Hliscs R, Mühlig P, Claussen U
Institute of Human Genetics and Anthropology, Friedrich-Schiller-University, Jena, Germany.
Cytogenet Cell Genet. 1997;76(3-4):167-71. doi: 10.1159/000134537.
In routine chromosome harvesting of blood lymphocytes it is well accepted that metaphase spreads are obtained from fixed mitotic cells which burst on the surface of slides during the dropping procedure. For confirmation and clarification, fixed mitotic cells were dropped onto coverslips and observed under an inverted microscope during the evaporation of the fixative. Fixed mitotic cells in the metaphase stage first stick onto the surface of the coverslip without changing their three-dimensional shape and they do not burst. Thereafter, when evaporation of the fixative occurs, they slowly flatten until they are spread. This slow process leads to a stretching of chromosomes which may be a prerequisite for high resolution banding patterns. Confocal laser scanning microscopic measurements of the length, thickness, and width of chromosomes after (i) short term evaporation of the fixative, (ii) evaporation of the fixative under routine harvesting conditions and (iii) a prolonged evaporation, confirmed the stretching of chromosomes. The humidity, the temperature, and the drying time of the fixative influence the dynamic flow of the remaining fixative on the slide. This dynamic flow leads to an intensive wash of the fixed mitotic cells with increasing concentrations of acetic acid which is primarily responsible for the better quality of the metaphase spread.
在常规的血液淋巴细胞染色体收获过程中，人们普遍认为中期染色体铺展是通过固定的有丝分裂细胞获得的，这些细胞在滴片过程中在载玻片表面破裂。为了进行确认和澄清，将固定的有丝分裂细胞滴到盖玻片上，并在固定剂蒸发过程中在倒置显微镜下观察。处于中期阶段的固定有丝分裂细胞首先粘附在盖玻片表面，而不改变其三维形状，并且它们不会破裂。此后，当固定剂蒸发时，它们会慢慢变平直至铺展。这个缓慢的过程导致染色体伸展，这可能是获得高分辨率带型的一个先决条件。共聚焦激光扫描显微镜对（i）固定剂短期蒸发后、（ii）常规收获条件下固定剂蒸发后以及（iii）长时间蒸发后染色体的长度、厚度和宽度进行测量，证实了染色体的伸展。固定剂的湿度、温度和干燥时间会影响载玻片上剩余固定剂的动态流动。这种动态流动会导致固定的有丝分裂细胞被浓度不断增加的乙酸强烈冲洗，而乙酸是中期铺展质量更好的主要原因。
