Microbiology (1999), 145, 1325–1333 Printed in Great Britain Flow cytometry and other techniques show that Staphylococcus aureus undergoes significant physiological changes in the early stages of surface-attached culture Ian Williams, 1 Frank Paul, 2 David Lloyd, 1 Robert Jepras, 2 Ian Critchley, 2 † Martin Newman, 2 John Warrack, 2 Theodora Giokarini, 1 Anthony J. Hayes 1 , Peter F. Randerson 1 and W. Alfred Venables 1 Author for correspondence : W. Alfred Venables. Tel :44 1222 874307. Fax : 44 1222 874305. e-mail : Venablescf.ac.uk 1 Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF1 3TL, UK 2 SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, 3rd Avenue, Harlow CM19 5AW, UK The techniques of flow cytometry, scanning and transmission electron microscopy, and confocal scanning laser microscopy were used to study the physiology of Staphylococcus aureus in the early stages of surface-attached culture, and to make direct comparisons with planktonic bacteria grown under the same conditions. Attached bacteria growing in nutrient-rich batch culture were found to go through the same growth phases as equivalent planktonic cultures, but with an exponential growth rate of about half that of the planktonic bacteria. Viability of attached bacteria was very high (around 100 %) throughout the first 24 h of growth. The size and protein content of attached bacteria varied with growth phase, and both measurements were always smaller than in planktonic bacteria at equivalent growth phases. Respiratory activity per bacterium, as measured by flow cytofluorimetry, and corrected for cell volume, peaked very early in attached cultures (before the first cell division) and declined from then on, whereas in planktonic bacteria it peaked in late exponential phase. Attached and planktonic bacteria showed thicker cell walls in stationary phase than in exponential phase. Membrane potentials of planktonic and attached bacteria were similar in stationary phase, but were much lower in exponential-phase attached cells than in the equivalent planktonic cells. It is apparent that a range of significant physiological adaptations occur during the early phases of attached growth. Keywords : Staphylococcus aureus, flow cytometry, attachment, biofilm INTRODUCTION In natural habitats most bacteria are found adhered to surfaces, either as individuals or as components of a biofilm (Marshall, 1976), and the great majority of problems caused by microbial growth (e.g. in industrial situations, disease states, or the colonization of implants ................................................................................................................................................. † Present Address : SmithKline Beecham Pharmaceuticals, 1250 South Collegeville Road, PO Box 5089, PA 19426-0989, USA. Abbreviations : CLSM, confocal laser scanning microscopy ; CTC, 5-cyano- 2,3-ditolyl tetrazolium chloride ; DiBAC 4 (3), bis-1,3-dibutylbarbituric acid trimethine oxonol ; FALS, forward angle light scatter ; FITC, fluorescein isothiocyanate ; SEM, scanning electron microscopy ; TEM, transmission electron microscopy. in human patients) involve bacteria in an adhered condition. Studies of the growth of adhered bacteria (or other microbes) have mostly concentrated on the prob- lems arising from such growth and on the means for its control or prevention, though there have also been numerous studies of the effects of adhesion on the genetic responses of the microbes (Goodman & Marshall, 1995). Whether the process of attachment to a surface itself directly triggers physiological change has not been addressed quantitatively. Thus, the most well- known property of adhered bacteria is their increased resistance to biocides (Costerton et al., 1987), but the extent to which this results from changes in bacterial physiology rather than a capacity of the polymer matrix of the biofilm to limit the rate of antibiotic diffusion is 0002-2976  1999 SGM 1325