We work with a difficult-to-stain Gram-negative bacterium. We need to stain the membrane and nucleoid with different dyes (unfortunately, we cant use the FM series of membrane dyes from thermofisher due to filter settings). The only way we can get the membrane to stain is by fixing our cells for 10 mins in 3.7% formalin. But does this affect overall morphology or the shape/condensation of the nucleoid? My literature searches say no, but if you have any paper/experience to share that would be great! Thanks in advance!
Hi Andrew,
If you use 3.7% formalin by diluting histology grade 37% formalin, please keep in mind that this also contains at least 10% of methanol and an unknown percentage of formic acid (depends on the age of the bottle)
see also: Fox, C H, F B Johnson, J Whiting, and P P Roller. “Formaldehyde Fixation.” Journal of Histochemistry & Cytochemistry 33, no. 8 (August 1985): 845–53. doi:10.1177/33.8.3894502.
Both methanol and formic acid will have an effect on the shape and condensation of the nucleoid.
A safer option would be to use 4% formaldehyde prepared from paraformaldehyde prills.
You could try Nile-red as an alternative membrane stain, membranes will appear in the red channel while more neutral lipids (like polybuterate) will show green/yellow fluorescence. It has also been used on unfixed bacteria and several strains apparently grow without issues in the presence of the dye
(see Spiekermann, P., Rehm, B., Kalscheuer, R. et al. Arch Microbiol (1999) 171: 73. https://doi.org/10.1007/s002030050681)
best,
Rob
Thanks Rob! I'll try switching to making my own fixative. About the Nile red, we have been using it and unfortunately the phb granules are so big in our species it sponges all the stain into them, making the membrane only very weakly stained. Good thoughts though :)
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