A SEMI-MICRO METHOD FOR THE DETERMINATION OF CRYSTAL VIOLET SORBED TO BIOLOGICAL MATERIAL

Abstract

A semi-micro method is reported to measure the degree of Gram positive behaviour, i.e. the amount of crystal violet sorbed by various proteinous materials. Part of the role supposedly played by "loosely bound" iodine as well as that by covalently bound iodine in the crystal violet uptake of intact wool (Gram negative) and alkali-degraded wool (Gram positive) has been determined. The influence of reversing the consecutive steps of staining and iodination on dye-uptake has been measured for wools. The iodine uptake prior to and after staining, as well as the dye-uptake prior to iodination, have been determined for the two types of wool as well as for Escherichia coli and Leuconostoc mesenteroides respectively. The dye-uptake of wools and of bacteria was also measured after treatment with 1-fluor-2,4-dinitrobenzene. The identical composition in non-sulphur containing amino acids of intact wool and alkalidegraded wool—prior to and after treatment with 1-fluor-2,4-dinitrobenzene—has been investigated by subjecting the wools to hydrolysis and one- as well as two-dimensional paper chromatography. Data are reported as to the crystal violet uptake prior to and after iodination of muscle and nerve-fibres, nail, ovalbumin, β-glucuronidase and pepsin. The theoretical implications of these and other data are discussed and attention is drawn to the manyfold aspects of the problem of Gram staining behaviour of proteins.