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Proceedings Paper

Accurate live and dead bacterial cell enumeration using flow cytometry (Conference Presentation)
Author(s): Fang Ou; Cushla McGoverin; Simon Swift; Frédérique Vanholsbeeck
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Paper Abstract

Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with particular characteristics of interest. However most FCM cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. The easiest, most reliable and inexpensive way of obtaining absolute counts with FCM is by using reference beads. We investigated a method of using FCM with reference beads to measure live and dead bacterial concentration over the range of 106 to 108 cells/mL and ratio varying from 0 to 100%. We believe we are the first to use this method for such a large cell concentration range while also establishing the effect of varying the live/dead bacteria ratios.

Escherichia coli solutions with differing ratios of live:dead cells were stained with fluorescent dyes SYTO 9 and propidium iodide (PI), which label live and dead cells, respectively. Samples were measured using a LSR II Flow Cytometer (BD Biosciences); using 488 nm excitation with 20 mW power. Both SYTO 9 and PI fluorescence were collected and threshold was set to side scatter. Traditional culture-based plate count was done in parallel to the FCM analysis. The concentration of live bacteria from FCM was compared to that obtained by plate counts. Preliminary results show that the concentration of live bacteria obtained by FCM and plate counts correlate well with each other and indicates this may be extended to a wider concentration range or for studying other cell characteristics.

Paper Details

Date Published: 21 April 2017
PDF: 1 pages
Proc. SPIE 10081, Frontiers in Biological Detection: From Nanosensors to Systems IX, 100810Q (21 April 2017); doi: 10.1117/12.2251346
Show Author Affiliations
Fang Ou, The Univ. of Auckland (New Zealand)
Cushla McGoverin, The Univ. of Auckland (New Zealand)
Simon Swift, The Univ. of Auckland (New Zealand)
Frédérique Vanholsbeeck, The Univ. of Auckland (New Zealand)


Published in SPIE Proceedings Vol. 10081:
Frontiers in Biological Detection: From Nanosensors to Systems IX
Amos Danielli; Benjamin L. Miller; Sharon M. Weiss, Editor(s)

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