Quantification of Staphylococcus aureus and Escherichia coli in the liquid medium by fluorimetry and its use in phagocytosis assay.
A fluorimetric technique was compared with the plate counting method for quantification of viable cells of Staphylococcus aureus and Escherichia coli in the liquid medium. The fluorimetric assay measures the release of fluorogenic 4-methylumbelliferone (4-MU) from 4-methylumbelliferyl phosphate by the bacterial phosphatases. The increase in fluorescence was dependent on the size of bacterial inocula. Setting the fluorescence threshold at the middle of the logarithmic growth phase resulted in good linear relationship between bacterial counts and fluorescence (r = 0.99 for both Staph. aureus and E. coli). There was also an excellent correlation between the fluorimetric assay and the plate counting method in quantifying viable bacteria in saline (r = 0.99). Both methods were further compared for evaluation of extracellular bacteria following phagocytosis. The fluorimetric technique, in general, gave a higher percentage of phagocytosis than the plate counting method with statistical significance for E. coli.
Fang
W
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Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Helsinki, Finland. Weihuan.Fang@Helsinki.Fi
D000818:Animals; D002417:Cattle; D015169:Colony Count, Microbial / Q000379:methods*; D003470:Culture Media; D004926:Escherichia coli / Q000254:growth & development*; D005069:Evaluation Studies as Topic; D005470:Fluorometry; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D066298:In Vitro Techniques; D007202:Indicators and Reagents / Q000378:metabolism; D007962:Leukocytes / Q000502:physiology; D010587:Phagocytosis; D013211:Staphylococcus aureus / Q000254:growth & development*; D013997:Time Factors
D003470:Culture Media; D007202:Indicators and Reagents; C005359:4-methylumbelliferyl phosphate; D006923:Hymecromone