Quantitative Studies of Differential Staining Reactions

It has been shown previously (Kennedy and Barbaro, 1953) that the amount of crystal violet adsorbed by bacteria can be measured quantitatively by either one of two procedures. One procedure involved micro-Kjeldahl analyses of bacterial cells before and after staining. The increase in total nitrogen in the stained cells represented the dye nitrogen adsorbed. The second procedure was based on micro-Kjeldahl analyses of the dye nitrogen in the stain solution before staining and in the supernatant fluids after staining and washing with m/15 phosphate buffer (pH 7.1). The decrease in dye nitrogen in the supernatants represented the dye nitrogen adsorbed by the celLs. The simultaneous use of both procedures provided added credence to the quantitative dye adsorption values obtained. Application of these procedures to the complete gram stain led to the development of a quantitative gram reaction (Barbaro and Kennedy, 1954). The investigations of Kennedy and Barbaro (1953), Barbaro and Kennedy (1954), and Barbaro (1954) on 13 species of bacteria showed that gram positive cells adsorbed and retained more crystal violet than gram negative cells and that gram variable species adsorbed an amount intermediate between the two. Bartholomew and Finkelstein (1954) studied the dye uptake of 7 species of bacteria and 1 species of Saccharomyces by colorimetricaily analyzing the dye content of a dilute stain solution before and after staining the organisms. Their cells were suspended in distilled water and stained at pH 6.0 to 6.5. Bartholomew and Finkelstein were unable to find a correlation between dye uptake ability and the gram stain.