Maintaining a relatively uniform concentration of ethanol in multiple NGM agar plates under the same conditions is difficult to achieve. Even when plates are wrapped in Parafilm, they seem to differ from each other in the concentration of ethanol in the NGM after a 24-hour period. The reason for this inter-plate variation is uncertain, although we know that ethanol evaporation occurs on each closed plate even at 20°C until liquid-gas phase equilibrium is reached. An effective strategy to lessen this variation may be to control the ethanol evaporation of each plate in a cohort by placing them under the same ethanol vapor pressure.
Ethanol (190 proof, USP grade, Sigma-Aldrich, St. Louis, MO, USA) was added to NGM agar plates seeded with E. coli OP50 to a final concentration of 100mM. The volume of ethanol added was determined by agar mass. The plates were placed unsealed in empty micropipette tip boxes (boxes from Fisherbrand Redi-Tip 0.5-10µL Ultra Micro) with an open petri dish plate filled with ethanol solution. The boxes were sealed with laboratory write-on tape and placed in an incubator at 20° ± 0.5°C. Using Ideal Gas Law and substituting the vapor pressure of ethanol at 20°C for P (0.0562 atm) (www.epa.gov/athens/learn2model/part-two/onsite/sparcproperties.html) and the volume of our box for V, the moles of ethanol needed in the open dish was determined to be 65.1 µmol, which was diluted to either 100 mM or 200 mM. Ethanol concentration was determined using a yeast alcohol dehydrogenase assay, reading NADH absorbance at 340 nm upon reaction completion. The study was done in triplicates.
Plates simply wrapped with Parafilm had an NGM agar ethanol sample standard deviation of 16.53 mM; plates placed in a closed system with 65.1 µmol ethanol at 100 mM concentration had a standard deviation of 13.26 mM; plates with 65.1 µmol ethanol at 200 mM concentration had a standard deviation of 6.50 mM. Our results show that compensating with 65.1 µmol ethanol at 200 mM concentration in our closed micropipette tip box decreases inter-plate variation by 60.7% compared to simply sealing plates with Parafilm.
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