Production of precise microbiology standards using flow cytometry and freeze drying

Production of precise microbiology standards using flow cytometry and freeze drying
Published Online: 31 Oct 2004
Received: 30 September 2003; Revised: 18 March 2004; Accepted: 21 April 2004
C. A. Morgan 1 2 *, P. Bigeni 1, N. Herman 1, M. Gauci 1, P. A. White 2, G. Vesey 1 3
Cytometry Part A
Volume 62A, Issue 2 , Pages 162 - 168
Wiley InterScience
1BTF Pty. Ltd., North Ryde, New South Wales, Australia
2School of Biotechnology and Biomolecular Sciences, Faculty of Science, University of New South Wales, Sydney, New South Wales, Australia
3Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, New South Wales, Australia

email: C. A. Morgan (contact@btfbio.com)
*Correspondence to C. A. Morgan, BTF Pty. Ltd., P.O. Box 599, North Ryde BC, NSW, 1670, Australia
Keywords
precision ? exact number of cells ? cell sorting ? bacteria ? standard reference materials ? BioBall
Abstract
Background
Quality control standards provide a quantity of microorganisms for routine use in microbiology to demonstrate the efficacy of testing methods and culture media. Standards are normally prepared by diluting a culture of microorganisms to obtain a suspension that contains an estimated number of colony-forming units per milliliter. The variability and inaccuracy of these standards increase the potential for false results. Flow cytometry has been used extensively to prepare precise standards of Cryptosporidium and Giardia that contain exact numbers of organisms in a volume of liquid ([1]). However, the same levels of accuracy have yet to be obtained for bacterial quality control standards.
Methods
A modification of a Becton Dickinson FACScalibur flow cytometer enabled 30 bacterial cells to be sorted into a single droplet, mixed with a cryoprotective solution within the droplet, and frozen in liquid nitrogen. The frozen droplets were then freeze dried for stable preservation of the viable bacterial cells.
Results
A freeze-dried sphere 3 mm in diameter was produced, which contained 30 microorganisms. The within-batch variation for these freeze-dried spheres was no greater than two standard deviations, and the between-batch variation was less than one standard deviation.
Conclusions
Bacterial reference controls can now be produced with consistent accuracy and unparalleled precision, thus enabling harmonization across the microbiological testing industry. ? 2004 Wiley-Liss, Inc.