Flow cytometry counting method for energetic thallus of tomato ulcer germs
A technology for tomato canker and viable bacteria, which is applied in the intersection of microbiology and plant protection, can solve the problems of thick cell wall, small cell size, inability to distinguish viable and inactive cells well, and has broad application prospects. , accurate quantitative, significant ecological and social benefits
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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 BD FACSCalibur, 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 is medium speed) Carry out the experiment of following step 1 and 2.
[0060] 1. Screen the nucleic acid dyes reported in the literature for the detection of bacterial viability. SYTO 9 and SYTO BC are used to stain tomato canker bacteria respectively. The final concentration of the dye in the bacterial solution is set to 50 μmol / l. ...
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