Root-associated bacteria are able to influence plant fitness and vigor. A key step in understanding the belowground plant-bacteria interactions is to quantify root colonization by the bacteria of interest. Probably, genetic engineering with fluorescence markers is the most powerful way to monitor bacterial strains in plant. However, this could have some collateral problems and some strains can be challenging to label. In this sense, bacterial inoculation under properly controlled conditions can enable reliable and reproducible quantification of natural bacterial strains. In this protocol, we describe a detailed procedure for quantification of root-associated bacteria. This method applies non-aggressive samples processed with morphological identification and PCR-based genetic

Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca2+]i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca2+ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca2+]i in the range of 0.1-39.8 µM in small cells like those of

Legionella pneumophila, a Gram-negative bacterium and the causative agent of Legionnaires’ disease, exports over 300 effector proteins/virulence factors, through its type II (T2SS) and type IV secretion systems (T4SS). One such T2SS virulence factor, ChiA, not only functions as a chitinase, but also as a novel mucinase, which we believe aids ChiA-dependent virulence during lung infection. Previously published protocols manipulated wild-type L. pneumophila strain 130b and its chiA mutant to express plasmid-encoded GFP. Similarly, earlier studies demonstrated that wheat germ agglutinin (WGA) can be fluorescently labeled and can bind to mucins. In the current protocol, GFP-labeled bacteria were incubated with type II and type III porcine stomach mucins, which were then labeled with