My upstairs GTA colleague and I were surprised that the Bioscreen growth curves in the previous post didn't show a dip in OD600 of the GTA-overproducer strain like that seen in manual (non-automated) growth curves.  This dip is thought to be caused by the lysis of GTA-producing cells as GTA production peaks when cells hit stationary phase.We thought part of the problem might be that I used the standard YPS medium which is based on…

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Here's the results of the Bioscreen growth curves I ran for Rhodobacter capsulatus strains:Each dot is the mean OD600 of 15 replicate wells, each containing 300 µl of culture, with ODs read every 20 minutes for 45 hours.  The cultures all grew up at about the same times, but I've shifted the X-axes so the curves don't overlap.  OD values below about 0.015 are not significantly above the backround absorption of the culture medium. The…

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In a few weeks I'll be headed for the Maritimes, for the final part of my sabbatical work on Gene Transfer Agent.  But before I leave here I want to run some detailed growth time courses on GTA-producing strains, taking advantage of the BioScreen machine belonging to the lab next door.I'll first do a trial run with all the strains I have,  to check the basic growth kinetics under the Bioscreen growth conditions.  Then I'll…

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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…

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Aacckk!  I've spent more than a month trying to get decent R. capsulatus phage plaques on R. capsulatus lawns.  Still no consistent success.  In one experiment I had much better plaques on cells of strain DE442 (a GTA overproducer), but that did not replicate.  I suspect that non-tiny plaques depend on exactly the right balance of the cells' physiological state, their density, the agar concentration, the culture medium, and other factors I haven't attempted to…

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I have a two-pronged plan to get a phage strain that gives good enough plaques for my GTA-as-vaccine experiments.I obtained reasonable titers of two phages, 'Titan' and 'Saxon'.  I'll invest a couple of weeks to see if I can get better and more reproducible plaques with either of these.  The genome sequences of these phages are not closely related.First, improve the plaquing conditions:  The researcher who isolated the phages recommends using for the lawn cells…

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My phage titering gave disappointing results.  Three of the five lysates gave no plaques at all, and the other two gave small indistinct plaques that couldn't be accurately counted or characterized. I took some photos of the plaques I did see.  The top photo is a section of one lawn, with several thousand tiny indistinct plaques.  (The blurry markings are the label on the bottom of the plate.)  The second photo is a closeup  of…

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Planning today's work:Titering the phage lysates should be a no-brainer, but it's been a long time since I worked with phage so I'd better think things through before I do it.I have 15 µl of each of 5 phage stocks ('lysates').  The original titers (plaque-forming units/ml, pfu/ml) are written on the tubes - they range from 6x10^5 pfu/ml to 2x10^11 pfu/ml.  But the lysates are probably quite old (maybe 2 years, maybe more), so their…

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Planning today's work:Titering the phage lysates should be a no-brainer, but it's been a long time since I worked with phage so I'd better think things through before I do it.I have 15 µl of each of 5 phage stocks ('lysates').  The original titers (plaque-forming units/ml, pfu/ml) are written on the tubes - they range from 6x10^5 pfu/ml to 2x10^11 pfu/ml.  But the lysates are probably quite old (maybe 2 years, maybe more), so their…

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This was going to be a post where I do the planning to titer my new lysates today, but it turned into an explanation of how microbiologists use plaques in lawns of bacteria to study phages.Wait, what's a 'lawn' and what's a 'plaque'?  A lawn is a thin layer of confluent bacterial growth, usually created by mixing a relatively large number of cells (≥10^6) with liquid agar solution ('top agar' or 'soft agar' and pouring…

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