Field of Science

Phage plaqueing still sucks - what to do now?

I feel like I've been sucked down a hole of trying to get consistently countable plaques from the Rhodobacter capsulatus phage I'm testing.  After seven weeks of plaqueing with various combinations of strains and agar concentrations and cell densities, I'm no closer to having a well-behaved phage I can use to test the GTA-as-vaccine hypothesis.

Along the way I've eliminated various sub-hypotheses:

1.  The plaques are tiny/faint/blurry/invisible because the phage capsids have long fibers that reduce diffusion through the top agar:  Test - use increasingly dilute top agar.  Top agar us usually 0.75% agar; I've taken this down to 0.3% (the lowest concentration that's still stable enough to handle). The first time I got somewhat larger plaques, but this was not reproducible.

2.  The plaques are tiny/faint/blurry/invisible because GTA gene products contribute to phage production:  Test:  Plaque phage on a GTA overproducer strain.  Result:  On the first try, plaques on the overproducer seemed larger.  But this was not reproducible.

3.  The plaques are tiny/faint/blurry/invisible because the GTA-as-vaccine hypothesis is true:  (Plaques can't grow because rapid diffusion of GTA particles allows surrounding cells to become CRISPR-resistant to the phage before the phage gets to them.)  Tested by plaqueing the phage on cells deleted for the entire GTA operon and for the separate endolysin.  Result:  Plaques on these '∆∆' strains are just as lousy (maybe more lousy) than on the GTA-producer parents.

4.  Variant (large) plaques contain mutations that increase infectivity or diffusion:  I made new lysates from a couple of big plaques that spontaneously appeared among the tiny plaques, but these lysates still gave tiny or no plaques

I know that the phage lysates do infect and kill the cells, and do produce progeny phages.   When I put a spot of sufficiently-concentrated phage onto a lawn, all the cells die, and when I make a lysate with this 'clear' top agar, I get way more phage then I put in.  Can I use the lysate to test the GTA-as-vaccine hypothesis even though I don't have countable plaques?

What would I do?  Here's an earlier blog post where I laid out a crude plan and a list of all the things I'd need to find out before actually doing the experiment that would test the hypothesis:

Luckily, after I wrote the above I made another grand attempt at titering the phages on the various strains.  Well, I made a sloppy attempt, learned from at and made a better attempt, which more-or-less worked. 

Basic test:  Pour lawns of the test strains, using cells concentrated from 400 µl of culture, in 1.5 ml of 0.4% top agar.  Put 10 µl spots of different dilutions of phage lysates onto these lawns, let the liquid absorb, and check the next day.  Yesterday I did this using photosynthetically grown cells (supposed to make better lawns) and today I've repeated it using cells grown aerobically in the dark.  Here's yesterday's result for one of the two phage and one of the six strains:

The central clear spot is undiluted lysate, and the other spots are 10-fold dilutions of that.  For undiluted, 10^-1, 10^-2 and 1-^-3, the spot is clear (all the cells have been lysed).  The 10^-4 spot still has patches of non-lysed lawn, and the 10^-5 and 10^-6 spots have distinguishable plaques.  Two of the four healthy strains gave countable plaques (27 and 29), which is nicely consistent.

I'll wait for tomorrow's results before proceeding.