International Journal of Cell Cloning, Vol 5, 472-479, Copyright © 1987 by AlphaMed Press
Tetrazolium staining by optical scanning overestimates colony size and number of colonies counted
MD Bregman, J Buckmeier and FL Meyskens Jr
Hipple Cancer Research Center, Dayton, Ohio.
We measured the effect that staining with 2-(P-iodophenyl)-3-(p-
nitrophenyl)-5-phenyl tetrazolium chloride (INT) had on the number and size
distribution of tumor colonies counted using an optical image analyzer (FAS
II). Staining increased the number of tumor colonies counted. By using
opaque tumor cells or pigmented melanoma cells and measuring colony growth
kinetics, we demonstrated that the use of INT staining to assist in
counting tumor colonies artificially increased the size of viable tumor
cell aggregates by adding a red precipitate to the outside surface of the
cells. Laboratories that are using the INT method for drug screening are
probably measuring colonies down to and below 42 microns in diameter. These
small colonies could result from as few as one or two divisions. Thus,
potentially useful drugs may be missed in the screen because of the
presence of abortive colonies: i.e., lethally damaged cells completing only
one or two divisions.
