Rapid In Situ Assay for Indoleacetic Acid Production by Bacteria Immobilized on a Nitrocellulose Membrane

TLDR

The assay uses L‑tryptophan‑supplemented medium, a nitrocellulose overlay, and Salkowski reagent on filter paper to produce red halos around colonies within 1 h. The assay rapidly and specifically detects IAA production, distinguishing producers from non‑producers within 1 h, with sensitivity down to 50 pmol, and enables high‑throughput screening of bacterial populations.

Abstract

We have developed a new assay that differentiates between indoleacetic acid (IAA)-producing and -nonproducing bacteria on a colony plate lift. Medium supplemented with 5 mM L-tryptophan is inoculated with isolates of interest, overlaid with a nitrocellulose membrane, and then incubated until bacterial colonies reach 1 to 2 mm in diameter. The membrane is removed to a filter paper saturated with Salkowski reagent and incubated until distinct red haloes form around the colonies. The colorimetric reaction to IAA is limited to a region immediately surrounding each colony, is specific to isolates producing IAA, occurs within 1 h after the membrane is placed in the reagent, and is sensitive to as little as 50 pmol of IAA in a 2-mm 2 spot. We have used this assay for quantifying epiphytic and endophytic populations of IAA-producing isolates of Pseudomonas syringae subsp. savastanoi and for detecting IAA-producing colonies of other pseudomonads and Erwinia herbicola . The assay provides a rapid and convenient method to screen large numbers of bacteria.