Abstract
Here we describe a protocol which we have used to study the homeostasis intracellular in vivo in lactic acid bacteria (LAB) using a fluorescent probe. This type of probes can be used for determining changes in the pH of cytoplasm with high sensitivity, temporal resolution and technical simplicity as well as accessing the rate of change of intracellular pH in response to a stimulus from kinetic measurements on short time scales (Breeuwer et al., 1996; Molenaar et al., 1991). This protocol has been designed to measure the intracellular pH using the pH-sensitive fluorescent probe 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) in LAB, Enterococcus faecalis (E. faecalis), Lactococcus lactis (L. lactis) and Lactobacillus casei (L. casei).
Keywords: Fluorescent probe, Loading cell, Ph measurement, Lactic acid bacteria, Intracellular acid-base homeostasis
Materials and Reagents
Equipment
Procedure
Notes
Recipes
Acknowledgments
This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and CONICET (PICT2010-1828 and PIP/2012). F.S. is a fellow of ANPCyT, P. M. is a fellow of CONICET, S. H. A. and C. M. are Career Investigators of the same institution.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.