Flow cytometry detection method for tetracycline resistance bacteria in drinking water

Abstract

The invention relates to a flow cytometry detection method for tetracycline resistance bacteria in drinking water. The method comprises the following steps of (1) adding a water sample to be detected into a brown test tube, adding tetracycline mother liquor, enabling the concentration of tetracycline in the water sample to be detected to be equal to an MIC value thereof, putting the water sample to be detected into a culture box, and culturing for 24h at 27 DEG C; (2) transferring the cultured water sample into a flow cytometry sample injection pipe, adding an SYBR Green I coloring agent and a propidium iodide coloring agent for coloring, and after fully vibrating, culturing in dark at 37 DEG C for 10 to 15min; (3) transferring the cultured flow cytometry sample injection pipe into a flow cytometry, starting to inject a sample, determining the number of the tetracycline resistance bacteria, and dividing by a sample injection volume so as to obtain the concentration of the tetracycline resistance bacteria in the drinking water. Compared with the prior art, the number of the tetracycline resistance bacteria is detected by utilizing the flow cytometry, so that the flow cytometry detection method has the advantages of quickness, accuracy and quantitation, overcomes the defect that the nonculturable resistance bacteria cannot be detected through a traditional culture method, and is more reliable in result.

Description

technical field

[0001] The invention relates to a detection method for tetracycline-resistant bacteria, in particular to a flow cytometer detection method for tetracycline-resistant bacteria in drinking water. Background technique

[0002] In recent years, due to the extensive use of antibiotics, the level of antibiotics in the environment has increased significantly compared with the 20th century, and the environmental pollution and ecological toxicity of antibiotics have become increasingly serious. At present, different kinds of antibiotics have been widely detected in groundwater, drinking water, surface water and agricultural soil. Tetracyclines are among the most common antibiotics in the environment. Antibiotics entering the environment will not only interfere with the normal metabolism and growth of other organisms in the environment, but also induce the production of a large number of antibiotic-resistant bacteria.

[0003] Antibiotic-resistant bacteria (Antibioti...

Examples

Embodiment 1

[0030] (1) Take 10ml of the water sample to be tested and put it into a brown test tube, add tetracycline mother solution (1.6g / L), so that the concentration of tetracycline in the water sample is equal to its MIC value;

[0031] (2) Place the test tube in an incubator and incubate at 27°C for 24 hours;

[0032] (3) Transfer 500 μL of the cultured water sample to the injection tube of the flow cytometer. In order to ensure the accuracy of the data, make 3 parallel samples and set a blank control sample. Only 10 mL of ultrapure water is added to the blank sample. Add SYBR Green I staining agent and PI staining agent to the parallel sample and the blank control sample respectively for staining. Before adding the SYBR Green I staining agent, use dimethyl sulfoxide (Sigma, USA) to dilute, and the dilution ratio is 1:100 ; First add 5 μL of diluted SYBR Green I staining agent, then add 1.5 μL of PI staining agent, shake fully on a vortex shaker for about 5 seconds, and then incubat...

Embodiment 2

[0036] (1) Take 10ml of the water sample to be tested in a 250ml beaker, and then add 90ml of phosphate buffer solution (34g of potassium dihydrogen phosphate, 175ml of sodium hydroxide solution (1mol / L), 825ml of ultrapure water, pH=7.2).

[0037] (2) Take 10 ml of the diluted water sample and add it to a brown test tube, add 100 μL (1.6 g / L) of tetracycline mother solution, so that the concentration of tetracycline in the water sample is equal to its MIC value;

[0038] (3) Place the test tube in an incubator and incubate at 27°C for 24 hours;

[0039] (4) Transfer 500 μL of the cultured water sample to the injection tube of the flow cytometer. In order to ensure the accuracy of the data, make 3 parallel samples and set a blank control sample. Only 10 mL of ultrapure water is added to the blank sample. Then add SYBR Green I staining agent and PI staining agent respectively for staining. Before adding SYBR Green I staining agent, use dimethyl sulfoxide (Sigma, the United Stat...

technical field

[0001] The invention relates to a detection method for tetracycline-resistant bacteria, in particular to a flow cytometer detection method for tetracycline-resistant bacteria in drinking water. Background technique

[0002] In recent years, due to the extensive use of antibiotics, the level of antibiotics in the environment has increased significantly compared with the 20th century, and the environmental pollution and ecological toxicity of antibiotics have become increasingly serious. At present, different kinds of antibiotics have been widely detected in groundwater, drinking water, surface water and agricultural soil. Tetracyclines are among the most common antibiotics in the environment. Antibiotics entering the environment will not only interfere with the normal metabolism and growth of other organisms in the environment, but also induce the production of a large number of antibiotic-resistant bacteria.

[0003] Antibiotic-resistant bacteria (Antibioti...

Examples

Embodiment 1

[0030] (1) Take 10ml of the water sample to be tested and put it into a brown test tube, add tetracycline mother solution (1.6g / L), so that the concentration of tetracycline in the water sample is equal to its MIC value;

[0031] (2) Place the test tube in an incubator and incubate at 27°C for 24 hours;

[0032] (3) Transfer 500 μL of the cultured water sample to the injection tube of the flow cytometer. In order to ensure the accuracy of the data, make 3 parallel samples and set a blank control sample. Only 10 mL of ultrapure water is added to the blank sample. Add SYBR Green I staining agent and PI staining agent to the parallel sample and the blank control sample respectively for staining. Before adding the SYBR Green I staining agent, use dimethyl sulfoxide (Sigma, USA) to dilute, and the dilution ratio is 1:100 ; First add 5 μL of diluted SYBR Green I staining agent, then add 1.5 μL of PI staining agent, shake fully on a vortex shaker for about 5 seconds, and then incubat...

Embodiment 2

[0036] (1) Take 10ml of the water sample to be tested in a 250ml beaker, and then add 90ml of phosphate buffer solution (34g of potassium dihydrogen phosphate, 175ml of sodium hydroxide solution (1mol / L), 825ml of ultrapure water, pH=7.2).

[0037] (2) Take 10 ml of the diluted water sample and add it to a brown test tube, add 100 μL (1.6 g / L) of tetracycline mother solution, so that the concentration of tetracycline in the water sample is equal to its MIC value;

[0038] (3) Place the test tube in an incubator and incubate at 27°C for 24 hours;

[0039] (4) Transfer 500 μL of the cultured water sample to the injection tube of the flow cytometer. In order to ensure the accuracy of the data, make 3 parallel samples and set a blank control sample. Only 10 mL of ultrapure water is added to the blank sample. Then add SYBR Green I staining agent and PI staining agent respectively for staining. Before adding SYBR Green I staining agent, use dimethyl sulfoxide (Sigma, the United Stat...

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