Bead-Based Flow-Cytometric Cell Counting of Live and Dead Bacteria
Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with characteristics of interest. Many flow cytometers cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. Here we describe the optimization and evaluation of a bead-based method for absolute cell counting applicable to basic flow cytometers without specialized counting features. Prior to the application of this method to an unknown concentration of a species of bacteria, a calibration experiment should be completed to characterize limits of detection and range of linearity with respect to the plate count method. To demonstrate the calibration process, mixtures of Escherichia coli or Staphylococcus aureus with proportions of live and dead cells ranging from 0% to 100% were prepared. These samples were stained using nucleic acid-binding dyes, and 6 μm reference beads were added (LIVE/DEAD® BacLight kit). The calibration samples were analyzed using bead-based FCM as well as the agar plate count method, and the results from both methods were compared.
Key wordsBacteria Bacterial cell enumeration Flow cytometry Counting beads Fluorescence Detection
We are grateful to the New Zealand Ministry of Business, Innovation and Employment for funding the Food Safe; real time bacterial count (UOAX1411) research programme. This work is in partial fulfilment of Fang Ou’s PhD thesis, who is grateful for the University of Auckland Doctoral Scholarship, the Todd Foundation Award for Excellence, the RHT Bates Scholarship and the Claude McCarthy Fellowship. Joni White is grateful for the Maurice and Phyllis Paykel Trust Research Scholarship in Health Sciences and the New Zealand Meat Industry Postgraduate Scholarship. The authors thank Stephen Edgar, Dr. Julia Robertson, Zak Whiting, and Janesha Perera for their laboratory support.
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