I plan to use cell sorting flow cytometer to enumerate E. coli in water samples
Oscar - This would be an unusual application for flow cytometry in most settings. The volume of liquid that can be interrogated in a give time is very small (
Hello Oscar. I agree with John. 1) By filtering environmental samples you can potentially get rid of big flocs and biofilm fragments. 2) Culturing is cheaper than flow cytometry. And I would also like to add that Flow Cytometry can not be applied to discriminate bacteria as well as when they are cultured in selective media and have their metabolism evaluated.
However, assuming you already know that, here are my thoughts on the subject... Since most flow cytometers allows one to evaluate particles as big as 50um, I believe that pre-filtering your samples considering that size will hardly eliminate bacteria from it in a representative amount. In addition to that, if you want a rapid and reliable method for counting and isolating bacteria, there are several protocols already standardized applying flow cytometry for both situations. You'll find literature and protocols using flow for quality control of Drinking Water and for assessment of the biodiversity in Environmental biology (including aquatic microbiology).
Answering your question, there are several dyes that can be applied to identify E. coli and other bacteria. The basic strategy here is to focus the staining procedure in their nucleic acids. The most frequently used dye for this purpose is SYBR green I, from Invitrogen. It allows total identification of microorganisms in your sample. Simultaneously, a viability dye can also be used to differentiate live and dead bacteria from the total detected cells. There are kits available that already offer both dyes in a single vial, using a nucleic acid staining dye for total cell identification and a viability dye for live/dead discrimination. BD has one called Cell Viability Kit which can be acquired with liquid counting beads to assess cell concentration in that given sample.
If you need some references of scientists that regularly use flow cytometry to evaluate aquatic samples and water quality, among others, search for papers from Dr. Daniel Vaulot (studies biodiversity and ecology in aquatic environments) and Dr. Frederik Hammes (microbiology and quality control). Both are members of Research Gate.
Hope this helps.
Regards,
R.
Method 1604 - Standard Methods for the Examination of Water & Wastewater. MI agar plates can be ordered through various vendors (Thomas Scientific) and the test is relatively simple. Filter sample (0.45 um filter) - (different dilutions can be used) and place on pre-made plates. Incubate for 24 hours @ 35C. Count blue colonies for E.Coli determination. Place under a 365nm UV light to count fluorescent Total Coliform colonies. Or method 9222B (m-Endo agar transferred to Nutrient Agar with MUG for 24 hours). Both of these media can be purchased dry and made in lab as needed.
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