Flow-cytometry-based method for rapidly measuring heterotrophic bacteria in eutrophic lake

A flow cytometry and rapid measurement technology, which is applied in the field of rapid detection of heterotrophic bacteria in large shallow lakes, can solve the problems of interference in the measurement process, long counting time, and large differences in results, and achieve accurate quantification, high precision, and high accuracy. short time effect

Inactive Publication Date: 2014-07-16
NANJING INST OF GEOGRAPHY & LIMNOLOGY
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Problems solved by technology

[0005] The present invention aims to overcome the defects of long counting time and large difference in results when the fluorescent microscope is used to detect heterotrophic bacteria in shallow water eutrophic lakes, and provides a method for quickly and accurately detecting

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  • Flow-cytometry-based method for rapidly measuring heterotrophic bacteria in eutrophic lake
  • Flow-cytometry-based method for rapidly measuring heterotrophic bacteria in eutrophic lake
  • Flow-cytometry-based method for rapidly measuring heterotrophic bacteria in eutrophic lake

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Embodiment 1

[0048] The following examples further illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0049] Example 1 Enumeration of Total Heterotrophic Bacteria in Large Shallow Water Eutrophic Lake Taihu

[0050] In February, May, August and November 2013, water sampling devices were used to collect surface water samples from each point in Taihu Lake, a total of 123 samples (such as Figure 3A ), collected into a pre-sterilized EP tube, added a fixative paraformaldehyde with a final concentration of 1%, and transported back to the laboratory in a refrigerated incubator.

[0051] Take at least 500ul sample, dilute it 1:10 with sterile water, and mix well.

[0052] Take a 5ml sample and use an ultrasonic cleaner (power 25W) for 5 minutes of ultrasonic action to break up the bacteria that are agglomerat...

Embodiment 2

[0061] Compared with Example 1, the difference is only: in the present embodiment, in step 1, the fixative formaldehyde of final concentration 1.5% is added in the sample to be tested; in step 2, ultrasonic treatment condition Es=P*t / V, The ultrasonic power P is 50W, the ultrasonic time t is 10min, the sample volume V is 20ml, and the Es is 1500KJ / L. Step 3: Stand in the dark for 15 minutes to color the DNA.

Embodiment 3

[0063] Compared with Example 1, the difference is only that: in this example, in step 1, the curing agent glutaraldehyde with a final concentration of 0.5% is added to the sample to be tested; in step 2, the ultrasonic treatment condition Es=P*t / V, wherein the ultrasonic power P is 100W, the ultrasonic time t is 2min, the sample volume V is 8ml, and Es is 1500KJ / L. In step 3, stand in the dark for 30 minutes to color the DNA.

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Abstract

The invention aims to develop a flow-cytometry-based method for rapidly measuring heterotrophic bacteria in a eutrophic lake. The method is a flow-cytometry-based bacterium counting method. The method comprises the following steps of fixing a sample, performing ultrasonic processing, filtering, performing SYBR Green I dyeing, detecting the heterotrophic bacteria by utilizing a flow cytometer, and acquiring forward and lateral scattering light, an SYBR Green I green fluorescence signal and a chlorophyll a red fluorescence signal of autotrophic plankton, wherein the SYBR Green I green fluorescence signal is used for setting a threshold value; and eliminating the influence of the nannoplankton on bacterium detection by utilizing the forward and lateral scattering light, separating the plankton from abiological particles and cellular debris according to the intensity of the SYBR Green I green fluorescence signal, and separating the bacteria from phytoplankton according to the intensity of the chlorophyll a red fluorescence signal of the autotrophic plankton, thereby obtaining the accurate number of the heterotrophic bacteria. The method has the time-saving and labor-saving effects, the large-scale ecological survey can be developed, and the counting precision and accuracy of the bacteria are improved.

Description

technical field [0001] The invention belongs to the field of environmental microbiology and relates to a detection method for heterotrophic bacteria in shallow lakes. By combining with flow cytometry, a rapid detection method for heterotrophic bacteria in large shallow lakes is established. Background technique [0002] Heterotrophic bacteria are an important part of the microfood web of the lake ecosystem, and play an important role in the biogeochemical cycle and energy flow of the lake ecosystem. They not only decompose organic compounds to release energy, but also use DOM that algae cannot absorb and convert it into POM for secondary production, improving the overall ecological efficiency of the lake ecosystem. Therefore, the quantification of heterotrophic bacteria in lake ecosystems has become a primary task in the study of lake ecosystems. [0003] A large number of literatures show that the DAPI fluorescence microscopy counting method has become a common bacterial c...

Claims

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Application Information

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IPC IPC(8): G01N15/14
Inventor 龚伊汤祥明高光王博雯胡洋
Owner NANJING INST OF GEOGRAPHY & LIMNOLOGY
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