For each 8 to 9 plates, melt 500ml of NZY agar in the microwave. Set the microwave to high power for 9 min (18 min for two bottles). Be certain to release the seal on the cap by turning counter-clockwise one full turn before turning on the microwave!
By careful visual inspection, and while swirling the contents, ensure that the agar is completely melted. Place the bottle(s) into an incubator at 55°C.
Allow the agar to equilibrate at 55°C, which takes several hours (unless it is an emergency in which case they can be largely pre-cooled with cool tap water prior to placing in the incubator).
If colonies are to be picked with the Gel-2-Well, the plates must be perfectly level. In this case use a 50ml graduated cylinder and pour plates in the Pure-Aire (white) sterile bench
Assuming (and ensure that this is truly the case) multiple bottles prepared at the same time are all safely at 55°C, then from one bottle pour a plate with 50ml of agar. This plate will be used as a control to ensure that the ampicillin is working as it should. If this assumption is not 100% safe, then prepare a control plate in this way from each bottle. Measure the 50ml with a sterile graduated cylinder.
To the bottle(s) from which 50ml of agar medium was removed add 675 μl of ampicillin stock at 100 mg/ml. To all other bottles add 750 μl of ampicil
lin. Mix well by swirling. Note that if the ampicillin is added when the medium is too warm, the ampicillin will be degraded and be unable to do its job.
Take one or two bottles at a time from the incubator and pour plates with 50ml of agar. Measure the agar medium with the 50ml graduated cylinder from step 5. Measure and pour carefully so that bubbles are minimized.
While the agar is still completely fluid, and as needed, pass the flame of a Bunsen burner very briefly over the surface to eliminate air bubbles. Note that it is not the heat but the CO2 that removes the bubbles, so do not heat the agar.
Allow the plates to cool and thus solidify uncovered in the sterile hood for 1 hr.
Cover with lids and store upside down. Normally they will be used on the day they are prepared. If to be used the next day, store overnight at 4°C.