Filamentous Fungi

1. Soil Sample Preparation

1. First, determine the initial moisture content of the soil by overnight drying of a known amount of the moist soil, and reweighing the dried soil. The equation to determine the initial moisture content of the soil is:
   
   (Equation 1)
   where:
   MC = moisture content
   W = net weight
   D = dry weight
2. Calculate the amount of water that must be added to 25 g of soil to increase the soil moisture content to 10% on a dry weight basis.
3. Next, add that amount of water to 25 g of the soil.
4. Cover the container with plastic wrap and puncture the film several times with a probe to allow aeration during incubation. Secure the film with a rubber band.
5. Weigh the soil and wrap, then incubate the sample at room temperature for one week.

2. Fungus Inoculation and Incubation

1. After the incubation is complete, weigh the soil sample with the wrap and rubber band. Calculate the weight loss due to moisture loss.
2. Calculate the new soil moisture content.
3. Next, prepare a 1/10-dilution series of the soils as shown in Figure 2. This will result in dilutions of 10^-1 (bottle A), 10^-2 (tube B), 10^-3 (tube C), 10^-4 (tube D), and 10^-5 (tube E) g soil per mL suspensions.
4. Prepare sterile Rose Bengal-streptomycin agar plates. Both the Rose Bengal and streptomycin inhibit bacterial growth. For very fertile soils where soil microbial populations are high, the chosen dilutions should be higher, i.e., 10^-3, 10^-4, and 10^-5, g soil per plate.
5. To each of the 10^-3 plates, add 0.1 mL from dilution tube 10^-2, and spread over the agar surface using an ethanol-flame sterilized glass spreader. Because a quantity of 0.1 mL is plated, this makes the effective dilution 10^-3. Repeat these steps for all dilutions.
6. Incubate plates at room temperature for one week.

3. Colony Counting and Examination by Microscopy

1. Make colony counts at one and only one dilution of each soil. The plates that are counted should have discreet countable colonies. Overgrown plates should not be counted. Likewise, plates with less than 10 colonies should not be counted. Note and describe the cultural characteristics of three different colonies.
2. Prepare pressure tape (transparent tape) mounts on slides for detailed microscope study using the following procedure:
   1. Deposit a drop of lactophenol mounting fluid at the center of a clean glass slide
   2. Cut a strip of clear cellophane tape about 3 cm long from the stock roll. To avoid contaminating the adhesive surface, use forceps when handling the tape. A dissecting needle will aid in freeing the tape from the forceps.
   3. The adhesive side of the tape is applied to the surface of a sporulating fungus colony. Take care to avoid excessive pressure on the tape or too dense a mass of hyphae and spores will be collected.
   4. Remove the tape from contact with the fungus colony and apply it, adhesive side down, to the drop of mounting fluid on the glass slide. Rub the tape gently with a smooth, flat instrument to express air bubbles.
   5. Examine the tape mount microscopically under the oil immersion objective with oil.
   6. Identify two different fungal genera using the supplied fungal identification key (Figure 4).

Figure 4. Fungal identification key.