Phloxine B (commonly known simply as phloxine) is a water-soluble red

dye A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution an ...

used for coloring

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s and cosmetics in the United States and coloring food in

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. It is derived from fluorescein, but differs by the presence of four bromine atoms at positions 2, 4, 5 and 7 of the xanthene ring and four chlorine atoms in the carboxyphenyl ring. It has an

absorption Absorption may refer to: Chemistry and biology * Absorption (biology), digestion **Absorption (small intestine) *Absorption (chemistry), diffusion of particles of gas or liquid into liquid or solid materials *Absorption (skin), a route by which ...

maximum around 540 nm and an

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maximum around 564 nm. Apart from industrial use, phloxine B has functions as an antimicrobial substance,

viability Viability is the ability of a thing (a living organism, an artificial system, an idea, etc.) to maintain itself or recover its potentialities. Viability or viable may refer to: Biology, medicine or ecology * Viability selection, the selection of ...

dye and

biological stain Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology (microscopic study of biological tissues), in cytology (microscopic study of cells), and in the m ...

. For example, it is used in hematoxylin-phloxine-saffron ( HPS) staining to color the cytoplasm and

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in shades of red.

Antimicrobial properties

Lethal dosage levels

In the presence of light, phloxine B has a

bactericidal A bactericide or bacteriocide, sometimes abbreviated Bcidal, is a substance which kills bacteria. Bactericides are disinfectants, antiseptics, or antibiotics. However, material surfaces can also have bactericidal properties based solely on their ...

effect on gram-positive strains, such as '' Bacillus subtilis'', '' Bacillus cereus'', and several methicillin-resistant ''

Staphylococcus aureus ''Staphylococcus aureus'' is a Gram-positive spherically shaped bacterium, a member of the Bacillota, and is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It is often positive ...

'' ( MRSA) strains. At a minimum inhibitory concentration of 25 μM, growth is reduced by 10-fold within 2.5 hours. At concentrations of 50 μM and 100 μM, growth is stopped completely and cell counts decrease by a factor of 10⁴ to 10⁵. For humans, the Food and Drug Administration deems phloxine B to be safe up to a daily dosage of 1.25 mg/kg.

Mechanism of action

Bacteria exposed to phloxine B die from oxidative damage. Phloxine B ionizes in water to become a

negatively charged Electric charge is the physical property of matter that causes charged matter to experience a force when placed in an electromagnetic field. Electric charge can be ''positive'' or ''negative'' (commonly carried by protons and electrons respectiv ...

ion that binds to positively charged cellular components . When phloxine B is subjected to light
debromination
occurs and

free radicals In chemistry, a radical, also known as a free radical, is an atom, molecule, or ion that has at least one unpaired valence electron. With some exceptions, these unpaired electrons make radicals highly chemically reactive. Many radicals spont ...

and

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are formed. These compounds cause irreversible damage to the bacteria, leading to growth arrest and cell death. Gram-negative bacteria are phloxine B-resistant due to the outer cell membrane that surrounds them. This

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-coated lipid bilayer creates a permeability barrier that prevents efficient uptake of the compound. Addition of EDTA, which is known to strip the lipopolysaccharides and increase membrane permeability, removes the phloxine B resistance and allows gram-negative bacteria to be killed as well.

Measure of viability

Phloxine B can be used to stain dead cells of several yeasts, including '' Saccharomyces cerevisiae'' and '' Schizosaccharomyces pombe''. When diluted in yeast growth media, the dye is unable to entere cell because of their membranes. Dead yeast cells lose membrane integrity, so phloxine B can enter and stain the intracellular cytosolic compounds. Therefore, staining is a measure of cell death. In cell counting assays, the number of fluorescent (i.e. dead) cells observed through a

haemocytometer The hemocytometer (or haemocytometer) is a counting-chamber device originally designed and usually used for counting blood cells. The hemocytometer was invented by Louis-Charles Malassez and consists of a thick glass microscope slide with a ...

can be compared to the total number of cells to give a measure of mortality. The same principle can be applied at higher throughput by fluorescence-activated

flow cytometry Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flo ...

( FACS), where all phloxine B-stained cells in a sample are counted. ote: some reports suggest that phloxine B is instead pumped out of live yeast cells but retained in dead/dying yeast cells. However, definitive evidence for either model is still needed.

References

{{reflist Organobromides Chloroarenes Lactones Fluorone dyes Spiro compounds