Differential staining of bacteria in clinical specimens using acridine orange buffered at low pH

Optimal conditions for acridine orange staining of air dried and methanol fixed bacteria on glass slides were studied. The pH of the staining buffer did not influence the fluorescence of an S. aureus and an E. coli strain at dye concentrations of 25-50 mg per litre. 81 bacterial strains representing 15 different species were stained with acridine orange under standard conditions, all strains showing orange fluorescence. The pH of the buffer influenced markedly the staining patterns of human cells and tissue materials, as represented by smears of peripheral blood, buccal scrapings, urethral secretions and tracheal exudates. The fluorescence obtained ranged from low intensity green at low pH values to bright orange at neutral and alkaline pH. This variability indicated a possibility of designing conditions for a differential staining method for the detection of bacteria in clinical specimens. The differential staining effect with a low pH in the buffer was confirmed on smears of buccal scrapings, cerebrospinal fluid samples and urethral secretions, showing orange fluorescence of the bacteria present and green-to-yellow fluorescence of background material, cells and tissue debris.