The common fixative used in cytogenetics (usually a combination between methanol and acetic acid) helps keep the cells in a "swollen" state, achieved after hypotonic treatment. It also helps break the cell membrane of the red blood cells and elutes some of the membrane lipids and proteins. The fixative solution makes the cell membrane more fragile and suitable for spreading flat on the slide when subjected to the drying techniques.
Regular 3:1 fixative
The conventional 3:1 methanol:acetic acid fixative worked well with all types of cells tested (lymphocytes, fibroblasts, tumor cells). Quality of slide preparations as well as banding and FISH depended more on the drying technique than on the proportion of methanol to acetic acid. However, as detailed below, G-banding contrast was influenced by the type of fixative (fig. 5, 6).
High acetic acid
2:1 and 1:1 methanol:acidic acid concentrations were also tested. Higher acid concentration generally improved spreading, probably by increasing even more the fragility of the cell membrane and by decreasing the speed of fixative evaporation. However, higher acetic acid concentrations are NOT necessary if the drying technique is properly mastered.
High methanol (6:1) fixative
A 6:1 methanol acetic acid can be used to spread pellets that tend to break too easily when conventional fixative and drying techniques are used. By increasing methanol proportion, drying/evaporation of the fixative is much faster, not allowing the cell membrane and the content of the cells to spread too far.
3:1 ehanol:acetic acid was compared with the regular fixative. Ethanol fixative worked well, metaphases were clean and the chromosomes were prevented from overspreading, due in part to the slower evaporation of ethanol compared to methanol. Apparently, a 3:1 ethanol:acetic acid works similarly to a methanol:acetic acid fixative between 3:1 and 6:1. GTG banding and FISH quality using ethanol:acetic acid fixed pellet was similar to regular fixative. Ethanol is much less toxic than methanol and can be successfully used, particularly on pellets that tend to overspread.