Preparation of Daphnia-parasites for light and electron microscopy and DNA extraction

Please cite as: Ebert, D. Web-guide to Daphnia parasites.

There is nothing like the ultimate method to preserve or fix your samples, but we use routinely the following methods and have good experience with them. We did not find a way to fix field sample for later investigation of host and parasite (e.g. for epidemiological studies). Formol-sugar, which is commonly used to preserve Daphnia and other plankton organisms is only good for the hosts. Microparasites cannot be detected easily in this preservative. The best way to screen mass samples of plankton for parasites is to use fresh material. Keeping it at 4°C will allow you to keep it for a few days. The following methods have all been used successfully for light- and electron-microscopy and DNA extraction.

DNA extraction

Later on, DNA is conveniently extracted
with a commercially availabe kit

Fixing samples for DNA analysis

  • Freeze animals at -20°C (or at -80°C). (This is always the best for DNA samples, but shipping is difficult)
  • Put animals in 100% (or 96%) ethanol.
  • Put animals in 70% ethanol and 30% destilled water (many people prefer this method over 100% ET-OH).
  • We usually do all three methods, to be on the safe side

Fixing for light microscopy

  • Place infected hosts in water with addition of formaldehyde (final concentration should be about 0.2%). Microsporidian parasites will survive and the hosts are preserved in sufficiently good condition to allow shipping. This material will be useful for squash preparations to be made.
  • Preserve some infected hosts in a histological fixative such Bouin (very good!) (recipe below) or 2-4 % formalin (also useful but less good).

Fixing for electron microscope

  • Place animals in 2.5 % glutaraldehyde.
  • The animal should be fixed the following day (i.e. wash them 2 to 3 times with the buffer used).
  • Animals can be shipped in buffer. If they are for too long in glutaraldehyde, there may be a risk that they will be overfixed.
  • If you fix large animals prick them with a needle to improve penetration (For Daphnia this is usually not needed).

Recipes

Bouin fixative

  • Saturated picric acid (75 ml)
  • Formaldehyde (40%) (25 ml)
  • Glacial acetic acid (5 ml)
  • Combine all three to make up 105 ml fixative.

Gluteraldehyde fixative

  • CaCl2 0.03 gram (33% more if you use CaCl2 × H2O)
  • 0.2 M Cacodylate buffer (50 ml) (see below for recipe)
  • 25 % Gluteraldehyde (10 ml)
  • Combine and add dest. water to make 100 ml fixative

Cacodylate buffer (0.2M)

Used in Gluteraldehyde fixative and as post fix buffer.

  • 100 ml NaCacodylate (0.4 M) (this is 8.561 gram/100ml)
  • 100 ml 0.2 M HCl
  • Combine to make 200 ml buffer (adjust pH to 7.0 to 7.2 if necessary).