I am using 1% Ga cacodylate-filtered buffer for quick hard fix of my phage sample to grid then perform negative staining by 2% uranyl acetate. But the phage picture look like empty. Does it mean no DNA inside phage head? Some published phage head look like white. Does it mean it fill with DNA? Or is it any technical issue?
Please find phage picture in the attached file.
What the phage?
The heads look as Myoviridae, why there aren't any tails?
Sometimes a stain penetrates to the capsid and it looks as "positive staining". This effect is well-known in microscopy of viruses. One of its causes is the osmotic destruction of the capsid - the stain penetrates through pores.
Try to use just conventional PBS without GA and decrease a time of staining (10-20 sec for 2%-water UA).
Good luck!
Hi Denis, thank you. It is one of the Podoviridae group phage. Tail is difficult to observed even in TEM analysis. You said it is well-known in viral microscopy, it mean in TEM analysis the stain enter into the capsid in some way/reasons.
Dear Shakhinur, there is no need for GA cacodylate buffer since UA acts as fixative for viruses. Try resuspending your phages in 0.1 M ammonium acetate (pH 7). Professor H.-W. Ackermann has published extensively on methods for the TEM examination of bacteriophage samples, see http://www.aslo.org/books/mave/MAVE_182.pdf. Best of luck!
Agree with Denis Korneev's explanation, "Sometimes a stain penetrates to the capsid and it looks as "positive staining".
Suggest using PTA (phosphotungstic acid) as more reliable negative stain.
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