Medium Preparation

1. To prepare 11 of MSR medium, mix 10 ml of solution 1 (macroelements) with 10 ml of solution 2 (calcium nitrate), 5 ml of solution 3 (vitamins), 5 ml of solution 4 (NaFeEDTA), 1 ml of solution 5 (microelements) and 10 g of sucrose.

2. After complete dissolution of the sucrose, adjust the volume to 1 l with demineralized water.

3. Adjust the pH to 5.5 with NaOH 1 M (rectify with HCl 1 M if necessary).

4. Add 3 g/l Gelgro (ICN Biochemicals, Cleveland, OH) or 5 g/l Phy-tagel™ or 8 g/l agar.

5. Autoclave for 15 min at 121 °C under 1 bar pressure.

6. After autoclaving, maintain the medium at 60 °C in a water bath.

Table 3. Medium quantities required in the different monoxenic systems

Proximal or root compartment

Slope

Distal-compartment

Used preferably for

Mono-compartmental Petri plates (9-cm diameter)

Fill with ~ 35 ml MSR

spora,

Glomus

Mono-compartmental Petri plates (squared 12x12cm)

Fill with ~ 100 ml MSR

No

No

Gigaspora

Bi-compartmental Petri plates (9-cm diameter)

Fill the proximal compartment with ~ 25 ml MSR until the top of the separation

Create a slope of 45 °C with ~ 5 ml MSR without sugar

Fill with ~12ml of MSR without sugar

Glomus

7. Pour the medium in the Petri plates under laminar flow, as indicated in Table 3.

For bi-compartmental Petri plates, it is important to promote spreading of mycelium from the proximal to the distal compartment. Two procedures can be used. In the first procedure, the proximal compartment is filled with the synthetic growth medium to the top of the separation wall. In the distal compartment, aslope(~ 45°)isrealizedwiththesamemediumbutlacking C. After solidification, the distal compartment is half-filled horizontally with the same medium lacking C (Fig. 2). In the second procedure, both compartments are filled 2 mm above the partition wall (Rufyikiri et al. 2003). After solidification, the medium in the distal compartment is cut along the partition wall with a scalpel and removed by using a spatula. The distal compartment is further replaced with a new medium lacking C at the level of the partition wall.

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