Dr. Khalid Al-Hussein asks: > I have a graduate student who is interested in counting and > determining the viability of bacteria (pesudomonas. sp, cultured in > different pH concentrations) using Flow. > > Can anyone suggest how we could do this?. > I would highly recommend the recent article on bacterial viability in Current Protocols in Cytometry (Davey HM, Weichart DH, Kell DB, Kaprelyants AS: Estimation of microbial viability using flow cytometry. In: Robinson JP et al (Eds): Current Protocols in Cytometry. Wiley-Liss, New York, 1999. 11.3.1-11.3.20). In general, there are two approaches to measurement of bacterial "viability" using flow. One examines the integrity of the cytoplasmic membrane; cells which allow propidium, TO-PRO-3, Sytox Green (the last two are from Molecular Probes) to enter, or which do not retain fluorescein or related compounds produced by intracellular hydrolysis of esters such as fluorescein diacetate, are considered to be nonviable. The second uses the presence of membrane potential as an indicator of bacterial viability, using lipophilic cationic dyes such as cyanines and rhodamine 123, or lipophilic anionic dyes, such as oxonols, as membrane potential indicators. The outer membrane of Gram-negative bacteria blocks entry of lipophilic compounds, and membrane potential cannot be estimated satisfactorily in most Gram-negative organisms unless the outer membrane is permeabilized using an agent such as EDTA. My experience with Pseudomonas aeruginosa is that the organisms do not survive very long (probably less than 30 minutes) after permeabilization, making it essential to do the preparation of cells and the flow cytometry on a strict time schedule. If you use an indicator of membrane integrity, such as propidium or Sytox Green, permeabilization is not necessary; cells which take up these dyes are likely to be nonviable. However, cultures may also contain nonviable cells which do not take up the dyes. The addition of fluorescent beads at known concentration to aliquots of sample allows you to get absolute counts of bacteria in fluorescence flow cytometers in much the same way that absolute T-cell counts are now obtained; both B-D and Coulter sell bead kits for this purpose, but it is also possible to make up your own dilutions of beads from other vendors and count them with a haemocytometer or, better still, with an impedance-based (Coulter) or optical cell counter. However, there may be a problem with Pseudomonas aeruginosa because its forward scatter signal amplitude is substantially smaller than those of other common bacteria, making it difficult to trigger on forward scatter signals. This could lead to underestimates of bacterial counts. Molecular Probes sells dye kits for bacterial viability estimation by flow; you might try one. Some people have had better luck with these than others. By correlating your flow results with plate counts for a few days' worth of experiments, you should be able to determine whether the kit will work with your organisms. -Howard Shapiro
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:53:29 EST