1) Sampling in the field:
In the field, samples are taken from the bare sediment scooping at least 50 ml of material from the upper layer (~1 cm) of sediment using the sample container itself. Sample containers are labeled according to the sample location and replicate number. Next to sampling bare sediment samples, patches with clear Vaucheria presence need to be sampled as positive control.
2) Sample preparation:
Prepare 2 tissue culture flasks and 1 Erlenmeyer per sample. One flask per sample will contain the potentially viable sample for incubation, while the other will be the negative control. Label the flasks accordingly.
If you work in batches because the autoclave can only handle a limited number of samples, make sure all Erlenmeyer flasks are labeled.
Take the samples from the freezer and let them thaw to room temperature.
Place the Erlenmeyer flask on a balance. Sterilise the metal spatula in the Bunsen flame. Bring ~50 gr of sample into the Erlenmeyer flask using the spatula. Add 2 ml of sterilised tap water and homogenise the sediment by stirring and gently shaking.
2.3) Add sample and medium to culture flask:
Place the tissue culture flask on a balance. Bring ~20 gr of homogenised sample into the flask using the spatula. Add 20 ml of MDV medium. Gently shake so the sediment gets evenly spread across the flask growth surface.
2.4) Autoclave sample:
Close the Erlenmeyer flask with cotton wool and sterilise the samples for 15 min at 121°C using the autoclave.
2.5) Add sample and medium to culture flask:
Place the tissue culture flask on a balance. Sterilise the metal spatula in the Bunsen flame. Bring ~20 gr of the sterilised sample into the flask using the spatula. Add 20 ml of MDV medium. Gently shake so the sediment gets evenly spread across the flask wall.
Above steps (2.2-5) are repeated for all samples, including the positive controls with Vaucheria present.
Place the tissue culture flask in the climate room for incubation for at least 6 weeks. Make sure the temperature and light conditions are set as described in: Equipment 3) Incubation
Gently shake the flasks once every couple of days to make sure nutrients are distributed well and gas exchange is facilitated.
Extend the incubation time with a few weeks, in case no clear visible signs of the filamentous algae are present. The positive controls are a good indicator of development.
4) Qualitative scoring:
First, the culture flasks are inspected for the development of green pigments (or brown in case of diatoms) on the sediment surface. Presence of pigments is an indication of good growth conditions for benthic algae and are therefore documented. If green pigments are present, the flask is more carefully observed for the presence of filaments. In case filaments are present it is likely the sample is positive for Vaucheria. In case the filaments are weakly developed a magnifying glass or microscope can be used to make sure the algae are present. To make sure the filaments are Vaucheria the flasks can be further inspected using magnifying glass or microscope.
Additionally, in well-developed samples Vaucheria can be identified at the species level. Therefore, the algae need to be sexually mature3. Moreover, it is possible to take pictures of the tissue culture flasks at the start and at the end of incubation.