Because of drawbacks in both tissue culture and para-nitroblue tetrazolium (PNBT) staining as methods for determining endothelial viability, a supravital stain (trypan blue) was evaluated as a possible means of accomplishing this. Rabbit and human corneas, fresh or stored, were bisected and stained with PNBT and trypan blue, and the viability estimated. In general, the two stains showed a good correlation, but the estimates from two different observers were of interest. Observer A found a 30% average difference between the PNBT and the trypan blue estimates; observer B found only a 10% average difference. This was probably due to the wide range of stain intensity with the PNBT method; all stained cells were called viable by observer A, while only those deeply stained were called viable by observer B. Trypan blue staining appears to give equal or better accuracy in evaluating endothelial viability, as well as being a simpler and quicker method.
STOCKER FW, KING EH, LUCAS DO, GEORGIADE N. A Comparison of Two Different Staining Methods for Evaluating Corneal Endothelial Viability. Arch Ophthalmol. 1966;76(6):833–835. doi:10.1001/archopht.1966.03850010835010
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