we used to incubate E.coli and S.aureus culture with FITC labelled peptides (for 30 minutes). and we will visualize the cells using confocal lser scanning microscope. but i am not sure about what is correct procedure to mount the cells in glass slides. i tried by adding 10ul culture in slide and placing the cover slip upon it. but i found cells are moving. i tried to fix with glycerol but i didn't get good image.
is there any fixative/mounting solution available for fixing bacterial cultures for confocal laser microscope.?
shall we use glycerol or formaldehyde as mounting agent like we used to do for fluorescence microscope?
Hello Dinesh, Ypu should use freshly prepared buffered para-formaldehyde for fixation
Choose fluorochromes for optimal excitation and minimal crosstalk
Use water soluble embedding media which polymerizes and contains anti-bleach-agent
Use cover glass set-ups (cover glass thickness of 0.17 mm)
Use immersion objectives (oil or glycerol immersion)
For observation of living cells
Use heatable table, clima chamber with CO2 gas control
If you use inverse microscope, then use glass bottom cell culture dishes with water or glycerol-immersion objectives
But if the microscope is an upright microscope, then use plastic dishes and dip-in objectives.
Agreed with previous answer Also a solution of nickel sulfate (stock solution 1% w/v for NiSO4) can be used for fixing of bacterial cells as well.
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