Hi,
I would like to look at bacteria devoid of their outer membrane by EM (negative staining).
I work on Spirochetes, and this would allow me to see the presence or absence of periplasmic flagella within individual cells.
I have found in a (very old) book the kind of picture I aim to obtain (see attached), but unfortunately, there is no Material & Methods section in the corresponding chapter. They only mention that the cells were treated with alpha-lytic protease.
Would anyone have some tips on what kind of protocol to use to achieve similar results?
Thank you for your help.
Cheers,
Lenka
Negative staining is an old, but very good method, though it offers less detail of the organism itself, being an outline only, than more direct methods might. I would suggest some research of what the outer membrane is made of. There may be a "solvent" that will act upon the membrane only (possibly a detergent at some dilution rate?). A well tuned, well aligned TEM can offer tremendous resolution of bacteria-sized organisms via direct bright-field mode when stained with Osmium and lead stain and uranel-acetate. Another method would be to fix, imbed and thin-section; then stain the sample for the maximum detail possible of both outer shell and internal organelles. The latter may require osmolarity adjustments to keep the bacteria life-like.
If you want to visualize only morphologically,you can mount the suspended bacterial sample directly on the C-coated grid performed negative staining with 2%heavy metal salt as uranyl acetate,lead citrate or phosphotungstic Acid.Air dried for 1 hr and visualize under TEm at low extraction voltage.But for anatomical study you have to performed a lot of steps as fixation,dehydration,resin infiltration,embedding and untrasectioning before TEM.
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