There are many ways of fixing and preserving 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, and suggestions of Tom Artois (Univ. Hasselt) for whole mount 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.
Whole mount preservation: