Acridine orange as an indicator of bacterial susceptibility to gentamicin

Abstract

We have studied the response of Escherichia coli NCTC10418 to gentamicin with flow cytometry. The susceptibility of individual bacterial cells to the antibiotic was assessed by differential staining using the metachromatic dye, acridine orange. Exponential phase cultures were exposed to the minimum bactericidal concentration of gentamicin and analysed at regular intervals over 90 min. Within 60 min of exposure to the drug, two sub-populations of organisms could be distinguished in cultures by their different acridine orange-associated fluorescence emissions of < 550 nm and > 550 nm. The number of bacteria exhibiting acridine orange-associated fluorescence at > 550 nm corresponded to counts of colony forming units.

Confocal Laser Scanning Microscopy Is Appropriate to Detect Viability of Enterococcus faecalis in Infected Dentin

“…The other dentin segment was washed with 100 L PBS and stained with 50 L 0.01% acridine orange; such dye has the ability to bind with bacterial RNA-emitting red fluorescence and to bind with bacterial DNA-emitting green fluorescence (20,21). This information allows analysis of the bacterial metabolism because cells in the log phase emit red fluorescence and those in the stationary phase emit a green fluorescence (11, 19 -22).…”

Use of multiparameter analysis for Vibrio alginolyticus viable but nonculturable state determination

“…The use of such processing software offers the feasibility of identifying as much as possible metabolic modifications, which are associated to cell shape modifications, allowing the evaluation of a density profile (metabolism) and cell morphology. In addition, the density profile derived from the Acridine Orange (AO) stained cells, also provides information on the metabolic activity (11,12). The dimension index was calculated by delineating the cells.…”

An Acridine Orange Spore Germination Fluorescence Microscopy Versus Spectral Paradox

“…As noted in the Materials and Methods section, leukocytes were stained with less AO (0.25 μM) [9] than the 20 μM AO which Bruno and Mayo originally used on germinating Bacillus spores [1] or the 66.3 μM AO final concentration used in the present studies. The author explored AO concentrations as low as 13.25 μM, but noted only green vegetative Bacillus cells at the 13.25 μM concentration (data not shown) perhaps because AO could not penetrate the vegetative cells to reach mRNA at this lower concentration without the aid of a detergent to permeabilize the cells [4]. Figure 2 illustrates the repeated observation of increased fluorescence in the orange-red region (>590 nm) from day 0 to days 1 and 2 post-germination induction with 2 % dextrose or TSB.…”

“…Mason and Lloyd [4] have shown that orange-red AO fluorescence beyond 550 nm is correlated with metabolic activity and bacterial viability. In the present work, the author reinvestigates this phenomenon, documents its existence, and attempts to explain the fluorescence microscopy versus spectrofluorometry paradox in terms of spectral emission overlap and the metachromatic nature of AO.…”

“…AO is primarily known as a differential fluorophore which intercalates into double-stranded (ds) DNA to produce green fluorescence emissions centered around 530 nm. Simultaneously, AO can stain single-stranded (ss) DNA or messenger (m)RNA by complexing with the ss nucleic acids and stacking in metabolically active cells to produce a red emission peak at~640 nm in addition to any 530 nm green fluorescence [1,3,4]. It is not entirely clear what structures stain green in or on Bacillus spores, but for uninduced dormant spores, the characteristic green fluorescent ring or "oval" staining pattern around the spore's perimeter strongly suggests surface or subsurface staining of the spore coat or cortex [5].…”