Method for quickly detecting extracellular respiratory activity of microorganisms

A microbial and external respiration technology, applied in the field of microbial detection, can solve the problems of detecting extracellular respiration activity and achieve high reliability

Inactive Publication Date: 2015-03-25
GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

A search of existing technologies found that there is currently no method for detecting extracellular respiration activity developed for microbial extracellular enzymes

Method used

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  • Method for quickly detecting extracellular respiratory activity of microorganisms
  • Method for quickly detecting extracellular respiratory activity of microorganisms
  • Method for quickly detecting extracellular respiratory activity of microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Microbial Cytochrome c Diffuse Reflectance Spectrum Scanning

[0047] (1) Preparation of microbial suspension

[0048] Inoculate the microbial preservation solution in a test tube containing 5ml of sterile LB medium at a ratio of 1:50, culture at 30°C with shaking at 180rpm until the logarithmic growth phase; centrifuge the culture solution at 7000g for 5min, and pour off the supernatant Collect the precipitate and wash it 3 times with HEPES buffer; adjust the concentration of the bacterial solution to OD600 of about 0.5 (about 2.5×10 8 CFU / mL), store at 4°C until use.

[0049] (2) Diffuse reflectance spectrum scanning

[0050] Take a quartz cuvette with 1cm optical path and 1ml volume, and use 1ml HEPES buffer as a blank to calibrate the diffuse reflectance absorption spectrum of the integrating sphere; Reflection Absorption Spectrum. According to the Kubelka-Munk equation F(R)=(1-R∞) 2 / (2R∞)=K / S Calculate the content of microbial cytochrome c. ...

Embodiment 2

[0051] Embodiment 2: the method for rapid detection microbial extracellular respiration activity

[0052] rapid detection of microorganisms ( Shewanella oneidensis MR-1, Shewanella putrefaciens SP200, Shewanella decolorationis S12, Pantoea agglomerans MFC-3, Aeromonas hydrophila HS01, Comamonas guandongensis CY01, Pseudomonas aeruginosa PAH-1, Fontibacter ferrireducens SgZ-2, Thauera humireducens SgZ-1, Escherichia coli K12 and Bacillus subtilis DMS10) Schematic diagram of the extracellular respiration activity method as shown in figure 1 As shown, the specific operation steps are:

[0053] 1) Add 100 μL to a 96-microwell plate to prepare the bacterial suspensions of the above-mentioned microorganisms according to the method described in Example 1;

[0054]2) Then add 100 μL of chromogenic solution (100 mM sodium citrate, 200 mM disodium hydrogen phosphate, 0.32 mM tetramethylbenzidine and 2 mM hydrogen peroxide, pH 4.3) to the above 96-well plate, r...

Embodiment 3

[0084] Example 3 The method for rapid detection of microbial extracellular respiration activity

[0085] 1) Preparation of the microbial suspension to be tested

[0086] microbes Shewanella oneidensis MR-1 preservation solution was inoculated in a test tube containing 5ml of sterile LB medium at a ratio of 1:50, cultured at 30°C with shaking at 180rpm to logarithmic growth phase; centrifuged at 7000g for 5min, and poured off the supernatant The precipitate was collected from the solution, and washed three times with normal saline; the concentration of the bacterial solution was adjusted to 2.5×10 8 About CFU / mL, which is the microbial suspension to be tested, stored at 4°C for later use;

[0087] 2) Add the chromogenic solution to the microbial suspension to be tested for color reaction

[0088] Add the above-prepared bacterial suspension into a 96-well plate, 100 μL per well; use an 8-channel pipette to absorb 100 μL of the chromogenic solution and add it to the 96-wel...

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Abstract

The invention discloses a method for quickly detecting the extracellular respiratory activity of microorganisms. The method comprises the following steps: adding color developing liquid containing tetramethyl benzidine and hydrogen peroxide into microbial suspension liquid for fully producing a color developing reaction; adding a sulfuric acid solution for stopping the color developing reaction, changing blue to yellow, and detecting the OD 450 nm value by an enzyme-linked immunosorbent assay detection instrument, wherein the OD value is the extracellular respiratory activity value of a microorganism sample. The method is simple and quick to carry out, a detection result can be most quickly obtained within 3 minutes, the method can be used for quickly detecting the microorganism sample and is very good in specificity, and the detection result is hardly influenced by other common microorganisms.

Description

technical field [0001] The invention belongs to the field of microorganism detection and relates to a method for rapidly detecting the extracellular respiration activity of microorganisms. Background technique [0002] Microbial extracellular respiration refers to the process in which microorganisms oxidize organic matter in the cell to release electrons under anaerobic conditions, and couple the respiratory chain to transfer the generated electrons to extracellular receptors for reduction. There are a wide variety of extracellular acceptors that can accept their electrons and be reduced, including water-soluble uranium, gold and silver ions, sulfates, iron-manganese minerals, and humic substances. In addition, the artificial electrodes can also receive electrons delivered to the outside of the cell during extracellular respiration, thereby generating an electric current. Therefore, microbial extracellular respiration has important application value, and shows important app...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31G01N21/78
Inventor 温俊林周顺桂陈俊华陆琴
Owner GUANGDONG INST OF ECO ENVIRONMENT & SOIL SCI
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