Dear  Anusha,

SEM is more suitable for visualization of biofilms. TEM is a powerful tool for examination of ultrastructure of a single cell.

What was the sample preparation technique? Probably, the spherical structures on the picture are the bacteria, but it is very difficult to say anything more... 

What want you to visualize by TEM? If your target is ultrastructure of the intercellular interface, TEM is a suitable tool (with ultrathin slices preparation). If you want to study the morphology of a biofilm in general, it is not good idea to use TEM.

Good luck!       

As maintained dr  Denis, SEM is more suitable for visualization of biofilms. You can mesure density of biofilm by using colorimetry. The crystal violet was dissolved in 300 mikro mili of DMSO by pipetting up and down. Absorbance was read at 600 nm using a microplate

reader.  The OD values  were obtain by subtracting the OD measure for pure DMSO.

Thanks for the wonderful answers.. Kenneth, Dennis and Farzin. I have not made any fixation on cryotomy in the sample preparation. I had simply loaded the biofilm on the copper grid. Biofilm formation was previously confirmed by crystal violet based colorimetric method. What are the special care required for sample preparation for TEM? 

When I loaded simple bacterial culture before biofilm formation, I could only find the rounded bacteria but after the biofilm formation I can also see the slighly opaue films apart from the completely opaue part (which I presume to be the individual bacteria). Any comments on that?

Dear Anusha,

The thickness of biofilms is approximately 1000 nm (1 micron). It is too much for transmission and you can see only the borders of bacteria with low quality of pictures.

There are two classical ways of biological sample preparation for TEM: negative staining (mainly for viruses) and ultrathin slices (thickness is about 50 nm) preparation by a special device - ultratome. The are a lot of books about those techniques and it is very simple to found them by Google.

What is your main goal? What you want to visualize by microscopy?

Probably, it is not necessary to use a TEM for your task and it will be enough to use light microscopy or/and SEM.

Good luck!

I want to check whether with the treatment of a particular compound, biofilm formation of a known bacteria gets hampered..We are getting positive results through crystal violet assay but I want to validate it with other techniques.

In this case the conventional (after the sample preparation) or environmental SEM is the best way.

TEM is a tool for the ultrastructure examination - subcellular level - organelles, etc. 

There are a lot of articles about biofilm examination by SEM. For example:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC127943/

Good luck!  

Thank you Dennis! Suggestion is really helpful.

Anusha, you are welcome :)

The other way for biofilm examination is AFM:

http://www.ncbi.nlm.nih.gov/pubmed/20648545

It is a very powerful tool for the surface study. AFM provides not only visualization, even mechanical properties of the surface in the nanometer scale.

Good luck! 

Just to follow on Denis' great answers, yes - SEM would be more appropriate for what you want to study!

I've done TEM of Staph aureus (only for visualisation of individual bacteria, not for any quantitative work) and they look very similar to what you show in your micrograph. However, you wouldn't be able to see many differences at the biofilm level with TEM - SEM or even confocal microscopy should work much beter. 

Regarding AFM, it may be really tricky to image the biofilm, specially as I know it can reach several microns in thickness at times. Yes, AFM is great for surface characteristics of individual bacterial cells or even a monolayer, but once you start growing proper biofilms both the thickness and mechanical properties of it can make the imaging technique extremely complicated.

Good luck!