When I used TEM for my MS thesis, I used formvar coated grids, so the question is, what grids are you currently using? 

I would also ask how you are applying the phage to the grid? I modified ultracentrifuge tubes by adding epoxy resin to the bottom and letting it dry. Thus, I had a flat surface. Then I would use double-sided tape on a small circle of filter paper where I would stick the edge of my grid to the tape. I would then ultracentrifuge my sample so that the particles were forced down onto the flat surface. I was doing this to visualize phage in freshwater samples. For your sample, if you were just putting a drop onto the grid, I could see that you may get clumping on the edges as it dries. 

I use Bacitracin as spreading and wetting agent on carbon coated pioloform filmed grids. I put the grids onto a drop of virus suspension (30 sec to 5 min) on Parafilm. I shortly drain the grids by tipping it vertically on a twofold  Kleenex cleansing tissue, then place the grid (film downward) on a drop of distilled water with 0.01-0,001% Bacitracin solved in it, after some seconds I drain the grid again on the Kleenex tissue and touch a drop of uranyl acetate with the film (downward), drain again on Kleenex, and let the grid dry. It works fine with plant viruses in plant sap or with purified virus suspensions.

Dear Kenneth,

thank you really for uploading this your "old" article providing description of "simple" technique together with explanation how and why it works. I enjoyed reading the paper and it reminded me a bit of how to produce formvar film(s) prior to mounting on grids.

(the glow discharging / e-bombardement step of glass slides' surface followed by storage of the decorated carbon film in a humidified petridish for a longer period prior to detachment on water surface is really interesting and might solve some problems in detaching formvar films more easily than I often experienced).

For convenience (and correct citation) the bibliographic data:

Kenneth M. Towe (1965): Carbon Films for Electron Microscopy: A Reliable Method for Stripping from Glass Surfaces; Rev. Sci. Instrum.*) 36, 1247 (1965)

*)  Review of Scientific Instruments

 which fortunately can be found also in your really admirable publication profile at https://www.researchgate.net/publication/241457548_Carbon_Films_for_Electron_Microscopy_A_Reliable_Method_for_Stripping_from_Glass_Surfaces.

Article Carbon Films for Electron Microscopy: A Reliable Method for ...

You just need to clean the isolated phage particles. I suggest purification of the phage particles  in a discontinuous CsCl gradient (e.g. according to Sambrook & Russel (2001)) and then dialysis of the sample against SM buffer.

Preethi,

Here is a thought, try using a small amount (0.01-0.1%) triton-x 100 in your suspension just before adsorbing to the grids.  I had to do this to separate clumped microspheres (100 nm) I used for virus quantitation.  Let us know what works.

@Alicia Schultz (Hanson):  I regret you faced anonymous downvoting hopefully for the first and last time. I couldn't follow exactly the procedure of sampling / collecting your phage in freshwater samples by means of an ' air fuge '  rather than an 'ultracentrifuge' ... (not knowing what  - in your answer - the negative criterion for downvoting really was).

But I know that ultracentrifugation (e. g. use of 'Cytospin') can be a valuable tool for concentrating few viral / phage particles (c. f. e. g. https://www.ncbi.nlm.nih.gov/pubmed/22113738  :  

Prata C, Ribeiro A, Cunha Â, Gomes NC, Almeida A.: Ultracentrifugation as a direct method to concentrate viruses in environmental waters: virus-like particle enumeration as a new approach to determine the efficiency of recovery in:  J Environ Monit. 2012 Jan;14(1):64-70. doi: 10.1039/c1em10603a. Epub 2011 Nov 24

Abstract (added for convenience): 

Some health important enteric viruses are considered to be emerging waterborne pathogens and so the improvement of detection of these viruses in the aquatic environment is one of the most important steps in dealing with these pathogens. Since these viruses may be present in low numbers in water, it is necessary to concentrate water samples before viral detection. Although there are several methods to concentrate viruses in environmental waters, all present some drawbacks and consequently the method should be chosen that, despite its limitations, is adequate to achieve the aim of each study. As the effectiveness of the concentration methods is evaluated by determining the efficiency of viral recovery after concentration, it is important to use a simple and effective approach to evaluate their recovery efficiency. In this work ultracentrifugation, usually used as a secondary step for virus concentration, was evaluated as the main method to concentrate directly viruses in environmental water samples, using the microscopic enumeration of virus-like particles (VLP) as a new approach to estimate the efficiency of recovery. As the flocculation method is currently employed to concentrate viruses in environmental waters, it was also used in this study to assess the efficiency of the ultracentrifugation as the main viral concentration method in environmental waters. The results of this study indicate that ultracentrifugation is an adequate approach to concentrate viruses directly from environmental waters (recovery percentages between 66 and 72% in wastewaters and between 66 and 76% in recreational waters) and that the determination of VLP by epifluorescence microscopy is a simple, fast and cheap alternative approach to determine the recovery efficiency of the viral concentration methods.

PMID: 22113738         DOI: 10.1039/c1em10603a  (retrieved via PubMed) Unfortunately free access to content /pdf only by personal membership or subscription/ free Royal Society of Chemistry publishing personal account @ http://pubs.rsc.org/en/content/articlelanding/2012/em/c1em10603a#!divAbstract

It would perhaps be helpful if you could post the method you used with the ultracentrifuge tubes using epon resin and double-sided tape and filter paper a bit more detailed or - if available - to add the reference / biobliographic data for the technique if you followed a previously published 'recipe'. Thank you for your understanding and perhaps coming back to this thread. Best regards, WM.

THANK YOU ALL FOR YOUR IMPORTANT SUGGESTIONS AND HELP. I shall try using these methods and get back to which method is more effective for my phage sample. 

Agree with Kenneth Towe's explanation, "You should try to use a carbon-coated grid that has been treated with glow-discharge prior to adding your suspension." The glow-discharge method which I learned from Robley C Williams  can also be used with former coated grids. 

Very unlikely to have any clumping of phage if initially purified/concentrated using CsCl equilibrium density gradient centrifugation. See attached article for methods.