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Method for measuring sizes of heterotrophic bacteria in shallow lake based on flow cytometry

A technology of flow cytometry and heterotrophic bacteria is applied in the field of rapid detection of the size of heterotrophic bacteria in large shallow lakes, and can solve the problems of flow cytometry interference, increased difficulty of flow cytometry analysis, and increased background noise. , to achieve the effect of short time, fast test speed and high accuracy

Inactive Publication Date: 2015-07-15
NANJING INST OF GEOGRAPHY & LIMNOLOGY
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Problems solved by technology

At present, the determination of the size of heterotrophic bacteria by flow cytometry is mostly used in marine ecosystems. There are no reports on the application of flow cytometry to lakes, especially shallow eutrophic lakes.
However, since there are a large number of particles and solid-phase substances in the lake water, including many non-biological particles, cell debris, etc., they will cause great interference to the flow cytometer, and the background noise will also increase accordingly. Difficulty is also increased, and the existing technology cannot be effectively used for the rapid retrieval of heterotrophic bacteria in eutrophic lakes. In view of this, the present invention is proposed

Method used

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  • Method for measuring sizes of heterotrophic bacteria in shallow lake based on flow cytometry
  • Method for measuring sizes of heterotrophic bacteria in shallow lake based on flow cytometry
  • Method for measuring sizes of heterotrophic bacteria in shallow lake based on flow cytometry

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Verification of the size of fluorescent microspheres

[0037] The flow cytometer is regulated and corrected with standard fluorescent beads, and the liquid flow system and optical system are adjusted so that the coefficient of variation of fluorescent signals detected by the standard fluorescent beads in each detector channel is lower than 3%.

[0038] Select 0.5um, 0.8um, 1um, 3um, 6um standard green microspheres (Polysciences, USA) with various particle sizes, and use flow cytometry to test separately, collect forward scattered light (FSC, reflecting cell size), side Scattered light (SSC, reflecting the internal structure of the cell), green fluorescence signal of FL1 channel. The flow rate was controlled within 1,000 cells per second, and a total of 20,000 cells were collected. By repeatedly measuring their FSC values, the relationship between cell particle size and FSC is established, the standard curve of bacterial size is made, the regression analysis e...

Embodiment 2

[0040] Example 2 Bacterial size in large shallow water eutrophication lake Taihu Lake

[0041] The present embodiment measures the size of bacteria in the large-scale shallow water eutrophic lake Taihu Lake, which specifically includes the following steps:

[0042] (1) In February 2014, a water collector was used to collect surface water samples from each point in Taihu Lake, a total of 32 samples (such as image 3 ), collected into a pre-sterilized EP tube, added formaldehyde as a fixative with a final volume concentration of 1%, and transported back to the laboratory in a refrigerated incubator.

[0043] (2) Take at least 500 μl of sample, dilute it with sterilized water 1:10 and mix well. Take 5ml of the sample and pass it through an ultrasonic cleaner (power 25W) for 5 minutes to break up the heterotrophic bacteria that are agglomerated and attached to other organisms. Pass through a 300-mesh sieve to remove large particles of impurities.

[0044] (3) Aspirate 1 ml of t...

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Abstract

The invention aims at disclosing a method for rapidly measuring the sizes of all heterotrophic bacteria in a shallow lake, and provides a method for measuring the sizes of heterotrophic bacteria in a shallow lake based on the flow cytometry. The method includes the steps that firstly, fixing, ultrasonic processing, filtering and SYBR Green I dyeing are carried out on a sample; secondly, the heterotrophic bacteria are detected through a flow cytometer, and the FSC value of the heterotrophic bacteria in the sample is measured according to FSC, for reflecting the sizes of cells, of the flow cytometer; thirdly, a standard curve of the sizes of the bacteria is drawn by building the relationship between the standard microballoon particle diameter and the FSC value, and a regression analysis equation is built; finally, the sizes of the heterotrophic bacteria in the sample are obtained by converting the FSC value of the heterotrophic bacteria in the sample.

Description

technical field [0001] The invention belongs to the field of environmental microbiology, and relates to a detection method for the size of heterotrophic bacteria in shallow lakes. By combining with flow cytometry, a rapid detection method for the size of heterotrophic bacteria in large shallow lakes is established. Background technique [0002] Bacteria are an important part of the food web and food chain in 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, estimating the biomass of bacteria is very important for the study of lake ecosystems. The cell size of bacteria refers to the volume of bacteria, and most of the calculation of bacterial biomass converts bacterial volume into biom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14
Inventor 龚伊高光汤祥明邵克强
Owner NANJING INST OF GEOGRAPHY & LIMNOLOGY
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