Leukocyte Culture of the Eel Without Autologous Serum

Abstract

Though the application of the leukocyte culture technique with phytohemagglutinin (PHA) to the chromosome study of vertebrates can be traced back more than 14 years, this technique was successful mainly with non-fish species. Ojima et al. (1970), andHeckman andBrubaker (1970), however, successfully cultivated leukocytes of freshwater cyprinid fishes, gold fish (Carassius auratus) and carp (Cyprinus carpio). Paying attention to some difference in species-specific growth factors between cells of cyprinid and salmonid species, a reproducible method of culturing leukocytes from rainbow trout (Salmo gairdneri) was developed by using a culture medium saturated with oxygen (Heckman et al. 1971).In the above two kinds of culture methods for fish leukocytes and in usual methods for other vertebrates, the autologous serum is simultaneously introduced into the culture medium when cells are inoculated.However, no significant toxic and inhibitory effects on the leukocyte growth are caused by the addition of autologous serum.We found that for successful leukocyte culture of the eel removal of autologous serum was an essential factor.A new technique of culturing leukocytes from Japanese eel (Anguilla japonica) for chromosome study is described in this paper with particular interest in removal of autologous serum.Technique: Six adult females of Japanese eel (Anguilla japonica) with body weight of 128.0-355.0 g and body length of 47.0-60.0 cm were collected in Korean fresh-water. The specimens were transported alive to our laboratory aquarium and were kept at 28°C until they were used.All fishes were thoroughly rinsed with 70% alcohol before sacrifice. Anticoagulant (0.2 ml, Dif co blood separation vial) was drawn into a sterile 5-ml syringe through a needle of a size commensurate with the size of the fishes. Usually, a 23-gauge (20 mm) needle was suitable.For convenience of blood aspiration, the pericardium was removed before blood collection, since it was rather rigid comparing with that of other fish species.About four milliliter of whole blood was then drawn by puncture of ventriculus cordis.The blood was then centrifuged at 350 rpm for 5 minutes to separate leukocytes in a refrigerated system. The supernatant of leukocyte-rich plasma was collected by 3 to 4 repeated sedimentations at 5-minute intervals. By this procedures

Distribution of C-band heterochromatin in the ZW sex chromosomes of European and American eels (Anguillidae, Teleostomi)

“…Lymphocytes were separated by Ficoll-Paque (Pharmacia Fine Chem icals) gradient centrifugation. The cells were washed twice with balanced salt solution to remove autologous serum (Kang and Park, 1975). Each culture contained about 1.5X10i; viable cells in 10 ml of Eagle's minimum essential medium with Hank's salts (Flow Labs) as previously described (Park and Kang, 1979).…”

A method for obtaining mitotic figures from blood leucocyte cultures of rainbow trout, Salmo gairdneri

“…However, blood leucocyte culture for the study of fish chromosomes has enjoyed only limited success. Methods of leucocyte culture have been described for the carp, C.vprinus carpio (Ojima et al, 1970), the goldfish, Curussius uurutus (Heckman & Brubaker, 1970), the Japanese eel, Anguilla juponica (Kang & Park, 1975), the Atlantic salmon, Salmo salur (Grammeltvedt, 1975) and the rainbow trout, Salmo gairdneri (Heckman et al, 197 1 ;Grammeltvedt, 1974;Thorgaard, 1976).…”

A comparison of methods used for the separation of fish lymphocytes

“…Some workers (Etlinger et al, 1976; Kang & Park, 1975;Ojima et al, 1970) have used a similar method, centrifuging the whole blood undiluted and collecting leucocyte-rich plasma fractions at intervals. This is more applicable when larger volumes are available.…”