Characteristics of sea-urchin mitotic apparatus isolated using a dimethyl sulphoxide/glycerol medium.

A method for isolating sea-urchin zygote mitotic apparatus (MA) is described which is based on the Filner-Behnke method of isolating brain microtubules. MA were isolated in 50 % (v/v) glycerol, 10% (v/v) dimethyl sulphoxide, 5 mM MgCl2, o-i mM EGTA, and 5 mM Sorensen's phosphate buffer at a final pH of 6-8. MA stored at room temperature in isolation medium had stable birefringence, stable microtubules, and stable solubility properties (in 0-5 M KC1) over a period of 10 days to 2 weeks. These MA also seem to have more dry matter per volume than do MA isolated using hexylene glycol. The biggest disadvantages of the method are that zygotes often are difficult to lyse, and that cytoplasmic debris the same size as the MA sometimes contaminates the MA pellet.