Abstract
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Vives-Rego J, Lebaron P, Nebe-von Caron G (2000) Current and future applications of flow cytometry in aquatic microbiology. FEMS Microbiol Rev 24(4):429–448
Berney M, Hammes F, Bosshard F, Weilenmann H-U, Egli T (2007) Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry. Appl Environ Microbiol 73(10):3283–3290
Duedu KO, French CE (2017) Two-colour fluorescence fluorimetric analysis for direct quantification of bacteria and its application in monitoring bacterial growth in cellulose degradation systems. J Microbiol Methods 135:85–92
He S, Hong X, Huang T, Zhang W, Zhou Y, Wu L, Yan X (2017) Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry. Methods Appl Fluoresc 5(2):024002
Stocks SM (2004) Mechanism and use of the commercially available viability stain BacLight. Cytometry A 61(2):189–195
ThermoFisher Scientific (2004) LIVE/DEAD ® BacLight™ bacterial viability and counting kit (L34856) Product Information. https://tools.thermofisher.com/content/sfs/manuals/mp34856.pdf. Accessed 22 Feb 2018
Gasol JM, Del Giorgio PA (2000) Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities. Sci Mar 64(2):197–224
Alsharif R, Godfrey W (2002) Bacterial detection and live/dead discrimination by flow cytometry. BD Biosciences. https://www.bdbiosciences.com/documents/Bacterial_Detection_Live_Dead.pdf. Accessed 3 Mar 2018
Peniuk GT, Schnurr PJ, Allen DG (2016) Identification and quantification of suspended algae and bacteria populations using flow cytometry: applications for algae biofuel and biochemical growth systems. J Appl Phycol 28(1):95–104
Ou F, McGoverin C, Swift S, Vanholsbeeck F (2017) Absolute bacterial cell enumeration using flow cytometry. J Appl Microbiol 123(2):464–477
Cooper GM (ed) (2000) The cell, 2nd edn. Sinauer Associates, Sunderland, MA
Brando B, Barnett D, Janossy G, Mandy F, Autran B, Rothe G et al (2000) Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. Cytometry 42(6):327–346
Wulff S (ed) (2006) Guide to flow cytometry. Dako Cytomation. http://gene-quantification.com/dako-facs-guide.pdf. Accessed 30 May 2017
Khan MMT, Pyle BH, Camper AK (2010) Specific and rapid enumeration of viable but nonculturable and viable-culturable gram-negative bacteria by using flow cytometry. Appl Environ Microbiol 76(15):5088–5096
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ou, F., McGoverin, C., White, J., Swift, S., Vanholsbeeck, F. (2019). Bead-Based Flow-Cytometric Cell Counting of Live and Dead Bacteria. In: Iovino, F. (eds) Streptococcus pneumoniae. Methods in Molecular Biology, vol 1968. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9199-0_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9199-0_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9198-3
Online ISBN: 978-1-4939-9199-0
eBook Packages: Springer Protocols