Objectives: Isolation of microfungi by dilution plating is widely employed in survey studies of the saprophytic soil microfungal flora. Because it is a commonly-used method, it is valuable in comparative studies. When the numbers of soil collections (sites) per community and isolates are standardized, the sample populations can be analyzed and compared by the quantitative methods used in higher plant ecology. The composite fungi obtained from a single community can be characterized by calculating frequency (sites of occurrence as a percent of total sites sampled) or tensity (absolute density: isolates per gram of soil; relative density: isolates as a percent of total isolates) for the individual species. Sample population from different communities can be compared by figuring 2w/a + b coefficients of similarity, where a = the number of species in one community, b = the number of species in the other community, and w = the number of species present in both. A coefficient of 1.0 (or X100, 100.0) indicates identical composition, and values below 1.0 indicate corresponding dissimilarity.

• Figure a dilution schedule which, in as few steps as possible, will yield a final concentration of 20-40 microfungal propagules per ml. Often a 1:10,000 dilution is satisfactory, but a trial run may save time and material.
  
  EXAMPLE:
  5 g soil in 125 ml sterile water = 5:125, a 1:25 dilution (dilution "1")
  1 ml dil. "1" in 39 ml sterile water = 1/25x1/40, a 1:1,000 dilution (dilution "2")
  1 ml dil. "2" in 19 ml sterile water = 2/1000x1/20, a 2:20,000 (or a 1:10,000) dilution
  
  
• When the plates and water blanks have been prepared, make the prescribed dilutions. Shake each of the suspensions vigorously to break up soil aggregates, and withdraw samples quickly while the suspension is in motion.
• Dispense the final dilution, 1 ml per plate, to 4-6 plates.
• After a 3-6 day incubation period at room temperature, count colonies per plate and average (for calculation of propagules in 1 gram of soil); then isolate a "sample population" by hyphal tip picks to tube slants of nutrient agar.

If you want 20 isolates from each site collection, begin at the plate trade mark and pick 20 sequential colonies. This will give you a random sample since the propagules presumably settled at random.

Calculation of Numbers Per Gram of Dry Soil: This calculation requires collection of a replicate soil sample in an airtight container. Weigh the fresh sample, oven-dry it (105°C for 72 hours), and reweigh it. Figure water content by the formula:

To obtain an estimate of number of colony sources per gram of dry soil, use the formula:

Sorting of Isolates and Data Tabulation: When the tube cultures have developed sufficiently (7-14 days), sort them into apparent taxonomic entities by macroscopic and/or microscopic examination. Then on data sheets, record sites-of-occurrence and numbers-of-isolates for all apparent entities.

          2w/a+b= 2(2)/4+6


                 = 4/10


                 = .40, or X100, 40.0 -- coefficient of similarity

Brown, J. C. 1958. Soil fungi of some British sand dunes in relation to soil type and succession. J. Ecology 46: 641-664.

Christensen, M., W.F. Whittingham and R.O. Novak. 1962. The soil microfungi of wet-mesic forests in southern Wisconsin. Mycologia 54: 374-388.

Orpurt, P.A. and J.T. Curtis. 1957. Soil microfungi in relation to the prairie continuum in Wisconsin. Ecology 38: 628-637.

Roswell, Thomas (ed.). 1973. Modern methods in the study of microbial ecology. Bulletins from the Ecological Research Committee, NFR, No. 17. Stockholm.

Tresner, H.D., M.P. Backus, and J.T. Curtis. l954. Soil microfungi in relation to the hardwood forest continuum in southern Wisconsin. Mycologia 46: 314-333.

Wicklow, D.T. 1973. Microfungal populations in surface soils of manipulated prairie stands. Ecology 54: 1302-1310.