e> Pergamon W<u.Sci. Tech. Vol. 39, No.7, pp. 269-272, 1999 C 1999IAWQ Published by Elsevier Science Lid PrinlCd in Great Bnlaln. All righlS rcserved 0273-1223/99 $20.00 + 0.00 PH: S0273-1 223(99)001 77-8 USE OF SOFT X-RAY MICROSCOPY FOR ANALYSIS OF EARLY-STAGE BIOFILM FORMATION E. S. Gilbert*, A. Khlebnikov*, W. Meyer-Ilse** and J. D. Keasling* · Department of Chemical Engineering. Universiry of California at Berkeley. Berkeley. CA 94720. USA .. Center for X-ray Optics. Lawrence Berkeley National Laboratory. Berkeley, CA 94720, USA ABSTRACT Soft X-ray microscopy (SXM) using syncbrolroll radiation is a newly-available technology for high resolution imaging of hydrated specimens, making it potentially valuable for the study of bioflims. In this research, SXM was used to investigate bacterial spatial disaibution and bioflim structure during the early slages of colonization of an exposed surface. heudomofIDS pwida DMP-I formed a clnscly-~ed monolayer on a silicon niaide substralWD after 24 hours of growth. The initial development of a multilayer, joterlocking structure was observed. The soft X-ray images were laken with the XM-I transmission X-ray microscope, located al the Center for X-ray Optics, Lawrence Berkeley National Laboratory. The results indicate the potential of SXM 10provide new insights for the study of biofilms. C 1999 JAWQ Published by Elsevier Science Lid. All rights reserved KEYWORDS Microscopy; imaging; colonization; spatial distribution; biofilm structure. INTRODUCTION The first cells to attach to the substratum during the colonization of an exposed surface have a substantial effect on the spatial distribution and the long-tenn survival of bacteria in multispecies biofilms (Eliashberg and Keasling, 1998). However, the process of colonization and the subsequent organization of cel1s into multilayered structures is not we)) characterized, primarily because of the difficulty involved in observing intact early-stage biofilms at high magnification. Surman and coworkers (1996) evaluated eight different microscopic techniques for the examination of microbial biofilms. Of the eight, three techniques were considered to be suitable for quantitative analysis: transmission electron microscopy (TEM), scanning confocal laser microscopy (SCLM), and episcopic differential interference microscopy (DIC) with fluorescence. It was concluded that none of these techniques could achieve the high magnification of hydrated samples that would be necessary for detailed in vivo study of early-stage biofilm formation. A newly available technology that could elucidate the initial steps of bacterial colonization of surfaces is soft X-ray microscopy (SXM). SXM pennits high magnification, high resolution imaging of hydrated specimens 269 ---