Crystal Violet Uptake Research Articles

Characterization of surfaces involved in adherence of Legionella pneumophila to Fischerella species.

Legionella pneumophila adheres to the slime coat of Fischerella spp. This was shown by microscopic examination and by a decline in L. pneumophila CFU in samples removed from coincubation mixtures of both organisms. Binding of partially purified Fischerella slime by L. pneumophila was most efficient by young, less hydrophobic L. pneumophila cells than by older, more hydrophobic cells. Uptake of crystal violet and partitioning into hexadecane were used to measure hydrophobicity of L. pneumophila. Purified soluble Legionella antigen also bound to Fischerella slime, as shown by indirect immunofluorescence. Adherence was not specific for L. pneumophila, since a variety of gram-negative, gram-positive, and acid-fast bacteria also bound to Fischerella slime.

Differences in crystal violet uptake and cation-induced death among yeast sterol mutants

Yeast sterol mutants exposed to crystal violet demonstrated greater dye uptake than the wild type. In addition, exposure for 10 min to hypertonic cation solutions showed a greater decrease in cell viability for mutants than for wild type.

Binding of Progesterone to Neisseria gonorrhoeae and Other Gram-Negative Bacteria

The binding of [1,2-(3)H]progesterone to progesterone-sensitive Neisseria gonorrhoeae CS-7 and the progesterone-insensitive Neisseria mucosa, Pseudomonas aeruginosa, and Salmonella typhimurium (rough and smooth strains) was investigated. The kinetics of binding to N. gonorrhoeae CS-7 demonstrated that the majority of the progesterone binding occurred and equilibrium was reached within the first 30 min. Despite the rapid binding of progesterone, only about 20% of the added steroid was bound at the cell concentration used throughout this study. Whole cells of progesterone-insensitive bacteria bound progesterone less efficiently than the progesterone-sensitive N. gonorrhoeae CS-7. N. mucosa bound low amounts of this steroid (20% of that bound by N. gonorrhoeae CS-7) whereas the other gram-negative bacteria exhibited little progesterone binding (<3% of that bound by N. gonorrhoeae CS-7). The outer membrane permeability of N. gonorrhoeae CS-7, as measured by crystal violet uptake and inhibition, was similar to the deep rough mutant of S. typhimurium TA 1535. The latter organism neither bound nor was inhibited by progesterone. However, isolated cell envelopes of N. gonorrhoeae and progesterone-insensitive bacteria all bound progesterone equally well. Cortisone and cholesterol, althouh structurally similar to progesterone, were not inhibitory to N. gonorrhoeae and did not bind to whole cells as well as progesterone. The major site of progesterone binding appeared to be the cytoplasmic membrane, which bound four times more progesterone than the outer membrane. In addition, isolated cytoplasmic membrane proteins bound more than three times more progesterone per milligram of protein than the intact membrane.

Altered crystal violet permeability and lytic behavior in antibiotic-resistant and -sensitive mutants of Neisseria gonorrhoeae.

Wild-type, antibiotic-resistant and hypersensitive isogenic strains of Neisseria gonorrhoeae were studied for uptake of crystal violet, rates of autolysis, and response to lysozyme. Total uptake of crystal violet was similar in all strains at 0 C but varied significantly at 37 C. Mutation at the nonspecific resistance locus ery resulted in relative impermeability to crystal violet at 37 C, as compared to wild type. The penetration barrier to crystal violet at 37 C was overcome by addition of 5 mM ethylenediaminetetraacetic acid. Mutation at ery also resulted in reduced rates of autolysis and reduced sensitivity to high concentrations of lysozyme under conditions of divalent cation (Mg2+) depletion. In contrast, mutation at the nonspecific drug hypersensitivity locus env resulted in increased uptake of crystal violet at 37 C, due to increased binding of dye to crude envelope as well as increased penetration into cytoplasm. The env mutants were also more rapidly autolytic and more sensitive to lysozyme than wild type in the absence of Mg2+. These results suggest that the cell envelopes of ery mutants are more stable and less permeable and those of env mutants are less stable and more permeable than wild-type strains.

Cell structure and quantitative gram stain of Bacillus megaterium.

SUMMARY In a strain of Bacillus megaterium the Gram reaction was investigated by comparing its effect on intact bacilli with its effects on bacilli treated with lysozyme in several different ways. The lysozyme-treated bacteria varied from bacilli showing only polar separation of the cell wall from the protoplasm to protoplasts free from cell wall. The uptake of the primary dye (crystal violet) by intact bacteria was higher than that of all lysozyme-depolymerized cell forms. Iodine uptake depended on the previous uptake of crystal violet. Therefore it was lower for the lysozyme-depolymerized forms. Cell wall took up crystal violet and iodine in small quantity. The same dye iodine complex was formed in all cell structures (cell wall and protoplasm). Gram differentiation was obtained with 95% (v/v) ethanol in water, this was found to extract much less of the dye iodine complex from intact bacteria than from the lysozyme-depolymerized forms. The latter lost about 80–90% of their net dye and iodine content, whereas intact bacilli lost only 20–45%. This was due to the cell wall which formed a barrier to the ethanol extraction of the dye iodine complex, for organisms whose cell walls had been depolymerized or even dissolved by lysozyme during or after the iodine step of the Gram reaction lost much more dye and iodine than intact bacilli. Chemical integrity of the cell wall is a prerequisite for Gram positivity.

A semi-micro method for the determination of crystal violet sorbed to biological material.

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.

Quantitative analysis of the Gram reaction.

SUMMARY: The crystal violet and iodine uptake by suspensions of Staphylococcus aureus and Escherichia coli was determined. It was found that equal amounts of intracellular dye-iodine complex were formed in these micro-organisms. The molar ratio dye to iodine was 1:2. The extractability of this complex with methanol and ethanol was compared with the relative solubility of dye-iodine precipitates. Results with E. coli suggest that the solvents pass freely into the cells and decolorize them after dissociation of the dye-iodine complex. Gram-positive micro-organisms may be arranged in a number of groups according to their extractability pattern with regard to the complex. These patterns suggest the presence of interaction between the cells and ethanol. This interaction may be located at the cell wall.

Influence of phosphate buffer on crystal violet uptake and retention by bacterial cells as related to the gram reaction.

Influence of phosphate buffer on crystal violet uptake and retention by bacterial cells as related to the gram reaction.