Multipurpose laminar‐flow adhesion cells for the study of bacterial colonization and biofilm formation

Abstract

The ability to reproducibly colonize substrata with concomitant monitoring of biofilm development is essential to laboratory studies of microbial activities at surfaces. A test system was developed whereby on‐line, non‐destructive measurements (open circuit potential [OCP], direct cell counts, bioluminescence, oxygen) of bacterial colonization and metabolic activity could be obtained from a series of laminar‐flow adhesion cells. The cells consisted of two high‐density polyethylene blocks 32 mm H × 65 mm W × 178 mm L, with a 1 mm deep flow channel milled in the top block. A glass viewing window enabled direct observation of a removable, flush‐mounted 25 x 50 mm coupon, which was recessed into the bottom block. Laminar‐flow conditions were validated at linear flow rates up to 1.3 cm s⁻¹ (20 ml min⁻¹). Reproducible colonization of Pseudomonas fluorescens monocultures was obtained, with 72 h direct‐counts and viable counts ranging from between 5.1 to 10.4 × 10⁷ and 1.4 to 2.2 × 10⁷ cells cm⁻², respectively, for replicated flow cells (n=20). In situ pulse‐labeling of intact biofilms with ¹⁴C‐acetate resulted in reproducible incorporation of the radiolabel into cell membrane lipids, with 60 min uptake values ranging from 1.0 to 1.8 × 10⁻⁵ DPM cell⁻¹. On‐line OCP measurements remained stable in sterile test systems, varying by less than 12 mV, for over 80 h. The flow cells provided a means for reproducibly colonizing various substrata under in situ environmental conditions without perturbing the developing biofilms.

Key words:

• On‐line biofilm monitoring
• flow‐cells
• colonization studies
• Pseudomonasfluorescens

Notes

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