Cell culture

WT strain MG1655 was grown in standard Terrific Broth (TB; Sigma) at 37°C and shaken at 200 rpm. An overnight culture was started from a frozen glycerol stock and was diluted 200× and grown for 5 hours until late-exponential phase [optical density at 600 nm (OD₆₀₀) of 0.6 ± 0.05].

Chemotaxis-gutted E. coli mutant RBB1050 (gift of R. Bourret, University of North Carolina) was grown in standard Tryptone Broth (TrB) supplemented with streptomycin (50 μg/ml; Sigma) for selection and shaken at 200 rpm. An overnight culture was grown from a frozen glycerol stock derived from a clonal population of nonchemotactic cells and was diluted 200× and grown for 6 hours to late-exponential phase (OD₆₀₀ of 0.4 ± 0.05).

To alter the average length of the E. coli cells, cephalexin (60 μg/ml) was added during the last 20 min to 2 hours of growth. To compute the average cell length for a given incubation time in cephalexin, still images were acquired using DIC microscopy and a 63×/1.4 numerical aperture (NA) objective at low cell densities. Using the image analysis software ImageJ (29), we measured the length of 200 to 400 individual cells for each cephalexin condition.

To alter the viscosity of the bacterial medium, a 20% stock solution of Ficoll PM-400 (Sigma) was prepared in TB by slowly dissolving 10 g of Ficoll powder in TB until reaching a final volume of 50 ml. This solution was filter sterilized and diluted with sterile TB to produce homogeneous solutions of the desired concentrations (5 to 10%). Solutions were stored at 4°C until time of imaging.

A quantity of 10 ml of stock solution was added to a No. 2 size Gilmont falling ball viscometer (Thermo Fisher Scientific, Waltham, MA) as per the manufacturer’s instructions. A glass bead was used for lower viscosity solutions (2 to 20 cP), and a stainless steel bead was used for higher viscosities (10 to 100 cP). The time required for the bead to drop a fixed distance was recorded. Three trials were performed for each sample, and the average time, t, was used to calculate the viscosity, μ, as per the following formulawhere K is a constant, ρ_c is the density of the bead, and ρ is the density of the solution. The drop time for the bead in water, which was assumed to have a viscosity of 1 cP, was used to determine the value of K. The same method was used to measure the viscosity of TB without Ficoll. We found that TB has a viscosity comparable to water.