CN105112497A - Method for separating and screening escherichia coli and staphylococcus aureus in estuary and nearshore marine environments and evaluating resistance of antibiotics - Google Patents
Method for separating and screening escherichia coli and staphylococcus aureus in estuary and nearshore marine environments and evaluating resistance of antibiotics Download PDFInfo
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Abstract
The invention discloses a method for separating and screening escherichia coli and staphylococcus aureus in estuary and nearshore marine environments and evaluating the resistance of antibiotics. The method comprises the following steps: immersing MI agar and VJ agar into screening antibiotics (tetracycline, sulfanilamide, carbostyril and the like) to prepare sensitive specific selective agar, quickly separating and counting antibiotic resistant escherichia coli (E. coli) and staphylococcus aureus (S. aureus) in the estuary and nearshore marine environments, and then evaluating the resistance level of the escherichia coli and the staphylococcus aureus. The method for separating and screening the escherichia coli and the staphylococcus aureus in the estuary and nearshore marine environments and evaluating the resistance of the antibiotics, disclosed by the invention, has the advantages of being simple, convenient and feasible in operation, high in accuracy, and safe and environment-friendly, and is suitable for rapid analysis of the escherichia coli and the staphylococcus aureus in water bodies and deposit samples of nearshore sea, estuaries and sea-entering drain outlets, and detecting the resistance level of the antibiotics.
Description
Technical field
The invention belongs to environmental monitoring technology field, relate to intestinal bacteria in a kind of river mouth and nearshore marine environment, streptococcus aureus separation screening and antibiotics resistance appraisal procedure.The method is applicable to coastal ocean, river mouth and enters the real-time analysis of intestinal bacteria and streptococcus aureus in extra large sewage draining exit water body and sediment sample and antibiotic resistance level detects.
Background technology
The detection method for typical pathogenic micro-organism intestinal bacteria and streptococcus aureus antibiotic resistance level is still lacked in Research of Environmental Sciences and monitoring field.
The mode of existing technology mainly by adopting fermenting experiment to combine with eosin methylene blue agar or violet red bile agar, carries out count detection to the intestinal bacteria in surrounding medium; Application Baird-Parker flat board and blood agar carry out count detection in conjunction with gramstaining experiment to streptococcus aureus.But because experimentation is loaded down with trivial details, cycle is longer, and need to carry out the multinomial Testing and appraisal test such as dyeing microscopic examination, coagulase test of blood plasma, cause it to be difficult to the true abundance level of two class pathogenic micro-organisms in authentic and valid reaction environment medium, more can not assess the antibiotic resistance level of two class pathogenic micro-organisms.
Summary of the invention
The invention provides intestinal bacteria in a kind of river mouth and nearshore marine environment, streptococcus aureus separation screening and antibiotics resistance appraisal procedure,
Present method is screened microbiotic (tsiklomitsin, sulfanilamide (SN), quinolone etc.) and make responsive specificity by immersing in MI agar and VJ agar and select agar, sharp separation also counts antibiotics resistance intestinal bacteria (E.coli) and streptococcus aureus (S.aureus) in river mouth and nearshore marine environment, and then assess its resistance level.
The present invention adopts following technical scheme:
Intestinal bacteria, streptococcus aureus separation screening and antibiotics resistance appraisal procedure in river mouth of the present invention and nearshore marine environment, is characterized in that: the concrete steps of described method are as follows:
(1) sterilizing:
Before experiment, filter membrane is placed between filter paper and wraps, the filter wrapped with kraft paper together with culture dish at 121 DEG C autoclaving 15min, each filter different sample before, clean funnel with alcohol and use flame calcination, eliminating sample room and disturb;
(2) preparation of substratum:
S.aureus: accurately take 60gVJ substratum (BectonDickinson, NewJersey), add 1L sterilized water, heating for dissolving also constantly stirs, boil sterilizing (103Kpa after a minute, 121 DEG C, 15min), water bath substratum through sterilizing being put into 45 DEG C ± 1 DEG C cools and adds 20ml (1%) potassium tellurite, the substratum prepared is sub-packed in triangular flask, a part is as streptococcus aureus screening VJ agar plate (not added with antibiotic), a part immerses microbiotic as antibiotics resistance screening VJ-R agar plate using minimum inhibitory concentration,
E.coli: accurately take 36.5gMI substratum (BectonDickinson, NewJersey), heating for dissolving is in 1L sterilized water, boil sterilizing (103Kpa after a minute, 121 DEG C, 15min), the water bath cooling of the substratum through sterilizing being put into 45 DEG C ± 1 DEG C is stand-by, prepare and add cefsulodin, its ultimate density is made to be 5 μ g/ml, the substratum prepared is sub-packed in triangular flask, a part is as intestinal bacteria screening MI agar plate (not added with antibiotic), a part immerses microbiotic as antibiotics resistance screening MI-R agar plate using minimum inhibitory concentration,
(3) antibiotic resistance experiment:
3.1 water body examples:
3.1.1 appropriate samples volume is chosen in the estimation of pickup area and pollution situation per sample, sample starting volume amasss when being less than 50ml with phosphate buffered saline buffer (0.1M, pH=6) supply and make 10 times of even liquid of gradient dilution according to this, complete to sample inoculation from preparing the even liquid of sample, whole process must not more than 15min;
3.1.2 the even liquid of sample of 3 serial dilution degree is chosen, by liquid to be measured good for homogeneous by 0.45 μm of aseptic filter membrane, take off filter membrane to move to the MI/VJ that solidified respectively and control in culture dish and MI-R/VJ-R resistance culture ware, each extent of dilution respectively do three groups parallel;
3.1.3 standard gold staphylococcus aureus (ATCC25923) the bacterium liquid of the standard E. coli (ATCC25922) of antibiotic-free resistance and antibiotic-free resistance to be diluted to after 0.5 Maxwell reduced turbidity according to the operation of 3.1.1 and 3.1.2 step and by sterilised membrane filter, to move in MI/VJ (positive control) and MI-R/VJ-R (negative control) culture plate as Quality Control control group;
3.1.4 after at every turn having filtered sample, got 100 milliliters of ultrapure waters or physiological saline with graduated cylinder, and be poured in funnel and rinse funnel twice, and cleaned funnel with alcohol and use flame calcination, disturbed to eliminate sample room;
3.1.5 hold aseptic nipper in film filter edge, lift filter membrane gently, then filter membrane is moved on agar plate, filter membrane slides on agar plate, uses scroll actions, to avoid occurring bubble between filter membrane and bottom-layer agar, if occur at non-wetting region bubble, then reappose filter membrane;
3.1.6 incubator constant temperature culture 24-36h under the condition of 35 DEG C ± 0.5 DEG C is put in culture dish upset, after cultivation, by MI culture dish under natural light to the enumeration of film blue, and under long wave ultraviolet (366nm) repeat count determination result, by VJ culture dish under natural light on film grey black circle enumeration;
3.2 sediment samples:
3.2.1 appropriate samples quality is chosen in the estimation of pickup area and pollution situation per sample, load weighted sediment sample is put into the sterile centrifugation tube filling 50mL sterilizing seawater, and vortex shakes after 3 minutes and leaves standstill 1 minute;
3.2.2 will leave standstill the liquid to be measured after 1 minute by 0.45 μm of aseptic filter membrane, take off filter membrane and move in the MI/VJ control culture dish and MI-R/VJ-R resistance culture ware solidified respectively, each sample respectively do three groups parallel;
3.2.3 standard gold staphylococcus aureus (ATCC25923) the bacterium liquid of the standard E. coli (ATCC25922) of antibiotic-free resistance and antibiotic-free resistance to be diluted to after 0.5 Maxwell reduced turbidity according to the operation of 3.1.1 and 3.1.2 step and by sterilised membrane filter, to move in MI/VJ (positive control) and MI-R/VJ-R (negative control) culture plate as Quality Control control group;
3.2.4 after at every turn having filtered sample, got ultrapure water or the physiological saline of 100 milliliters with graduated cylinder, and be poured in funnel and rinse funnel twice, and cleaned funnel with alcohol and use flame calcination, disturbed to eliminate sample room;
3.2.5 hold aseptic nipper in film filter edge, lift filter membrane gently, then filter membrane is moved on agar plate, filter membrane slides on agar plate, uses scroll actions, to avoid occurring bubble between filter membrane and bottom-layer agar, if occur at non-wetting region bubble, then reappose filter membrane;
3.2.6 incubator constant temperature culture 24-36h under the condition of 35 DEG C ± 0.5 DEG C is put in culture dish upset, after cultivation, by MI culture dish under natural light to the enumeration of film blue, and under long wave ultraviolet (366nm) repeat count determination result, by VJ culture dish under natural light on film grey black circle enumeration;
(4) data analysis and calculating:
In calculation sample, intestinal bacteria (E.coli) should meet following primary condition with streptococcus aureus (S.aureus) abundance and resistance level:
4.1 dull and stereotyped total colony number≤200/plate;
4.2 dull and stereotyped upper target colony number≤100/plate (20 ~ 80 is best);
4.3. intestinal bacteria (E.coli) and streptococcus aureus (S.aureus) abundance calculation formula:
4.4. intestinal bacteria (E.coli) and streptococcus aureus (S.aureus) antibiotics resistance rate calculation formula:
During sample collecting, bleeding iron and related reagent material should prepare in advance, sterilizing, water and sediment sample are collected in aseptic leakproof polypropylene specimen container, lab analysis is sent back to as early as possible in 1-4 DEG C of refrigeration, sample should be analyzed in 12 hours after acquisition, and simple sample analytic process is no more than 15 minutes.
In step 3.1.1, original volume is selected: river mouth and enter extra large sewage draining exit: 10ml, coastal ocean: 300 ~ 500ml.
In step 3.2.1, sample initial mass is selected: river mouth and enter extra large sewage draining exit: 0.1 ~ 0.3g, coastal ocean: 0.3 ~ 0.5g.
MI substratum utilizes specific enzyme reaction, detects the intestinal bacteria in water.Containing two kinds of enzyme substrates: MUGal (fluorescence) and IBDG (color) in substratum, coliform can produce beta-galactosidase enzymes, with MUGal reaction, generate a kind of compound (366nm) under long wave ultraviolet and send fluorescence; E.coli can produce beta-glucosidase, and IBDG reaction, generates blue compound, and under natural light, bacterium colony is blue.The growth of the Gram-negative bacteria of gram-positive microorganism and all the other non-coliforms is suppressed by adding certain density cefsulodin in the medium.In addition, by invading antibiotic-screening antibiotic resistant microbes by minimum inhibitory concentration in the medium.
Vogel-Johnson substratum is the reducing power and the adaptability to Repone K that utilize streptococcus aureus to tellurium, Gram-negative bacteria and all the other non-staphylococcus aureus growths is suppressed by the Repone K in nutrient agar and potassium tellurite, add Sodium.alpha.-ketopropionate simultaneously and repair damaged cell stimulation staphylococcus aureus growth, to improve recall rate.Streptococcus aureus reductive tellurium makes bacterium colony be grey black.
Positively effect of the present invention is as follows:
In river mouth of the present invention and nearshore marine environment, intestinal bacteria, streptococcus aureus separation screening and antibiotics resistance appraisal procedure have easy to operation, accuracy rate is high, the advantage of safety and environmental protection, is applicable to coastal ocean, river mouth and enters the real-time analysis of intestinal bacteria and streptococcus aureus in extra large sewage draining exit water body and sediment sample and antibiotic resistance level is monitored.
Embodiment
The following examples describe in further detail of the present invention.
Embodiment 1
1. Method And Principle
MI substratum utilizes specific enzyme reaction, detects the intestinal bacteria in water.Containing two kinds of enzyme substrates: MUGal (fluorescence) and IBDG (color) in substratum, coliform can produce beta-galactosidase enzymes, with MUGal reaction, generate a kind of compound (366nm) under long wave ultraviolet and send fluorescence; E.coli can produce beta-glucosidase, and IBDG reaction, generates blue compound, and under natural light, bacterium colony is blue.The growth of the Gram-negative bacteria of gram-positive microorganism and all the other non-coliforms is suppressed by adding certain density cefsulodin in the medium.In addition, by invading antibiotic-screening antibiotic resistant microbes by minimum inhibitory concentration in the medium.
2. safety
Experimenter must understand and observe Microbiological Lab's safety criterion, safety preparation, use and dispose reagent and material; Forbid to inhale transfer pipet with mouth; Meanwhile, under avoiding being exposed to long wave or ultraviolet germicidal lamp for a long time; Experiment terminates all pollution plates of rear autoclaving and material.
3. equipment and articles for use
Except Microbiological Lab's conventional sterilant and culture device, other equipment and material as follows:
A. constant incubator: 36 DEG C ± 1 DEG C.
B. refrigerator: 2 DEG C ~ 5 DEG C.
C. constant water bath box: 37 DEG C ~ 65 DEG C.
D. electronic balance: sensibility reciprocal 0.01g.
E. turbula shaker.
F. aseptic straw: 1mL (tool 0.01mL scale), 10mL (tool 0.1mL scale) or micropipet and suction nozzle.
G. aseptic Erlenmeyer flask: capacity 100mL, 500mL.
H. sterile petri dish: diameter 90mm.
I. syringe: 0.5mL.
J.pH meter or pH colorimetric cylinder or accurate pH test paper.
K. ultraviolet lamp: wavelength 360nm ~ 366nm, power≤6W.
4. reagent and standard
A.MI substratum
B. phosphate buffer soln
C. sterilizing seawater
D. cefsulodin
5. sample collecting, preserves and stores
Bleeding iron and related reagent material should prepare in advance, sterilizing.Water and sediment sample are collected in aseptic leakproof polypropylene specimen container, send lab analysis back to as early as possible in 1-4 DEG C of refrigeration.Sample should be analyzed in 12 hours after acquisition, and simple sample analytic process is no more than 15 minutes.
6. calibration and stdn
Check incubator temperature, twice daily, to guarantee the range of operation specified.
Check thermometer, at least annual a NIST certification is carried out to thermometer.
7. quality control
7.1 substratum Quality Controls: carry out pretest to every batch cultivation base, guarantee correct enzyme reaction occurs.
7.2 filter membrane Quality Controls: be directly placed on agar plate by filter membrane, cultivate 24 hours, if without colony growth, illustrate that filter membrane is aseptic at 35 DEG C.
7.3 diluent Quality Controls: by 50ml diluent by sterilizing filter membrane, and be positioned on culture plate, cultivate 24 hours, if without colony growth, illustrate that diluent is aseptic at 35 DEG C.
8. experimental procedure
8.1 sterilizing
Before experiment, filter membrane is placed between filter paper and wraps, the filter wrapped with kraft paper together with culture dish at 121 DEG C autoclaving 15min.Before the different sample of each filtration, clean funnel with alcohol and use flame calcination, eliminating sample room interference.
The preparation of 8.2 substratum
E.coli: accurately take 36.5gMI substratum (BectonDickinson, NewJersey), heating for dissolving, in 1L sterilized water, boils sterilizing after a minute (103Kpa, 121 DEG C, 15min).The water bath cooling of the substratum through sterilizing being put into 45 DEG C ± 1 DEG C is stand-by.Prepare and add cefsulodin, making its ultimate density be 5 μ g/ml.Be sub-packed in triangular flask by the substratum prepared, a part is as intestinal bacteria screening MI agar plate (not added with antibiotic), and a part immerses microbiotic as antibiotics resistance screening MI-R agar plate using minimum inhibitory concentration.
8.3 antibiotic resistance experiments
8.3.1 water body example:
(1) estimation of pickup area and pollution situation is chosen appropriate samples volume (suggestion sample starting volume is long-pending to be selected: river mouth and enter extra large sewage draining exit: 10ml per sample, coastal ocean: 300 ~ 500ml), supply with phosphate buffered saline buffer (0.1M, pH=6) when original volume is less than 50ml and make 10 times of even liquid of gradient dilution (selecting the even liquid of sample of suitable 3 serial dilution degree to test) according to this.Complete to sample inoculation from preparing the even liquid of sample, whole process must not more than 15min.
(2) filter, suction pump and filter flask are connected.Take off the funnel on filter top, carefully get the filter membrane of a slice sterilizing with aseptic tweezers, have one of lattice to face up, be placed in the plane of filter and (be careful during operation, pollute funnel inner) then by funnel reset, and be fixed.
(3) choose the even liquid of sample of 3 suitable serial dilution degree, by liquid to be measured good for homogeneous by aseptic 0.45 μm of filter membrane, take off filter membrane and move in the MI control culture dish and resistance culture ware solidified respectively, each extent of dilution respectively do three groups parallel.
(4) to be diluted to after proper concn by sterilised membrane filter with standard E. coli (ATCC25922) the bacterium liquid of antibiotic-free resistance according to aforesaid operations, to move in MI and MI-R culture plate as Quality Control control group.
(5) after at every turn having filtered sample, got ultrapure water (100 milliliters) or the physiological saline of proper volume with graduated cylinder, and be poured in funnel and rinse funnel twice, and cleaned funnel with alcohol and use flame calcination, disturbed to eliminate sample room.
(6) hold aseptic nipper in film filter edge, lift filter membrane gently, then filter membrane is moved on agar plate.Filter membrane slides on agar plate, uses scroll actions, to avoid occurring bubble between filter membrane and bottom-layer agar.If occur at non-wetting region bubble, then reappose filter membrane.
(7) incubator constant temperature culture 24h under the condition of 35 DEG C ± 0.5 DEG C is put in culture dish upset.After cultivation, by MI culture dish under natural light to the enumeration of film blue, and under long wave ultraviolet (366nm) repeat count determination result.
8.3.2 sediment sample:
(1) estimation of pickup area and pollution situation is chosen appropriate samples quality (suggestion sample initial mass is selected: river mouth and enter extra large sewage draining exit: 0.1 ~ 0.3g per sample, coastal ocean: 0.3 ~ 0.5g), load weighted sediment sample is put into the sterile centrifugation tube filling 50mL sterilizing seawater, vortex shakes after 3 minutes and leaves standstill 1 minute.
(2) filter, suction pump and filter flask are connected.Take off the funnel on filter top, carefully get the filter membrane of a slice sterilizing with aseptic tweezers, have one of lattice to face up, be placed in the plane of filter and (be careful during operation, pollute funnel inner) then by funnel reset, and be fixed.
(3) will the liquid to be measured after 1 minute be left standstill by 0.45 μm of aseptic filter membrane, takes off filter membrane and move in the MI control culture dish and resistance culture ware solidified respectively, each sample respectively do three groups parallel.
(4) to be diluted to after proper concn by sterilised membrane filter with standard E. coli (ATCC25922) the bacterium liquid of antibiotic-free resistance according to aforesaid operations, to move in MI and MI-R culture plate as Quality Control control group.
(5) after at every turn having filtered sample, got ultrapure water (100 milliliters) or the physiological saline of proper volume with graduated cylinder, and be poured in funnel and rinse funnel twice, and cleaned funnel with alcohol and use flame calcination, disturbed to eliminate sample room.
(6) hold aseptic nipper in film filter edge, lift filter membrane gently, then filter membrane is moved on agar plate.Filter membrane slides on agar plate, uses scroll actions, to avoid occurring bubble between filter membrane and bottom-layer agar.If occur at non-wetting region bubble, then reappose filter membrane.
(7) incubator constant temperature culture 24h under the condition of 35 DEG C ± 0.5 DEG C is put in culture dish upset.After cultivation, by MI culture dish under natural light to the enumeration of film blue, and under long wave ultraviolet (366nm) repeat count determination result.
9. data analysis and calculating
In calculation sample, intestinal bacteria (E.coli) abundance and resistance level should meet following primary condition:
1. dull and stereotyped total colony number≤200/plate;
2. dull and stereotyped upper target colony number≤100/plate (20 ~ 80 is best);
3. intestinal bacteria (E.coli) abundance calculation formula:
4. intestinal bacteria (E.coli) antibiotics resistance rate calculation formula:
10. waste management
The waste produced in experimentation process should be consistent with all applicable rules of laboratory waste management method and regulations, reduce to greatest extent and control the pollution that causes from experimental implementation, and observe solid and hazardous waste disposal regulations, answer autoclave sterilization before all infectious waste treatments.
Embodiment 2
1. Method And Principle
Vogel-Johnson substratum is the reducing power and the adaptability to Repone K that utilize streptococcus aureus to tellurium, Gram-negative bacteria and all the other non-staphylococcus aureus growths is suppressed by the Repone K in nutrient agar and potassium tellurite, add Sodium.alpha.-ketopropionate simultaneously and repair damaged cell stimulation staphylococcus aureus growth, to improve recall rate.Streptococcus aureus reductive tellurium makes bacterium colony be grey black.
2. safety
Experimenter must understand and observe Microbiological Lab's safety criterion, safety preparation, use and dispose reagent and material; Forbid to inhale transfer pipet with mouth; Meanwhile, under avoiding being exposed to long wave or ultraviolet germicidal lamp for a long time; Experiment terminates all pollution plates of rear autoclaving and material.
3. equipment and articles for use
Except Microbiological Lab's conventional sterilant and culture device, other equipment and material as follows:
A. constant incubator: 36 DEG C ± 1 DEG C.
B. refrigerator: 2 DEG C ~ 5 DEG C.
C. constant water bath box: 37 DEG C ~ 65 DEG C.
D. electronic balance: sensibility reciprocal 0.01g.
E. turbula shaker.
F. aseptic straw: 1mL (tool 0.01mL scale), 10mL (tool 0.1mL scale) or micropipet and suction nozzle.
G. aseptic Erlenmeyer flask: capacity 100mL, 500mL.
H. sterile petri dish: diameter 90mm.
I. syringe: 0.5mL.
J.pH meter or pH colorimetric cylinder or accurate pH test paper.
K. ultraviolet lamp: wavelength 360nm ~ 366nm, power≤6W.
4. reagent and standard
E.VJ substratum
F. phosphate buffer soln
G. sterilizing seawater
H. potassium tellurite
5. sample collecting, preserves and stores
Bleeding iron and related reagent material should prepare in advance, sterilizing.Water and sediment sample are collected in aseptic leakproof polypropylene specimen container, send lab analysis back to as early as possible in 1-4 DEG C of refrigeration.Sample should be analyzed in 12 hours after acquisition, and simple sample analytic process is no more than 15 minutes.
6. calibration and stdn
Check incubator temperature, twice daily, to guarantee the range of operation specified.
Check thermometer, at least annual a NIST certification is carried out to thermometer.
7. quality control
7.1 substratum Quality Controls: carry out pretest to every batch cultivation base, guarantee correct enzyme reaction occurs.
7.2 filter membrane Quality Controls: be directly placed on agar plate by filter membrane, cultivate 24 hours, if without colony growth, illustrate that filter membrane is aseptic at 35 DEG C.
7.3 diluent Quality Controls: by 50ml diluent by sterilizing filter membrane, and be positioned on culture plate, cultivate 24 hours, if without colony growth, illustrate that diluent is aseptic at 35 DEG C.
8. experimental procedure
8.1 sterilizing
Before experiment, filter membrane is placed between filter paper and wraps, the filter wrapped with kraft paper together with culture dish at 121 DEG C autoclaving 15min.Before the different sample of each filtration, clean funnel with alcohol and use flame calcination, eliminating sample room interference.
The preparation of 8.2 substratum
S.aureus: accurately take 60gVJ substratum (BectonDickinson, NewJersey), adds 1L sterilized water, and heating for dissolving also constantly stirs, and boils sterilizing after a minute (103Kpa, 121 DEG C, 15min).Water bath substratum through sterilizing being put into 45 DEG C ± 1 DEG C cools and adds 20ml (1%) potassium tellurite.Be sub-packed in triangular flask by the substratum prepared, a part is as streptococcus aureus screening VJ agar plate (not added with antibiotic), and a part immerses microbiotic as antibiotics resistance screening VJ-R agar plate using minimum inhibitory concentration.
8.3 antibiotic resistance experiments
8.3.1 water body example:
(1) estimation of pickup area and pollution situation is chosen appropriate samples volume (suggestion sample starting volume is long-pending to be selected: river mouth and enter extra large sewage draining exit: 10ml per sample, coastal ocean: 300 ~ 500ml), supply with phosphate buffered saline buffer (0.1M, pH=6) when original volume is less than 50ml and make 10 times of even liquid of gradient dilution (selecting the even liquid of sample of suitable 3 serial dilution degree to test) according to this.Complete to sample inoculation from preparing the even liquid of sample, whole process must not more than 15min.
(2) filter, suction pump and filter flask are connected.Take off the funnel on filter top, carefully get the filter membrane of a slice sterilizing with aseptic tweezers, have one of lattice to face up, be placed in the plane of filter and (be careful during operation, pollute funnel inner) then by funnel reset, and be fixed.
(3) the even liquid of sample of 3 suitable serial dilution degree is chosen, by liquid to be measured good for homogeneous by 0.45 μm of aseptic filter membrane, take off filter membrane to move to the VJ that solidified respectively and control in culture dish and VJ-R resistance culture ware, each extent of dilution respectively do three groups parallel.
(4) to be diluted to after proper concn by sterilised membrane filter with standard gold staphylococcus aureus (ATCC25923) the bacterium liquid of antibiotic-free resistance according to aforesaid operations, to move in VJ and VJ-R culture plate as Quality Control control group.
(5) after at every turn having filtered sample, got ultrapure water (100 milliliters) or the physiological saline of proper volume with graduated cylinder, and be poured in funnel and rinse funnel twice, and cleaned funnel with alcohol and use flame calcination, disturbed to eliminate sample room.
(6) hold aseptic nipper in film filter edge, lift filter membrane gently, then filter membrane is moved on agar plate.Filter membrane slides on agar plate, uses scroll actions, to avoid occurring bubble between filter membrane and bottom-layer agar.If occur at non-wetting region bubble, then reappose filter membrane.
(7) incubator constant temperature culture 36h under the condition of 35 DEG C ± 0.5 DEG C is put in culture dish upset.After cultivation, VJ culture dish is justified enumeration to grey black on film under natural light.8.3.2 sediment sample:
(1) estimation of pickup area and pollution situation is chosen appropriate samples quality (suggestion sample initial mass is selected: river mouth and enter extra large sewage draining exit: 0.1 ~ 0.3g per sample, coastal ocean: 0.3 ~ 0.5g), load weighted sediment sample is put into the sterile centrifugation tube filling 50mL sterilizing seawater, vortex shakes after 3 minutes and leaves standstill 1 minute.
(2) filter, suction pump and filter flask are connected.Take off the funnel on filter top, carefully get the filter membrane of a slice sterilizing with aseptic tweezers, have one of lattice to face up, be placed in the plane of filter and (be careful during operation, pollute funnel inner) then by funnel reset, and be fixed.
(3) will the liquid to be measured after 1 minute be left standstill by 0.45 μm of aseptic filter membrane, takes off filter membrane and move in the VJ control culture dish and VJ-R resistance culture ware solidified respectively, each sample respectively do three groups parallel.
(4) to be diluted to after proper concn by sterilised membrane filter with streptococcus aureus (ATCC25923) the bacterium liquid of antibiotic-free resistance according to aforesaid operations, to move in VJ and VJ-R culture plate as Quality Control control group.
(5) after at every turn having filtered sample, got ultrapure water (100 milliliters) or the physiological saline of proper volume with graduated cylinder, and be poured in funnel and rinse funnel twice, and cleaned funnel with alcohol and use flame calcination, disturbed to eliminate sample room.
(6) hold aseptic nipper in film filter edge, lift filter membrane gently, then filter membrane is moved on agar plate.Filter membrane slides on agar plate, uses scroll actions, to avoid occurring bubble between filter membrane and bottom-layer agar.If occur at non-wetting region bubble, then reappose filter membrane.
(7) incubator constant temperature culture 36h under the condition of 35 DEG C ± 0.5 DEG C is put in culture dish upset.After cultivation, VJ culture dish is justified enumeration to grey black on film under natural light.
9. data analysis and calculating
In calculation sample, golden yellow grape alveolar sphere bacterium (S.aureus) abundance and resistance level should meet following primary condition:
1. dull and stereotyped total colony number≤200/plate;
2. dull and stereotyped upper target colony number≤100/plate (20 ~ 80 is best);
3. golden yellow grape alveolar sphere bacterium (S.aureus) abundance calculation formula:
4. golden yellow grape alveolar sphere bacterium (S.aureus) antibiotics resistance rate calculation formula:
10. waste management
The waste produced in experimentation process should be consistent with all applicable rules of laboratory waste management method and regulations, reduce to greatest extent and control the pollution that causes from experimental implementation, and observe solid and hazardous waste disposal regulations, before all infectious waste treatments should at 121 DEG C autoclaving 15min.
Although illustrate and describe embodiments of the invention, for the ordinary skill in the art, be appreciated that and can carry out multiple change, amendment, replacement and modification to these embodiments without departing from the principles and spirit of the present invention, scope of the present invention is by claims and equivalents thereof.
Claims (4)
1. intestinal bacteria, streptococcus aureus separation screening and an antibiotics resistance appraisal procedure in river mouth and nearshore marine environment, is characterized in that: the concrete steps of described method are as follows:
(1) sterilizing:
Before experiment, filter membrane is placed between filter paper and wraps, the filter wrapped with kraft paper together with culture dish at 121 DEG C autoclaving 15min, each filter different sample before, clean funnel with alcohol and use flame calcination, eliminating sample room and disturb;
(2) preparation of substratum:
S.aureus: accurately take 60gVJ substratum (BectonDickinson, NewJersey), add 1L sterilized water, heating for dissolving also constantly stirs, boil sterilizing (103Kpa after a minute, 121 DEG C, 15min), water bath substratum through sterilizing being put into 45 DEG C ± 1 DEG C cools and adds 20ml (1%) potassium tellurite, the substratum prepared is sub-packed in triangular flask, a part is as streptococcus aureus screening VJ agar plate (not added with antibiotic), a part immerses microbiotic as antibiotics resistance screening VJ-R agar plate using minimum inhibitory concentration,
E.coli: accurately take 36.5gMI substratum (BectonDickinson, NewJersey), heating for dissolving is in 1L sterilized water, boil sterilizing (103Kpa after a minute, 121 DEG C, 15min), the water bath cooling of the substratum through sterilizing being put into 45 DEG C ± 1 DEG C is stand-by, prepare and add cefsulodin, its ultimate density is made to be 5 μ g/ml, the substratum prepared is sub-packed in triangular flask, a part is as intestinal bacteria screening MI agar plate (not added with antibiotic), a part immerses microbiotic as antibiotics resistance screening MI-R agar plate using minimum inhibitory concentration,
(3) antibiotic resistance experiment:
3.1 water body examples:
3.1.1 appropriate samples volume is chosen in the estimation of pickup area and pollution situation per sample, sample starting volume amasss when being less than 50ml with phosphate buffered saline buffer (0.1M, pH=6) supply and make 10 times of even liquid of gradient dilution according to this, complete to sample inoculation from preparing the even liquid of sample, whole process must not more than 15min;
3.1.2 the even liquid of sample of 3 serial dilution degree is chosen, by liquid to be measured good for homogeneous by 0.45 μm of aseptic filter membrane, take off filter membrane to move to the MI/VJ that solidified respectively and control in culture dish and MI-R/VJ-R resistance culture ware, each extent of dilution respectively do three groups parallel;
3.1.3 standard gold staphylococcus aureus (ATCC25923) the bacterium liquid of the standard E. coli (ATCC25922) of antibiotic-free resistance and antibiotic-free resistance to be diluted to after 0.5 Maxwell reduced turbidity according to the operation of 3.1.1 and 3.1.2 step and by sterilised membrane filter, to move in MI/VJ (positive control) and MI-R/VJ-R (negative control) culture plate as Quality Control control group;
3.1.4 after at every turn having filtered sample, got 100 milliliters of ultrapure waters or physiological saline with graduated cylinder, and be poured in funnel and rinse funnel twice, and cleaned funnel with alcohol and use flame calcination, disturbed to eliminate sample room;
3.1.5 hold aseptic nipper in film filter edge, lift filter membrane gently, then filter membrane is moved on agar plate, filter membrane slides on agar plate, uses scroll actions, to avoid occurring bubble between filter membrane and bottom-layer agar, if occur at non-wetting region bubble, then reappose filter membrane;
3.1.6 incubator constant temperature culture 24-36h under the condition of 35 DEG C ± 0.5 DEG C is put in culture dish upset, after cultivation, by MI culture dish under natural light to the enumeration of film blue, and under long wave ultraviolet (366nm) repeat count determination result, by VJ culture dish under natural light on film grey black circle enumeration;
3.2 sediment samples:
3.2.1 appropriate samples quality is chosen in the estimation of pickup area and pollution situation per sample, load weighted sediment sample is put into the sterile centrifugation tube filling 50mL sterilizing seawater, and vortex shakes after 3 minutes and leaves standstill 1 minute;
3.2.2 will leave standstill the liquid to be measured after 1 minute by 0.45 μm of aseptic filter membrane, take off filter membrane and move in the MI/VJ control culture dish and MI-R/VJ-R resistance culture ware solidified respectively, each sample respectively do three groups parallel;
3.2.3 standard gold staphylococcus aureus (ATCC25923) the bacterium liquid of the standard E. coli (ATCC25922) of antibiotic-free resistance and antibiotic-free resistance to be diluted to after 0.5 Maxwell reduced turbidity according to the operation of 3.1.1 and 3.1.2 step and by sterilised membrane filter, to move in MI/VJ (positive control) and MI-R/VJ-R (negative control) culture plate as Quality Control control group;
3.2.4 after at every turn having filtered sample, got ultrapure water or the physiological saline of 100 milliliters with graduated cylinder, and be poured in funnel and rinse funnel twice, and cleaned funnel with alcohol and use flame calcination, disturbed to eliminate sample room;
3.2.5 hold aseptic nipper in film filter edge, lift filter membrane gently, then filter membrane is moved on agar plate, filter membrane slides on agar plate, uses scroll actions, to avoid occurring bubble between filter membrane and bottom-layer agar, if occur at non-wetting region bubble, then reappose filter membrane;
3.2.6 incubator constant temperature culture 24-36h under the condition of 35 DEG C ± 0.5 DEG C is put in culture dish upset, after cultivation, by MI culture dish under natural light to the enumeration of film blue, and under long wave ultraviolet (366nm) repeat count determination result, by VJ culture dish under natural light on film grey black circle enumeration;
(4) data analysis and calculating:
In calculation sample, intestinal bacteria (E.coli) should meet following primary condition with golden yellow grape alveolar sphere bacterium (S.aureus) abundance and resistance level:
4.1. dull and stereotyped total colony number≤200/plate;
4.2. dull and stereotyped upper target colony number≤100/plate (20 ~ 80 is best);
4.3. intestinal bacteria (E.coli) and streptococcus aureus (S.aureus) abundance calculation formula:
4.4. intestinal bacteria (E.coli) and streptococcus aureus (S.aureus) antibiotics resistance rate calculation formula:
2. intestinal bacteria, streptococcus aureus separation screening and antibiotics resistance appraisal procedure in river mouth as claimed in claim 1 and nearshore marine environment, it is characterized in that: during sample collecting, bleeding iron and related reagent material should prepare in advance, sterilizing, water and sediment sample are collected in aseptic leakproof polypropylene specimen container, lab analysis is sent back to as early as possible in 1-4 DEG C of refrigeration, sample should be analyzed in 12 hours after acquisition, and simple sample analytic process is no more than 15 minutes.
3. intestinal bacteria, streptococcus aureus separation screening and antibiotics resistance appraisal procedure in river mouth as claimed in claim 1 and nearshore marine environment, it is characterized in that: in step 3.1.1, original volume is selected: river mouth and enter extra large sewage draining exit: 10ml, coastal ocean: 300 ~ 500ml.
4. intestinal bacteria, streptococcus aureus separation screening and antibiotics resistance appraisal procedure in river mouth as claimed in claim 1 and nearshore marine environment, it is characterized in that: in step 3.2.1, sample initial mass is selected: river mouth and enter extra large sewage draining exit: 0.1 ~ 0.3g, coastal ocean: 0.3 ~ 0.5g.
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| CN114544846A (en) * | 2022-02-24 | 2022-05-27 | 河海大学 | Resistance gene research method under influence of tide in coastal region and solid phase extraction instrument |
| US11959122B2 (en) | 2022-06-01 | 2024-04-16 | Nanjing Institute Of Environmental Sciences, Mee | Method for assessing microbial drug resistance multi-level risks of antibiotic residues in water environment |
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| US11959122B2 (en) | 2022-06-01 | 2024-04-16 | Nanjing Institute Of Environmental Sciences, Mee | Method for assessing microbial drug resistance multi-level risks of antibiotic residues in water environment |
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