Influence of Fluorescence of Bacteria Stained with Acridine Orange on the Enumeration of Microorganisms in Raw Milk

Abstract

The staining of gram-positive and gram-negative cultures with acridine orange in metabolically active and inactive states was investigated using a Bactoscan, direct epifluorescent filter technique (DEFT), and standard plate count as the reference method. The evaluation of the bacterial cultures in the Bactoscan revealed a linear relationship between Bactoscan counts (pulses) and the quantity of pure culture suspension used. But the proper detection of bacteria with the fluorescence optic methods was dependent on the type of microorganism and the physiological state of the cells. The Bactoscan and DEFT underestimated the bacterial counts of gram-negative cultures as compared with standard plate counting. When stained with acridine orange, metabolically active bacteria showed more orange fluorescence and a lower percentage of green fluorescent cells as compared with inactive bacteria. Bactoscan pulse height analysis (PHA) diagrams, graphs of the detected pulses and their intensity, showed low pulses of inactive bacteria. Many of these weak pulses were eliminated from counting because of their faint fluorescent staining. In contrast, PHA diagrams of metabolically active microorganisms showed bright staining and, therefore, high pulses. A complete count of these bacteria was possible. These investigations point out that discrepancies between the fluorescence optical counting methods and the standard plate count depend strongly on the staining of the cultures with acridine orange and, therefore, on the type of microorganism and the metabolic state of the cells measured.

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).…”

A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

“…In the dairy industry, the IDF standard plate count method is widely used as the reference method for rapid microbiological methods (Frundzhyan et al 1999;Rapposch et al 2000;Beloti et al 2002;Firstenberg-Eden et al 2002;). The number of colony-forming units counted with the IDF standard plate count method depends on parameters such as the bacterial species, culturing conditions, aggregates, the physiological status of the bacteria and sublethal injuries (Suhren and Reichmuth 2000).…”

OmcF, a Putative c -Type Monoheme Outer Membrane Cytochrome Required for the Expression of Other Outer Membrane Cytochromes in Geobacter sulfurreducens

“…Growth of fumarate cultures was assessed by measuring turbidity at 600 nm with a Genesys 2 spectrophotometer (Spectronic Instruments, Rochester, NY). Cell densities of Fe(III)-grown cultures were determined by epifluorescence microscopy using acridine orange staining (19,47). G. sulfurreducens cells were quantified using the SimplePCI software, version 5.3 (CImaging Systems, Compix Inc., Mars, PA).…”