I have been having problems getting TEM images of clostridium tetani phages. I have been using Formvar/carbon coated grids and can barely find any phage particles. I have also tried using 0.22 um filtered phage lysate (in thioglycollate medium) as well as PEG-8000 precipitated phage particles. In one instance, grids were glow discharged in order to increase hydrophilicity. Any suggestions would be most appreciated.
Prepare a sheet of mica and cover it with a thin layer of carbon by evaporation (i.e. prepare a carbon coated mica).Put a drop of your phage uspension on a plastic surface. cut a small piece (3x3 mm) from your carbon cotaed sheet of mica and float the carbon off from the surface of the mica onto the surface of the phage suspension (move the sheet of mica at an angle towards the surface of the drop). It you just touch it, the carbon will not float off from the mica surface. The carbon will now float on the drop of your phage suspension. Keep it floating for a a short time (5 to 60 seconds, depending on the phage concentration in your suspension). Now lift the floating carbon film off from the drop of phage suspension with the piece of mica you had used before and transfer the attached carbon film to a drop of negative stain (uranyl acetate). Float the carbon film on the surface of the drop of negative stain for 30 seconds. Now take an uncovered cupper grid (treated for a few seconds in alcolhol, let it dry) and lift the floating carbon film from underneath upwards. Blot the grid nearly dry with the edge of a piece of filter paper. Too much dry-blotting will remove too much of the negative stain. Remnants should remain around the phage particles.
Maybe your phage suspension has some problems. Is it high titer? is it purified? You can read this article: Basic phage electron microscopy.Ackermann, H.-W, 2009, Methods Mol Biol 501, 113–126.
Actually got in touch with Hans Ackermann for some tips on Phage EM. His recommendations were very helpful. Found glow discharge to not help so much as it was easier to coat the grids with poly lysine and add a little bacitracin to my phage sample
Dr. Cohen, Could you please share the method that you used that was successful? Please email me at [email protected]. Thank you
Dr. Cohen, I noticed that I provided the wrong/misspelled email address. Could you please share the method that you used that was successful? Please email me at [email protected]. Thank you
Hi. I did, back in 2015. I can resend the protocol that I used if you need it.
Dr Cohen, can I have the protocol? Could you please send it me at [email protected]
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating