Currently, I am struggling to obtain quality SEM images to assist in the classification of phages obtained from sewage samples from hosts E.coli and K.pneumoniae. I have followed the standard protocols for isolating and propagating phages. PEG precipitation was used to concentrate the phages. I sent 10ul of phages (106 to 107 titre) which were used to fix (air fixed) phages to glow-discharged grids, followed by a 1% uranyl acetate stain. What was noticeable in the images is that there was plenty of cellular debris, although samples were filtered through 0.2 micron filters, making it difficult to distinguish clear phage morphology. Out of my 9 samples I could only distinguish morphologies similar to Tectiviridae, Microviridae and Siphoviridae.
CsCl gradient centrifugation could give me better results but such a method may not be feasible for me. Is there any advice to providing cleaner sample for better quality images or is the method of imaging not suitable for the clarity of image I am looking for?
Hi Aaron,
I think you should purify phages. If you want to good quality picture, you need to go through CsCl gradient centrifugation method. You may try Phosphotungstic acid for negative staining. I hope you got the better picture in TEM.
First of Phages images are possible by TEM because SEM has not that much resolution. I think these are also TEM pics.
1.PEG method is good but give washings for more than 3 times,so before starting make it sure phage titer should be very high because we lose some of the phages in washing step . Washing ,purity and High titer are most important factors to get good images. I
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