Symbiotic Fungi: Principles and Practice (Soil Biology)

94 I.M. van Aarle
l Usually 20–50 ml is used for mycelium, 40–100 ml for root sections and
100–500 ml for root pieces.
4. Let the reaction develop for 20 min:
l Optimal reaction times should be determined for each type of samples.
Ideally this is done by immersion of the sample in a couple of drops of ELF
substrate-buffer solution on a glass microscopy slide. The sample is monitored
continuously under the microscope for the occurrence of ELF crystals
(bright yellow-green fluorescence), and the time of appearance of first specific
ELF crystals as well as that of background staining in the medium is measured.
The fluorescent precipitate is very photostable and supports long periods of
visualisation.
5. Wash the sample with wash buffer in order to remove excess substrate.
This washing increases the preservation time of the slides:
l Normally three changes of wash buffer from 5 min each with gentle agitation
are sufficient, but sometimes more changes are needed in order to remove all
excess substrate.
l The pH of the wash buffer must not be above 8.0 otherwise the crystals may
dissolve.
l In order not to lose too many hyphae or not to damage the root sections during
this washing step the samples could be washed in mini-sieves. Such sieves
can be made from 2 ml Eppendorf tubes, by cutting off the bottom, heating
the cut edge in a flame and immediately put the heated edge on a piece of
nylon mesh. Once the edge is cooled the mesh is sealed to the tube, resulting
in a mini-sieve.
6. Carefully remove the wash buffer without drying the samples.
7. Mount the sample in the mounting medium provided with the kit. Put one or
two drops of mounting medium on the microscopy slide, put the sample in
the mounting medium. Hyphae and intraradical mycelium can be carefully
distributed with fine forceps. Cover the samples with a cover slip:
l Leave the mounted slides on a flat surface to dry. After several hours the
slides can be sealed with nail varnish.
l The fluorescent signal of the ELF precipitate is stable for a long time;
however, it is advisable to store the samples in the dark. After several days
of storage the mounting medium might start to crack, especially if the
mounting medium was viscous/aged at the time the slides were made.
l Store the mounting medium upside down. In this way the tube does not have
to be inversed when applying the mounting medium. Inversion of the tubes
readily leads to the formation of air bubbles, especially when the mounting
medium gets older and more viscous.
l Do not use glycerol to mount the samples, since the ELF crystals will dissolve
within hours.