Enumeration of phagocytosed Staphylococcus aureus inside cells of the mouse macrophage cell line J774 by bioluminescence, fluorescence and viable counting techniques.
In order to quantify intracellular Staphylococcus aureus within a macrophage-like cell line by a bioluminescence technique, the mouse cell line J774 and opsonized Staphylococcus aureus were incubated together to allow phagocytosis to occur. Experiments using UV microscopy and fluorescent stained S. aureus were performed to determine an estimate of the mean intracellular bacterial numbers. For enumeration of intracellular bacteria by a bioluminescence technique, extracellular bacteria were removed by washing, the macrophages lysed mechanically and osmotically and treated with apyrase to remove somatic ATP. Bacterial cells were washed and the intracellular ATP measured by firefly luciferase bioluminescence in a luminometer. This new method of enumerating intracellular bacteria was compared to the conventional method of viable counts and found to correlate (r = 0.78). The bioluminescence assay developed was found to be a relatively rapid alternative method to the techniques currently used to enumerate intracellular bacteria and could prove advantageous in studies of intracellular killing and effects of antimicrobial agents on intracellular pathogens.
Department of Medical Microbiology, City Hospital, Birmingham, UK.
J
J
Sunderland
N
N
Brenwald
E
E
Smith
J M
JM
Andrews
R
R
Wise
D
D
Honeybourne
D000818:Animals; D002452:Cell Count / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D008163:Luminescent Measurements; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D010587:Phagocytosis; D013211:Staphylococcus aureus
D005456:Fluorescent Dyes; D016650:Fluorescein-5-isothiocyanate