Absolute quantification method for fluorescence in-situ hybrid cell counting

A technology of fluorescence in situ hybridization and cell counting, which is applied in the field of fluorescence in situ hybridization detection, can solve the problems of inability to connect and distinguish microbial community structures, and achieve the effects of simple and accurate calculation methods, extended application depth, and strong applicability

Inactive Publication Date: 2012-12-26
BEIJING UNIV OF TECH
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Problems solved by technology

For the treatment process with both biofilm and suspension, it is impossible to analyze the connection and difference of microbial community structure between the two under a quantitative standard

Method used

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  • Absolute quantification method for fluorescence in-situ hybrid cell counting
  • Absolute quantification method for fluorescence in-situ hybrid cell counting
  • Absolute quantification method for fluorescence in-situ hybrid cell counting

Examples

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preparation example Construction

[0038] Ⅰ Preparation of 4% paraformaldehyde-PBS solution

[0039] Take about 5ml of ultrapure water in a beaker, heat it in a microwave oven for 20s until it feels hot to the touch but not hot, pour 0.4g of paraformaldehyde (PFA) weighed in advance into the beaker, add a few drops of 2molL -1 NaOH, until the PFA is completely dissolved, then add 3333 μL 3×PBS, place the beaker in an ice bath to cool, and use 2molL -1 Adjust the pH to about 7.2 with HCl. Pour the solution into a previously iced 10mL volumetric flask, add ice ultrapure water to volume, filter through a 0.22μm membrane, and transfer to a previously iced reagent bottle. Store in refrigerator at 4°C and use within 24 hours.

[0040] Ⅱ Cell Fixation and Storage

[0041]Take 30mg of centrifuged sludge, dissolve it in 1000mL ultrapure water, and centrifuge at 15000rppm×5min. Discard 700 μL of supernatant, leave 300 μL, shake to resuspend the sludge. Add 900 μL of 4% PFA in PBS, that is, 1 volume of sludge + 3 vol...

example

[0058] In order to investigate the effect of carbon-nitrogen ratio on synchronous nitrification and denitrification, four groups of deposited bed biofilm reactors were set up in the experiment, the filler was BX filler, DO was 1-3mg / L, and the carbon-nitrogen ratio was 4, 8, 12, 15. Other conditions are the same.

[0059] 1. When the four groups of reactors are stable, take mud samples from the biofilm and suspension in each reactor, centrifuge at 12000rmp for 3 minutes, take 30mg, and take the same sample and dry it at 120°C for 2h to weigh.

[0060] 2. Select eubacterial gene probe EUB338 (5'-GCTGCCTCCCGAGGAT-3') (5' end Cy3 modification), ammonia oxidizing bacteria gene probe NS0190 (5'-CGATCCCTGCTTTTCTCC-3'.) (5' end FITC labeling) , nitrite oxidizing bacteria gene probe NIT3 (5'-CCTGTGCTCCATGCTCCG-3'.) (HEX mark at the 5. terminal) was used as fluorescence in situ hybridization analysis probes for total bacteria, ammonia oxidizing bacteria and nitrite oxidizing bacteria ...

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Abstract

Belonging to the technical field of fluorescence in-situ hybridization detection, the invention relates to an absolute quantification method for fluorescence in-situ hybrid cell counting. The absolute quantification method mentioned in the invention adds a weight quantification step in a fluorescence in-situ hybridization process, makes improvements to sampling, sample pretreatment, experimental treatment, late-stage result analysis and other stages of an original experimental scheme, so that the complete set of method is established. The method is characterized in that: when quantifying a biological membrane and other solid samples, a quantitative high speed centrifuged sample can be adopted, high temperature drying is performed on the same sample at the same time, then the sample is subjected to fluorescence in situ hybridization and observation under a microscope as well as calculation, thus finally obtaining the microorganism concentration of dry sludge with certain weight. The method realizes comparison of different forms of samples in a same reactor in terms of microbial community numbers, and at the same time, puts forward an absolute quantification means able to establish a standard method.

Description

technical field [0001] The invention belongs to the technical field of fluorescence in situ hybridization detection, relates to the improvement of the absolute quantification of the fluorescence in situ hybridization technology, and specifically relates to an absolute quantitative method applicable to both suspension liquid and biofilm mud samples. Background technique [0002] Fluorescent positional hybridization (FISH) is to label DNA (or RNA) probes with special fluorescent dyes, and then directly hybridize the probes to the chromosomes or RNA of biological samples to qualitatively, quantitatively, Positioning and other detection. FISH technology has the characteristics of strong specificity, high sensitivity, and short analysis time, so it is also widely used in the field of sewage treatment. [0003] The research of fluorescence in situ hybridization in quantification mainly focuses on two aspects. One is relative quantification, which mainly uses mixed probes to obser...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06G01N21/64
Inventor 张岩杨正阳王丽丽刘焕光朱敏
Owner BEIJING UNIV OF TECH
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