External ID: CHOL_MOT

Protocol: Vibrio cholerae motility assay; Soft agar motility assay Methods
Fifty nl of test compound was dispensed to a dry assay plate with an Echo550. Then 50ul of LB containing 0.3% agar was added to the plates with a Matrix/Thermo Wellmate. The soft agar containing assay plates were allowed to solidify at room temperature for at least 4 hours. Bacterial inoculation was done with an Echo 555 which was used to transfer 2.5 nl of bacterial culture, containing approximately 20 bacteria, to the soft agar plate. The 2.5 nl drop of bacterial culture was delivered to the corner of the well and was accomplished by adjusting the well offset for the destination plate on the Echo. Plates were incubated at 30C for 14 hours then read using absorbance at 615nm on a Perkin Elmer Envision. The read location in the well was offset diagonally to allow maximum distance between the inoculation site and the read location. The primary read was absorbance at 615 to monitor bacterial growth and motility from the inoculation site to the read location. To differentiate between non-motile and non-viable, alamarBlue was added following the absorbance read. Plates were incubated for 60 minutes at 30C and then fluorescence intensity was determined by reading with an excitation wavelength of 535 and an emission wavelength of 590nm using a top read and a well center location. Non-motile viable colonies convert the alamarBlue while non-viable wells remained blue allowing the discrimination between antimicrobial compounds and motility inhibitors.

Comment: Possible artifacts in the V. cholerae motility assay include, but are not limited to, compounds that auto fluoresce (false positive for viability read) and compounds that absorb in the 500-600 nm range (false negative), or precipitate.
Outcome: Compounds that inhibited motility (OD615 assay) but that did not inhibit bacterial viability (alamarBlue assay) were identified as actives. A total of 18 compounds were ordered screened in the confirmatory (dose response assay) at concentrations ranging from 50 to 0.1 uM. Two compounds showed IC50s of less than 12uM in the motility inhibition assay and were inactive in the alamarBlue bacterial viability inhibition assay. Two additional compounds had a selectivity index of greater than 3.5, were also considered "Active". Compound that were active in the primary screen that were not tested in the confirmatory set were labeled with an Outcome of "Inconclusive".
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score.
Compounds in the primary screen are scored on a scale of 0-40 based on the difference % inhibition of the motility and growth inhibition results. In the interests of time, the compounds were selected on first run values. The second primary screen replicate was used for statistical analysis of the reproducibility of the primary assay.