CN105176884A - Method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli - Google Patents
Method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli Download PDFInfo
- Publication number
- CN105176884A CN105176884A CN201510679353.3A CN201510679353A CN105176884A CN 105176884 A CN105176884 A CN 105176884A CN 201510679353 A CN201510679353 A CN 201510679353A CN 105176884 A CN105176884 A CN 105176884A
- Authority
- CN
- China
- Prior art keywords
- riemerella anatipestifer
- intestinal bacteria
- mixed culture
- substratum
- culture
- 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.)
- Granted
Links
Abstract
The invention discloses a method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli. The method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli comprises the following steps of (1) performing strain recovery; (2) screening riemerella anatipestifer and escherichia coli with antibiotics sensitivity differences; (3) inoculating the riemerella anatipestifer and the escherichia coli into a culture medium for mixed culture at a certain time interval or different diluting concentration; (4) measuring the minimum inhibitory concentration of antibiotics in the second step on the screened riemerella anatipestifer and escherichia coli; (5) preparing an antibiotic plate culture medium; (6) separating mixed culture bacterium liquid. The method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli has the advantages that two kinds of bacteria are used outside duck poultry bodies and can jointly grow for more than 20 generations; conditions are provided for studying drug resistance transfer and relevant mechanisms of the two kinds of bacteria under the symbiosis condition; important significance is realized on disease prevention and control of duck breeding industry.
Description
(1) technical field
The invention belongs to biological technical field, particularly a kind of riemerella anatipestifer and intestinal bacteria mixed culture and the method that is again separated.
(2) background technology
Riemerella anatipestifer is the pathogenic bacteria causing Riemerella anatipestifer disease, Riemerella anatipestifer disease causes the acute or chronic septic of young duck to infect, be a kind of principal disease that duck industry is supported in the serious harm whole world, and intestinal bacteria are the pathogenic agent causing bacteriosis that poultry farming financial loss is the most serious.
Riemerella anatipestifer and the normal polyinfection of intestinal bacteria clinically; two kinds of bacterium all can make the duck infection morbidity of different varieties, age in days and sex; fall ill with fibrinous pericarditis, serohepatitis, airsacculitis and meningitis for characteristic pathological changes; clinical diagnosis is difficult to both to make a distinction; and the duck group mortality ratio infecting two diseases is high; mortality is high, causes very big financial loss to foster duck industry.
Often find to there is resistance transfer phenomena between two kinds of bacterium clinically, but in vitro in culturing process, owing to just forcing riemerella anatipestifer to stop producing with its competitive edge in the intestinal bacteria short period of time, and then block the process of the two syntrophism, caused furtheing investigate.This may be the mechanism having it special because of environment of bacteria growth in vivo, causes the two long-term syntrophism and then produces resistance transfer.How creating a kind of environment in vitro allows the two can grow 20 generations more than, thus makes resistance transmission (or amalgamation research) research become possibility, has important Research Significance.
(3) summary of the invention
The present invention in order to make up the deficiencies in the prior art, provide a kind of simple to operate, for inquiring into the method that riemerella anatipestifer shifts with the resistance of intestinal bacteria under symbiosis conditions and related mechanism provides the riemerella anatipestifer of condition and intestinal bacteria mixed culture and is again separated.
The present invention is achieved through the following technical solutions:
Riemerella anatipestifer and intestinal bacteria mixed culture and the method be again separated, comprise the steps:
(1) bacterial classification recovery:
The riemerella anatipestifer that freeze-drying is preserved is inoculated in corresponding substratum with strain Escherichia coli respectively, carries out bacterial classification recovery, the riemerella anatipestifer after obtained recovery and intestinal bacteria;
(2) riemerella anatipestifer and the intestinal bacteria of Antibiotic Sensitivity difference are screened:
Riemerella anatipestifer after the recovery obtain step (1) and intestinal bacteria, adopt medicament sensitivity test to filter out riemerella anatipestifer to certain antibiotic sensitive sex differernce and intestinal bacteria;
(3) mixed culture:
The riemerella anatipestifer filter out step (2) and intestinal bacteria are inoculated in substratum, can carry out mixed culture 9-12h to riemerella anatipestifer and intestinal bacteria, and obtained mixed culture bacterium liquid;
(4) riemerella anatipestifer that in determination step (2), selected microbiotic filters out step (2) and colibacillary minimal inhibitory concentration:
(5) microbiotic plate substratum is prepared:
The selected microbiotic choosing mensuration in step (4), to the antibiotic concentration between riemerella anatipestifer and colibacillary minimal inhibitory concentration, prepares microbiotic plate culture medium, for subsequent use;
(6) mixed culture bacterium liquid is separated:
After the mixed culture bacterium liquid that step (3) is obtained is diluted, adopt spread plate method, in the microbiotic plate culture medium that step (5) is obtained, cultivation 20-24h is carried out to the riemerella anatipestifer in dilution bacterium liquid and intestinal bacteria, can be separated with intestinal bacteria riemerella anatipestifer.
In described step (1), the concrete grammar of bacterial classification recovery is:
(1-1) riemerella anatipestifer bacterial classification recovery:
Measure 1ml physiological saline, the riemerella anatipestifer bacterial classification appropriate freeze-drying preserved dissolves and mixes in physiological saline, obtained riemerella anatipestifer suspension, after obtained riemerella anatipestifer suspension inoculation is in the tryptose soya agar substratum containing 4% serum, is placed in CO
220-24h is cultivated at 37 DEG C in incubator, then from substratum the single colony inoculation of picking in containing 4% serum pancreas peptone soybean broth substratum in, rotating speed be 150r/min, temperature is shaking culture 20-24h on the shaking table of 37 DEG C, the riemerella anatipestifer after obtained recovery;
Described riemerella anatipestifer bacterial strain is provided by Domestic Fowls Inst., Shandong Academy of Agricultural Sciences.
(1-2) intestinal bacteria type strain bacterial classification recovery:
Measure 1ml physiological saline, the strain Escherichia coli appropriate freeze-drying preserved dissolves and mixes in physiological saline, obtained intestinal bacteria suspension, by obtained intestinal bacteria suspension inoculation in nutrient broth medium, rotating speed be 150r/min, temperature is shaking culture 16-20h on the shaking table of 37 DEG C, the intestinal bacteria after obtained recovery;
Described strain Escherichia coli is purchased from China Veterinery Drug Inspection Office.
The method that described step (2) medicament sensitivity test adopts is agar diffusion method of the paper, the microbiotic tested in drug sensitive test paper used is any one in gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin, and in corresponding drug sensitive test paper, the content of gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin is respectively 10 μ g, 5 μ g, 5 μ g, 10 μ g; Wherein, judge that the standard of riemerella anatipestifer to gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin sensitivity is respectively antibacterial circle diameter and is less than or equal to 12mm, 15mm, 12mm, 12mm, judge that the standard of intestinal bacteria to gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin sensitivity is respectively antibacterial circle diameter and is more than or equal to 15mm, 21mm, 16mm, 17mm.
In described step (3), the concrete grammar of mixed culture is:
First measuring the pancreas peptone soybean broth that riemerella anatipestifer that step (2) filters out is seeded to containing 4% serum supports in base, rotating speed be 150r/min, temperature is shaking culture 14-18h on the shaking table of 37 DEG C, when seeing muddiness to substratum, again the intestinal bacteria that step (2) filters out are inoculated in above-mentioned substratum and mix, rotating speed be 150r/min, temperature is shaking culture 9-12h on the shaking table of 37 DEG C, obtained mixed culture bacterium liquid.
In described step (3), the concrete grammar of mixed culture is:
(3-1) riemerella anatipestifer and intestinal bacteria that step (2) filters out is measured, by the riemerella anatipestifer measured, the pancreas peptone soybean broth be seeded to containing 4% serum is supported in base, is seeded to by the intestinal bacteria measured in pancreas peptone soybean broth substratum;
(3-2) by inoculate respectively in step (3-1) riemerella anatipestifer and colibacillary substratum respectively rotating speed be 150r/min, temperature is shaking culture 16-20h on the shaking table of 37 DEG C;
(3-3) the riemerella anatipestifer bacterium liquid that step (3-2) shaking table shaking culture obtains is diluted to 15 × 10
8cFU/ml, Escherichia coli bacteria liquid is diluted to 1.5 × 10
3cFU/ml;
(3-4) Escherichia coli bacteria liquid after the riemerella anatipestifer bacterium liquid after 50 obtained in aspiration step (3-3) μ l dilute and 10 μ l dilute, and Simultaneous vaccination contains in the pancreas peptone soybean broth substratum of 4% serum in 4ml, rotating speed be 150r/min, temperature is shaking culture 9-12h on the shaking table of 37 DEG C, obtained mixed culture bacterium liquid.
It is the double dilution method of test tube to the test method of antibiotic minimal inhibitory concentration that described step (4) measures the riemerella anatipestifer and intestinal bacteria that filter out.
The microbiotic plate culture medium that described step (5) obtains is the tryptose soya agar substratum containing 4% serum, each microbiotic that correspondence is selected, add this antibiotic concentration in substratum to be respectively: gentamicin 10 μ g/ml, Ciprofloxacin 3 μ g/ml, Ofloxacine USP 23 6 μ g/ml, norfloxicin 6 μ g/ml.
The concrete grammar that described step (6) is separated mixed culture bacterium liquid is:
The appropriate mixed culture bacterium liquid that step (3) is obtained is diluted to 3 × 10
3-6 × 10
3after CFU/ml, draw 100 μ l diluents, coat maconkey agar substratum, containing the tryptose soya agar substratum of 4% serum and step (5) obtained containing in the microbiotic plate culture medium of gentamicin, be placed in CO
2cultivate 20-24h at incubator 37 DEG C, can the riemerella anatipestifer in mixed culture bacterium liquid be separated with intestinal bacteria.
Riemerella anatipestifer of the present invention and intestinal bacteria mixed culture and the method be again separated, the riemerella anatipestifer to certain antibiotics resistance is filtered out by medicament sensitivity test, and the intestinal bacteria to this antibiotic sensitive, and the riemerella anatipestifer adopting the double By Dilution of test tube to filter out and intestinal bacteria are to this antibiotic minimal inhibitory concentration, then by the riemerella anatipestifer filtered out and intestinal bacteria at a certain time interval or different concentration be inoculated in the pancreas peptone soybean broth substratum containing 4% serum, mixed culture can be carried out to riemerella anatipestifer and intestinal bacteria.The dilution of the bacterium liquid of mixed culture is coated respectively maconkey agar substratum, containing 4% serum tryptose soya agar substratum and obtained to cultivate containing in antibiotic microbiotic plate culture medium, can implement again be separated to these two kinds of bacteriums.A kind of riemerella anatipestifer provided by the invention and intestinal bacteria are mixed culture and the method that is again separated in vitro, simple to operate, enable more than two kinds of bacterium syntrophism 20 generations duck fowl is external, for inquire into both under symbiosis conditions resistance transfer and related mechanism provide condition, the diseases prevention and treatment of foster duck industry are had great importance.
(4) embodiment
Below in conjunction with embodiment, the invention will be further described, but the present invention is not limited thereto, and the preparation method in embodiment is customary preparation methods, no longer describes in detail.
Embodiment 1:
This riemerella anatipestifer and intestinal bacteria mixed culture and the method be again separated, adopt following steps:
(1) bacterial classification recovery:
(1-1) riemerella anatipestifer bacterial classification recovery:
Measure 1ml physiological saline, the riemerella anatipestifer bacterial classification appropriate freeze-drying preserved dissolves and mixes in physiological saline, obtained riemerella anatipestifer suspension, after obtained riemerella anatipestifer suspension inoculation is in the tryptose soya agar substratum containing 4% serum, is placed in CO
220-24h is cultivated at 37 DEG C in incubator, then from substratum the single colony inoculation of picking in containing 4% serum pancreas peptone soybean broth substratum in, rotating speed be 150r/min, temperature is shaking culture 20-24h on the shaking table of 37 DEG C, the riemerella anatipestifer after obtained recovery;
Described riemerella anatipestifer bacterial strain is provided by Domestic Fowls Inst., Shandong Academy of Agricultural Sciences.
(1-2) intestinal bacteria type strain bacterial classification recovery:
Measure 1ml physiological saline, the strain Escherichia coli appropriate freeze-drying preserved dissolves and mixes in physiological saline, obtained intestinal bacteria suspension, by obtained intestinal bacteria suspension inoculation in nutrient broth medium, rotating speed be 150r/min, temperature is shaking culture 16-20h on the shaking table of 37 DEG C, the intestinal bacteria after obtained recovery;
Described strain Escherichia coli is purchased from China Veterinery Drug Inspection Office.
(2) riemerella anatipestifer and the intestinal bacteria of Antibiotic Sensitivity difference are screened:
Riemerella anatipestifer after the recovery that step (1) is obtained and intestinal bacteria, adopt agar diffusion method of the paper to filter out riemerella anatipestifer to certain antibiotic sensitive sex differernce and intestinal bacteria, concrete grammar is:
Choose gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin four kinds of microbiotic, agar diffusion method of the paper is adopted to carry out medicament sensitivity test, the content testing gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin in drug sensitive test paper used is respectively 10 μ g, 5 μ g, 5 μ g, 10 μ g, riemerella anatipestifer after recovery is seeded on the tryptose soya agar plate culture medium containing 4% serum, puts CO
216-20h cultivated by incubator 37 DEG C; Intestinal bacteria after recovery are seeded on plain agar flat board and cultivate, and put in 37 DEG C of thermostat containers and cultivate 16-18h.Wherein, judge that the standard of riemerella anatipestifer to gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin sensitivity is respectively antibacterial circle diameter and is less than or equal to 12mm, 15mm, 12mm, 12mm, judge that the standard of intestinal bacteria to gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin sensitivity is respectively antibacterial circle diameter and is more than or equal to 15mm, 21mm, 16mm, 17mm.
Test records riemerella anatipestifer and intestinal bacteria to above-mentioned four kinds of antibiotic sensitive data in table 1.
Table 1 riemerella anatipestifer and intestinal bacteria are to the susceptibility (antibacterial circle diameter: mm) of chemicals
(3) mixed culture:
First measure the 5 μ l riemerella anatipestifers that step (2) filters out and be seeded to 4ml containing in the pancreas peptone soybean broth substratum of 4% serum, rotating speed be 150r/min, temperature is shaking culture 14-18h on the shaking table of 37 DEG C, when seeing muddiness to substratum, 5 μ l intestinal bacteria step (2) filtered out again are inoculated in above-mentioned substratum and mix, rotating speed be 150r/min, temperature is shaking culture 6h, 9h, 12h, 18h on the shaking table of 37 DEG C, obtained mixed culture bacterium liquid;
In order to verify that riemerella anatipestifer and the intestinal bacteria of the present embodiment after the process of step (1)-(3) achieve mixed culture in the pancreas peptone soybean broth substratum containing 4% serum, after bacterium liquid after mixed culture 6h, 9h, 12h, 18h is carried out the dilution of different concns by contriver respectively, riemerella anatipestifer and colibacillary bacterium colony number in bacterium liquid are added up, in table 2.
After table 2 mixed culture 6h, 9h, 12h, 18h, the riemerella anatipestifer of different weaker concn and Escherichia coli bacteria liquid bacterium colony number are added up
From table 2, data can be seen, riemerella anatipestifer after the process of step (1)-(3) and intestinal bacteria are energy syntrophism 6h, 9h, 12h in the pancreas peptone soybean broth substratum containing 4% serum, achieve two kinds of bacterium mixed culture in vitro, for inquiring into riemerella anatipestifer and the resistance of intestinal bacteria under symbiosis conditions shifts and related mechanism provides condition.
(4) choose gentamicin to test, the riemerella anatipestifer adopting test tube double By Dilution gentamicin to filter out step (2) and colibacillary minimal inhibitory concentration are respectively 40ug/ml, 5ug/ml;
(5) microbiotic plate substratum is prepared:
Choose the gentamicin of mensuration in step (4) to the antibiotic concentration 10 μ g/ml between riemerella anatipestifer and colibacillary minimal inhibitory concentration, the microbiotic plate culture medium of preparation containing gentamicin;
(6) mixed culture bacterium liquid is separated:
The appropriate mixed culture bacterium liquid that step (3) is obtained is diluted to 3 × 10 respectively
3cFU/ml, 6 × 10
3cFU/ml, draws 100 μ l diluents, and the microbiotic plate culture medium containing gentamicin coat maconkey agar substratum, obtaining containing tryptose soya agar substratum and the step (5) of 4% serum, is placed in CO
2cultivate 20-24h at incubator 37 DEG C, can the riemerella anatipestifer in mixed culture bacterium liquid be separated with intestinal bacteria.
Embodiment 2:
This riemerella anatipestifer and intestinal bacteria mixed culture and the method be again separated, adopt following steps:
(1) bacterial classification recovery:
1. riemerella anatipestifer bacterial classification recovery:
Measure 1ml physiological saline, the riemerella anatipestifer bacterial classification appropriate freeze-drying preserved dissolves and mixes in physiological saline, obtained riemerella anatipestifer suspension, after obtained riemerella anatipestifer suspension inoculation is in the tryptose soya agar substratum containing 4% serum, is placed in CO
220-24h is cultivated at 37 DEG C in incubator, then from substratum the single colony inoculation of picking in containing 4% serum pancreas peptone soybean broth substratum in, rotating speed be 150r/min, temperature is shaking culture 20-24h on the shaking table of 37 DEG C, the riemerella anatipestifer after obtained recovery;
Described riemerella anatipestifer bacterial strain is provided by Domestic Fowls Inst., Shandong Academy of Agricultural Sciences.
2. intestinal bacteria type strain bacterial classification recovery:
Measure 1ml physiological saline, the strain Escherichia coli appropriate freeze-drying preserved dissolves and mixes in physiological saline, obtained intestinal bacteria suspension, by obtained intestinal bacteria suspension inoculation in nutrient broth medium, rotating speed be 150r/min, temperature is shaking culture 16-20h on the shaking table of 37 DEG C, the intestinal bacteria after obtained recovery;
Described strain Escherichia coli is purchased from China Veterinery Drug Inspection Office.
(2) riemerella anatipestifer and the intestinal bacteria of Antibiotic Sensitivity difference are screened:
Riemerella anatipestifer after the recovery that step (1) is obtained and intestinal bacteria, adopt agar diffusion method of the paper to filter out riemerella anatipestifer to certain antibiotic sensitive sex differernce and intestinal bacteria, concrete grammar is:
Choose gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin four kinds of microbiotic, agar diffusion method of the paper is adopted to carry out medicament sensitivity test, the content testing gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin in drug sensitive test paper used is respectively 10 μ g, 5 μ g, 5 μ g, 10 μ g, riemerella anatipestifer after recovery is seeded on the tryptose soya agar plate culture medium containing 4% serum, puts CO
216-20h cultivated by incubator 37 DEG C; Intestinal bacteria after recovery are seeded on plain agar flat board and cultivate, and put in 37 DEG C of thermostat containers and cultivate 16-18h.Wherein, judge that the standard of riemerella anatipestifer to gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin sensitivity is respectively antibacterial circle diameter and is less than or equal to 12mm, 15mm, 12mm, 12mm, judge that the standard of intestinal bacteria to gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin sensitivity is respectively antibacterial circle diameter and is more than or equal to 15mm, 21mm, 16mm, 17mm.
Test records riemerella anatipestifer and intestinal bacteria to above-mentioned four kinds of antibiotic sensitive data in table 1.
(3) mixed culture:
(3-1) riemerella anatipestifer and intestinal bacteria that step (2) filters out is measured, by the riemerella anatipestifer measured, the pancreas peptone soybean broth be seeded to containing 4% serum is supported in base, is seeded to by the intestinal bacteria measured in pancreas peptone soybean broth substratum;
(3-2) by inoculate respectively in step (3-1) riemerella anatipestifer and colibacillary substratum respectively rotating speed be 150r/min, temperature is shaking culture 16-20h on the shaking table of 37 DEG C;
(3-3) the riemerella anatipestifer bacterium liquid that step (3-2) shaking table shaking culture obtains is diluted to 15 × 10
8cFU/ml, Escherichia coli bacteria liquid is diluted to 1.5 × 10
3cFU/ml;
(3-4) Escherichia coli bacteria liquid after the riemerella anatipestifer bacterium liquid after 50 obtained in aspiration step (3-3) μ l dilute and 10 μ l dilute, and Simultaneous vaccination contains in the pancreas peptone soybean broth substratum of 4% serum in 4ml, rotating speed be 150r/min, temperature is shaking culture 9-12h on the shaking table of 37 DEG C, obtained mixed culture bacterium liquid;
(4) choose gentamicin to test, the riemerella anatipestifer adopting test tube double By Dilution gentamicin to filter out step (2) and colibacillary minimal inhibitory concentration are respectively 40ug/ml, 5ug/ml;
(5) microbiotic plate substratum is prepared:
Choose the gentamicin of mensuration in step (4) to the antibiotic concentration 10 μ g/ml between riemerella anatipestifer and colibacillary minimal inhibitory concentration, the microbiotic plate culture medium of preparation containing gentamicin;
(6) mixed culture bacterium liquid is separated:
The appropriate mixed culture bacterium liquid that step (3) is obtained is diluted to 3 × 10 respectively
3cFU/ml, 6 × 10
3cFU/ml, draws 100 μ l diluents, coat maconkey agar substratum, containing the tryptose soya agar substratum of 4% serum and step (5) obtained containing on the microbiotic plate culture medium of gentamicin, be placed in CO
2cultivate 20-24h at incubator 37 DEG C, can the riemerella anatipestifer in mixed culture bacterium liquid be separated with intestinal bacteria.
Claims (8)
1. riemerella anatipestifer and intestinal bacteria mixed culture and the method that is again separated, is characterized in that: comprise the steps:
(1) bacterial classification recovery:
The riemerella anatipestifer that freeze-drying is preserved is inoculated in corresponding substratum with strain Escherichia coli respectively, carries out bacterial classification recovery, the riemerella anatipestifer after obtained recovery and intestinal bacteria;
(2) riemerella anatipestifer and the intestinal bacteria of Antibiotic Sensitivity difference are screened:
Riemerella anatipestifer after the recovery obtain step (1) and intestinal bacteria, adopt medicament sensitivity test to filter out riemerella anatipestifer to certain antibiotic sensitive sex differernce and intestinal bacteria;
(3) mixed culture:
The riemerella anatipestifer filter out step (2) and intestinal bacteria are inoculated in substratum, can carry out mixed culture 9-12h to riemerella anatipestifer and intestinal bacteria, and obtained mixed culture bacterium liquid;
(4) riemerella anatipestifer that in determination step (2), selected microbiotic filters out step (2) and colibacillary minimal inhibitory concentration:
(5) microbiotic plate substratum is prepared:
The selected microbiotic choosing mensuration in step (4), to the antibiotic concentration between riemerella anatipestifer and colibacillary minimal inhibitory concentration, prepares microbiotic plate culture medium, for subsequent use;
(6) mixed culture bacterium liquid is separated:
After the mixed culture bacterium liquid that step (3) is obtained is diluted, adopt spread plate method, in the microbiotic plate culture medium that step (5) is obtained, cultivation 20-24h is carried out to the riemerella anatipestifer in dilution bacterium liquid and intestinal bacteria, can be separated with intestinal bacteria riemerella anatipestifer.
2. riemerella anatipestifer according to claim 1 and intestinal bacteria mixed culture and the method that is again separated, is characterized in that: in described step (1), the concrete grammar of bacterial classification recovery is:
(1-1) riemerella anatipestifer bacterial classification recovery:
Measure 1ml physiological saline, the riemerella anatipestifer bacterial classification appropriate freeze-drying preserved dissolves and mixes in physiological saline, obtained riemerella anatipestifer suspension, after obtained riemerella anatipestifer suspension inoculation is in the tryptose soya agar substratum containing 4% serum, is placed in CO
220-24h is cultivated at 37 DEG C in incubator, then from substratum the single colony inoculation of picking in containing 4% serum pancreas peptone soybean broth substratum in, rotating speed be 150r/min, temperature is shaking culture 20-24h on the shaking table of 37 DEG C, the riemerella anatipestifer after obtained recovery;
Described riemerella anatipestifer bacterial strain is provided by Domestic Fowls Inst., Shandong Academy of Agricultural Sciences.
(1-2) intestinal bacteria type strain bacterial classification recovery:
Measure 1ml physiological saline, the strain Escherichia coli appropriate freeze-drying preserved dissolves and mixes in physiological saline, obtained intestinal bacteria suspension, by obtained intestinal bacteria suspension inoculation in nutrient broth medium, rotating speed be 150r/min, temperature is shaking culture 16-20h on the shaking table of 37 DEG C, the intestinal bacteria after obtained recovery;
Described strain Escherichia coli is purchased from China Veterinery Drug Inspection Office.
3. riemerella anatipestifer according to claim 1 and intestinal bacteria mixed culture and the method that is again separated, it is characterized in that: the method that described step (2) medicament sensitivity test adopts is agar diffusion method of the paper, the microbiotic tested in drug sensitive test paper used is any one in gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin, and in corresponding drug sensitive test paper, the content of gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin is respectively 10 μ g, 5 μ g, 5 μ g, 10 μ g; Wherein, judge that the standard of riemerella anatipestifer to gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin sensitivity is respectively antibacterial circle diameter and is less than or equal to 12mm, 15mm, 12mm, 12mm, judge that the standard of intestinal bacteria to gentamicin, Ciprofloxacin, Ofloxacine USP 23, norfloxicin sensitivity is respectively antibacterial circle diameter and is more than or equal to 15mm, 21mm, 16mm, 17mm.
4. riemerella anatipestifer according to claim 1 and intestinal bacteria mixed culture and the method that is again separated, is characterized in that: in described step (3), the concrete grammar of mixed culture is:
First measuring the pancreas peptone soybean broth that riemerella anatipestifer that step (2) filters out is seeded to containing 4% serum supports in base, rotating speed be 150r/min, temperature is shaking culture 14-18h on the shaking table of 37 DEG C, when seeing muddiness to substratum, again the intestinal bacteria that step (2) filters out are inoculated in above-mentioned substratum and mix, rotating speed be 150r/min, temperature is shaking culture 9-12h on the shaking table of 37 DEG C, obtained mixed culture bacterium liquid.
5. riemerella anatipestifer according to claim 1 and intestinal bacteria mixed culture and the method that is again separated, is characterized in that: in described step (3), the concrete grammar of mixed culture is:
(3-1) riemerella anatipestifer and intestinal bacteria that step (2) filters out is measured, by the riemerella anatipestifer measured, the pancreas peptone soybean broth be seeded to containing 4% serum is supported in base, is seeded to by the intestinal bacteria measured in pancreas peptone soybean broth substratum;
(3-2) by inoculate respectively in step (3-1) riemerella anatipestifer and colibacillary substratum respectively rotating speed be 150r/min, temperature is shaking culture 16-20h on the shaking table of 37 DEG C;
(3-3) the riemerella anatipestifer bacterium liquid that step (3-2) shaking table shaking culture obtains is diluted to 15 × 10
8cFU/ml, Escherichia coli bacteria liquid is diluted to 1.5 × 10
3cFU/ml;
(3-4) Escherichia coli bacteria liquid after the riemerella anatipestifer bacterium liquid after 50 obtained in aspiration step (3-3) μ l dilute and 10 μ l dilute, and Simultaneous vaccination contains in the pancreas peptone soybean broth substratum of 4% serum in 4ml, rotating speed be 150r/min, temperature is shaking culture 9-12h on the shaking table of 37 DEG C, obtained mixed culture bacterium liquid.
6. riemerella anatipestifer according to claim 1 and intestinal bacteria mixed culture and the method that is again separated, is characterized in that: it is the double dilution method of test tube to the test method of antibiotic minimal inhibitory concentration that described step (4) measures the riemerella anatipestifer and intestinal bacteria that filter out.
7. riemerella anatipestifer according to claim 1 and intestinal bacteria mixed culture and the method that is again separated, it is characterized in that: the microbiotic plate culture medium that described step (5) obtains is the tryptose soya agar substratum containing 4% serum, each microbiotic that correspondence is selected, add this antibiotic concentration in substratum to be respectively: gentamicin 10 μ g/ml, Ciprofloxacin 3 μ g/ml, Ofloxacine USP 23 6 μ g/ml, norfloxicin 6 μ g/ml.
8. riemerella anatipestifer according to claim 1 and intestinal bacteria mixed culture and the method that is again separated, is characterized in that: the concrete grammar that described step (6) is separated mixed culture bacterium liquid is:
The appropriate mixed culture bacterium liquid that step (3) is obtained is diluted to 3 × 10
3-6 × 10
3after CFU/ml, draw 100 μ l diluents, coat maconkey agar substratum, containing the tryptose soya agar substratum of 4% serum and step (5) obtained containing in antibiotic microbiotic plate culture medium, be placed in CO
2cultivate 20-24h at incubator 37 DEG C, can the riemerella anatipestifer in mixed culture bacterium liquid be separated with intestinal bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510679353.3A CN105176884B (en) | 2015-10-19 | 2015-10-19 | The method that riemerella anatipestifer is mixed with Escherichia coli and detaches again |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510679353.3A CN105176884B (en) | 2015-10-19 | 2015-10-19 | The method that riemerella anatipestifer is mixed with Escherichia coli and detaches again |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105176884A true CN105176884A (en) | 2015-12-23 |
| CN105176884B CN105176884B (en) | 2018-10-16 |
Family
ID=54899318
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510679353.3A Active CN105176884B (en) | 2015-10-19 | 2015-10-19 | The method that riemerella anatipestifer is mixed with Escherichia coli and detaches again |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105176884B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108938661A (en) * | 2018-07-27 | 2018-12-07 | 四川农业大学 | The application in the drug for inhibiting Riemerellosis Anatipestifer is being prepared in conjunction with the gyrA gene PNA joint antibiotic of cell-penetrating peptide |
| CN110904185A (en) * | 2019-12-25 | 2020-03-24 | 武汉市农业科学院 | Quick detection kit for drug sensitivity of duck pathogenic bacteria based on iodine nitro tetrazole color development |
| CN110964781A (en) * | 2019-12-25 | 2020-04-07 | 武汉市农业科学院 | ATP bioluminescence-based quick detection kit for drug sensitivity of duck pathogenic bacteria |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199653A (en) * | 2010-03-28 | 2011-09-28 | 海南大学 | Method for rapidly screening and evaluating antibiotic bioactivator |
| CN102634562A (en) * | 2012-04-13 | 2012-08-15 | 天津大学 | Method for detecting nutrition environment change in subculture process of vitamin C production strain |
| CN104623650A (en) * | 2015-01-14 | 2015-05-20 | 广东永顺生物制药股份有限公司 | Preparation method and product of riemerella anatipestifer and escherichia coli combined deactivated vaccine |
-
2015
- 2015-10-19 CN CN201510679353.3A patent/CN105176884B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102199653A (en) * | 2010-03-28 | 2011-09-28 | 海南大学 | Method for rapidly screening and evaluating antibiotic bioactivator |
| CN102634562A (en) * | 2012-04-13 | 2012-08-15 | 天津大学 | Method for detecting nutrition environment change in subculture process of vitamin C production strain |
| CN104623650A (en) * | 2015-01-14 | 2015-05-20 | 广东永顺生物制药股份有限公司 | Preparation method and product of riemerella anatipestifer and escherichia coli combined deactivated vaccine |
Non-Patent Citations (4)
| Title |
|---|
| 倪粒琼 等: "鸭疫里默氏杆菌与大肠杆菌融合株的构建", 《中国兽医学报》 * |
| 李翠 等: "鸭疫里默氏杆菌与大肠杆菌混合培养及重新分离方法初探", 《家禽科学》 * |
| 杨凤霞 等: "环境中抗生素抗性基因的水平传播扩散", 《应用生态学报》 * |
| 黄兵 等: "放线菌与枯草芽孢杆菌的共培养及其对活性次生代谢产物的影响", 《生物工程学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108938661A (en) * | 2018-07-27 | 2018-12-07 | 四川农业大学 | The application in the drug for inhibiting Riemerellosis Anatipestifer is being prepared in conjunction with the gyrA gene PNA joint antibiotic of cell-penetrating peptide |
| CN108938661B (en) * | 2018-07-27 | 2020-09-15 | 四川农业大学 | Application of gyrA gene PNA combined antibiotic combined with cell-penetrating peptide in preparation of drug for inhibiting riemerella anatipestifer |
| CN110904185A (en) * | 2019-12-25 | 2020-03-24 | 武汉市农业科学院 | Quick detection kit for drug sensitivity of duck pathogenic bacteria based on iodine nitro tetrazole color development |
| CN110964781A (en) * | 2019-12-25 | 2020-04-07 | 武汉市农业科学院 | ATP bioluminescence-based quick detection kit for drug sensitivity of duck pathogenic bacteria |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105176884B (en) | 2018-10-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109207384A (en) | modified yeast cell wall and its preparation method and application | |
| CN105176884A (en) | Method for mixed culture and re-separation of riemerella anatipestifer and escherichia coli | |
| CN104789500A (en) | Pullorum staining agglutination antigen as well as preparation method and application thereof | |
| CN108118033A (en) | One plant of cracking performance salmonella bacteriophage and its separation method | |
| CN104745672A (en) | Method for rapidly identifying black shank resistance of tobaccos | |
| Zahra et al. | Isolation and characterization of small-colony variants of Ornithobacterium rhinotracheale | |
| Zahid et al. | In vitro and in vivo pathogenicity tests of local isolates APEC from naturally infected broiler in Baghdad | |
| US11033032B2 (en) | Leclercia adecarboxylata biocontrol strain efficiently inhibiting production of aflatoxins by Aspergillus flavus and application thereof | |
| CN104611255B (en) | One plant height coherency Pediococcus pentosaceus and its application in purifying water body in vibrio parahemolyticus | |
| CN109486714A (en) | A kind of weak malicious separation strains of avian infectious bronchitis nephritis virus and its application | |
| CN101638625B (en) | Method for culturing orange myxobacter JCH-04 and antibiosis metabolic product | |
| CN112961804A (en) | Salmonella typhimurium and application thereof | |
| CN100360680C (en) | Preparing method for cow mammitis streptococcus culture fluid and its use | |
| Lochhead et al. | Aspects of antagonisms between micro-organisms in soil | |
| CN110747251A (en) | Rapid screening method of potential probiotic strains for inhibiting pathogenic bacteria | |
| CN105200116B (en) | A kind of method of Paenibacillus polymyxa content in quick measure complex micro organism fungicide | |
| CN107287275B (en) | Culture medium, kit containing culture medium and application of culture medium | |
| CN104561234A (en) | Antibiotic residue detection kit and application thereof | |
| CN105296406A (en) | Culture method and inactivation method of haemophilus parasuis | |
| CN104789499A (en) | Pullorum agar gel precipitating antigen as well as preparation method and application thereof | |
| CN103275897B (en) | Method for quickly screening antibiotics-producing actinomycetes from soil | |
| CN104212874B (en) | The selective medium of a kind of detection by quantitative Verticillium lecanii and application thereof | |
| CN112680372B (en) | Enterococcus faecalis and application thereof in broiler feed | |
| CN1858235A (en) | Preparing method for cow mammitis E.coli culture fluid and its use | |
| NL2033150B1 (en) | Application of limonium wrightii naphthoquinone in preparing anti- haemophilus parasuis drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200820 Address after: Luogushan Middle Road, Chengbei Industrial Park, Yishui County, Linyi City, Shandong Province Patentee after: SHANDONG GREENLAND ANIMAL PHARMACEUTICAL Co.,Ltd. Address before: 250023 No. 1 Jiao Tong Road, Tianqiao District, Shandong, Ji'nan Patentee before: POULTRY INSTITUTE, SHANDONG ACADEMY OF AGRICULTURAL SCIENCE |