The spreading of metaphases is a slow process which leads to a stretching of chromosomes

Abstract

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.

B chromosomes of Korean field mouse <i>Apodemus peninsulae</i> (Rodentia, Murinae) analysed by microdissection and FISH

“…The ends of the chromosomes 9B1, 9B2 and 9B8 remained "uncondensed" at metaphase. Chromosome compaction at the interphase -metaphase transition involves multiple DNA-protein interactions and leads to establishment of connections between neighboring chromatin loops and scaffold associated regions (SARs) and then between the neighboring coils of chromatin fiber (Hliscs et al, 1997). Probably B arm specific type 2 repeats that were present in chromosomes 9B1, 9B2 and 9B8 did not contain the binding sites for these proteins.…”

A new method for improving metaphase chromosome spreading

“…In contrast, Henegariu et al (2) specified no ambient conditions and used hot steam to moisten the slides and then put them on a 65-75°C metal plate, which, according to our own tests, dries the slides in 10 s. Whereas some researchers dropped cells on dry slides (4,5), others suggested that having a thin layer of water on the slides immediately before adding the cells improves spreading (1,2,6). Dropping cells from a height reportedly enhanced chromosome spreading (1,3), but others have disagreed (2,7). Aware of the "mysterious aspects" of chromosome spreading, Barch et al (10) suggested that it is more of an "art" than science.…”

Comparison of different ploidy detection methods in Oncorhynchus mykiss, the rainbow trout

“…Chromosome preparation is known to be very random (Deng et al 2003). There are too many factors to consider such as relative humidity (Spurbeck et al 1996), cell dropping height (Barch et al 1997;Hlics et al 1997), and flame vs. air drying method of the slide (Karami et al 2015). Still, even taking into account all these aspects, getting a well-spread metaphase is overly time consuming and not always rewarding.…”