ARI Volume 2 Number 1.pdf - Zoo-unn.org

Cytogenetic variations in Clarias species 279caped plastic test tube. The content wascentrifuged at 2,700 rpm for 3 minutesusing micro angle centrifuge.2. Hank’s balance salt solution plusphytohaemagglutinin separation: 1ml of HBSS and 0.2 ml of 1 mg ml -1 PHAwere added to 1 ml of anticoagulatedblood. The contents were mixed andcentrifuged at 2, 700 rpm for 3 minutes toensure proper settling out of erythrocytes.3. Phytohaemagglutinin separation: 1ml of anticoagulated blood was dilutedwith 0.2 ml of 1 mg ml -1 PHA. This wasmixed by gentle inversion in a sterile 5 mlcapped plastic test tube. The content wascentrifuged at 2,700 rpm for 3 minutes.In each case, the leucocyte richsupernatant was used as inoculum.Blood Leucoyte Culture Techniques: Thecomplete culture medium used composed of 20 mlof HBSS, 2 ml of Eargle’s Essential CultureMedium. Laboratory prepared phytohaemagglutininwas added to make up either 5 % of theentire culture medium. Furthermore, freshlyprepared rabbit sera were added to make up 20 %of the entire culture medium. The completemedium was aseptically filtered using membranefilter. The complete culture medium (5 ml) wasplaced in sterile tube. Various glass and plasticsterile test tubes were tried for culturing of fishleuocytes. About 0.05 ml of leucocyte richsupernatant was aseptically placed on the bottomof the culture tube containing 5 ml of the completeculture medium using a sterile 1 ml syringe fittedwith 26-gauge 1¼-inch needle. Culture vials wereincubated at 36 ± 1 0 C for 72 hours.Chromosome Harvesting and SlidePreparation Techniques: The cultured leucocytecells were arrested 3 – 6 hours with 0.3 µg ml -1 ofcolchicine solution before the end of the 72 hoursculture period to give good mitotic chromosomespread for karyotyping (Eyo, 1997). At the end ofincubation and metaphase arresting period, thecells were harvested by centrifuging at 1000 rpmfor 5 minutes. The supernatants were discardedand the white button-like residue given 5 ml of0.075 M KCl hypotonic treatment for 10 minutesand expirated using a micropipette. The residueswere obtained through centrifugation and thesupernatants discarded. 2 ml of freshly preparedcold Carnony’s fixative (absolute ethanol andglacial acetic acid (3:1)) was added to the residuecells, expirated to disperse the cell pellets and leftstanding for 15 minutes. The fixation process wasrepeated twice at 15 minutes intervals. After thefinal centrifugation, the fixative was decantedleaving about 3 drops in which to re-suspend thecells through expiration. The chromosome spreadwas accomplished using a modification of Mellman(1965) air-dried technique.Thoroughly clean grease free slides(commercially pre-cleaned slides) were dipped intodeionized water and frozen. The chilled slideswere observed for cleanliness by adherence ofwater film. Excess fluid was shaken off and onedrop of cells suspension was placed on the centerof the wet slide inclined at an angle of 45 0 . Ablotter placed underneath the slides removedexcess fluid. Drying was completed immediatelyby blowing the slides to promote evaporation andby gentle warming under 80 watts electric bulb.The quality of slides was checked using ahand lens. Inadequate spreading was correctedby re-fixation of the cells. Slides were stained in10 % buffered Giemsa stain for 5 minutes, anddehydrated for 1 min each in two jars of acetone,one jar of acetone and xylene solution (1:1) andfinally one jar of xylene. Slides were observed todestain in acetone when shaken and or agitated.Thus, shaking and agitation was avoided. Slideswere scanned for metaphase chromosomes usinga binocular light microscope at X 1500magnification. Slides with good metaphasechromosomes were processed into permanentmounts. The chromosomal arm lengths weremeasured to the nearest micron using ocularmicrometer.Chromosome Analysis Nomenclature: Tenslides per species showing good metaphasechromosome were analysed. The frequency ofdiploid chromosomes number per species wasrecorded. The maximum or minimum mean andmodal chromosome number were computed foreach species. F-LSD was employed to separatethe differences in modes. The arm ratio r, (longarm L/ short arm S) and the centromeric index i(100 x short arm/total length of chromosome)were calculated. Furthermore, each chromosomewas classified into any of the six groups; M, m,sm, st, t, and T based on Levan et al. (1964)classification (Table 1).Table 1: Chromosome arm nomenclaturerelevant to Clariid cytotaxonomyTerm Location d value r valueM Median point M 00 1.0m median region m 0.0 – 2.5 1.01 -1.7Sm Submedian region Sm 2.5-5.0 1.7-3.0St subterminal region st 5.0-7.5 3.0-7.0t(a) terminal region t 7.5-10 7.0-00T(a) Terminal point T 10-0 00