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Published in Ronald Fayer, Lihua Xiao, Cryptosporidium and Cryptosporidiosis, 2007
Human endometrial carcinoma cell line RL95-2 supported optimal development of a C. parvum bovine isolate when cells were cultured for 7 days before parasite inoculation onto coverglass disks (Rasmussen et al., 1993). Oocysts were treated with 2% sodium hypochlorite, washed five times with PBS, excysted at 37°C in 0.75% sodium taurocholate and 0.25% trypsin (in PBS), washed with PBS, suspended in culture medium, filtered through 3-µm polycarbonate filters, and inoculated onto host cells in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, supplemented with 10% FBS, 2 g/L sodium bicarbonate, 10-mM HEPES, 5-µg bovine insulin, 100 IU/mL penicillin G, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B. After variable culture periods, cells on coverglass disks were washed in phosphate buffer, fixed with Bouin’s fixative for 2 h, washed with ethanol, and stained with Giemsa stain. Apparent asexual and sexual stages were observed by bright-field microscopy, and possible oocysts via scanning electron microscopy.
Genotoxic risk in occupational exposure to petrol and its amelioration by vitamin C and vitamin E
Published in Archives of Environmental & Occupational Health, 2022
Amrin Shaikh, Puranjay Chandel, Divya Chandel
Whole blood cultures were setup according to the method of Fenech9 with slight modifications to perform Micronucleus assay, where 7 mL of RPMI 1640 media (pre-supplemented with 10% fetal calf serum) was taken and 0.1 mL of phytohemagglutinin was added followed by 0.5 mL of heparinized whole blood. At 44 h, 10 µL of cytochalasin-B was added and the cultures were harvested at 72 h. Culture tubes were centrifuged at 2000 rpm for 15 min and the supernatant was discarded. The cells in the pellet were suspended by adding 6 mL of hypotonic solution (0.56% KCl) and incubated in a water bath at 37 °C for 20 min. Cells were fixed in chilled fixative (1:3, acetic acid:methanol) and culture tubes were kept in an ice bath for 20–30 min. After two changes of fixative, cells were suspended in a small volume of fixative and slides were prepared by gently dropping this cell suspension onto pre-cleaned, chilled, wet slides, dried on a slide warmer, and stained with 2% Giemsa Stain.