Acid‐Fast Staining and Petroff‐Hausser Chamber Counting of Mycobacterial Cells in Liquid Suspension

Robin Treuer1, Shelley E. Haydel2

1 School of Life Sciences, Arizona State University, Tempe, Arizona, 2 The Biodesign Institute Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10A.6
DOI:  10.1002/9780471729259.mc10a06s20
Online Posting Date:  February, 2011
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Accurate and rapid cell counts of mycobacterial species in culture are difficult to obtain. Here, a method using modified Kinyoun acid‐fast staining was adapted for use with a Petroff‐Hausser sperm and bacteria cell‐counting chamber by using a liquid suspension staining technique. Cell counts obtained by this method were compared to viable cell counts by agar plate counting, revealing accurate correlation. Curr. Protoc. Microbiol. 20:10A.6.1‐10A.6.6. © 2011 by John Wiley & Sons, Inc.

Keywords: acid‐fast staining; mycobacteria; microscope; hemacytometer

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

Table of Contents

  • Basic Protocol 1: Carbolfuchsin Staining and Cell Counting of Acid‐Fast‐Stained Mycobacteria in Liquid Suspension
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Carbolfuchsin Staining and Cell Counting of Acid‐Fast‐Stained Mycobacteria in Liquid Suspension

  Materials
  • Mycobacterial liquid culture
  • TB carbolfuchsin KF (BD Diagnostics)
  • Sterile water
  • TB decolorizer TM (BD Diagnostics)
  • 5‐µm syringe filters
  • 1.5 ml microcentrifuge tubes
  • Microcentrifuge
  • Petroff‐Hausser sperm and bacteria counting chamber
  • Light microscope with 100× oil immersion objective
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Allen, J.L. 1992. A modified Ziehl‐Neelsen stain for mycobacteria. Med. Lab. Sci. 49:99‐102.
   Gilkerson, S.W. and Kanner, O. 1963. Improved technique for the detection of acid‐fast bacilli by fluorescence. J. Bacteriol. 86:890‐891.
   Kinyoun, J.J. 1915. A note on Uhlenhuths method for sputum examination, for tubercle bacilli. Am. J. Public Health 5:867‐870.
   Lukey, P.T. and Hooker, E.U. 2001. Macrophage virulence assays. In Methods in Molecular Medicine: Mycobacterium tuberculosis Protocols (T. Parish and N.G. Stoker, eds.) pp. 271‐280. Humana Press, Totowa, N.J.
   Neelsen, F. 1883. Ein casuistischer Beitrag zur Lehre von der Tuberkulose. Centrabl. Med. Wissenschaften 28:497‐501.
   Selvakumar, N., Rahman, F., Rajasekaran, S., Narayanan, P.R., and Frieden, T.R. 2002. Inefficiency of 0.3% carbol fuschin in Ziehl‐Neelsen staining for detecting acid‐fast bacilli. J. Clin. Microbiol. 40:3041‐3043.
   Selvaraju, S.B., Khan, I.U.H., and Yadav, J.S. 2008. Specific detection and quantification of culturable and non‐culturable mycobacteria in metalworking fluids by fluorescence‐based methods. Lett. Appl. Microbiol. 47:451‐456.
   Weyer, K. 1998. Laboratory Services in Tuberculosis Control. Part II. Microscopy. WHO Technical Bulletin 98.258. World Health Organization, Geneva.
   Zahrt, T. and Deretic, V. 2000. An essential two‐component signal transduction system in Mycobacterium tuberculosis. J. Bacteriol. 182:3832‐3838.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library