Spores
AMF spore extraction is one of the routine activities at CICG. The method used to extract spores from monospecific cultures, trap cultures or from field soil samples is wet sieving (Gerdemann & Nicolson, 1968) followed by centrifugation in a sucrose gradient (20% and 60%).
The soil sample will differ according to its origin: a) a small part (ca. 30 ml) of the substrate is removed from one side of the tubes with the help of scissors to analyze monospecific cultures, b) one or two metallic cylinders (ca. 50 ml) are taken from pots to analyze trap cultures, and c) soil aliquot (50 or 100 ml) of samples from the field or accessions that are sent to be deposited at the CICG. It is important in all cases that samples include roots as some AMF species produce intraradicular spores.
The sample is placed in a commercial blender with a capacity of 2 L and suspended in tap water (ca. 1.7 L). Two quick pulses in the blender are turned on to put the spores in suspension. Afterwards, the suspension is passed through two overlapping sieves, taking care that most of the soil particles are retained in the blender cup.
The bottom sieve has an opening of 45 µm where most of the spores will be retained. Other sieves with openings of 53 µm and 25 µm can also be used. The top sieve has an opening of 710 µm. In this sieve, larger caliber roots, organic debris and larger soil particles (sand) and large spores and sporocarps will be retained.
The material retained in the 710 µm sieve is transferred to a large Petri dish and inspected under a dissecting microscope for observation and collection of large spores and sporocarps. The material retained in the 45 µm sieve is initially collected in a becker with the help of a “rubber policeman” attached to a glass stick and then transferred to 50 ml Falcon tubes containing a gradient of 20% and 60% sucrose (15 ml of each of the solutions). Falcon tubes are then centrifuged for 1 minute at 2000 rpm (CELM brand centrifuge - model LS-3plus). In this process, the soil and other particles settle to the bottom and the spores and fine organic particles are suspended in sucrose.
The supernatant from each tube is then quickly decanted into a 45 µm stainless steel sieve. This sieve has the same opening as the sieve used in wet sieving, but it is smaller in size. The material collected in this sieve is washed for 1 minute with tap water to remove the excess of sucrose and transferred to Petri dishes. The spores are observed under the dissecting microscope and collected manually with a Pasteur pipette with extruded tip. Petri dishes and spores are stored in a refrigerator (4o C) and processed within a one to two weeks period.
To speed up the process of spore picking, a measurement of spore size of the various morphotypes under the dissecting microscope (for spores from trap cultures or field collected spores). Afterwards, the material collected in the 45 µm sieve (greater than 45 µm and less than 710 µm) can be passed through several sieves with different openings (300, 250, 180, 125 and 90 µm). This speeds up the process of pull out spores from the Petri dishes since morphotypes (and organic debris) are separated by different size.
The final and most laborious step is to separate the spores of each morphotype (except if pure cultures) to be used for various purposes (mount spores on slides, inoculate plants for experiment or for establishment of monospecific cultures, extract DNA, germination experiments, etc.). To sort spores into morphotypes, knowledge and skills in taxonomy is necessary, but if the spores are in good condition, anyone who is a good observer can sort the spores into morphotypes.
AMF spores from monospecific cultures, trap cultures or from field soil are mounted on permanent slides. The mounting medium used is Polyvinyl-Lactic Acid-Glycerol (PVLG) and PVLG mixed with Melzer's reagent.
On a slide, gently place one drop of PVLG and another drop of PVLG+Melzer. Afterwards, place the spores using a Pasteur pipette with an extruded tip, taking care not to dispense too much water along with the spores. With a needle, mix the spores well together with the mounting medium so the spores are placed in the center of the drop. Afterwards, let the slide rest for about 5 minutes to let the drop surface dry a little (this will help not to have bubbles when placing the coverslip).
Gently place the coverslip over the droplet with PVLG and press down. Place another coverslip over the drop with PVLG+Melzer and press gently.
Under the dissecting scope, break the spores individually using a needle or the tip of a pencil, pressing directly onto the coverslip. With this procedure, each spore can be broken up into a C shape (or like the “pac man”), thus exposing the germinal walls. If necessary, add a little more of the mounting medium along the sides of the coverslips. Allow the slide to dry at room temperature for 5 days and then place in an oven at 35-40o C for 3 days for the mounting medium to harden. There is no need to seal the coverslip as the mounting medium will harden and the slides become permanent.
A label must always accompany each slide and must contain the following information: a) genus name and specific epithet if known, b) date, c) culture code (or sample). Slides are stored on aluminum trays in oak boxes and organized by genera and species.