Phloxine
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| Verifiedfields = changed
| Watchedfields = changed
| verifiedrevid = 396687735
| ImageFile=Phloxine.png
| ImageSize=200px
| PIN=Disodium 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-3-oxo-3H-spiro[
| OtherNames=Cyanosin; Cyanosine; Eosine bluish; Eosine Blue; Cyanosin B; Eosin Blue; Phloxine P; Phloxin B; Eosine I Bluish; Acid red 92; C.I. 45410; D & C Red no. 28
|Section1={{Chembox Identifiers
| CASNo_Ref = {{cascite|correct|CAS}}
| CASNo=18472-87-2
| UNII_Ref = {{fdacite|correct|FDA}}
| UNII = 767IP0Y5NH
| PubChem=6433718
| ChEBI_Ref = {{ebicite|changed|EBI}}
| ChEBI = 87192
| SMILES=C1=C2C(=C(C(=C1Br)[O-])Br)OC3=C(C(=C(C=C3C24C5=C(C(=C(C(=C5Cl)Cl)Cl)Cl)C(=O)O4)Br)[O-])Br.[Na+].[Na+]
}}
|Section2={{Chembox Properties
| C=20 | H=2 | Br=4 | Cl=4 | Na=2 | O=5
| Appearance=Red to brown powder
| Density=
| MeltingPt=
| BoilingPt=
| Solubility=Soluble
}}
|Section3={{Chembox Hazards
| MainHazards=
| FlashPt=
| AutoignitionPt =
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Phloxine B (commonly known simply as phloxine) is a water-soluble red dye used for coloring drugs and cosmetics in the United States{{cite book|last1=Food and Drug Administration|title=The Code of Federal Regulations of the United States of America, Title 21, Part 74.1328|date=2001|page = 296|publisher=U S Government Printing Office|url=https://play.google.com/books/reader?id=ziM6AAAAIAAJ&printsec=frontcover&output=reader&hl=en_GB&pg=GBS.PP1|accessdate=15 April 2016}} and coloring food in Japan.{{cite journal|last1=Kamikura|first1=M|title=Thin Layer Chromatography of Synthetic Dyes (X)|journal=Food Hygiene and Safety Science (Shokuhin Eiseigaku Zasshi)|date=1970|volume=11|issue=4|pages=242–248|doi=10.3358/shokueishi.11.242|doi-access=free}} 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.{{cite journal|last1=Duarte|first1=Paulo|last2=Ferreira|first2=Diana P.|last3=Ferreira Machado|first3=Isabel|last4=Filipe|first4=Luis|last5=Ferreira|first5=Vieira|last6=Rodríguez|first6=Hernan B.|last7=San Román|first7=Enrique|title=Phloxine B as a Probe for Entrapment in Microcrystalline Cellulose|journal=Molecules|date=2012|volume=17|issue=2|pages=1602–1616|doi=10.3390/molecules17021602|pmid=22314381|pmc=6268435|doi-access=free}} It has an absorption maximum around 540 nm and an emission maximum around 564 nm.{{cite journal|last1=Coppeta|first1=J.|last2=Rogers|first2=C.|title=Dual emission laser induced fluorescence for direct planar scalar behavior measurements|journal=Experiments in Fluids|date=1998|volume=25|issue=1|pages=1–15|doi=10.1007/s003480050202|bibcode=1998ExFl...25....1C|s2cid=37649159}} Apart from industrial use, phloxine B has functions as an antimicrobial substance, viability dye and biological stain.{{cite journal|last1=Rasooly|first1=Avraham|last2=Weisz|first2=Adrian|title=In Vitro Antibacterial Activities of Phloxine B and Other Halogenated Fluoresceins against Methicillin-Resistant Staphylococcus aureus|journal=Antimicrobial Agents and Chemotherapy|date=2002|volume=46|issue=11|pages=3650–3653|doi=10.1128/AAC.46.11.3650-3653.2002|pmid=12384384|pmc=128710}} For example, it is used in hematoxylin-phloxine-saffron (HPS) staining to color the cytoplasm and connective tissue in shades of red.{{cite web|last1=Borgerink|first1=Hermina|title=HPS stain|url=http://histonet.utsouthwestern.narkive.com/CdlDznhK/hps-stain|website=Narkive Mailing List Archive|accessdate=18 April 2016}}
Antimicrobial properties
= Lethal dosage levels =
In the presence of light, phloxine B has a bactericidal effect on gram-positive strains, such as Bacillus subtilis, Bacillus cereus, and several methicillin-resistant Staphylococcus aureus (MRSA) strains.{{cite journal |last1=Rasooly |first1=Reuven |title=Expanding the bactericidal action of the food color additive phloxine B to gram-negative bacteria |journal=FEMS Immunology & Medical Microbiology |date=August 2005 |volume=45 |issue=2 |pages=239–244 |doi=10.1016/j.femsim.2005.04.004 |pmid=15949926 |doi-access=free }} 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 ion that binds to positively charged cellular components {{citation needed|date=January 2019}}. When phloxine B is subjected to light, [https://en.wiktionary.org/wiki/debromination debromination] occurs and free radicals and singlet oxygen 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 polysaccharide-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,{{cite journal |last1=Leive |first1=Loretta |last2=Kollin |first2=Virginia |title=Controlling EDTA treatment to produce permeable escherichia coli with normal metabolic processes |journal=Biochemical and Biophysical Research Communications |date=July 1967 |volume=28 |issue=2 |pages=229–236 |doi=10.1016/0006-291x(67)90434-2 |pmid=4166571 }} removes the phloxine B resistance and allows gram-negative bacteria to be killed as well.
Measure of viability
{{further|Viability assay}}
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 can be compared to the total number of cells to give a measure of mortality.{{cite book |doi=10.1016/S0076-6879(08)03202-3 |chapter=Chapter 2 Viability Assays to Monitor Yeast Autophagy |title=Autophagy: Lower Eukaryotes and Non-Mammalian Systems, Part A |series=Methods in Enzymology |year=2008 |last1=Noda |first1=Takeshi |volume=451 |pages=27–32 |pmid=19185710 |isbn=9780123745484 }} The same principle can be applied at higher throughput by fluorescence-activated flow cytometry (FACS), where all phloxine B-stained cells in a sample are counted.{{cite journal |last1=Guérin |first1=Renée |last2=Beauregard |first2=Pascale B. |last3=Leroux |first3=Alexandre |last4=Rokeach |first4=Luis A. |title=Calnexin Regulates Apoptosis Induced by Inositol Starvation in Fission Yeast |journal=PLOS ONE |date=16 July 2009 |volume=4 |issue=7 |pages=e6244 |doi=10.1371/journal.pone.0006244 |pmid=19606215 |pmc=2705804 |bibcode=2009PLoSO...4.6244G |doi-access=free }}
[Note: some reports suggest that phloxine B is instead pumped out of live yeast cells but retained in dead/dying yeast cells.{{cite journal |last1=Kwolek-Mirek |first1=Magdalena |last2=Zadrag-Tecza |first2=Renata |title=Comparison of methods used for assessing the viability and vitality of yeast cells |journal=FEMS Yeast Research |date=September 2014 |volume=14 |issue=7 |pages=1068–1079 |doi=10.1111/1567-1364.12202 |pmid=25154541 |doi-access=free }}{{cite journal |last1=Minois |first1=Nadège |last2=Frajnt |first2=Magdalena |last3=Wilson |first3=Chris |last4=Vaupel |first4=James W. |title=Advances in measuring lifespan in the yeast Saccharomyces cerevisiae |journal=Proceedings of the National Academy of Sciences of the United States of America |date=11 January 2005 |volume=102 |issue=2 |pages=402–406 |doi=10.1073/pnas.0408332102 |pmid=15625107 |pmc=544282 |bibcode=2005PNAS..102..402M |doi-access=free }} However, definitive evidence for either model is still needed.]