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Flow Cytometry Enumeration Method for Viable Bacteria of Tomato Canker Pathogen

A technology for tomato ulcers and viable bacteria, which is applied in the field of microbiology and plant protection, can solve the problems of small cells, thick cell walls, and inability to distinguish viable and inactivated cells well, and achieve accurate quantitative, The effect of broad application prospects, significant ecological and social benefits

Active Publication Date: 2016-06-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the small size and thick cell wall of P. tomato canker sores, using the existing kits and following the method provided in the instructions, it is not possible to distinguish viable and inactivated bacteria.

Method used

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  • Flow Cytometry Enumeration Method for Viable Bacteria of Tomato Canker Pathogen
  • Flow Cytometry Enumeration Method for Viable Bacteria of Tomato Canker Pathogen
  • Flow Cytometry Enumeration Method for Viable Bacteria of Tomato Canker Pathogen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the absolute count of tomato canker bacterium

[0046] 1. Pick a single colony of activated tomato canker sores, add it to 10mL LB liquid medium, place it at 28°C, 140rpm and shake it for 22h to the late logarithmic growth stage;

[0047]2. Take 1ml of the bacterial solution obtained in step 1 and centrifuge it at 12000rpm for 3min, collect the bacterial body, wash twice with 0.85g / 100ml NaCl aqueous solution, and then use 0.85g / 100ml NaCl aqueous solution to prepare different dilutions of canker sore bacteria bacterial suspension;

[0048] 3. Take 300 μl of the bacterial suspension of each dilution, use the plate counting method for plate counting, place the plated LB plates at 28°C for 3 days, and select plates with appropriate concentrations for counting. The concentration conversion formula is as follows:

[0049]

[0050] 4. Add the remaining 700 μl of the diluent to the absolute counting tube, and use the flow cytometer to detect the cells. By ad...

Embodiment 2

[0057] Embodiment 2, the live bacteria detection of tomato canker bacterium

[0058] Under different staining conditions, the method of Example 1 is used to carry out absolute counting of tomato canker bacterium, and compare with the result of plate counting method, determine suitable dye type, dye ratio, staining time, flow cytometer according to correlation parameters etc.

[0059] Utilize the flow cytometry detection condition of embodiment 1 (the model of flow cytometer is BDFACSCalibur, and the detection parameter of flow cytometer is set to: FSC voltage mode is E00, SSC voltage is 440V, amplification mode is Log type, sample injection speed For medium speed) carry out the experiment of following steps 1 and 2.

[0060] 1. Screen the nucleic acid dyes reported in the literature for the detection of bacterial viability. SYTO9 and SYTOBC are used to stain tomato canker sores respectively. for 10 7 CFU / ml (10 5 -10 7 CFU / ml is acceptable), the staining time is set to 20...

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Abstract

The invention discloses a flow cytometry counting method for energetic thalluses of tomato ulcer germs. The invention discloses a method for detecting energetic thalluses of a sample to be detected. The method comprises the following steps: performing SYTO9 dying on the sample to be detected, and counting by using an absolute counting tube, thereby obtaining the total cell number or total cell concentration of the sample to be detected. By adopting the method that the flow cytometry is combined with the absolute counting tube, rapid and accurate quantification of the tomato ulcer germs is achieved, the problem of quantitative detection on the energetic tomato ulcer germs is solved, and the method can be both used in basic microbiology study on tomato ulcer germs and used in detection on pathogen, disease diagnosis and plant quarantine in agriculture production, and has wide application prospect in the field of microbiology and plant protection.

Description

technical field [0001] The invention relates to a method for counting viable bacteria, in particular to a flow cytometry method for counting viable bacteria of tomato canker sores, and belongs to the cross field of microbiology and plant protection. Background technique [0002] Tomato canker is one of the most serious and devastating diseases in tomato production. The disease was first discovered in 1909 on greenhouse tomatoes in Michigan, USA. In the 1930s, 1960s, and 1980s, the disease became prevalent in the Midwest of the United States, North Carolina, and Ontario, Canada, causing yield losses of up to 80%. During the period 1943-1946, the disease became a pandemic in the UK, which seriously affected the canned tomato industry. At present, more than 60 countries in the world have reported the occurrence and harm of the disease, covering the Americas, Europe, Asia, Africa and Oceania. [0003] In 1954, my country recorded tomato canker for the first time. At present,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N15/14
Inventor 罗来鑫蒋娜李健强许新刘西莉曹永松阮小珀
Owner CHINA AGRI UNIV
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