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Applied and Environmental Microbiology, August 2004, p. 4980-4988, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4980-4988.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Model System for Growing and Quantifying Streptococcus pneumoniae Biofilms In Situ and in Real Time

R. M. Donlan,1* J. A. Piede,2 C. D. Heyes,3 L. Sanii,3 R. Murga,1 P. Edmonds,2 I. El-Sayed,4 and M. A. El-Sayed3

Biofilm Laboratory, Division of Healthcare Quality Promotion, National Center for Infectious Diseases, Centers for Disease Control and Prevention,1 School of Biology,2 School of Chemistry and Biochemistry, Laser Dynamics Laboratory, Georgia Institute of Technology, Atlanta, Georgia,3 Department of Otolaryngology— Head and Neck Surgery, University of California at San Francisco, San Francisco, California4

Received 18 December 2003/ Accepted 26 April 2004

Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 105 cells per cm2, while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.

* Corresponding author. Mailing address: Biofilm Laboratory, ELB/DHQP/NCID, Centers for Disease Control and Prevention, Mail Stop C-16, 1600 Clifton Rd., N.E., Atlanta, GA 30333. Phone: (404) 639-2322. Fax: (404) 639-3822. E-mail: rld8{at}cdc.gov.

Applied and Environmental Microbiology, August 2004, p. 4980-4988, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4980-4988.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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