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Applied and Environmental Microbiology, July 1999, p. 2877-2894, Vol. 65, No. 7
School of Microbiology and Immunology,
University of New South Wales, Sydney, 2052, Australia
Received 25 September 1998/Accepted 31 March 1999
Cell surface analysis often requires manipulation of cells prior to
examination. The most commonly employed procedures are centrifugation
at different speeds, changes of media during washing or final
resuspension, desiccation (either air drying for contact angle
measurements or freeze-drying for sensitive spectroscopic analysis,
such as X-ray photoelectron spectroscopy), and contact with hydrocarbon
(hydrophobicity assays). The effects of these procedures on
electrophoretic mobility, adhesion to solid substrata, affinity to a
number of Sepharose columns, structural integrity, and cell viability
were systematically investigated for a range of model organisms,
including carbon- and nitrogen-limited Psychrobacter sp.
strain SW8 (glycocalyx-bearing cells), Escherichia coli
(gram-negative cells without a glycocalyx), and Staphylococcus
epidermidis (gram-positive cells without a glycocalyx). All of
the cell manipulation procedures severely modified the physicochemical
properties of cells, but with each procedure some organisms were more
susceptible than others. Considerable disruption of cell surfaces
occurred when organisms were placed in contact with a hydrocarbon
(hexadecane). The majority of cells became nonculturable after air
drying and freeze-drying. Centrifugation at a high speed (15,000 × g) modified many cell surface parameters significantly,
although cell viability was considerably affected only in E. coli. The type of washing or resuspension medium had a strong
influence on the values of cell surface parameters, particularly when
high-salt solutions were compared with low-salt buffers. The values for
parameters obtained with different methods that allegedly measure
similar cell surface properties did not correlate for most cells. These results demonstrate that the methods used to prepare cells for cell
surface analysis need to be critically investigated for each microorganism so that the final results obtained reflect the nature of
the in situ microbial cell surface as closely as possible. There is an
urgent need for new, reliable, nondestructive, minimally manipulative cell surface analysis techniques that can be used in situ.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cell Surface Analysis Techniques: What Do Cell
Preparation Protocols Do to Cell Surface Properties?
*
Corresponding author. Present address:
Laboratório de Microbiologia Ambiental, Instituto de
Ciências Biomédicas, Universidade de São Paulo,
Cidade Universitaria, Av. Prof. Lineu Prestes, 1374, CEP 05508-900 São Paulo/SP, Brazil. Phone: 55 11 818 7205. Fax: 55 11 818 7354. E-mail: schneide{at}icb.usp.br.
Applied and Environmental Microbiology, July 1999, p. 2877-2894, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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