Slide preparation
This protocol is extracted from research article:
Swimming bacteria power microspin cycles
Sci Adv, Dec 19, 2018; DOI: 10.1126/sciadv.aau0125

Slides were prepared following the procedure described in (15). Five milliliters of cell culture was centrifuged for 10 min at 2000g. Medium was poured from the tube, and the pellet was dried. The pellet was then gently resuspended (carefully avoiding scratching the pellet with the pipette tip) in TB, TB-Ficoll stock, or TrB. d-Mannose (RPI Corp.) was added to TrB for RBB1050 resuspension medium at a final concentration of 0.2% to prevent bacteria from clumping together. The volume of medium used to resuspend the pellet ranged from 0 to 50 μl to modulate volume filling fractions from 0.85 to 0.41. Twenty-five microliters of volume fraction–adjusted culture was transferred to a 1.5-ml microcentrifuge tube. An emulsion was created by pipetting and mixing the culture with 100 μl of mineral oil (Sigma) containing diphytanoyl phosphatidylcholine (DiPhyPC; 10 mg/ml; Avanti) to prevent droplets from coalescing. The microcentrifuge tube was then rolled, upright, between thumb and forefinger for ~10 s to create a gradient in droplet size across the height of the tube. Fifteen microliters of emulsion was pipetted from the top of the tube, to select for small droplets, and was sandwiched between two coverslips. Coverslips were rendered hydrophobic with silane (Sigma) ~1 hour before imaging to create droplets that were widest equidistant from the coverslips. This method produced many pancake-shaped droplets with heights of approximately 25 μm and diameters from 10 to 200 μm.

Because oxygen consumption by the bacteria limits the amount of time droplets remain motile (15), the time between all steps of slide preparation was minimized and monitored carefully. Setting time = 0 at the completion of centrifugation, the pellet was dried and resuspended until time = 60 s. Emulsion pipetting then lasted until time = 120 s. Slides were plated and rushed to the prepared microscope to be imaged immediately.

Volume fraction, ϕ, was calculated as per the following formulaEmbedded Imagewhere ϕ0 is the volume fraction of the pellet, V0 is the pellet volume, and d is the volume of fluid in which the pellet was resuspended. Using the maximum packing density of circles, 0.9, to set an upper limit to our estimate, we estimated the value of ϕ0 to be 0.85. Using a pipette, V0 was measured for three E. coli and three B. subtilis cultures. V0 for B. subtilis was 56 ±14 μl and for E. coli was 47± 2 μl. Volume fraction was then modulated by changing d during slide preparation.

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