Problems associated with the morphometric measurement of transverse skeletal muscle fibers: I. Analysis of frozen sections.

The aim of this study was to evaluate the effects of a number of reported procedures which precede freezing on the cross sectional area of skeletal muscle fibers. Probable sources of variation were recognised as: Within blocks, same animal, same procedure. Variability in the size of muscle fibers from area-based measurements was determined for the anterior head of the biceps femoris muscle of the rat. Fibers were measured within areas selected at random from sections taken from the same block of tissue. Variance within and between sections was subsequently established. Between blocks from rats having undergone the same procedure. Variability in the size of muscle fibers measured within areas selected at random from sections taken from different blocks of tissue within the same animal. Between rats, same or equivalent procedure. Fibers were measured from randomly selected areas within sections taken from blocks of tissue derived from different animals. The muscles were subjected to the same or equivalent treatment prior to freezing. Between procedures. Restraining muscles prior to and during freezing did not significantly affect the results except the procedure involving holding the muscle between two pairs of forceps. However, the variability was high within and between sections and blocks from the same muscle. It is postulated that the variability is of technical rather than biological origin and may be the result of small focal contractions which occur along the fibers during freezing.