Fluorescence Microscopic Studies on the Trematodes Eggs: On the Discrimination of the Dead from the Live Eggs of Paragonimus westermani and Clonorchis sinensis

In this report it was tried to discriminate the dead from the live eggs of trematodes (Paragonimus westermani and Clonorchis sinensis) by means of fluorescence microscopy. In the beginning, the attitude of the eggs of the trematodes toward the acridine orange vital staining was examined comparing with that of nematodes eggs (Ascaris, Ancylostoma etc.). As the result, it was recognized that the eggs of the both trematodes had two characters seemed to be inconsistent each orther: (1) They were liable to be injured by acridine orange solution in contrast to the eggs of nematodes, because the contents (germs and yolk cells or miracidium) of the live eggs, immersed in aqueous solution of acridine orange for several days, emitted secondary fluorescence in yellowish green. (2) On the other hand, the trematodes eggs were more resistant to the staining with acridine orange than the nematodes eggs, because by the treatment with acridine orange in short time the contents of the dead eggs did not emit secondary fluorescence. These characters, differed from that of nematodes, were more remarkable in the eggs of Paragonimus westermani than Clonorchis sinensis. As a result of the experiments, the condition of the acridine orange vital staining, suitable for the discrimination of the dead from the live eggs of the trematodes, was decided as follows: In case of acridine orange concentration in 1:5, 000 and temperature at 26°C, the time for the staining was in need of 5∼6 hours for the Paragonimus eggs, and (10∼) 60 minutes for the Clonorchis eggs. When the trematodes eggs were killed by various methods (boiling, drying, action of chemicals etc.) and then observed under the fluorescence microscope after the treatment with acridine orange according to the above mentioned condition, the contents of the live eggs did not fluoresce, but the most of the dead ones emitted secondary fluorescence in yellowish green. Therefore, it was proved that on the eggs of the trematodes it was able to discriminate the dead from the live ones according to the observation whether their contents fluoresce or not. And the observation of autofluorescence in the contents of eggs was also helpful for the discrimination.