Differences in Absorbance (OD₆₂₀) of Various Strains of Staphylococcus aureus as Determined by Direct Microscopic...

A modification of the Breed direct microscopic method for counting Staphylococcus aureus and its use in preparing standard curves with the spectrophotometer is described. Eight strains of S. aureus were grown on various media. The organisms were formalin killed, diluted in phosphate buffered saline (PBS), and mildly sonicated to disperse clumped organisms. Each suspension was further diluted in PBS to yield absorbance (OD₆₂₀) readings of 0.60 to 1.60 at OD₆₂₀. After measuring absorbance, each dilution of S. aureus was further diluted in a solution containing crystal violet, bovine sera, 70% ethanol, and 0.01M PBS pH 6.8, to a concentration of approximately 10⁵ cells/ml. Ten microliters of the S. aureus -dye suspension was uniformly spread on a somatic cell slide circle and allowed to dry. The film dried evenly due to the lower surface tension caused by the ethanol. S. aureus appeared dark purple against a light purple background. A fixed diameter of each circle was counted using a lens reticle at a magnification of 1000X. Care was taken to grow and handle the organisms in a repeatable fashion to avoid loss of exopolysaccharide and shrinkage or swelling of cells, which was shown to cause changes in absorbance. The effect of trypsin treatment on the standard curves was examined in four of the strains.