Abstract: AbstractObjective: By continuing to inoculate the supernatant of the amniotic fluid cultured for 7～9 days into the in situ culture box, the harvest steps of the original supernatant culture method were simplified, and the optimal temperature and humidity conditions for controlling the karyotype dispersion of the in situ slide method were explored. Methods: The amniotic fluid samples of 50 pregnant women with prenatal diagnosis were selected as the study objects, and the amniotic fluid was cultured in situ with double lines. About 7～9 days after the growth of multiple spindle cell clones attached to the wall, fresh media was replaced. One box of supernatant was transferred to the cell culture bottle for further culture, and the other box was directly inoculated into the new in situ culture box. The collection of the amniotic fluid was considered according to the cell formation during the division period 1～3 days after the fluid exchange. The time of fluid exchange and harvest time of the supernatant were the same as that of the amniotic fluid. The supernatant suspension of amniotic fluid harvested by the culture bottle method was prepared by dropping tablets, baking tablets and G-banding. 100 glass slides of amniotic fluid and supernatant of amniotic fluid harvested by the in-situ method were randomly divided into 4 groups. After the last fixation, the glass slides were placed under the corresponding temperature and humidity conditions for karyotype dispersion. Group B: 20 ℃, 50%; Group C: 23 ℃, 40%; Group D: After baking at 23 ℃, 50%, at 80 ℃ for 2 h or overnight at 55 ℃, G banding was digested by pancreatic enzyme. The number of cell clones, karyotypes andhigh-quality karyotypes after G banding were compared between the in situ method of supernatant and the culture bottle method of supernatant. At the same time, the differences in chromosome dispersion between different groups were compared according to the temperature and humidity.