about 60 ℃ nutrient solution after will sterilizing at last and poured in the liquid storage funnel of automatic skimmer, caught the 25ml nutrient solution with the culture dish of 90mm, lain against the super clean bench internal cooling and all be frozen into solid medium to nutrient solution, each packing is no more than 200.Open the ultraviolet lamp of super clean bench in the cooled and solidified process.After solidifying, in super clean bench, the lid of culture dish is covered, behind the back-off culture dish is taken out, put into vacuum packaging bag and vacuumize.Each vacuum-packed band contains a packaged culture dish that solid medium is arranged.
3) make the drug sensitive test paper of totally 16 kinds of medicines such as amoxycilline Trihydrate bp, kantlex, gentamicin, Xin Meisu, paraxin, erythromycin, PXB, lincomycin, trimethoprim-sulfamethoxazole SMZ/TMP, doxycycline, Norxin, Rifampin, Cephradine, Ofloxacine USP 23, Enrofloxacin and sulphafurazole with the method for K-B paper disk method.Drug sensitive test paper is sticked in the front of storage medicine circle respectively according to 1～16 numbering, put into vacuum packaging bag and vacuumize.Each vacuum-packed band is adorned the 1 packaged susceptibility storage medicine circle that drug sensitive test paper is arranged.
A kind of susceptibility test kit that uses carries out the method that susceptibility detects,
A, preparation: hand, scissors and dissection container are cleaned up, and with forms such as 75% medical alcohol, poach or baked wheaten cakes to scissors with dissect sterilizing containers.
B, sampling: select individualities such as the typical dying fishes and shrimps of illness, clear water is cleaned the region of anatomy; Dissect with scissors, observe pathology, sterilize once more behind the scissors, the clip typical focus of soya bean size is approximately directly put into the centrifuge tube that enrichment culture medium is housed, and screw centrifuge tube pipe lid, if detect water sample, then directly get the 5-10 sample that drips and splash in the centrifuge tube that enrichment culture medium is housed, and screw centrifuge tube pipe lid.
C, liquid culture: place the back for several times with the forced oscillation centrifuge tube, and should avoid the sunlight direct projection, afterwards every half hour to 1 hour for several times with the forced oscillation centrifuge tube, up to the liquid muddiness.Storage period is relevant with temperature, and temperature is low more, and storage period should be long more.Reference time is: need 8-13 hour in the time of 30-37 ℃; In the time of 25-30 ℃, need 13-18 hour
D, coating: centrifugal liquid in pipe is poured into the culture dish centre that solid medium is housed, should avoid pouring into the throw out at the bottom of the centrifuge tube, on solid medium, liquid is smeared evenly lightly with spreading rod then, and should be avoided the solid culture primary surface is scratched.
E, subsides susceptibility are store the medicine circle: the spacious lid of culture dish is kept flat dry, to surperficial substantially dry, take out drug allergy ring then, drug sensitive test paper is put into the cultivation box down, the light pressure, the scraps of paper are fully contacted with media surface, cover back-off behind the lid of culture dish, be statically placed in temperature local 8-15 hour more than 20 degree.
F, result judge:
1), the medicine that occurs in the susceptibility paster of clear area all can be used as drug candidate, medicine in the bigger susceptibility paster in clear area can be used as preferred agents, take all factors into consideration pharmacopoeia class commonly used, medication history, medicine valency, the administering mode of this class disease, the factor of import-export regulations, select only microbiotic;
2), be mottled distribution, do not show that then disease may not be to be caused by infectation of bacteria as growing bacterium or bacterium.
3), be covered with whole cultivation box as bacterium, but the clear area do not occur, it is invalid to pathogenic bacterium then to show when medicine in all the susceptibility pasters on susceptibility storage medicine circle is at the standard using dosage, advises that other products select medicine again.
The comparison test
1. enrichment culture medium enriching effect experiment
It is 10 that experimental strain is transferred to concentration after activated
7Behind the cfu/ml order of magnitude, be inoculated in respectively in L1 (common nutrient broth), L2 (the present invention's enrichment culture medium) and L3 (distilled water) substratum.Inoculum size 1%, 25 ℃ of culture temperature, incubator rotating speed 200r/min, incubation time is 18h.Spectrophotometer method is surveyed bacterium and is increased bacterium front and back concentration.Every group 3 parallel.
Multiplication factor=every group increases bacterium cultivate back bacterial concentration/every group and increase bacterium and cultivate before bacterial concentration.
The result of liquid enriching effect is as shown in table 1:
Table 1
Find out from above result: substratum L2 is suitable for strain growth most.Promptly selected this enrichment culture medium of this research is as enrichment liquid.
2. solid medium culture effect experiment
Bacterium liquid 10 after liquid nutrient medium increased bacterium and cultivate
3Get 100 μ l behind the cfu/ml order of magnitude, separate application is in S1 (ordinary nutrient agar), on S2 (the present invention's solid medium) and the inverted flat board of S3 (TCBS substratum) solid medium.Cultivate 18h for 25 ℃.The counting clone forms the quantity situation.
The actual theoretical number * 100% of the preceding thalline of number/coating that forms of cloning efficiency=clone.
The solid medium culture effect is as shown in table 2:
Table 2
Through t check back three's difference not remarkable (P＞0.1).The promptly selected substratum S1 of this research is as solid medium.
3. the comparison of test kit drug sensitivity tests
Adopt the K-B method, respectively pathogenic bacteria is carried out the drug susceptibility experiment with above 16 kinds of medicines.
Adopt the method for this test kit specification sheets, respectively 3 kinds of pathogenic bacterias are carried out the drug susceptibility experiment with above 16 kinds of medicines.
Table 3
Annotate: the S-sensitivity; The R-resistance; The M-medium sensitivity.
As can be seen from the above results, adopt two kinds of methods to carry out drug sensitive experiment, this test kit contrasts with the K-B method, and only two results of lactic norfloxacini and sarafloxacin hydrochloride are slightly different.Other result is all identical.Illustrate that this test kit can reach the accuracy that conventional K-B method is carried out drug sensitive experiment substantially.
Claims (3)
1. the preparation method of a susceptibility test kit, it is characterized in that described susceptibility examination box comprise centrifuge tube that enrichment culture medium is housed, be equipped with solid medium culture dish and susceptibility storage medicine circle;
The preparation of the centrifuge tube of enrichment culture medium is housed:
A, be that the peptone of 5-15 part, the yeast extract of 3-8 part, the sodium chloride of 5-15 part are poured in the beaker that 600-1000 part distilled water is housed, stir weight proportion;
B, in beaker, pour hydrochloric acid or sodium hydroxid into, the solution pH value in the beaker is adjusted to 7.3 ± 0.1, be mixed with enrichment culture medium;
C, pour the solution in the beaker into adjustable skimmer, enrichment culture medium is injected in the centrifuge tube, lid is screwed on but do not screw, stay half-turn to a circle by adjustable skimmer;
D, the centrifuge tube that enrichment culture medium will be housed are placed on the test-tube stand, put into the automatic high pressure Autoclave again, autoclaving 10-20 minute;
After e, sterilization finish centrifuge tube is taken out, screw the pipe lid of centrifuge tube then, put into true packing bag and vacuumize;
Be equipped with solid medium the preparation of culture dish:
A, be that the peptone of 5-15 part, the yeast extract of 3-8 part, the sodium chloride of 5-15 part, the agar of 10-15 part are poured in the Erlenmeyer flask that 600-1000 part distilled water is housed, stir weight proportion;
B, in Erlenmeyer flask, pour hydrochloric acid or sodium hydroxid into, the solution pH value in the beaker is adjusted to 7.3 ± 0.1, make nutrient solution;
C, Erlenmeyer flask is put into the automatic high pressure Autoclave, autoclaving 10-20 minute;
D, the liquid storage funnel to automatic skimmer in super clean bench are sterilized, and the nutrient solution that will be cooled to the 55-65 degree is then poured in the liquid storage funnel of automatic skimmer, divides to install in the culture dish;
E, culture dish is lain against on the super clean bench, and get to the super clean bench ultraviolet lamp, make the nutrient solution cooled and solidified become solid medium;
F, the lid of culture dish is covered, the vacuum packaging bag of packing into then is evacuated;
The preparation of susceptibility storage medicine circle:
Make the drug sensitive test paper of 15-20 kind medicine, drug sensitive test paper is pasted on the front of storing the medicine circle, the vacuum packaging bag of packing into then is evacuated.
2. the preparation method of susceptibility test kit according to claim 1, after it is characterized in that in the step b of the preparation of the centrifuge tube that enrichment culture medium is housed, adding sea crystal or concentrated seawater, adjust salinity between the 24-28, between the pH6.8-7.2, be mixed with enrichment culture medium; Be equipped with solid medium the step b of preparation of culture dish in add sea crystal or concentrated seawater after, adjust salinity between the 24-28, between the pH6.8-7.2, be mixed with nutrient solution.
3. the susceptibility detection method according to the susceptibility test kit of claim 1 and 2 preparations is characterized in that
A, preparation: hand, scissors and dissection container are cleaned up, and with forms such as 75% medical alcohol, poach or baked wheaten cakes to scissors with dissect sterilizing containers;
B, sampling: select individualities such as the typical dying fishes and shrimps of illness, clear water is cleaned the region of anatomy; Dissect with scissors, observe pathology, sterilize once more behind the scissors, the clip typical focus of soya bean size is approximately directly put into the centrifuge tube that enrichment culture medium is housed, and screw centrifuge tube pipe lid, if detect water sample, then directly get the 5-10 sample that drips and splash in the centrifuge tube that enrichment culture medium is housed, and screw centrifuge tube pipe lid;
C, liquid culture: place the back for several times with the forced oscillation centrifuge tube, and should avoid the sunlight direct projection, afterwards every half hour to 1 hour for several times with the forced oscillation centrifuge tube, up to the liquid muddiness;
D, coating: centrifugal liquid in pipe is poured into the culture dish centre that solid medium is housed, should avoid pouring into the throw out at the bottom of the centrifuge tube, on solid medium, liquid is smeared evenly lightly with spreading rod then, and should be avoided the solid culture primary surface is scratched;
E, subsides susceptibility are store the medicine circle: the spacious lid of culture dish is kept flat dry, to surperficial substantially dry, take out drug allergy ring then, drug sensitive test paper is put into the cultivation box down, the light pressure, the scraps of paper are fully contacted with media surface, cover back-off behind the lid of culture dish, be statically placed in temperature local 8-15 hour more than 20 degree;
F, result judge:
1), the medicine that occurs in the susceptibility paster of clear area all can be used as drug candidate, medicine in the bigger susceptibility paster in clear area can be used as preferred agents, take all factors into consideration pharmacopoeia class commonly used, medication history, medicine valency, the administering mode of this class disease, the factor of import-export regulations, select only microbiotic;
2), be mottled distribution, do not show that then disease may not be to be caused by infectation of bacteria as growing bacterium or bacterium;
3), be covered with whole cultivation box as bacterium, but the clear area do not occur, it is invalid to pathogenic bacterium then to show when medicine in all the susceptibility pasters on susceptibility storage medicine circle is at the standard using dosage, advises that other products select medicine again.
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| CN102505040A (en) * | 2011-11-07 | 2012-06-20 | 山西卫氏鱼康实业有限公司 | A method for rapidly screening bacterial disease prevention medicine of the aquatic animals |
| CN108004134A (en) * | 2017-12-21 | 2018-05-08 | 武汉轻工大学 | A kind of susceptibility kit and preparation method thereof and bacterial susceptibility detection method |
| CN108570488A (en) * | 2017-03-13 | 2018-09-25 | 乌鲁木齐市动物疾病控制与诊断中心 | Veterinary clinic quickly increases bacterium and drug sensitive test culture medium and its preparation and application method |
| CN109022539A (en) * | 2018-08-31 | 2018-12-18 | 泰州市春达动物药业饲料有限公司 | A kind of susceptibility detection method applied to aquaculture susceptibility kit |
| CN109112064A (en) * | 2018-08-31 | 2019-01-01 | 泰州市春达动物药业饲料有限公司 | A kind of susceptibility kit and preparation method thereof applied to aquaculture |
| CN110317854A (en) * | 2019-04-11 | 2019-10-11 | 天津市水产技术推广站 | A kind of susceptibility detection kit |
| CN110592178A (en) * | 2019-11-05 | 2019-12-20 | 广西壮族自治区水产科学研究院 | Drug sensitivity detection kit for bacterial diseases of freshwater fishes and application thereof |
| CN111304057A (en) * | 2020-03-23 | 2020-06-19 | 浙江圣达生物研究院有限公司 | Kit and method for evaluating antibacterial effect based on microorganism test piece |
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| CN109022539A (en) * | 2018-08-31 | 2018-12-18 | 泰州市春达动物药业饲料有限公司 | A kind of susceptibility detection method applied to aquaculture susceptibility kit |
| CN109112064A (en) * | 2018-08-31 | 2019-01-01 | 泰州市春达动物药业饲料有限公司 | A kind of susceptibility kit and preparation method thereof applied to aquaculture |
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