Reliability and validity of the physical disector method for estimating neuron number

The physical disector was proposed as an unbiased and efficient means to estimate neuron number; however, the validity and reliability of this method have been examined only infrequently. Estimates of neuron number in the dorsal root ganglia (DRG) of bullfrogs (Rana catesbeiana) were compared to nucleolar counts based on 3-dimensional reconstructions. Accuracy of disector estimates were not affected by size of the animal. Similarly, disector estimates were not systematically altered when area measurements were limited to cellular regions of the DRG versus inclusion of the entire cross-sectional area. However, the recommended protocol for applying the disector resulted in sampling errors that introduced considerable variability in repeated estimates of neuron number from a single ganglion. In addition to this lack of reliability, disector estimates were consistently lower than those obtained by means of a nucleolar counting method that was calibrated against 3-dimensional reconstructions of neuronal profiles. The systematic error of the disector method was greater when ganglia were cut parallel to the long axis of the DR than when they were cut perpendicular to this axis. Increasing the sample size beyond what was recommended increased the reliability of estimates obtained with the disector; however, the bias associated with the plane of section was not reduced. These results emphasize the need for empirical validation of methods used to estimate neuron number in the tissue to which they are to be applied.