There are many ways of fixing free-living flatworms. The protocols I give here are only suggestions, which are largely based on suggestions of Willi Salvenmoser (Univ. Innsbruck) for histological preservation.
Fixation for histology:
Generally, the quality of the fixation in these tiny animals is very important, and fixation should always be done with fresh fixatives. Moreover, the procedures are a little different for marine, brackish and freshwater species, because the higher osmolarity of sea water affects both the relaxation and fixation steps.
Relaxation: Before fixation the worms should be relaxed with magnesium chloride solution (7.14 g/100mL of MgCl2 *6H2O in distilled water or tap water), so that they do not contract and assume unnatural postures when placed into the fixative.
- Marine (25-40‰): place the worm into about 1ml of mixture of about 1/3 of the water in which the worms were collected or extracted and 2/3 of magnesium chloride solution.
- Freshwater (0-2‰): place the worm into about 1mL of the water in which the worm was held and add one to a few drops of magnesium chloride solution. Different species react quite differently to this, and you will have to experiment a bit.
- Brackish (2-25‰): again, you will have to experiment a bit. The higher the salinity, the more resistant the worms are to the magnesium chloride solution.
Fixation: I 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) (2.14g/100mL).Such worms could later be postfixed with osmium textroxide (if one wants to investigate them with transmission electron microscopy). For marine samples I add 10% sucrose to the fixative, which increases the osmolarity and which improves the quality of the fixation. For field work I usually prepare one fixative with sucrose and one without, and then I mix the two fixatives for brackish water samples in approximately the right relationship. Worms are then rinsed in buffer (of the same osmolarity) and transferred to 70% ethanol, where they can be stored for some time.