Cell binding studies were conducted to determine the association of the isothiazolone biocide with microbial cells. Chemostat-grown cells of S. cerevisiae were used in these studies in the presence of CMIT , MITand DCOIT biocides. Cells were treated with radioabeled biocide and radioactivity levels were used to determine the uptake of the biocide into the cells.
Fungal cultures were grown in New Brunswick Scientific BioFlow chemostats at 30 C and an agitation rate of 100 revolutions per minute. S. cerevisiae was grown in SDB+ 0.4% glucose at a generation rate of 5.6 h. Cells were harvested in sterile 50 mL Oak Ridge tubes, washed, and resuspended to an absorbance (660 nm) of 0.8 in 10 mM PO4 buffer (pH 7.2). Samples were frozen and protein content was determined.
Protein was determined using the Bio-Rad automated microassay in 96-well microplates using the Biomek 2000 Workstation. The plates were incubated at room temperature for 5 to 60 minutes. Optical density (OD) at 650 nm was measured using a Molecular Devices Thermomax MicroPlate Reader. Data was analyzed using the Molecular Devices SoftMax program.