There are many ways of fixing free-living flatworms. The protocols I give here are only suggestions, which are essentially based on suggestions of Willi Salvenmoser (Univ. Innsbruck) and Tom Artois (Univ. Hasselt). Generally, the quality of the fixation in these tiny animals is very important, and fixation
The procedures are a little different for marine, brackish and freshwater species. The higher osmolarity of sea water affects both the relaxation and fixation step.
Fixation:
Before fixation the worms should be relaxed, so that they do not contract and assume unnatural postures when placed into the fixative.
Marine (30-40‰): place the worm into a mixture of about 1/3 the water in which the worms were collected or extracted and 2/3 magnesium chloride solution (71.4 g/L of MgCl2 *6H2O in distilled water or tap water).
Brackish (5-30‰): place the worm into a mixture of about 1/3 the water in which the worms were collected or extracted and 2/3 magnesium chloride solution (71.4 g/L of MgCl2 *6H2O in distilled water or tap water).
We usually fix worms for 1h in 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (adjusted to pH 7.2) at 4°C (i.e. in the refrigerator).
Such worms could later be postfixed with Osmium Textroxide, if one want to investigate them with transmission electron microscopy.
fixed for 1 h in 2.5% glutaraldehyde
in 0.1 M cacodylate buffer (pH 7.2) containing 10% sucrose,
postfixed for 1 h with 1% osmium tetroxide in 0.1 M
cacodylate buffer,