Fluorometric assessment of gram-negative bacterial permeabilization

Ilkka Helander (Corresponding Author), Tiina Mattila-Sandholm

Research output: Contribution to journalArticleScientificpeer-review

186 Citations (Scopus)

Abstract

Uptake of the fluorescent probe 1‐N‐phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram‐negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram‐negative bacteria.
Original languageEnglish
Pages (from-to)213 - 219
Number of pages7
JournalJournal of Applied Microbiology
Volume88
Issue number2
DOIs
Publication statusPublished - 2000
MoE publication typeA1 Journal article-refereed

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Magnesium Chloride
Escherichia coli O157
Bacteria
Pseudomonas aeruginosa
Permeability
Food Preservatives
Salmonella typhimurium
Fluorescent Dyes
Edetic Acid
Citric Acid
Reading
Phospholipids
Fluorescence
Membranes
sodium polymetaphosphate
sodium citrate

Cite this

Helander, Ilkka ; Mattila-Sandholm, Tiina. / Fluorometric assessment of gram-negative bacterial permeabilization. In: Journal of Applied Microbiology. 2000 ; Vol. 88, No. 2. pp. 213 - 219.
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abstract = "Uptake of the fluorescent probe 1‐N‐phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram‐negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram‐negative bacteria.",
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Fluorometric assessment of gram-negative bacterial permeabilization. / Helander, Ilkka (Corresponding Author); Mattila-Sandholm, Tiina.

In: Journal of Applied Microbiology, Vol. 88, No. 2, 2000, p. 213 - 219.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Fluorometric assessment of gram-negative bacterial permeabilization

AU - Helander, Ilkka

AU - Mattila-Sandholm, Tiina

PY - 2000

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AB - Uptake of the fluorescent probe 1‐N‐phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram‐negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram‐negative bacteria.

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