1. It is difficult to remove the agar layer without disturbing the surface with a spatula, or if aspirating, small pieces of gel sometimes remain. One protocol online says that you can gently rinse the agar layer off with warm tap water and it will easily slide off. Has anyone ever tried this? Does it actually work, or are the fixed cells disturbed?
2. There are so many different protocols for the crystal violet stain.
My original recipe: 0.1% cryst. viol. + 0.2% formaldehyde + in PBS, which was a weak stain (no ethanol?)
Another says to use a working soln. of 1.0% crystal violet in 20% ethanol and dH2O (2), and others add methanol in addition to the ethanol. Could anyone share what works best for them?
Thanks! -Cheri
0.1% CV is sufficient. You have to first fix cells using methanol for 15 min. Stain for 10 min. Rinse. See cell under microscope after rinsing and drying.
If you want to quantify add 95% ethanol.
Instead of using agar, you may want to try either CMC (carboxymethyl cellulose) or Avicel which do not solidify and can just be aspirated from the wells.
As for your staining method, either formalin or EtOH or EtOH/MeOH mixtures should work. There isn't much difference. Also concentration of crystal violet should be sufficient to stain cells.
Not sure if it matters anymore given month later, but I've done both washing agar plugs off and pulling them off. In my case I believe I was doing influenza plaque assays. Either way I would use a 1:1 mixture of 2X EMEM and 1.6% agarose (Final 0.8%) and overlay the plate with 2 mL of EMEM-agarose mixture.
For washing off I would have a steady stream of luke warm water (hard to explain speed, but definitely don't want it to be going too fast). Sometimes smacking the side of the plate (using hand or against bench) helps loosen the plugs. And then you let the water just hit the outer edge of the plugs and they should just come off if done right.
I've had much better luck just peeling off the plug using a pipet tip (10-200 ul). I circle the edge of the plug with the tip and then gently work up one side, should come off.
For the crystal violet, either concentration should be fine. But the critical point is to mix the crystal violet powder in 100% alcohol first (otherwise it doesn't go into solution well and staining turns out poor) then dilute it to the final volume. I usually fix the monolayer first, but shouldn't really matter that much.
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