Dear colleagues,
I am working with MBEC device to grow pathogenic periodontal bacteria. Though I have read papers that used MBEC device and performed different microscopy techniques on biofilms, they do not explain:
- What they used to broke the peg from the lid without damage the biofilm
- How much time did they fix the biofilm without damage it
- What they used to cut the peg with the biofilm in order to do the SEM
Particularly, I am interested in analyze the biofilms using SEM (Scanning electron microscopy). Can you help me to solve my questions? Any other suggestion regarding the protocol for SEM or any other microscopy technique?
Thank you very much for your time to read this.
Best regards,
Here you can find manufacturers manual:
http://www.innovotech.ca/documents/MBEC-Procedural-Manual-v1.1.pdf
But it is just awful in SEM section. People do not know what they are talking about. (They even recommend to use epoxy to mount pieces on SEM stub!).
I have not used MBEC lids, but I have worked with dental biofilms. I would recommend to use the same type of wells (but fresh ones) for fixation and dehydration of biofilm:
Place lid with pegs on fresh wells with buffer (to wash out growth media) for 3 min.
Replace buffer with 2.5% gluteradehyde (fixative) for 1 hour (or overnight in a refrigerator).
Wash in water.
Dehydrate in graded solutions of alcohol (like 33%, 66%, 85%, 95%, 2x100%, 20 min in each) with final treatment in HMDS. Brake pegs needle-nosed pliers (as recommended by manufacturer), affix them with double sided carbon tape on Al stubs, coat in sputter coater, observe.
If it is too much trouble, you may try just to wash pegs in water (to get rid of media) and air dry them. Dental biofilms are pretty tough, you can observe them without special specimen preparation, but some changes in morphology will occur.
Dear Vladimir,
Thank you very much for answer my enquiries. In addition, I would like to know wether it is better to break the pegs before fix them, because I want to evaluate pre-treatment biofilm Vs post expousure to the antimicrobial. Do you think it is a good idea? do you think that by doing that I could damage the biofilms?
Thank you,
Edison - this is a great technical question. The plates are so great for growing biofilms, but just terrible for trying to do SEM on the surface. Two points -
1) Breaking the pegs from the lid of the plate is likely to really damage the biofilms you're trying to image. What we've done (see the pub below) is use a heated filament (in the form of a disposable electrocautery pen - http://www.tigermedical.com/Products/Disposable-Opthalmic-FineTip-Cautery__BOVAA00.aspx?gclid=CjwKEAjwlfO3BRDR4Pj_u-iO2U0SJAD88y1SrWrf28MhqJxxoUS6xng3ZtjHk7rd4pTKMNIjfVLOSBoCARHw_wcB) to melt lid around the individual peg. This allows you to cut out a peg without destroying it. It's not ideal, but it works.
2) You can indeed glue a single peg to an SEM stub, but it's important to use a cement with a suspension of graphite to be sure that you have electrical connectivity between the stub and the peg. If you're generous with the graphite cement, you should be able to sputter coat the peg without too much difficulty.
Lastly, as I'm sure you know - SEM is almost always useful for confirmatory work - it's very difficult to do quantitative assessments on peg-based biofilms by any imaging method.
Article Complement C5a Generation by Staphylococcal Biofilms
With needle-nosed pliers you may damage some areas of biofilm, but I believe there will be a lot of it left untouched for observation.
Of course tool advised by John G Younger should be very handy for this work. I do not have one, but I used (quite a few times) just heated on an alcohol burner scalpel blade for cutting out bottoms of wells with cell cultures on them. Works good.
Dear John and Dear Vladimir, I really appreciate your advises. I will follow your suggestions and I let you know how was the experiment.
Thank you both very much. Kind Regards!
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