I followed given methodology:
Collected phage buffer containing phage sample 10ml , centrifuged at 12000 rpm for 10 min, top supernatant 10µl collected and diluted 10-5 of phage sample 100 µl added on grid and allow dry for 30-40 min at 18 °C under A/C then analyzed by TEM.
Here I attached my TEM photos, please suggest me for same.
Stalin,
You will need to negative stain the sample, either with 1 % uranyl acetate (if there is no phosphate present, or 1% phosphotungstic acid (if there is phosphate present). The grids should be glow discharged, or coated with poly-L-lysine to make them hydrophilic. Float grid (adsorb to sample) on 30 ul drop of phage, wait 2 min then blot sample with filter paper. Float grid through two consecutive drops of stain, then blot the grid dry from the side. Allow to completely dry in grid box then view. .All stains should be filtered.
good luck,
Michael Delannoy
Johns Hopkins SOM Microscope Facility
Dear Michael
I had appropriate TEM micograph for MRSA. I picked up a plaque and dropped it into 400 microliter of Cacl2 and transferred it to TEM lab.
We used carbon coated grid and glutaraldehyde for fixation and uranyl acetate for staining.
But in this way, there is no phage for P.aeruginosa in TEM. May my phages died during transferring. What is your idea???
Reza,
Fixed cells, phage will not stick to a glow discharged grid (hydrophilic). If fixation is necessary I generally attach live sample to my grid (if you can't glow discharge then float the carbon grid on 10% Poly-l-lysine. Rinse with DH20, then attach live sample.
(2 min) afterwards pass/float grid over fix (5 min) then rinse and finally your stain.
You can also place your plaque in Tris as this is acceptable with UA. If you can get some phosphotungstic acid (1% PTA)-(pH 7.0 filtered), then you can pick up plaques in PBS or phosphate buffer as this is compatible with PTA.
sincerely,
Michael Delannoy
Johns Hopkins SOM Microscope Facility
Correction: The Poly-L-Lysine concentration should be 0.01%. The 10% number is for charging coverslips.
Michael Delannoy As my research on Phage resistance mechanism, So I want to see the morphological feature how phage can not attach with bacteria. can you tell me how many phage I have to add with bacteria sample ?
Michael Delannoy Can I concentrate my phages at around 18000 rpm? That's the maximum I have in my lab. Your help will be greatly appreciated.
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