Evaluation of an indirect method for estimating myofiber number in transverse sections of skeletal muscle

The total number of myofibers contained in the transverse sections of 6 canine pectineus muscles was counted, using direct- and indirect-counting methods. For the direct-counting method, the transverse section of each muscle was photographed, from which a montage of each section was constructed. Then, the total number of myofibers contained in each montage was counted. The number obtained by the direct-counting method was used to determine the accuracy of the indirect-counting method. For the indirect-counting method, 40 randomly selected areas in each section were photographed. Then, the total number of myofibers contained in the randomly selected areas of each section was counted. From these counts, the total number of myofibers contained in each transverse section was calculated after the entire cross-sectional area of each section was determined by planimetry. The indirect-counting method was performed 3 times for each section. By direct count, the number of myofibers in a section was between 12,368 and 163,223. Generally, the indirect-counting method tended to overestimate the number of myofibers per section. The mean percentage error of estimates plus or minus standard error for all muscles and trials was 3.89 plus or minus 1.17; the range was minus 6.17 to +11.97. The percentage errors of the indirect counts were significantly correlated with the number of myofibers/sq mm and total number of myofibers/section; for every 1,000 myofibers/sq mm or 100,000 myofibers/section, an error of 2 or 5%, respectively, can be anticipated. The indirect-counting method utilized 10 to 20% of the man-hour effort required by the direct-counting method.