When root tip sets are fixed in acetic ethanol or squashed in drops of glacial acetic acid at different durations or concentrations, there is often an apparent variation in sizes of chromosomes observed for different root tip sets
"Why does exposure of root tip cells to glacial acetic acid during fixing or during squashing affect the observed size of chromosomes?"
Acetic acid has very low pH, which weakens hydrogen and ionic bonds.
In young cell walls, pectins are largely acidic and then prone to establish electrostatic interactions with cellulose and hemicuellulose. In older tissues pectins are methyl-esterified -- more hydrophobic -- and harden at acid pH.
Chromatin behaves roughly the same way. Interactions between histones and DNA are mostly electrostatic, so weaken by pH lowering.
Weakened interactions between histones and DNA result in softer chromosomes that can be easily squashed, turning them from bread to pancakes.
Glacial acetic acid and ethanol dehydrate cells and their components including chromosomes.
Traditional fixatives are based on the observation that acetic acid causes swelling of the protoplast, while Ethanol causes shrinkage, so most fixatives play with the ratio of the two to try to get good images. The most common fixative for light microscopy of plant tissues is FAA - (Formalin, Acetic acid, and Alcohol). It is prepared as 90 ml of 50% Ethanol, 5ml of glacial acetic acid, and 5ml of commercial formalin.
Dear Philip,
can we squash root tips with drops of glacial acetic acid (i.e. 99-100%)? Will 99-100% (as commercially available) swell chromosomes?
"Why does exposure of root tip cells to glacial acetic acid during fixing or during squashing affect the observed size of chromosomes?"
Acetic acid has very low pH, which weakens hydrogen and ionic bonds.
In young cell walls, pectins are largely acidic and then prone to establish electrostatic interactions with cellulose and hemicuellulose. In older tissues pectins are methyl-esterified -- more hydrophobic -- and harden at acid pH.
Chromatin behaves roughly the same way. Interactions between histones and DNA are mostly electrostatic, so weaken by pH lowering.
Weakened interactions between histones and DNA result in softer chromosomes that can be easily squashed, turning them from bread to pancakes.
Dear Olivier,
You have put across the answer in a simple and perfect manner. Thanks a lot.
Oliver is right. Acetic acid soften the tissue.. So squashing is easy d get perfect spread.
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