I do this at an industrial scale. It gets really annoying as you scale up to hundreds / thousands of different strains, all of which need pickable colonies.
A serial dilution 3 or 4 times seems to always do the trick. Typically on a robotic workstation you have to aspirate 6.5uL, then slowly dispense 5.5uL above the Petri dish (sbs format) and then stab into the agarose. Makes lovely perfectly-sized and separated wells, so 96 cell lines fit on only 3 or 4 plates.
With better plate reading you can get that down to 1 or 2 plates but it’s less reliable
Countable is a relative term in microbiology. I like that the author stuck to the phrase "countable colonies", since colony forming units are not really "countable as cells".
Ah, brings me back to the countless nights I spent counting plate after plate of HEK293 cells using a Haemocytometer [0], a light microscope, and a mechanical counter [1].
At least with HEK293 cells you could mostly tell if they were dead through the microscope (dead cells are darker).
I've made the same transition and feel the same way. I miss the bench, but I don't miss the late hours, vending machine dinners, and Christmas day cell culture maintenance.
For growing up phage you start by mixing different dilutions of phage with the host and a thin top agar, then plate it. On those plates with a countable number of plaques you work backwards to your plaque forming units (PFU) per mL. Once you know this you can produce "lacy plates." You add enough PFUs so that you expect the plaques to just touch. This produces a plate that has the appearance of lace. The web of surviving bacteria provide evidence that it actually grew, while delivering a high yield. Finally you scrape off the top agar, suspend it and run it through a filter. Good times.
look at the 1:10 plate. there's colonies that isn't yeast there. luckily, it got diluted out, or, maybe that plating was a bit more careless than the others.
i didnt mean to imply that all of the colonies aren't yeast.
I do this at an industrial scale. It gets really annoying as you scale up to hundreds / thousands of different strains, all of which need pickable colonies.
A serial dilution 3 or 4 times seems to always do the trick. Typically on a robotic workstation you have to aspirate 6.5uL, then slowly dispense 5.5uL above the Petri dish (sbs format) and then stab into the agarose. Makes lovely perfectly-sized and separated wells, so 96 cell lines fit on only 3 or 4 plates.
With better plate reading you can get that down to 1 or 2 plates but it’s less reliable