Iguana vitro subsp. culture

Dib, Lais Verdan, Barbosa, Alynne da Silva, Correa, Lais Lisboa, Torres, Breno da Silva, Pissinatti, Alcides, Moreira, Silvia Bahadian, Teixeira, Rodrigo Hidalgo Friciello, Andre, Costa, Luíz Mota da, Jos, Muniz, e Augusto Pereira Carneiro, Junglos, Amauri Michel, Hirano, Zelinda Maria Braga & Amendoeira, Maria Regina Reis, 2024, Morphological and molecular characterization of parabasilids isolated from ex situ nonhuman primates and their keepers at different institutions in Brazil, International Journal for Parasitology: Parasites and Wildlife 24, pp. 100946-100946 : 100946-

2.4. In vitro culture of protozoa

All fecal samples that showed motile flagellate trophozoites on direct examination were inoculated into of modified Pavlova xenic culture medium ( Jones, 1946; Pavlova, 1938) and of TYSGM-9 medium ( Diamond, 1982) in glass test tubes (15 × 1.8 cm) with screw caps. To make a 1000 ml of Pavlova medium was used 1.292 g of sodium hydrogen phosphate, 0.42 g of potassium dihydrogen phosphate, 7.27 g of sodium chloride, and 1, 46 g of yeast extract. A total of 970 mL of TYSGM-9 medium was made by a nutrient broth composed of 2g of casein peptone, 1g of yeast extract, 7.5 g of sodium chloride, 2.8 g of anhydrous dipotassium hydrogen phosphate, 0.4 g of potassium dihydrogen phosphate and 2 g of porcine gastric mucin. Both xenic culture media were autoclaved.

A total of 200 μl of fresh stool solution and buffered saline solution used for direct examination was inoculated into each tube of a set of four tubes. Each tube had 8 mL of medium. Two of these tubes contained modified Pavlova medium, one supplemented with horse serum and the other supplemented with fetal bovine serum. The two remaining tubes contained the TYSGM-9 medium with the respective sera and 500 μl of Tween 80 solution 5%.

In addition, antibiotic solutions with 10,000 U/μl streptomycin and penicillin and a drop of rice starch suspension (dilution: 1 g of starch in 16 ml of the medium to be used) were added to the xenic medium. Once inoculated, each isolate was incubated in a bacteriological oven at 36 ◦ C, and the sediment in the tube was observed every day at 24-h intervals for seven days. From the seventh day on, all samples that contained viable parasites, i.e., moving parasites, were considered to be successfully isolates. In vitro maintenance of the protozoa was performed at

2

48–72 h intervals of subculture, i.e., the isolate was inoculated into fresh medium.