The initial solutions were prepared at the following concentration: 100 mg/mL. Then, in order to obtain a homogeneous emulsion, sonication was carried out using an ultrasonic homogenizer (Hielscher Ultrasound Technology, Teltow, Germany).
The research involved the use of Rhabditis sp. nematodes, which live in soil in the natural environment. The culturing nematodes and research methodology of a nematocidal properties assessment was developed by the Department and Chair of Biology and Genetics of the Medical University of Lublin, Poland, patent no. 232918, Bogucka-Kocka A, Kołodziej P. The Rhabditis sp. nematodes were cultured in sterile 6-well plates on an agar medium enriched with bovine serum. After 4–5 days of culturing at room temperature, the growth and development of all the nematode development stages were observed.
Rhabditis sp. nematodes were eluted from the agar solid medium using 0.6% NaCl. They were then transferred to new sterile 24-well plates for culturing on a liquid medium. The tested compounds and albendazole were added to the culture prepared in 2 experimental concentrations: 5.56 mg/mL and 11.12 mg/mL. The respective new thiosemicarbazide derivatives were tested in a concentration corresponding to a survival rate of 50% for albendazole (LC50) and half of the former. The culture of nematodes without the addition of the tested compounds constituted the negative control, while the positive control was the culture with the addition of the following reference substance with anthelmintic activity: albendazole (Sigma, Munich, Germany).
After a 24 h exposure, the culture of Rhabditis sp. nematodes was observed in terms of development, deformity, damage, and motility using a stereoscopic and light microscope (Olympus, Tokyo, Japan). To determine the viability of nematodes, they were stained with methylene blue. The live and dead specimens were counted in counting chambers.
The research involved the use of Rhabditis sp. nematodes, which live in soil in the natural environment. The culturing nematodes and research methodology of a nematocidal properties assessment was developed by the Department and Chair of Biology and Genetics of the Medical University of Lublin, Poland, patent no. 232918, Bogucka-Kocka A, Kołodziej P. The Rhabditis sp. nematodes were cultured in sterile 6-well plates on an agar medium enriched with bovine serum. After 4–5 days of culturing at room temperature, the growth and development of all the nematode development stages were observed.
Rhabditis sp. nematodes were eluted from the agar solid medium using 0.6% NaCl. They were then transferred to new sterile 24-well plates for culturing on a liquid medium. The tested compounds and albendazole were added to the culture prepared in 2 experimental concentrations: 5.56 mg/mL and 11.12 mg/mL. The respective new thiosemicarbazide derivatives were tested in a concentration corresponding to a survival rate of 50% for albendazole (LC50) and half of the former. The culture of nematodes without the addition of the tested compounds constituted the negative control, while the positive control was the culture with the addition of the following reference substance with anthelmintic activity: albendazole (Sigma, Munich, Germany).
After a 24 h exposure, the culture of Rhabditis sp. nematodes was observed in terms of development, deformity, damage, and motility using a stereoscopic and light microscope (Olympus, Tokyo, Japan). To determine the viability of nematodes, they were stained with methylene blue. The live and dead specimens were counted in counting chambers.
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