tétrazolium (NBT) (Sigma Chemical Co. Ltd., London) as the electron acceptor.
Sections were stained for Ca^ ^ activated ATPase activity following alkaline preincubation at pHlO.4 (Johnston et al., 1974). Pre
incubation was for 2 - 2 0 mins in a medium of, for the tench, 1 mMCaC^^» 100 mM Tris-HCL (pH 10.4) and for the catfish 18 mM CaCC^, 50 mM
2-amino-2 methyl-1 propanol (pH 10.4) (221 buffer. Sigma Poole, Dorset). Sections were subsequently incubated in, for the tench, an incubation medium containing 5 mM ATP, 1 mM CaCCg, 30 mM KCÊ,
10 mM Tris HCL (pH 9.4) and for the catfish, 20 mM KC£, 8 mM CaCC^, 30 mM ATP and 80 mM 221 buffer (pH 9.2) for 15 mins, washed in water and successively treated with 3% Cobalt chloride (3 min.) washed in water again and finally stained with 1% ammonium sulphide solution. Controls were performed in which ATP was omitted from the incubation medium. Preincubations of 1 - 5 mins result in inactivation of slow muscle myosin. Longer periods of alkaline preincubation result in a progressive loss of staining of fast glycolytic fibres, whereas fast aerobic fibres stain intensely for myofibrillar ATPase even after 20 mins of preincubation at alkaline pH.
Staining for acetylcholinesterase activity.
Muscle endplates and pre-terminal axons were localised by staining fibres for acetylcholinesterase activity. Small bundles (3-4 myotomes, 2mm^ x 20mm long) were dissected complete with
dorsal fin and pinned on to cork strips at their resting lengths in situ. Samples were fixed at room temperature for 2 - 8 hrs in 10% formalin, 100 mM acetate buffer, pH 5.2, then thoroughly washed in distilled water. The strips were subsequently incubated for 6 - 8 hrs at 37°C in a freshly prepared solution containing 2.5% CuSOjSH 0 (0.2 ml); 3.7% glycine (0.2 ml); 0.2N sodium acetate buffer, pH 5.2 and the clear-supernatent from 15 mg acetylcholine iodide, 0.3 ml of 2.5% copper sulphate and 0.7 ml water (Naik, 1963). Samples were then stained in 1% ammonium sulphide, washed in water and cleared in glycerol for48 - 72 hrs at room temperature. Small fibre bundles were teased out under a binocular microscope (X7) and mounted in glycerol on glass slides.
Electron Microscopy
Fixation of muscle samples.
Small strips (3 x 5 x 20 mm) of superficial red muscle were dissected from the myotomes immediately posterior to the dorsal fin in the region of the lateral line nerve (Fig. 2:4 (a) ). White muscle strips of similar size were excised from the underlying muscle. Care was taken to exclude the fibres on the boundary between the red and white muscle zones as these represent a distinct fibre type. Strips were pinned to cork board at their resting lengths in situ and immersed in 3% gluteraldehyde, O.IM phosphate buffer, pH 7.2
at room temperature. Initial fixation was for 2 - 5 hr. Subsequently, small fibre bundles (100 - 150 slow fibres and 20 - 50 fast fibres) were dissected from the superficial layers of the strips and left overnight in a fresh change of the same fixative. Fibre bundles were then washed in 0.12M phosphate buffer, pH7.2 at room temperature then post fixed in 1% osmium tetroxide, 0.12M phosphate buffer,
and embedded in araldite resin, CY 212) EM Scope, Trent, England). Semithin (lyam) and ultrathin (400Â) sections were cut on an OM U2 Reichert Ultramicrotome. Semithin sections were stained in 1% toluidine blue stain and viewed under a light microscope (XlO-100) to assess the orientation and potential of the block. Ultrathin sections were mounted on formvar-coated, 150 mesh, copper grids, double stained with uranyl acetate and lead citrate and viewed with a Philips 301 electron microscope.
Morphometric Techniques
Quantification of muscle fibre components
The numbers involved for the separate species and groups of fish studies are shown in Table 2:1.
Equal numbers of fish were sampled for each condition (eg acclimated to either aerated or hypoxic water). For each fibre type (red or white) a number of transversly and longitudenally orientated blocks were prepared. The blocks from each fish were kept separately in the case of the carp and tench and randomly mixed for the catfish.
An extremely detailed investigation of the composition of the red and white fibres in aerated and hypoxic water acclimated fish was made only in the case of the tench. For the carp and catfish the method described below was used to determine the volume fraction occupied by the mitochondria only.
The volume fraction occupied by myofibrils, mitochondria and other cellular components was determined from micrographs of transversely
blocks that were selected at random (eg red fibres from fish acclimated to aerated water). Each section contained approximately 50 - 80
red (slow) fibres or 20 - 30 white (fast) fibres. From each of these sections three to four whole fibres were photographed at
random (magnification x 2000 - 3400). In the case of white fibres it was often necessary to photograph two or three overlapping fields in order to reconstruct photomicrographs of the whole fibre. Negatives of micrographs of whole fibres were projected so that the image overlaid a square test grid. Reconstructed photomicrographs were placed under a transparent acetate test grid. The projected images or photomicrographs were magnified such that the mean mitochondrial size was similar in all cases (magnification x 2.6 - 4.4). Volume fractions of mitochondria and myofibrils etc. were determined using a point counting method (Weibel, 1980). The test system
consisted of a coherant grid composed of discrete line segments (1 cm) with endpoints arranged in a regular square lattice. Magnification was such that the test lines corresponded to a distance of 2 . 3 jsm on
the image which is equivalent to between 1 and 1.5 times the mean diameter of the mitochondria (Mathieu et al., 1980). The volume fractions (V^) of each component were determined from:
\
where Pi = number of points falling on a component and Pt = total number of points falling on the fibre. The area outside a line drawn around the myofibrils was considered to represent the subsarcolemmal zone. Mitochondria were scored as falling either in the subsarcolemmal or intermyofibrillar zones. There were at least 200 test points on each fibre. Areas and perimeters of fibres and the subsarcolemmal
zone were determined from traced outlines of the fibres (magnified