Microfluorimetry of cells stained with fluorescent antibody

1. 1. The amount of fluorescein bound to protein was determined by spectrophotometric and fluorimetric measurements of free dye occurring in the dialysate of a labeled conjugate, using the labeling compound, fluorescein isothiocyanate, as a standard. Two samples of fluorescein and one of fluorescein amine were unsuitable as standards for this purpose. 2. 2. The amount of bound dye could also be determined by spectrophotometric comparison of the conjugate with fluorescein isothiocyanate solutions. 3. 3. The average value for bound fluorescein was 54 μg of dye per mg protein when the initial reaction mixture contained 100 μg of dye per mg of protein. When the initial mixture contained 50 μg of dye per mg of protein, the average amount bound was 23.8 μg. 4. 4. The fluorescence of bound fluorescein isothiocyanate was about 10 per cent of that of the free dye in buffered saline solution. 5. 5. Microfluorimetry of individual coliform bacteria showed that the average amount of antibody protein reacting with a cell was 1.15 × 10 −7 μg carrying 3.25 × 10 −9 μg of fluorescein.