The technique of single muscle-fiber cultures has already proven valuable in extending knowledge of myogenesis, stem cell heterogeneity, the stem cell niche in skeletal muscle, and satellite cell activation. This report reviews the background of the model and applications, and details the procedures of muscle dissection, fiber digestion and isolation, cleaning the fiber preparation, plating fibers, and extensions of the technique for studying activation from stable quiescence of satellite cells, mRNA expression by in situ hybridization and regulation of satellite cell activation in zebrafish muscle by nitric oxide, hepatocyte growth factor.