CN104611261A - Method for recovering sensitivity of ESBLs-producing multiple resistant bacteria - Google Patents
Method for recovering sensitivity of ESBLs-producing multiple resistant bacteria Download PDFInfo
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Abstract
The invention discloses a method for recovering the sensitivity of ESBLs-producing multiple resistant bacteria. The method comprises the following steps: (1) clinically selecting original strains, and preserving for the future use; (2) preparing a bacterial suspension from the various original strains through bacterial propagation and separation; (3) subcultivating and carrying out passage through a blood bacterial propagation broth tube; and (4) determining a drug sensitivity to obtain the drug sensitivity recovery condition of the bacteria obtained through blood bacterial propagation broth tube subculture. The method for recovering the sensitivity of ESBLs-producing multiple resistant bacteria disclosed by the invention is practicable, and the result has the advantages of accuracy and reliability.
Description
Technical field
The present invention relates to a kind of method that resistant organism bacterium susceptibility is recovered, be specifically related to a kind of method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered.
Background technology
The current whole world enters the epoch after microbiotic, and the recall rate of domestic multi-drug resistant bacteria is more than 50%; Within 2012, " China infects and learns magazine " publishes: the multi-drug resistant bacteria positive rate producing super wide spectrum enzyme is 52.4%; " pharmacoepidemiology magazine " reports that the positive rate of the super wide spectrum enzyme of kerekou pneumonia uncle product is 49.3%, and the positive rate that intestinal bacteria produce super wide spectrum enzyme is 45.4%; Present multi-drug resistant bacteria infects the health having injured children, 2010 national monitoring networks reports: 0-14 year the children Streptococcus Cray uncle positive rate that produces super wide spectrum enzyme be 64.9%, the positive rate that intestinal bacteria produce super wide spectrum enzyme is that the positive rate children ratio that 69.9%. data presentation produces super wide spectrum enzyme is grown up high.
Secondly, hospital is owing to using microbiotic and thimerosal, in hospital environment, major part is multi-drug resistant bacteria, the newborn infant of hospital's birth, bacterium when it sets up normal microflora at first may be exactly just multi-drug resistant bacteria, so newborn infant is once sick, when cultivating, about 3/4ths is all multi-drug resistant bacteria, and exploring the method recovering multiple drug resistant bacteria susceptibility has become a brand-new problem.
Summary of the invention
The invention provides a kind of method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered, display multi-drug resistant bacteria goes down to posterity at nutritious substratum can recuperation section antibiotics sensitivity, method of the present invention is practical, and its result has accurately, reliable advantage.
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered, comprises the steps:
(1) multi-drug resistant bacteria choosing clinical product ESBLs is original strain, and preservation is for subsequent use;
(2) described original strain is prepared into bacteria suspension through increasing bacterium, separation;
(3) described bacteria suspension transferred species is increased bacterial context soup pipe to a blood, transferred species is separated plate to one simultaneously, and Secondary Culture 18-24h, forms bacterium colony in described separation plate respectively; Then observe the described bacterium colony be separated in plate, if described bacterium colony is pollution-free merely, illustrate that this goes down to posterity qualified, if described bacterium colony has pollution, then re-start this and go down to posterity, until described bacterium colony is pollution-free; Then described blood is increased bacteria suspension transferred species in bacterial context soup pipe to increase in bacterial context soup pipe and transferred species is separated in plate to another simultaneously to another blood; Repeat above step at least to 20 generation;
(4) the bacteria suspension transferred species that last blood step (3) obtained increases in bacterial context soup pipe carries out separation and Culture 18-24h in hemoculture ware, produce colonies typical, drug sensitive test is carried out to described colonies typical, compare with the susceptibility of described original strain simultaneously, show that blood increases the drug susceptibility recovery situation of the bacterium that bacterial context soup pipe Secondary Culture obtains.
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered of the present invention, wherein, the multi-drug resistant bacteria of described product ESBLs is Klebsiella Pneumoniae ESBL (Klebsiella peneumoniae ESBL) or escherichia coli ESBL (Escherichiacoli ESBL); Described Klebsiella Pneumoniae ESBL, preserving number CCTCC NO:M 2014594, preservation date: on November 25th, 2014, be now preserved in China typical culture collection center, address: Wuhan, China Wuhan University; Described escherichia coli ESBL, preserving number CCTCC NO:M 2014593, preservation date: on November 25th, 2014, be now preserved in China typical culture collection center, address: Wuhan, China Wuhan University.
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered of the present invention, wherein, described step (1) specifically comprises the steps:
The colonies typical getting described original strain is inoculated in the Eppendorf tube of the sharp end containing fungi preservation liquid,-20 DEG C of cryopreservation, described fungi preservation liquid is massfraction is 15% glycerine pancreas casein soybean soup, its concrete composition is: pancreas casein 17g/L, soya peptone 3g/L, sodium-chlor 5g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, all the other are distilled water.
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered of the present invention, wherein, described step (2) specifically comprises the steps:
First the bacterial strain in the Eppendorf tube of the sharp end of described refrigeration through Zengjing Granule, described Zengjing Granule adds in Zengjing Granule pipe for the described bacterial strain that takes a morsel, be placed in 35 ± 1 DEG C cultivate 18-24h; And then carry out separation and Culture, described separation and Culture concrete steps are as follows: dip the bacteria suspension after described Zengjing Granule with transfering loop, streak inoculation is on nutrient agar medium plate, be placed in 35 ± 1 DEG C and cultivate 18-24h, visible single bacterium colony, judges that described bacterial strain has pollution-free, and as pollution-free, this Zengjing Granule thing is test bacteria suspension, if there is pollution, again do above-mentioned steps, till pollution-free.
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered of the present invention, wherein, described step (3) specifically comprises the steps:
A, described bacteria suspension transferred species is increased bacterial context soup pipe to described blood, transferred species is to described separation plate simultaneously, and distinguish Secondary Culture, described Secondary Culture, for cultivate 18-24h under 35 ± 1 DEG C of conditions, forms bacterium colony in described separation plate;
B, the bacterium colony observed in described separation plate, if described bacterium colony is pollution-free merely, illustrate that this goes down to posterity qualified, if described bacterium colony has pollution, then re-start this and go down to posterity, until described bacterium colony is pollution-free;
C, described blood increased bacterial context soup pipe and be separated plate and be numbered A respectively
1and B
1;
By described A
1the bacteria suspension that blood increases in bacterial context soup pipe goes down to posterity according to described step a and b, and the blood of transferred species is increased bacterial context soup pipe and be separated plate and be numbered A respectively
2and B
2;
By described A
2the bacteria suspension that blood increases in bacterial context soup pipe goes down to posterity according to described step a and b, and the blood of transferred species is increased bacterial context soup pipe and be separated plate and be numbered A respectively
3and B
3;
By described A
3the bacteria suspension that blood increases in bacterial context soup pipe goes down to posterity according to described step a and b, and the blood of transferred species is increased bacterial context soup pipe and be separated plate and be numbered A respectively
4and B
4;
Continuous passage 30 times according to the method described above, the blood gone down to posterity for the last time increases bacterial context soup pipe and is separated plate and is numbered A respectively
30and B
30.
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered of the present invention, wherein, described step (4) specifically comprises the steps:
By the A that step (3) obtains
30the bacteria suspension transferred species that blood increases in bacterial context soup pipe carries out separation and Culture 18-24h in hemoculture ware, produce colonies typical, drug sensitive test is carried out to described colonies typical, compare with the susceptibility of described original strain simultaneously, show that blood increases the drug susceptibility recovery situation of the bacterium that bacterial context soup pipe Secondary Culture obtains.
Middle mensuration susceptibility instrument used is French Mei Liai automatic bacterial qualification/Analysis of Drug Susceptibility instrument, model: ViETK 2Compact.
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered of the present invention, wherein, its blood described increases in bacterial context soup pipe and increases bacterial context soup containing blood, and its component is: peptone 10.0g/L; Extracted beef powder 3.0g/L; Sodium-chlor 5.0g/L; Glucose 1.0g/L; Sodium Citrate 3.0g/L; Dipotassium hydrogen phosphate 2g g/L; Para-amino benzoic acid 0.05g/L; Magnesium sulfate 2.4g/L; Phenol red 0.024g/L; All the other are distilled water; It is 7.3 ~ 7.5 that blood increases bacterial context soup pH value; Through 121 DEG C of sterilizing 15min and 35 DEG C, 24h sterility test is for subsequent use.
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered of the present invention, wherein, described fungi preservation liquid method for making is: be mixed even by each composition of described fungi preservation liquid, heating for dissolving obtains mixed solution, after correcting pH value to 7.0 ~ 7.5 of described mixed solution, filter described mixed solution and filtrate be sub-packed in 1.5ml point end Eppendorf tube, often pipe 0.6ml, autoclaving 15 minutes under 121 DEG C of conditions.
Answer Attention question as follows in the inventive method operating process:
Decontaminate: untapped blood increases bacterial context soup pipe and is put into incubator preculture in advance 2 days, to get rid of the pollution of meat soup pipe, barrier gown, through autoclaving, uses disposable breathing mask and cap.
The integrity of checking experiment equipment: 35 ± 1 DEG C of incubators; With 75% alcohol disinfecting wiping incubator before test, the special desk-top autoclave of Bacteriology Room; France Mei Liai automatic bacterial qualification/Analysis of Drug Susceptibility instrument etc.;
The preparation of operating environment: test and carry out at two stage biological safety cage, first open the disinfection by ultraviolet light more than 40 minutes of room and super clean bench before each experiment, before sterilization, in advance the multi-drug resistant bacteria bacteria suspension (encoded) of transfering loop, inoculating needle, the clinical product ESBLs detected is put into super clean bench; Strict aseptic technique: wash hands, wears sterilizing barrier gown, band disposable breathing mask, cap, band sterile gloves.
Compared with prior art, the inventive method tool has the following advantages:
The inventive method explores multi-drug resistant bacteria to recover antibiotic susceptibility, reliable drug sensitivity test information is provided for clinical, microbiotic is instructed to apply, the inventive method blood Zengjing Granule pipe goes down to posterity 20-30 generation to multi-drug resistant bacteria transferred species, understands multi-drug resistant bacteria in the antibiotic high nutrient environment of disengaging to antibiotic susceptibility recovery situation.
The inventive method multi-drug resistant bacteria in blood increasing bacterial context soup pipe, go down to posterity continuously by 20-30 generation, prove to produce ESBLs Klebsiella Pneumoniae by experiment, escherichia coli departs from microbiotic environment, increase bacterial context soup pipe at nutritious blood to cultivate, the antibiotic susceptibility of part has been recovered, a little multi-drug resistant bacteria that can be remaining in body after predict human affected treatment, or at the neonatal multi-drug resistant bacteria carried that hospital produces, after departing from the microbiotic environment one long period, its susceptibility can be recovered.
The going down to posterity to increase in bacterial context soup pipe at blood and carry out (similar human body environment) of bacterium in the inventive method, the data obtained are more true and reliable, have directive significance to the antibiotic use of the mankind from now on.
Embodiment
Embodiment 1
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered, comprises the steps:
(1) multi-drug resistant bacteria choosing clinical product ESBLs is original strain, and preservation is for subsequent use;
(2) described original strain is prepared into bacteria suspension through increasing bacterium, separation;
(3) described bacteria suspension transferred species is increased bacterial context soup pipe to a blood, transferred species is separated plate to one simultaneously, and Secondary Culture 18-24h, forms bacterium colony in described separation plate respectively; Then observe the described bacterium colony be separated in plate, if described bacterium colony is pollution-free merely, illustrate that this goes down to posterity qualified, if described bacterium colony has pollution, then re-start this and go down to posterity, until bacterium colony is pollution-free; Then described blood is increased bacteria suspension transferred species in bacterial context soup pipe to increase in bacterial context soup pipe and transferred species is separated in plate to another simultaneously to another blood; Repeat above step at least to 20 generation;
(4) the bacteria suspension transferred species that last blood step (3) obtained increases in bacterial context soup pipe carries out separation and Culture 18-24h in hemoculture ware, produce colonies typical, drug sensitive test is carried out to described colonies typical, compare with the susceptibility of described original strain simultaneously, show that blood increases the drug susceptibility recovery situation of the bacterium that bacterial context soup pipe Secondary Culture obtains.
Embodiment 2
The method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered, comprises the steps:
(1) choosing product Klebsiella Pneumoniae ESBL or escherichia coli ESBL is original strain, and preservation is for subsequent use: the multi-drug resistant bacteria of described product ESBLs is Klebsiella Pneumoniae ESBL or escherichia coli ESBL; Described Klebsiella Pneumoniae ESBL, preserving number CCTCC NO:M 2014594, preservation date: on November 25th, 2014; Described escherichia coli ESBL, preserving number CCTCC NO:M 2014593, preservation date: on November 25th, 2014;
The colonies typical getting described original strain is inoculated in the Eppendorf tube of the sharp end containing fungi preservation liquid,-20 DEG C of cryopreservation, described fungi preservation liquid is 15% glycerine pancreas casein soybean soup, its concrete composition is: pancreas casein 17g/L, soya peptone 3g/L, sodium-chlor 5g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, all the other are distilled water;
Fungi preservation liquid method for making is: be mixed even by each composition of described fungi preservation liquid, heating for dissolving obtains mixed solution, after correcting pH value to 7.0 ~ 7.5 of described mixed solution, filter described mixed solution and filtrate be sub-packed in 1.5ml point end Eppendorf tube, often pipe 0.6ml, autoclaving 15 minutes under 121 DEG C of conditions;
(2) first the bacterial strain in the Eppendorf tube of the sharp end of described refrigeration through Zengjing Granule, described Zengjing Granule adds in Zengjing Granule pipe for the described bacterial strain that takes a morsel, be placed in 35 ± 1 DEG C cultivate 18-24h; And then carry out separation and Culture, described separation and Culture concrete steps are as follows: dip the bacteria suspension after described Zengjing Granule with transfering loop, streak inoculation is on nutrient agar medium plate, be placed in 35 ± 1 DEG C and cultivate 18-24h, visible single bacterium colony, judges that described bacterial strain has pollution-free, and as pollution-free, this Zengjing Granule thing is test bacteria suspension, if there is pollution, again do above-mentioned steps, till pollution-free;
Increase bacterial context soup containing blood in described increasing bacterial context soup pipe, its component is: peptone 10.0g/L; Extracted beef powder 3.0g/L; Sodium-chlor 5.0g/L; Glucose 1.0g/L; Sodium Citrate 3.0g/L; Dipotassium hydrogen phosphate 2g g/L; Para-amino benzoic acid 0.05g/L; Magnesium sulfate 2.4g/L; Phenol red 0.024g/L; All the other are distilled water; It is 7.3 ~ 7.5 that blood increases bacterial context soup pH value; Through 121 DEG C of sterilizing 15min and 35 DEG C, 24h sterility test is for subsequent use;
(3) a, described bacteria suspension transferred species is increased bacterial context soup pipe to described blood, transferred species is to described separation plate simultaneously, and distinguish Secondary Culture, described Secondary Culture, for cultivate 18-24h under 35 ± 1 DEG C of conditions, forms bacterium colony in described separation plate;
B, the bacterium colony observed in described separation plate, if described bacterium colony is pollution-free merely, illustrate that this goes down to posterity qualified, if described bacterium colony has pollution, then re-start this and go down to posterity, until described bacterium colony is pollution-free;
C, described blood increased bacterial context soup pipe and be separated plate and be numbered A respectively
1and B
1;
By described A
1the bacteria suspension that blood increases in bacterial context soup pipe goes down to posterity according to described step a and b, and the blood of transferred species is increased bacterial context soup pipe and be separated plate and be numbered A respectively
2and B
2;
Continuous passage 30 times according to the method described above, the blood gone down to posterity for the last time increases bacterial context soup pipe and is separated plate and is numbered A respectively
30and B
30;
By described A
2the bacteria suspension that blood increases in bacterial context soup pipe goes down to posterity according to described step a and b, and the blood of transferred species is increased bacterial context soup pipe and be separated plate and be numbered A respectively
3and B
3;
By described A
3the bacteria suspension that blood increases in bacterial context soup pipe goes down to posterity according to described step a and b, and the blood of transferred species is increased bacterial context soup pipe and be separated plate and be numbered A respectively
4and B
4;
This experiment strict aseptic technique, and take to decontaminate before experiment, using is all aseptic materials, therefore pollution condition does not occur in succeeding generations;
(4) by A that step (3) obtains
30blood increases bacterial context soup pipe transferred species and cultivates in hemoculture ware, gets the colonies typical cultivating generation and carries out drug sensitive test, compare simultaneously with the susceptibility of original strain, show that blood increases the drug susceptibility recovery situation of the bacterium that bacterial context soup pipe Secondary Culture obtains; The instrument measuring susceptibility used is French Mei Liai automatic bacterial qualification/Analysis of Drug Susceptibility instrument, model: ViETK 2Compact.
It is as follows that the present embodiment records result:
Produce the escherichia coli of ESBLs go down to posterity before and after drug sensitive test result
Produce the escherichia coli different strains numbering Y of ESBLs
1 ~y
18the escherichia coli Secondary Culture of the product ESBLs of clinical detection 30 times, the escherichia coli of ESBLs is produced in 13 strains, go down to posterity through blood-bouillon, 10 strains are wherein had to add up to 25 kinds of antibiotics sensitivities to recover, 4 strains have 2 kinds of microbiotic (levofloxacin, ammonia benzyl/relax) by quickly become resistance, the phenomenon that its susceptibility to different antibacterials is recovered is in table 1.
The escherichia coli blood that table 1 produces ESBLs increase bacterial context soup go down to posterity before and after drug sensitive test results contrast
Remarks: R represents resistance, I represents medium sensitivity, and S represents sensitivity, and 0 representative is not done.
Produce the Klebsiella Pneumoniae of ESBLs go down to posterity before and after drug sensitive test result
Produce the different strains numbering Y of the Klebsiella Pneumoniae of ESBLs
2~ Y
19in Klebsiella Pneumoniae Secondary Culture 23 generation of the product ESBLs of clinical detection, the Klebsiella Pneumoniae of ESBLs is produced in 5 strains, go down to posterity through blood-bouillon, 3 strains are wherein had to add up to 9 kinds of antibiotics sensitivities to recover, 1 strain have 2 kinds of microbiotic (tobramycin, Ciprofloxacin) by quickly become resistance, the phenomenon that its susceptibility to different antibacterials is recovered is in table 2.
The Klebsiella Pneumoniae blood that table 2 produces ESBLs increase bacterial context soup go down to posterity before and after drug sensitive test results contrast
That produces that the escherichia coli of ESBLs goes down to posterity that front and back susceptibility changes the results are shown in Table 3
Susceptibility before and after the escherichia coli that table 3 produces ESBLs goes down to posterity changes statistics
Produce the Klebsiella Pneumoniae of ESBLs go down to posterity before and after drug sensitive test change the results are shown in Table 4
Susceptibility before and after the Klebsiella Pneumoniae that table 4 produces ESBLs goes down to posterity changes statistics
The escherichia coli of ESBLs is produced in 13 strains, goes down to posterity for 30 generations through blood-bouillon, wherein has 10 strains to add up to 25 kinds of antibiotics sensitivities to recover, 4 strains have 2 kinds of microbiotic (levofloxacin, ammonia benzyl/relax) by quickly become resistance; The Klebsiella Pneumoniae of ESBLs is produced in 5 strains, goes down to posterity for 30 generations through blood-bouillon, wherein has 3 strains to add up to 9 kinds of antibiotics sensitivities to recover, 1 strain have 2 kinds of microbiotic (tobramycin, Ciprofloxacin) by quickly become resistance.Add up to 18 strains to produce in ESBLs to have the antibiotics sensitivity of 13 strain 34 kinds time to recover, have the microbiotic of 5 strain 6 kinds time by quickly become resistance.
At present due to antibiotic unreasonable use cause multi-drug resistant bacteria to infect extend over the entire globe, particularly medical personnel's contact sterilizing liquid, microbiotic is frequent, it is 6 times of population that multi-drug resistant bacteria carries, and infant's multi-drug resistant bacteria detects higher than grownup.For exploring the method design the present invention recovering multiple drug resistant bacteria susceptibility, the inventive method can disclose produces the recovery situation that there is part antibiotic drug sensitive in super wide spectrum enzyme bacterial strain continuous 20-30 generation after blood increasing bacterial context soup pipe goes down to posterity, this experimental result also can disclose: after people's interior therapeutic, multi-drug resistant bacteria that is remaining or that carry is at the antibiotic environment of disengaging, can recover in vivo through breeding of long time (time of test is shorter) the more antibiotic susceptibility that goes down to posterity; The inventive method provides reliable drug sensitivity test information for clinical, instructs microbiotic to apply.
Above-described embodiment is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; under not departing from the present invention and designing the prerequisite of spirit; the various distortion that those of ordinary skill in the art make technical scheme of the present invention and improvement, all should fall in protection domain that claims of the present invention determines.
Claims (8)
1. the method making the multi-drug resistant bacteria susceptibility of product ESBLs recover, is characterized in that, comprise the steps:
(1) multi-drug resistant bacteria choosing clinical product ESBLs is original strain, and preservation is for subsequent use;
(2) described original strain is prepared into bacteria suspension through increasing bacterium, separation;
(3) described bacteria suspension transferred species is increased bacterial context soup pipe to a blood, transferred species is separated plate to one simultaneously, and Secondary Culture 18-24h, forms bacterium colony in described separation plate respectively; Then observe described bacterium colony, if described bacterium colony is pollution-free merely, illustrate that this goes down to posterity qualified, if described bacterium colony has pollution, then re-start this and go down to posterity, until described bacterium colony is pollution-free; Then described blood is increased bacteria suspension transferred species in bacterial context soup pipe to increase in bacterial context soup pipe and transferred species is separated in plate to another simultaneously to another blood; Repeat above step at least to 20 generation;
(4) the bacteria suspension transferred species that last blood step (3) obtained increases in bacterial context soup pipe carries out separation and Culture 18-24h in hemoculture ware, produce colonies typical, drug sensitive test is carried out to described colonies typical, compare with the susceptibility of described original strain simultaneously, show that blood increases the drug susceptibility recovery situation of the bacterium that bacterial context soup pipe Secondary Culture obtains.
2. the method making the multi-drug resistant bacteria susceptibility of product ESBLs recover according to claim 1, is characterized in that, the multi-drug resistant bacteria of described product ESBLs is Klebsiella Pneumoniae ESBL or escherichia coli ESBL; Described Klebsiella Pneumoniae ESBL, preserving number CCTCC NO:M 2014594, preservation date: on November 25th, 2014; Described escherichia coli ESBL, preserving number CCTCC NO:M 2014593, preservation date: on November 25th, 2014.
3. the method making the multi-drug resistant bacteria susceptibility of product ESBLs recover according to claim 2, is characterized in that, described step (1) specifically comprises the steps:
The colonies typical getting described original strain is inoculated in the Eppendorf tube of the sharp end containing fungi preservation liquid,-20 DEG C of cryopreservation, described fungi preservation liquid is 15% glycerine pancreas casein soybean soup, its concrete composition is: pancreas casein 17g/L, soya peptone 3g/L, sodium-chlor 5g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L, all the other are distilled water.
4. the method making the multi-drug resistant bacteria susceptibility of product ESBLs recover according to claim 3, is characterized in that, described step (2) specifically comprises the steps:
First the bacterial strain in the Eppendorf tube of the sharp end of described refrigeration through Zengjing Granule, described Zengjing Granule adds in Zengjing Granule pipe for the described bacterial strain that takes a morsel, be placed in 35 ± 1 DEG C cultivate 18-24h; And then carry out separation and Culture, described separation and Culture concrete steps are as follows: dip the bacteria suspension after described Zengjing Granule with transfering loop, streak inoculation is on nutrient agar medium plate, be placed in 35 ± 1 DEG C and cultivate 18-24h, visible single bacterium colony, judges that described bacterial strain has pollution-free, and as pollution-free, this Zengjing Granule thing is test bacteria suspension, if there is pollution, again do above-mentioned steps, till pollution-free.
5. the method making the multi-drug resistant bacteria susceptibility of product ESBLs recover according to claim 4, is characterized in that, described step (3) specifically comprises the steps:
A, described bacteria suspension transferred species is increased bacterial context soup pipe to described blood, transferred species is to described separation plate simultaneously, and distinguish Secondary Culture, described Secondary Culture, for cultivate 18-24h under 35 ± 1 DEG C of conditions, forms bacterium colony in described separation plate;
B, the bacterium colony observed in described separation plate, if described bacterium colony is pollution-free merely, illustrate that this goes down to posterity qualified, if described bacterium colony has pollution, then re-start this and go down to posterity, until described bacterium colony is pollution-free;
C, described blood increased bacterial context soup pipe and be separated plate and be numbered A respectively
1and B
1;
By described A
1the bacteria suspension that blood increases in bacterial context soup pipe goes down to posterity according to described step a and b, and the blood of transferred species is increased bacterial context soup pipe and be separated plate and be numbered A respectively
2and B
2;
By described A
2the bacteria suspension that blood increases in bacterial context soup pipe goes down to posterity according to described step a and b, and the blood of transferred species is increased bacterial context soup pipe and be separated plate and be numbered A respectively
3and B
3;
By described A
3the bacteria suspension that blood increases in bacterial context soup pipe goes down to posterity according to described step a and b, and the blood of transferred species is increased bacterial context soup pipe and be separated plate and be numbered A respectively
4and B
4;
Continuous passage 30 times according to the method described above, the blood gone down to posterity for the last time increases bacterial context soup pipe and is separated plate and is numbered A respectively
30and B
30.
6. the method making the multi-drug resistant bacteria susceptibility of product ESBLs recover according to claim 5, is characterized in that, described step (4) specifically comprises the steps:
By the A that step (3) obtains
30the bacteria suspension transferred species that blood increases in bacterial context soup pipe carries out separation and Culture 18-24h in hemoculture ware, produce colonies typical, drug sensitive test is carried out to described colonies typical, compare with the susceptibility of described original strain simultaneously, show that blood increases the drug susceptibility recovery situation of the bacterium that bacterial context soup pipe Secondary Culture obtains.
7. the method making the multi-drug resistant bacteria susceptibility of product ESBLs recover according to claim 6, is characterized in that, described blood increases in bacterial context soup pipe and increases bacterial context soup containing blood, and its component is: peptone 10.0g/L; Extracted beef powder 3.0g/L; Sodium-chlor 5.0g/L; Glucose 1.0g/L; Sodium Citrate 3.0g/L; Dipotassium hydrogen phosphate 2g g/L; Para-amino benzoic acid 0.05g/L; Magnesium sulfate 2.4g/L; Phenol red 0.024g/L; All the other are distilled water; It is 7.3 ~ 7.5 that described blood increases bacterial context soup pH value; Through 121 DEG C of sterilizing 15min and 35 DEG C, 24h sterility test is for subsequent use.
8. make the method that the multi-drug resistant bacteria susceptibility of product ESBLs is recovered according to claim 7, it is characterized in that, described fungi preservation liquid method for making is: be mixed even by each composition of described fungi preservation liquid, heating for dissolving obtains mixed solution, after correcting pH value to 7.0 ~ 7.5 of described mixed solution, filter described mixed solution and filtrate be sub-packed in 1.5ml point end Eppendorf tube, often pipe 0.6ml, autoclaving 15 minutes under 121 DEG C of conditions.
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| 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 |
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