A method for chromosome preparations from large fish specimens using in vitro short-term treatment with colchicine

Abstract

Abstract. This paper describes a new technique for preparing mitotic fish chromosomes using short-term in vitro treatment with colchicine. The results show that a large number of good quality metaphases (many suitable for chromosome banding) can be obtained by this technique, which requires an average of 1 h and 30 min for all steps. The procedure considerably reduces the time normally required for chromosome preparations in fish.Key words. Fish cytogenetics; mitotic chromosomes; technique.Fish chromosome data have great importance in studies on evolution, systematics and mutagenesis, and in aquaculture. Although many techniques employing the cell culture technique to obtain chromosome preparations have been described 3'4, they are usually expensive and time consuming, a fact which limits their use in many laboratories. Direct preparations are less expensive and quicker to prepare but require injection of mitotic inhibitors into and killing of the animals 5-7. The air-drying technique, the most common procedure used in chromosome preparations, was initially developed for mammalian chromosome studies a and was first used in fishes by Ojima and Hitotsumachi 9. Although the basic steps remain the same, many small modifications have been added to this technique in recent years 1~ The present paper describes a modified method for obtaining fish chromosome preparations using the air-drying technique preceded by short-term colchicine treatment of the cells in vitro. The method results in a reasonable quantity and quality of chromosome spreads and is particularly useful for large fish specimens. Material and methodsMitotic chromosome preparations were obtained from cephalic kidney or gills or regenerated fin epithelium according to the following procedures: The animals were anaesthetized by immersion in a solution of 0.01% benzocaine. As soon as respiratory activity stopped, tissue was obtained either from the gills, by opening the operculum and pulling a forceps over the filaments of the branchial arches, or by cutting a piece of the fin epithelium, or by dissecting out pieces of cephalic kidney. Tissues were washed once in a Petri dish containing Hanks' saline solution (HSS) to remove any adhering fat or blood, and minced in 5-8 ml of clean HSS at room temperature. After discarding residual tissue lumps, the clean cell suspension was placed into a 20-ml conical centrifuge tube and 1 drop of 0.03% colchicine was added. The suspension was stirred lightly and the tube placed in an incubator at 37 ~ for 15 min. After colchicine treatment, the cell suspension was centrifuged at 800-1000 rpm for 7 min. The supernatant was removed by suction and the pellet resuspended by vigorous tapping of the tube, before adding 7 ml of a 0.075 M KC1 hypotonic solution. The tube was then stirred lightly and placed in an incubator at 37 ~ for 30-40 min. After hypotonization, 5 drops of a freshly prepared ice-cold fixative (3 methanol: 1 acetic acid) were added to the tube and the cell suspension stirred gently at room temperature. After 5 mi...

Karyotypic variation of Glanidium ribeiroi Haseman, 1911 (Siluriformes, Auchenipteridae) along the Iguazu river basin

“…The specimens were deposited in the fish collection of the Museum of Zoology of the University of São Paulo, and were cataloged (MZUSP 109797). The preparation of cell suspensions of mitotic chromosomes was obtained from the anterior kidney cells (Foresti et al, 1993). The fundamental number (FN) was calculated considering metacentric (m), submetacentric (sm) and subtelocentric (st) chromosomes as having two arms, and acrocentric (a) chromosomes as having only one arm.…”

Cytogenetics of Imparfinis schubarti (Siluriformes: Heptapteridae) from the Piumhi drainage, a diverted river in Minas Gerais State, Brazil

“…The mitotic metaphases were obtained according to BERTOLLO et al (1978) andFORESTI et al (1993). Chromosome morphology was determined according to the arm size relation proposed by LEVAN et al (1964).…”

Nuclear DNA content determination in Characiformes fish (Teleostei, Ostariophysi) from the Neotropical region

“…Mitotic chromosome preparations were obtained from kidney and gill cells using the air-drying technique described by Foresti et al (1993). Individual relative DNA content was determined according to the technique described by Gold and Price (1985) with some minor modifications.…”