A rapid colorimetric assay for evaluating Legionella pneumophila growth in macrophages in vitro.
A rapid colorimetric technique for in vitro quantitation of Legionella pneumophila intracellular proliferation in macrophages is described. The assay is based on the electron transport activity of metabolically active L. pneumophila. The yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is cleaved by the mitochondrial activity of viable L. pneumophila, forming a dark formazan derivative with an absorption spectrum different from that of the native compound. The MTT method for measuring intracellular growth of L. pneumophila closely correlated with the CFU assay. The ability of macrophages from the A/J mouse strain to support intracellular growth of L. pneumophila and the ability of desferrioxamine to restrict L. pneumophila intracellular proliferation were confirmed by both methods. The MTT assay offers the advantages of rapidity, simplicity, and cost efficiency over the CFU assay, since it can be performed in the same flat-bottom microtiter plate with measurement in an enzyme-linked immunosorbent assay reader, allowing efficient processing of large numbers of samples.
Gebran
S J
SJ
Department of Medical Microbiology and Immunology, University of South Florida, Tampa 33612-4799.
Newton
C A
CA
Yamamoto
Y
Y
Klein
T W
TW
Friedman
H
H
D000818:Animals; D003124:Colorimetry / Q000379:methods*; D003676:Deferoxamine / Q000494:pharmacology; D004579:Electron Transport; D005260:Female; D005562:Formazans / Q000032:analysis; D007501:Iron / Q000494:pharmacology; D016952:Legionella pneumophila / Q000187:drug effects / Q000254:growth & development*; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D008815:Mice, Inbred Strains; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
D005562:Formazans; D013778:Tetrazolium Salts; D013844:Thiazoles; D007501:Iron; C022616:thiazolyl blue; D003676:Deferoxamine