NEW POSTER: Application of fluorochromes for assessment of cell viability and damage

Friday, 19 December 2008 14:59

Along traditional culture based methods more rapid methods for detection of viability of microbes are needed. Several fluorescent stains are on the market e.g. for indication of metabolic activity and viability of microbes or for the measurement of integrity of cell membranes. Applications include assessment of viability of beneficial microbes as well as revealing the mode of action of antimicrobial agents against harmful bacteria. Fluorescence of the fluorochromes can be detected with various methods, e.g. epifluorescence microscope, flow cytometer or fluorescence microplate reader.

Alakomi et al. investigated the applicability of a microplate scale fluorochrome assay for predicting the cell state of freeze-dried Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis preparations. LIVE/DEAD BacLight -Bacterial Viability Kit was used to viability assessment and DiBAC4(3) for the kinetic measurement of changes in bifidobacterial cell membrane functions during exposure to low pH. The developed microplate scale fluorochrome assay offered a rapid and robust tool for the viability assessment of probiotic preparations.

Microbicidal acids and several antimicrobial compounds belong to the membrane-active substances. Whether these compounds possess outer membrane (OM) disrupting activity against Gram-negative bacteria can be studied by using fluorochromes. Measurement of an increase of the uptake of a hydrophopic probe, such as 1-N-phenylnaphthylamine (NPN), is a widely applied method to monitor OM permeability.

Alakomi et al. showed that lactic acid was a powerful permeabilizer at concentrations of 5-10 mM, the effect being significantly stronger than that of a mineral acid (HCl) at similar pH values. In addition, organic acids present in berries, such as malic acid, sorbic acid and benzoic acid, were shown to be efficient permeabilizers of Salmonella as shown by increase in the NPN uptake assay and by LPS release.


Reference:

Alakomi H-L, Saarela M. Application of fluorochromes for assessment of cell viability and damage - detection of cell membrane integrity. 1st MoniQA International Conference. "Increasing Trust in Rapid Analysis for Food Quality and Safety", Rome, Italy, October 8-10, 2008.