I performed TEM analysis for my bacteriohage.repetitively I got only salts images how to avoid the presence of salts during TEM analysis.
Hi Nattan,
With the information you provided I could think of the following solutions only.
Are you sure that you are using a high titer phage stock ? also some phages are unstable in CsCl. In such cases sucrose gradient could be an alternate choice. did you try making the grids hydrophilic before applying the sample ?
Dear Moumita Dutta,
Thank you for your answering, and I followed bellow given method.
The necessitate phages (titer 10-8-10-9) were filtered by syringe filter (0.22µm), moreover further concentrated by centrifugation at 20,000 for 60 min, 4°C, followed by re suspension of the pellet washed twice in (0.1M) Neutral Ammonium Acetate of the phage concentrates were placed on Formvar-carbon-coated copper grid, allowing the phages to adsorb for 20 min. The grids were then stained with 2% Uranyl Acetetate or 10 µl 2% Potassium Phosphotungstate (pH 7.4; 0.22-µm filters) for 10 min, and the grid was washed with filtered (0.22µm) several drops of Milli Q water and allowed to dry for 15 min. The grids were observed at 60-100 kV (H.W.Ackermann., 2011).
Dear Nattan,
Why don't you try any other buffer for re-suspension of purified phage particles like TM buffer. If you have a glow discharge unit available you can use it to make the grids hydrophilic and apply sample 3-4 ul, wait for 1 min, withdraw the excess liq with filter paper, apply UA and wait maximum 1 min, try without washing once. when you examine the grid go to lower mag and first try to search for a square with intact carbon and then search for good stain patches.
I hope this helps....
Thank you for your help,
Please give me detailed procedure with reference articles
You can search for negative staining of virus. it is very common technique. your staining time looks very long. you can also check my paper for detailed of bacteriophage staining.
Nattan Stalin
Hi .. you can use 0.9% normal saline for avoid the SALTS in your sample .. the first you must be centrifuge your sample at 7000 rpm for 30 m then you take your suspension and put it in the 0.9 NaCl and you have to use the formvar- grid carbon and uranyl acetate staining . this is protocols that i read in deference articles but when i took electron microscopic image for my samples i just pick up a plaque and then centrfuge at 4000 rpm for 10 m then i filtered throgh 0.2M AND stained with 2% uranyl acetate i took agood results .. read this article that i download for you i wish this can help you
I'm exactly on your way (@Moumita Dutta ) of preparation. Also, I can't change my buffer and it contains HEPES, NaCl and sucrose. The problem is these crystals! Any other way to remove crystals? Or any one knows that what can I do when doing preparation of samples to avid these crystals.
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