Jun 02, 2026
Ethanol Tolerance Assay Experiment in 96-well Plates
- Daniel Olson1,
- Marybeth Maloney2,
- Kevin He3
- 1Dartmouth College;
- 2Research Assistant in Olson Lab;
- 3Undergraduate Student at Thayer School of Engineering
- Plate Cell Growth Ethanol Tolerance Assay Protocol
Protocol Citation: Daniel Olson, Marybeth Maloney, Kevin He 2026. Ethanol Tolerance Assay Experiment in 96-well Plates. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6okwzlqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 02, 2026
Last Modified: June 02, 2026
Protocol Integer ID: 318400
Keywords: ethanol tolerance assay experiment, evaluating ethanol tolerance, ethanol tolerance, specific responses to ethanol stress, throughput ethanol inhibition, ethanol stress, defined ethanol concentration gradient, ethanol concentration gradient, anaerobic fermentation condition, identification of ethanol, multiple ethanol concentration, microorganisms under anaerobic fermentation condition, metabolic engineering study, ethanol, engineered thermophilic biofuel, thermophilic biofuel, engineered strain, biofuel production research, dependent kinetic assay, strain characterization, producing microorganism, metabolic activity, maximum reaction velocity, tolerant phenotype
Disclaimer
Adapted from Marybeth Maloney's protocol and imported by Kevin He from the Olson Lab
Abstract
This protocol describes a high-throughput ethanol inhibition assay for evaluating ethanol tolerance in engineered thermophilic biofuel-producing microorganisms under anaerobic fermentation conditions. Cultures are exposed to a defined ethanol concentration gradient (0–35 g/L) in 96-well plates, and metabolic activity is quantified using an NADH-dependent kinetic assay measured at 340 nm. Maximum reaction velocity (Vmax) values can be calculated and normalized to untreated controls to compare strain-specific responses to ethanol stress. The protocol enables rapid screening of engineered strains, identification of ethanol-tolerant phenotypes, and quantitative assessment of inhibition across multiple ethanol concentrations. This method can be adapted for strain characterization, metabolic engineering studies, and biofuel production research.
Materials
- Ethyl alcohol, pure. Sigma # 459836 100ml
- Sodium pyrophosphate (aka sodium diphosphate) Sigma 221368
- Glycine Sigma G7126
- Semicarbazide HCL Sigma S2201
- NAD Sigma N6522
- K2HPO4
- KH2PO4
- BSA SigmaA3059
- Alcohol dehydrogenase enzyme Sigma A3263
Before start
Several days or a week prior: Make sure to have all the chemicals needed.
Protocol
Three days before:
Check plasticware, combi tips, 96-well growth plate, pipette tips along with film covers (inside the anaerobic chamber and re-supply if needed. Make sure to slightly open the corners of packages/wrappers before bringing plasticware through the airlock to ensure that it is anaerobic.
Check if you have sufficient growth media. If not, make fresh media, and bring into anaerobic chamber to ensure oxygen is removed.
Check for mid-log freezer stocks of the strains you want to test, frozen in the same type of media that you will use to run the growth plate. If you do not already have, start overnight cultures of strains to be tested.
Make duplicates of fresh ethanol standard solutions in water to be used during the growth (anaerobic) and the ethanol final measurement (Aerobic).
At your lab bench take a needle and syringe to collect a 1 – 1.5 mL aliquot of pure ethyl alcohol from stock bottle. Dispense into a 1.5 mL or 2 mL tube.
Two days before:
Prepare the anaerobic chamber. Fill chamber with gas mix and nitrogen until hydrogen concentration is e 2%. At this point, the oxygen concentration readings in the chamber become accurate. If oxygen concentrations are high, vacuum air out of chamber, and then refill with nitrogen and gas mix. Keep topping off the chamber with gas mix as it deflates. Repeat process if needed. The goal is to minimize oxygen concentrations and achieve hydrogen concentrations greater than or equal to 2%.
Check if you have buffer solutions (glycine buffer, ADH (Enzyme solution), and NAD stock solution) for the ethanol concentration assay. If not, make fresh buffers use milli-Q water, and store at the appropriate temperatures as indicated in the accompanying excel sheet.
Make aliquot(s) of media with or without antibiotics that will be needed to fill the plate. If using media with antibiotics, recommend adding excess antibiotics such that 35 µL of this concentrated antibiotic media yields the final desired antibiotic concentration in 200 µL media.
The day of:
Check that anaerobic chamber still has e 2% hydrogen, and 0 ppm oxygen. If not, continue to prepare the chamber as above.
Bring frozen, mid-log, working stocks of strains to be tested into anaerobic chamber, along with a repeat pipette, 1.0-10.0 µL multichannel pipette and a 96-well plate with 50ul dH20 with a sealing film for collecting the Initial Ethanol Assay samples.
Start the growth curve protocol on the computer adjacent to the anerobic chamber to let the plate reader get up to 55°C while you fill the plate.
Before opening the software check the signup sheet. Then open software (GEN5 3.11) and press Read Now on the pop-up window.
Select growth curve protocol (Dan 1 growth curve protocol 120hrs).
Press “Ok” at the next pop-up box.
Pop up box will report current temperature and show progress toward heating to 55°C starting point.
Set up your growth plate in the anaerobic chamber. Fill each well in the 96-well plate with 200 µL liquid as follows:
Before removing sample from the freezer vial rinse the combi-tip going up and down with plain media at least 3x. Now begin by filling the strain designated wells with 2.0 ul frozen culture using a multi-channel pipet using a 100ul combi tip so you can visually see the drop being added to the bottom of the well. Now put freezer vial back on ice and discard when assay is finished and running in the plate reader.
Very carefully add media to wells in columns 2 – 11. Add 200 µL of growth media. When using the 2.5 mL Combi tip set the multi-channel pipettor dispensing to #6 no greater. Then very carefully without touching the side of the well and holding the pipettor at an angle, add the media to the side of the well, to avoid splashing over into adjacent wells. If fewer than 10 strains are being tested, leave the additional wells empty.
Columns #1 and #12 remain empty.
Collecting Ethanol Assay samples in the anaerobic chamber. Set up the 96-well plate with 50ul dH20 / well with the cover. Remove sealing film.
Adding Ethanol Assay samples to wells. Using a multi-channel pipet add 5.0ul of each sample into the corresponding well. Gently place a plate film, not too firmly because you will be removing it to add the standard samples at your lab bench.
Seal the initial test plate with an RTS film in the anaerobic chamber, use a rubber roller or roll of tape to make sure the film is sealed to the wells, and set the plate into the plate holder (without plastic lid). Be sure to insert the plate into the reader with A1 corner of the plate in the back, right corner of the plate holder. Start the run by pressing “Ok” in the pop-up box with the temperature on the computer screen. This will move the plate into the machine and begin your growth curve.
During the anaerobic growth curve:
Check on the plate reader and software periodically to make sure the software is still running and has not aborted.
An hour before the growth curve is complete, defrost ethanol assay aliquots (NAD and ADH) on ice.
After the growth plate has completed (5 days).
Start the ethanol assay protocol on the computer connected to the aerobic plate reader so that the instrument can warm up to 30°C. Make sure to leave a sign on the plate reader that you plan to use it, so no one turns it off on you because this assay is very time sensitive. Immediately after you have added the assay buffer you want to hurry to the plate reader to read your samples.
Setting up the ethanol concentration assay plate:
(Aerobic) Mix assay buffer components in a 50 mL falcon tube on ice, keep on ice until ready to use.
Add 5.0ul standard sample to the appropriate well. Use columns 1 and # 12 to make a standard curve.
Using a multi-channel pipet carefully add 190 µL of the assay buffer to all wells in a new 96-well plate, to avoid spillovers since the wells will be very full.
Note: Work as quickly as possible in the next two steps to minimize reaction progression in some wells before you finish filling the plate.
Cover the plate with Sealing film use a use a rubber roller to make sure the film is sealed to the wells.
Place the plate in the plate reader holder, again with A1 in the back, right corner. Click “Ok” in the pop-up box on the screen to start the read.
Acknowledgements
Speak with Dan about retrieving and analyzing data.