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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.

Cell Surface Analysis Techniques: What Do Cell Preparation Protocols Do to Cell Surface Properties?

Richard S. Pembrey, Kevin C. Marshall, and René P. Schneider*

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.

* 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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