Begin with a multi-well plate containing growth media pre-inoculated with cellulose-producing bacteria.

In the treated well, the media is supplemented with 2-chloroethylphosphonic acid (CEPA).

Seal the plate to prevent gas exchange and incubate.

During incubation, CEPA hydrolyzes and releases ethylene, a gaseous molecule.

Ethylene activates bacterial cellulose synthase, which utilizes cytosolic UDP-glucose to synthesize cellulose.

Over time, the bacteria extrude cellulose through their membrane.

This promotes bacterial self-assembly at the air-liquid interface, forming a floating cellulose-based biofilm, or pellicle.

Following incubation, transfer the pellicles to a fresh multi-well plate. Add an alkaline solution and incubate at a higher temperature to lyse the cells.

Remove the alkaline solution. Repeatedly wash with agitation to remove cellular debris while retaining the cellulose.

Dry and weigh the pellicles. A higher dry weight in the CEPA-treated samples compared to the control indicates enhanced bacterial cellulose production in response to ethylene.

The pellicle assay is a standard analysis tool for bacterial cellulose production. PH and morphological analysis are detailed in text protocol. In preparation, harvest cells from starter cultures in triplicate and quantify the cells using a Petroff-Hauser Counting Chamber.

Then, prepare four medium master mixes with different CEPA concentrations. For each master mix, aliquot 60 milliliters of pH 7 SH medium and add 120 microliters of either 0, 5, 50 or 500 millimolar CEPA stock solution to obtain final CEPA concentrations of 0, 0.01, 0.1, or 1.0 millimolar. Mix these media by swirling the flasks.

One set of media is needed for each of the triplicates and a fourth set is needed to function as a sterile control. So, divide each 60 milliliter medium preparation into four 14 milliliter aliquots. Then, pre-cool the 14 milliliter aliquots before inoculating with their respective starter cultures for a final concentration of 100,000 cells per milliliter.

Keep all the inoculated tubes on ice to prevent cellulose production. Use the remaining 14 milliliter aliquots for sterile control wells. Now, from each 14 milliliter master mix, load two milliliter aliquots into six wells of a sterile 24 well plate for a total of four loaded plates per starter culture analyzed.

Carefully seal the plates with paraffin film and incubate them statically for seven days at 30 degrees Celsius. To collect a pellicle from the plate, depress one side of the pellicle to elevate the opposing edge and pick it up with forceps.

Next, individually transfer the pellicles into the wells of a six well plate. Treat them with 12 milliliters of 0.1 normal sodium hydroxide at 80 degrees Celsius for 20 minutes to lyse the cells. Then, remove the sodium hydroxide and neutralize the treated pellicles by thoroughly washing them with 12 milliliters of ultra pure water for 24 hours with agitation in a six well plate.

During this incubation, change the water every six hours. When the incubation is over, the pellicles should be white. Then, place the pellicles on silicon mats and dry them at 50 degrees Celsius for 48 hours to prepare them for dry weight measurement.