Use of Image‐Based Flow Cytometry in Bacterial Viability Analysis Using Fluorescent Probes

Youwen Pan1, Laura Kaatz1

1 Technology Resources/Sterility Assurance, Baxter Healthcare Corporation, Round Lake, Illinois
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 2C.5
DOI:  10.1002/9780471729259.mc02c05s27
Online Posting Date:  November, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This protocol was developed to utilize imaging flow cytometry (IFCM) in combination with fluorescent dyes to both enumerate and analyze morphological features of live and dead cells in a mixed live/dead bacterial sample. The fluorescent dyes used in this protocol include 5(6)‐carboxyfluorescein diacetate (CFDA), which indicates the functional activity of esterase inside viable bacterial cells, and DRAQ7, a dye that exploits membrane‐compromised bacterial cells to enter and stain the cell. The live cell population stained with CFDA emits a fluorescent green color while the dead cell population stained with DRAQ7 emits a fluorescent red color, which allows the two populations to be distinctively separated by the IFCM system. Additionally, the cytometer captures a clear image of each object, which can then be analyzed for morphology features. The IFCM system is able to reliably, accurately, and precisely determine a bacterial cell concentration as long as the concentration of cells in a sample is no less than 1 × 103 cells/ml. The two dyes, CFDA and DRAQ7, have been demonstrated to be an effective stain combination for bacterial viability analysis. Curr. Protoc. Microbiol. 27:2C.5.1‐2C.5.11. © 2012 by John Wiley & Sons, Inc.

Keywords: image; flow cytometer; bacteria; CFDA; DRAQ7

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Instrument Decontamination, Initiation, and Calibration
  • Basic Protocol 2: Preparation of Bacterial Samples for Image‐Based Flow Cytometry
  • Basic Protocol 3: Data Acquisition
  • Basic Protocol 4: Data Processing and Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Instrument Decontamination, Initiation, and Calibration

  Materials
  • SpeedBead Calibration Reagent (Amnis, cat. no. 40040)
  • Debubbler: 70% isopropyl alcohol (IPA)
  • Sterilizer: 1:100 diluted bleach (4% to 6% sodium hyperchlorite)
  • Cleanser: Coulter Clenz (Beckman Coulter, cat. no. 8546929)
  • Rinse: Purified/distilled/deionized water
  • Sheath: PBS (Lonza, cat. no. 17‐515Q)
  • ImageStreamX (ISX) imaging flow cytometer (Amnis), equipped with a LED illuminator for the brightfield of an image, a 488‐nm blue laser (power range 10 to 100 mW), a 658‐nm red laser (0 to 120 mW), and a 785‐nm laser (0 to 70 mW) for a scatter signal
  • ImageStreamX imaging flow cytometer software: INSPIRE for sample acquisition and IDEAS for data processing and analysis
  • INSPIRE ImageStream system software user's manual

Basic Protocol 2: Preparation of Bacterial Samples for Image‐Based Flow Cytometry

  Materials
  • Escherichia coli strain K12 (ATCC #15222; or desired bacterial strain)
  • Brain heart infusion (BHI) agar and BHI broth medium (BD Difco, cat. no. 241830, or equivalent)
  • Purified water
  • 70% isopropyl alcohol (IPA)
  • Ice
  • 5(6)‐Carboxyfluorescein diacetate (CFDA; λex = 492 nm, λem = 517 nm; Sigma‐Aldrich, cat. no. 21879, or equivalent): Dissolve the powder in DMSO to make a stock solution at 20 mM
  • DRAQ7, 0.3 mM (λex = 599/644 nm, λem = 678 nm/694 nm; Biostatus Limited, cat. no. DR71000)
  • 0.5 M EDTA, pH 8.0
  • 1 M phosphate buffer, pH 8.0: Weigh and dissolve 14.2 g Na 2HPO 4 and 0.64 g KH 2PO 4 in purified water to make 100 ml of final solution
  • Pipets (multiple sizes able to deliver various volumes)
  • Incubator (capable of 30° to 35°C)
  • Inoculating loop
  • 15‐ml tubes (Corning, cat. no. 430791 or equivalent)
  • Shaker (capable of 800 to 1000 rpm)
  • Spectrophotometer
  • Centrifuge (capable of 3000 × g for 10 min)
  • Vortex mixer
  • Water bath (capable of 80°C)
  • Ice bucket
  • Sterile microcentrifuge tubes (1.5‐ml, particulate‐free)
NOTE: Filter all reagents through 0.22‐µm filters prior to use (except stains).

Basic Protocol 3: Data Acquisition

  • 1% (v/v) bleach (∼500 ppm sodium hypochlorite) prepared with purified water, adjust to pH 7 with acetic acid
  • 1% (w/v) sodium thiosulfate (Na2SO3) prepared with purified water

Basic Protocol 4: Data Processing and Analysis

  Materials
  • ImageStreamX imaging flow cytometer software: IDEAS for data processing and analysis
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bisha, B. and Brehm‐Stecher, B.F. 2009. Flow‐through imaging cytometry for characterization of Salmonella subpopulations in alfalfa sprouts, a complex food system. Biotechnol. J. 4:880‐887.
   Hammes, F., Berney, M., and Egli, T. 2011. Cultivation‐independent assessment of bacterial viability. Adv. Biochem. Engin./Biotechnol. 124:123‐150.
   Hammes, F. and Egli, T. 2010. Cytometric methods for measuring bacteria in water: Advantages, pitfalls and applications. Anal. Bioanal. Chem. 397:1083‐1095.
   Hoefel, D., Grooby, W.L., Monis, P.T., Andrews, S., and Saint, P.T. 2003. Enumeration of water‐borne bacteria using viability assays and flow cytometry: A comparison to culture‐based techniques. J. Microbiol. Methods 55:585‐597.
   Joux, F. and Lebaron, P. 2000. Use of fluorescent probes to assess physiological functions of bacteria at single‐cell level. Microbes Infect. 2:1523‐1535.
   Porter, J., Robinson, J., Pickup, R., and Edwards, C. 1995. Recovery of a bacterial sub‐population from sewage using immunoflourescent flow cytometry and cell sorting. FEMS Microbiol. Lett. 133:195‐199.
   Valm, A.M., Mark Welch, J.L., Rieken, C.W., Hasegawa, Y., Sogin, M.L., Oldenbourg, R., Dewhirst, F.E., and Borisy, GG. 2011. Systems‐level analysis of microbial community organization through combinatorial labeling and spectral imaging. Proc. Natl Acad. Sci. U.S.A. 108:4152‐4157.
   Wang, Y., Hammes, F., De Roy, K., Verstraete, W., and Boon, N. 2010. Past, present and future applications of flow cytometry in aquatic microbiology. Trends Biotechnol. 28:416‐424.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library