Application of vital staining with Acridine Orange (A.O.) to investigation of embryonic growth at the stage of organogenesis

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In this study, normal and abnormal growth of post-implantation embryos was investigated by means of vital staining with acridine orange using fluorescence microscope. Embryos from mother mice injected with AO, emitted intense secondary fluorescence five minutes after injection and still did emit weak secondary one 24 hrs. after. But 48 hrs. after, embryos did illuminate only auto-fluorescence without the secondary one.Pregnant mice were administered with Mitomycin C and Caffeine simultaneously on 9th day of gestation. This treatment has been well known to cause digital malformation of the embryo at a high incidence. Then, on the successive 10th and 11th day of gestation, one hr. after AO injection into mother mice, embryos were taken out of uterine horns and investigated by fluorescence microscope.From the time course investigation, we confirmed that the secondary fluorescence existed longer in damaged tissue cells of embryos by treatment with Mitomycin C and caffeine than in control ones, as living embryonic cell can excrete AO while damaged cells can not.There were many granule-like cell masses with intense yellowish-green fluorescence diffusely on the whole body of embryo and those specifically existed with high density at the mesenchymal tissue of limb buds without formation of digital rays.From the results, above mentioned, normal and abnormal embryonic growth can be investigated in detail by color and intensity of fluorescence using fluorescence microscopy through AO vital staining.Further investigation in combination with whole embryo culture is expected to bring us a fruitful result about normal and abnormal embryonic growth.