I'm having trouble getting clear images of my bacteriophage under TEM. The process I've followed thus far is described below. Any help would be appreciated.
I've isolated bacteriophage active against Staphylococcus aureus bacteria from wastewater.
I grew these to high titer by infecting liquid cultures (S. aureus grown to OD600=0.1) with a low dose of bacteriophage (Multiplicity of infection = 0.1 to 0.05) and incubated until lysis was observed ~3-5 hours later.
These lysates were spun down at 5000g for 5 minutes and filtered through 0.22µm filters.
Chloroform was added (0.1 volumes / 1ml in 10ml), mixed, and left to incubate at room temp for 15-20 minutes with inversions every so often.
These were then centrifuged at 3220g for 10 minutes and care was taken to remove the lysate without touching the cell debris and chloroform layers.
We then used ultrafiltration columns (Cytiva: Vivaspoin 20, 100,000 MWCO) to resuspend the phages (trapped on the column membrane) in pure SM-buffer.
30µl of high titer phage was then fixed using paraformaldehyde and sent for TEM.
TEM images are stained with Uranyl acetate.
Please advise.
Thanks, Josh.
A few things come to mind. FIrst it may be your phage are just aggregating for some reason under the conditions you use. You could try adding something to reduce the aggregation (a touch of tween) or maybe some BSA. It could also be that you have a bit of cell debris left and the cell fragments have receptors for the phage to bind to. But I would think in this case you would see some free phages in certain fields and some aggregates like the above.
Are these the only things you see?
I agree with Michael J. Benedik . Just a thought: it looks like the darkened area is in the region of the highest phage aggregation. Maybe it is the reason for higher charging? Please note, that the resolution in that region is also affected - drastically reduced.
For me it looks like negative stain, not contamination. Just a bit too much of uranyl acetate.
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