Generation of Mycelial Disks see also Fig steps

To obtain small pieces of vegetatively growing mycelium of equal size, a floating "mat of mycelium" (known as a "hyphal mat" or "mycelial mat") is grown in standing liquid culture, from which small disks can be cut for experimental purposes (10,11).

3.1.1. Preparation of Mycelial Mat

Two days prior to the intended start of the experiment:

1. Make sure to have one or two fresh slants (3-10 d old) for each Neurospora strain to be used (see Note 4).

2. Add 30 mL of Vogel's minimal medium to a sterile Petri dish or cell culture dish (see Note 5). Depending on the scale of the experiment use one to three dishes for each Neurospora strain.

3. Add 1 to 2 mL Vogel's minimal medium to each slant, replace cotton wool plug, and vortex vigorously.

4. Take off spore suspension with a sterile filtered pipet tip and transfer to a 1.5-mL Eppendorf tube.

Fig. 1. RNA isolation from Neurospora. Schematic overview of the processes involved in extracting total RNA from Neurospora mycelium. 1. Fresh slants; 2. Mycelial mats; 3. Mycelial disks; 4. The circadian experiment; 5. Harvest; 6. Homogenization of mycelium; 7. Extraction of total RNA. Conidia are harvested from fresh slants and used to inoculate liquid medium to produce mycelial mats, from which mycelial disks are cut. These segments of vegetatively growing mycelium are subjected to experimental procedures and are subsequently harvested and snap-frozen. RNA is then extracted from frozen, ground-up mycelium.

Fig. 1. RNA isolation from Neurospora. Schematic overview of the processes involved in extracting total RNA from Neurospora mycelium. 1. Fresh slants; 2. Mycelial mats; 3. Mycelial disks; 4. The circadian experiment; 5. Harvest; 6. Homogenization of mycelium; 7. Extraction of total RNA. Conidia are harvested from fresh slants and used to inoculate liquid medium to produce mycelial mats, from which mycelial disks are cut. These segments of vegetatively growing mycelium are subjected to experimental procedures and are subsequently harvested and snap-frozen. RNA is then extracted from frozen, ground-up mycelium.

5. Measure the optical density (OD)530 from a dilution of the spore suspension (e.g., use 2.5 ^L in 1 mL H2O). OD530 = 1 equals approx 3 x 106 spores/mL.

6. Vortex the spore suspension vigorously and transfer approx 1 x 108 spores into the liquid in each cell culture dish and pipet slowly up and down to distribute the spores evenly (see also Note 6).

7. Leave cultures on the lab bench or incubate at 25°C or 30°C (static incubation under constant light; see also Note 6). After 12 to 18 h a mycelial mat will form, floating on the liquid. To get a good mycelial mat of even thickness, care should be taken not to disturb the dishes when the mat is still thin and fragile.

8. If necessary, vary the growth conditions in order to obtain a thick and rigid nonsporulating mycelial mat on the intended starting day of the experiment (see Note 6).

Fig. 2. Cutting mycelial disks. Left: Mycelial disks have been cut from a mycelial mat and transferred to a fresh dish, ready for inoculation of liquid cultures. Middle and right: Another mycelial disk is cut using a flamed cork borer. Pictures by C. Heintzen, University of Manchester, UK.

3.1.2. Cutting of Mycelial Disks On the starting day of the experiment:

1. Check to make sure the mycelial mat is thick, quite rigid, and not overgrown or sporulating. Only then proceed to the next step (see Note 7).

2. Make sure flasks for inoculation have been prepared (see Subheading 3.2.1.).

3. Cut small pieces of mycelium of equal size, so-called mycelial disks, from the mycelial mat using a flamed cork borer (Fig. 2) (see also Note 8). Avoid cutting disks in the peripheral areas of the mycelial mat where aerial hyphae are present or areas where fungal spores may have developed (see Note 7).

4. Transfer mycelial disks to a fresh dish, containing a small volume of Vogel's minimal medium, using a pair of flamed pointed forceps (Fig. 2).

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