I have developed biofilm from marine bacteria on the glass coverslips and in the process to view it under the scanning electron microscope. However, from the readings of some studies, I want to ask about the need to use 2.5% gluteraldehyde prepared in artificial sea water for fixation. Is the use of the sea water is necessary since it is a marine bacteria?
Secondly, the post-fixation step. Some have washed the fixed cover slips with 0.1M sodium acetate buffer (pH 7.3), or used the osmium tetroxide but many studies just neglect this two steps. Can I directly jump to the dehydration step with a series of ethanol and the Hexamethyldisilazane (HMDS) after the fixation step? Will it affect the biofilm structure?
Thanks!
Hey Afifah - We've done a fair amount of bacterial and particle SEM on coverslips or similar structures. The one thing I'd ask first is what you're growing on the cover slip. If you have pure culture of some 'marine bacterium that you're cultivating in the lab, then it will be a slightly different than if you're working with biofilms established by organisms in actual sea water, where many species (some known, some not) and even eukaryotic organisms may be present in the film.
If you're working with just a culture that you know only includes one or more bacterial species, then proceed like this -
1) Very gently rinse nonadherent material from the coverslip with any lab neutral buffer (PBS is actually fine).
2) Then, immediately (but still gently) immerse the coverslip in 2.5% glutaraldehyde in PBS or, if you prefer, cacodylate buffer. Cacodylate is used as a fixative when you need a strong buffer without phosphate. For bacteria alone, this choice probably doesn't matter much. Immerse for at least an hour, and as long as over night.
3) Dehydrate the sample with progressively concentrated ethanol. We usually do immersions of 50%, then 75%, then 85%, then 95%, then 99% EtOH. The final immersion should be in 100% EtOH. Then you can remove and let the sample dry.
4) For bacteria and biofilm EPS, no HDMS is necessary.
If you have a sample where you may have more interesting creatures (e.g., amoebas, algae, etc.) then you should do the following -
1) Definitely use glutaraldehyde in cacodylate buffer.
2) After the EtOH immersions, do a final HDMS immersion as well.
The silane immersion at the end of the dehydration is intended to be a very strongly dehydrating step. When bacteria are glutaraldehyde fixed and submitted to the deep vacuum of the SEM stage, their cell walls are strong enough to stay intact. However, some eukaryotic structures that have a any remaining water within may be damaged when the water vaporizes as the sample is placed under vacuum.
Hope that's helpful, and definitely send pictures when you get it to work.
SEM in my experience is more fun that it is actually difficult, and you should just jump in and adjust your protocol as you go. Once you're started, you'll start to have an intuition for how to adjust your method.
Best, John
Hi John, really thankful for the steps and explanations you gave. It is more simpler than other resources. Will try to follow the steps first and see how the result come out.
Thanks a lot!
how 2% sodium acetate help to fixed biofilm formation in clinically samples?
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