CN105349616A - Evaluation method of veterinary drug or disinfectant through microscale drug sensitive tests - Google Patents
Evaluation method of veterinary drug or disinfectant through microscale drug sensitive tests Download PDFInfo
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- CN105349616A CN105349616A CN201510797094.4A CN201510797094A CN105349616A CN 105349616 A CN105349616 A CN 105349616A CN 201510797094 A CN201510797094 A CN 201510797094A CN 105349616 A CN105349616 A CN 105349616A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2304/00—Chemical means of detecting microorganisms
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Abstract
The present invention discloses an evaluation method of a veterinary drug or a disinfectant through microscale drug sensitive tests, the evaluation method is as follows: 1, preparation of a sample loading bacteria liquid; 2, preparation of disinfectants of different concentrations; 3, sample loading for incubation; and 4, result determination, to be more specific, (1) comparison of MIC, namely, minimal inhibitory concentration, (2) OD450 bacteriostasis trend comparison, (3), effective inhibitory concentration cost comparison, and final selection of the best disinfectant by synthetical comparison and appraisement of the MIC, OD450 bacteriostasis trend and effective inhibitory concentration cost; a drug which is highly-efficient or effective to a farm can be evaluated by microscale drug sensitive evaluation, and an effective veterinary drug for disease prevention is provided. Through the evaluation method, the veterinary drug or the disinfectant for disease prevention in the farm can be evaluated in a lab by the microscale drug sensitive tests, and a certain reference for the proper use of the drug is provided.
Description
Technical field
The present invention relates to the evaluation method of veterinary drug or sterilizing agent, especially a kind of by the evaluation method of micro-drug sensitive test to veterinary drug or sterilizing agent.
Background technology
At present, before purchasing market veterinary drug and Herb of Common violet, plant generally judges that whether medicine is effective by rule of thumb, and this exists certain uncertainty, for plant's epidemic prevention and control brings certain risk.
Summary of the invention
The object of this invention is to provide a kind of by the evaluation method of micro-drug sensitive test to veterinary drug or sterilizing agent, the method be simple, accurately, quick.
For achieving the above object, method of the present invention comprises the steps:
One, loading bacterium solution preparation:
By separating Escherichia coli in water sample, the single bacterium colony of picking is mixed in the TSB of 5mL, 37 DEG C of baking oven stationary incubation 2h, now bacterium liquid is muddy, gets 100 μ L bacterium liquid 10 times dilution, 8 ~ 10 gradients, is evenly applied in TSA, count after 37 DEG C of standing 24h, be then adjusted to 10 according to the bacterial concentration of counting
5cFU/mL, for loading bacterium liquid;
Two, different concns sterilizing agent preparation:
According to minimum working concentration and the highest working concentration of operation instruction on sterilizing agent, doubling dilution experiment concentration need comprise minimum and the highest working concentration, is respectively 6 gradients: 0.5 times, 1 times, 2 times, 4 times, 8 times, 16 times of minimum concentrations, and unit is g/L or mL/L;
Three, loading is hatched:
1. the 1st front 6 holes of row in 96 microwell plates are added 6 kinds of different concns sterilizing agent 100 μ L respectively, the 7th hole adds loading bacterium liquid 200 μ L as positive control, and the 8th hole adds this sterilizing agent 200 μ L;
2. the 1st front 6 holes of row add 100 μ L loading bacterium liquid again, cause every Kongzui end-body to be 200 μ L;
3. the 2nd row is as the parallel control of the 1st row's sterilizing agent, and other sterilizing agents also with after aforesaid way application of sample, build plate lid; Then be placed in 37 DEG C, stationary incubation, respectively get 4 points respectively at 3 ~ 6h and 12 ~ 24h and observe MIC and measure OD450 value;
Four, result judges:
1. the comparison of MIC and minimal inhibitory concentration: the MIC of sterilizing agent compares with recommended density in its operation instruction or minimum working concentration, if lower than recommended density, then sterilizing agent effect is better, otherwise then poor; Different sterilizing agent MIC compares, and MIC is lower, but first must be less than recommended density, and sterilizing agent effect is better;
2. the antibacterial trend comparison of OD450: for the identical sterilizing agent of MIC value, by the antibacterial trend of OD450, analyzes its bacteriostatic action time and stability, judges good and bad;
3. effectively Mlc expense compares: according to effective Mlc and MIC, more each sterilizing agent dilutes the same water yield, the expense spent;
Finally, in conjunction with the antibacterial trend of MIC and OD450 and effectively Mlc expense synthetical comparison and assessment, optimum sterilizing agent is selected;
Above-mentioned mL is: milliliter; μ L is: microlitre; DEG C be: degree Celsius; H is: hour; CFU/mL is: colony-forming unit/milliliter; G/L is: grams per liter; ML/L is: milliliter/liter; TSA is: Tryptic Soy Agar; TSB is: trypticase soya broth; MIC is: minimal inhibitory concentration; OD450 is: 450 nanometer optical densities values.
Beneficial effect:
The present invention is evaluated efficiently by micro-susceptibility or for this plant active drug, also can provide effective veterinary drug to Prevention and cure of epidemic situation simultaneously.
The present invention adopts micro-drug sensitive test to carry out laboratory evaluation for plant's disease preventing and treating veterinary drug used or sterilizing agent, for proper use of medicine provides certain reference frame.
Embodiment
Embodiment 1:
This by the evaluation method of micro-drug sensitive test to veterinary drug or sterilizing agent, comprise the steps:
One, loading bacterium solution preparation:
By separating Escherichia coli in water sample, the single bacterium colony of picking is mixed in the TSB of 5mL, 37 DEG C of baking oven stationary incubation 2h, now bacterium liquid is muddy, gets 100 μ L bacterium liquid 10 times dilution, 8 gradients, is evenly applied in TSA, count after 37 DEG C of standing 24h, be then adjusted to 10 according to the bacterial concentration of counting
5cFU/mL, for loading bacterium liquid;
Two, different concns sterilizing agent preparation:
According to minimum working concentration and the highest working concentration of operation instruction on sterilizing agent, doubling dilution experiment concentration need comprise minimum and the highest working concentration, is respectively 6 gradients: 0.5 times, 1 times, 2 times, 4 times, 8 times, 16 times of minimum concentrations, and unit is g/L or mL/L;
Three, loading is hatched:
1. the 1st front 6 holes of row in 96 microwell plates are added 6 kinds of different concns sterilizing agent 100 μ L respectively, the 7th hole adds loading bacterium liquid 200 μ L as positive control, and the 8th hole adds this sterilizing agent 200 μ L;
2. the 1st front 6 holes of row add 100 μ L loading bacterium liquid again, cause every Kongzui end-body to be 200 μ L;
3. the 2nd row is as the parallel control of the 1st row's sterilizing agent, and other sterilizing agents also with after aforesaid way application of sample, build plate lid; Then be placed in 37 DEG C, stationary incubation, respectively get 4 points respectively at 3h and 12h and observe MIC and measure OD450 value;
Four, result judges:
1. the comparison of MIC and minimal inhibitory concentration: the MIC of sterilizing agent compares with recommended density in its operation instruction or minimum working concentration, if lower than recommended density, then sterilizing agent effect is better, otherwise then poor; Different sterilizing agent MIC compares, and MIC is lower, but first must be less than recommended density, and sterilizing agent effect is better;
2. the antibacterial trend comparison of OD450: for the identical sterilizing agent of MIC value, by the antibacterial trend of OD450, analyzes its bacteriostatic action time and stability, judges good and bad;
3. effectively Mlc expense compares: according to effective Mlc and MIC, more each sterilizing agent dilutes the same water yield, the expense spent;
Finally, in conjunction with the antibacterial trend of MIC and OD450 and effectively Mlc expense synthetical comparison and assessment, optimum sterilizing agent is selected;
Above-mentioned mL is: milliliter; μ L is: microlitre; DEG C be: degree Celsius; H is: hour; CFU/mL is: colony-forming unit/milliliter; G/L is: grams per liter; ML/L is: milliliter/liter; TSA is: Tryptic Soy Agar; TSB is: trypticase soya broth; MIC is: minimal inhibitory concentration; OD450 is: 450 nanometer optical densities values.
Embodiment 2:
By the evaluation method of micro-drug sensitive test to veterinary drug or sterilizing agent, comprise the steps:
One, loading bacterium solution preparation:
By separating Escherichia coli in water sample, the single bacterium colony of picking is mixed in the TSB of 5mL, 37 DEG C of baking oven stationary incubation 2h, now bacterium liquid is muddy, gets 100 μ L bacterium liquid 10 times dilution, 10 gradients, is evenly applied in TSA, count after 37 DEG C of standing 24h, be then adjusted to 10 according to the bacterial concentration of counting
5cFU/mL, for loading bacterium liquid;
Two, different concns sterilizing agent preparation:
According to minimum working concentration and the highest working concentration of operation instruction on sterilizing agent, doubling dilution experiment concentration need comprise minimum and the highest working concentration, is respectively 6 gradients: 0.5 times, 1 times, 2 times, 4 times, 8 times, 16 times of minimum concentrations, and unit is g/L or mL/L;
Three, loading is hatched:
1. the 1st front 6 holes of row in 96 microwell plates are added 6 kinds of different concns sterilizing agent 100 μ L respectively, the 7th hole adds loading bacterium liquid 200 μ L as positive control, and the 8th hole adds this sterilizing agent 200 μ L;
2. the 1st front 6 holes of row add 100 μ L loading bacterium liquid again, cause every Kongzui end-body to be 200 μ L;
3. the 2nd row is as the parallel control of the 1st row's sterilizing agent, and other sterilizing agents also with after aforesaid way application of sample, build plate lid; Then be placed in 37 DEG C, stationary incubation, respectively get 4 points respectively at 6h and 24h and observe MIC and measure OD450 value;
Four, result judges:
1. the comparison of MIC and minimal inhibitory concentration: the MIC of sterilizing agent compares with recommended density in its operation instruction or minimum working concentration, if lower than recommended density, then sterilizing agent effect is better, otherwise then poor; Different sterilizing agent MIC compares, and MIC is lower, but first must be less than recommended density, and sterilizing agent effect is better;
2. the antibacterial trend comparison of OD450: for the identical sterilizing agent of MIC value, by the antibacterial trend of OD450, analyzes its bacteriostatic action time and stability, judges good and bad;
3. effectively Mlc expense compares: according to effective Mlc and MIC, more each sterilizing agent dilutes the same water yield, the expense spent;
Finally, in conjunction with the antibacterial trend of MIC and OD450 and effectively Mlc expense synthetical comparison and assessment, optimum sterilizing agent is selected;
Above-mentioned mL is: milliliter; μ L is: microlitre; DEG C be: degree Celsius; H is: hour; CFU/mL is: colony-forming unit/milliliter; G/L is: grams per liter; ML/L is: milliliter/liter; TSA is: Tryptic Soy Agar; TSB is: trypticase soya broth; MIC is: minimal inhibitory concentration; OD450 is: 450 nanometer optical densities values.
Claims (1)
1., by the evaluation method of micro-drug sensitive test to veterinary drug or sterilizing agent, it is characterized in that: comprise the steps:
One, loading bacterium solution preparation:
By separating Escherichia coli in water sample, the single bacterium colony of picking is mixed in the TSB of 5mL, 37 DEG C of baking oven stationary incubation 2h, now bacterium liquid is muddy, gets 100 μ L bacterium liquid 10 times dilution, 8 ~ 10 gradients, is evenly applied in TSA, count after 37 DEG C of standing 24h, be then adjusted to 10 according to the bacterial concentration of counting
5cFU/mL, for loading bacterium liquid;
Two, different concns sterilizing agent preparation:
According to minimum working concentration and the highest working concentration of operation instruction on sterilizing agent, doubling dilution experiment concentration need comprise minimum and the highest working concentration, is respectively 6 gradients: 0.5 times, 1 times, 2 times, 4 times, 8 times, 16 times of minimum concentrations, and unit is g/L or mL/L;
Three, loading is hatched:
1. the 1st front 6 holes of row in 96 microwell plates are added 6 kinds of different concns sterilizing agent 100 μ L respectively, the 7th hole adds loading bacterium liquid 200 μ L as positive control, and the 8th hole adds this sterilizing agent 200 μ L;
2. the 1st front 6 holes of row add 100 μ L loading bacterium liquid again, cause every Kongzui end-body to be 200 μ L;
3. the 2nd row is as the parallel control of the 1st row's sterilizing agent, and other sterilizing agents also with after aforesaid way application of sample, build plate lid; Then be placed in 37 DEG C, stationary incubation, respectively get 4 points respectively at 3 ~ 6h and 12 ~ 24h and observe MIC and measure OD450 value;
Four, result judges:
1. the comparison of MIC and minimal inhibitory concentration: the MIC of sterilizing agent compares with recommended density in its operation instruction or minimum working concentration, if lower than recommended density, then sterilizing agent effect is better, otherwise then poor; Different sterilizing agent MIC compares, and MIC is lower, but first must be less than recommended density, and sterilizing agent effect is better;
2. the antibacterial trend comparison of OD450: for the identical sterilizing agent of MIC value, by the antibacterial trend of OD450, analyzes its bacteriostatic action time and stability, judges good and bad;
3. effectively Mlc expense compares: according to effective Mlc and MIC, more each sterilizing agent dilutes the same water yield, the expense spent;
Finally, in conjunction with the antibacterial trend of MIC and OD450 and effectively Mlc expense synthetical comparison and assessment, optimum sterilizing agent is selected;
Above-mentioned mL is: milliliter; μ L is: microlitre; DEG C be: degree Celsius; H is: hour; CFU/mL is: colony-forming unit/milliliter; G/L is: grams per liter; ML/L is: milliliter/liter; TSA is: Tryptic Soy Agar; TSB is: trypticase soya broth; MIC is: minimal inhibitory concentration; OD450 is: 450 nanometer optical densities values.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510797094.4A CN105349616A (en) | 2015-11-19 | 2015-11-19 | Evaluation method of veterinary drug or disinfectant through microscale drug sensitive tests |
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|---|---|---|---|
| CN201510797094.4A CN105349616A (en) | 2015-11-19 | 2015-11-19 | Evaluation method of veterinary drug or disinfectant through microscale drug sensitive tests |
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| CN105349616A true CN105349616A (en) | 2016-02-24 |
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| CN201510797094.4A Pending CN105349616A (en) | 2015-11-19 | 2015-11-19 | Evaluation method of veterinary drug or disinfectant through microscale drug sensitive tests |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103120689A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Application of pyrazol compound as mycobacterium tuberculosis inhibitor |
| CN104198658A (en) * | 2014-09-10 | 2014-12-10 | 广西扬翔农牧有限责任公司 | Method for carrying out laboratory evaluation on veterinary medicines or disinfectants for cultivation |
-
2015
- 2015-11-19 CN CN201510797094.4A patent/CN105349616A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103120689A (en) * | 2011-11-21 | 2013-05-29 | 华中农业大学 | Application of pyrazol compound as mycobacterium tuberculosis inhibitor |
| CN104198658A (en) * | 2014-09-10 | 2014-12-10 | 广西扬翔农牧有限责任公司 | Method for carrying out laboratory evaluation on veterinary medicines or disinfectants for cultivation |
Non-Patent Citations (1)
| Title |
|---|
| 王俊丽: "18种中药对猪大肠杆菌的体外抑菌活性的测定方法比较", 《安徽农业科学》 * |
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Address after: Gangnan District Jiangnan Industrial Park Avenue and East Industrial Road 537100 the Guangxi Zhuang Autonomous Region Guigang City, the northeast corner of the intersection Applicant after: GUANGXI YANGXIANG AGRICULTURE AND ANIMAL HUSBANDRY CO., LTD. Address before: No. 844 Hong Kong Road 537100 the Guangxi Zhuang Autonomous Region Gangbei District, Guigang City Applicant before: GUANGXI YANGXIANG AGRICULTURE AND ANIMAL HUSBANDRY CO., LTD. |
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| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20160224 |
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| WD01 | Invention patent application deemed withdrawn after publication |