The Measurement of Bacillus mycoides Spore Adhesion Using Atomic Force Microscopy, Simple Counting Methods, and a Spinning Disk Technique W. Richard Bowen, Adam S. Fenton, Robert W. Lovitt, Chris J. Wright Centre for Complex Fluids Processing, Department of Chemical and Biological Process Engineering, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK; telephone: 44-1792-295862; fax: 44-1792-295862; e-mail: r.bowen@swansea.ac.uk Received 18 February 2000; accepted 10 June 2001 DOI: 10.1002/bit.10321 Abstract: An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hy- drophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydro- phobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be ¯exible and to promote the adhesion of the spore by in- creasing the area of spore contact with the surface. Re- sults from counting procedures using light microscopy matched the density of spores observed on the hydro- phobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify di- rectly the interactions of B. mycoides spores at the two surfacesinanaqueousenvironment.Themeasurements used ``spore probes'' constructed by immobilizing a sin- glesporeattheapexofatiplessAFMcantilever.Thedata showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydro- philic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractiveforceonthesporeswasmeasuredasthespores approached the hydrophobic-coated surface. At the hy- drophilic glass surface, only repulsive forces were mea- sured during the approach of the spores. The AFM force measurements were in qualitative agreement with the resultsofahydrodynamicshearadhesionassaythatused a spinning disk technique. Quantitatively, AFM mea- surements of adhesive force were up to 4 ´ 10 3 times larger than the estimates made using the spinning disk data.Thisisaconsequenceofthedifferenttypesofforces appliedtothesporeinthedifferentadhesionassays.AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells. ã 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 170± 179, 2002. Keywords: adhesion; atomic force microscopy; Bacil- lus mycoides; hydrodynamic shear INTRODUCTION The adhesion of microorganisms is an important phe- nomenon in both natural and industrial environments. In bioprocessing and food technology equipment, the adhesion of microbial cells to surfaces may lead to fouling of systems or the contamination of products. However, cell adhesion and bio®lm formation are es- sential in immobilized cell bioreactors for the ecient entrapment of the biocatalyst (Casey et al., 2000; Lele et al., 1996). In agriculture, the adhesion of microbes is also essential for ecient biological control of insect pest species (Sosa-Gomez et al., 1998) and weeds (Alt- man et al., 1990). In medicine and dentistry, the adhe- sion of bacteria and yeast to human tissue is an initial step in the onset of infection. In the case of implanted biomaterial surfaces, the adhesion of microorganisms can lead to the formation of bio®lms that result in pa- tient morbidity or mortality (An and Friedman, 1998). Once established at a surface, a microbial population is more able to withstand chemical or physical attackÐfor example, from reactor cleaning regimes or clinical anti- biotic treatmentÐcompared with a planktonic popula- tion (Bower and Daeschel, 1999). Thus, the ability to prevent microbial attachment to a surface has obvious bene®ts. Many microorganisms are able to form spores to aid population dispersion and survival. It is often these microbial vectors that compromise cleaning re- gimes rather than vegetative cells of the species. Con- sequently, for some microbial species, the control of spore adhesion is of greater relevance to the mainte- nance of sterility (Andersson et al., 1995; Brown, 2000). The attachment of a microbiological cell or spore and subsequent development of a surface population occurs in three phases. The ®rst phase, as the cell approaches the surface, is controlled by nonspeci®c physiochemical forces (as usually described by the DLVO theory). Once in intimate contact, the second phase of attachment Correspondence to: W. R. Bowen Contract grant sponsor: UK Biotechnology and Biological Sciences Research Council ã 2002 Wiley Periodicals, Inc.