Fungal Cultures and Protein Extraction


In order to extract DNA, RNA, or protein from fungi, it is necessary to grow fungal cultures (so that enough of the substance of interest can be extracted). There are two types of medium used to grow fungal cultures: solid medium and liquid medium. There are, however, different recipes for media used to grow different different species -- although many species can grow on similar media. The standard medium used in our fungus lab is referred to as "organic medium" (OM) -- see below for the protocol. The only difference in the preparation of solid and liquid OM is the addition of agar -- agar is used as a solidifying agent. Contamination is always a concern, therefore media preparations and solutions are sterilized by autoclaving (an autoclave is like an oven, but uses steam at high temperature and pressure for sterilization -- e.g. 15 psi and 120 degrees C).

Solid OM is prepared, sterilized, poured onto (sterile) petrie plates, and allowed to cool (and therefore, solidify). Liquid OM is simply prepared and poured into separate flasks, which are then autoclaved and allowed to cool. OM plates are really just used to continue the fungal lines -- it is the liquid OM that is used to grow fungal cultures for extraction procedures (e.g. extraction of DNA, RNA, or protein).

There are, in fact, a wide variety of culture media that can be used for growing fungal cultures -- it really depends on what the species is (i.e. different species may prefer different nutrients). For example, V8 agar (V8 referring to "V8 juice") can be used for fungi that are plant pathogens (e.g. Cladosporium fulvum, however, I have found that other fungi (e.g. soil fungi) can grow on V8 agar just as well as -- and in some cases, better than -- standard OM. Fungal spores may also be preserved in silica gel (silica gel is essentially the same material as fish-tank gravel).

The time it takes to grow enough of a fungal culture in liquid OM, depends on the species. Some species grow very fast (e.g. Penicillium and Neurospora), while others grow much slower (e.g.Psilocybe). Once enough culture is grown, the liquid is removed by suction-filtering and the fungal mycelium is ground up in liquid nitrogen. The extraction buffer is added immediately after grinding. Frozen ground cultures can also be stored at -80 degrees C before extraction, however, I prefer to do the extraction right after grinding mycelium in liquid nitrogen. The protocol I use for extraction of total protein from fungal cultures is provided below.

Organic Medium (OM)
1% glucose (10 g/L)
0.1% Peptone (1g/L)
0.01% Yeast Extract (0.1g/L)
0.1% KH2PO4 (1 g/L)
0.03% MgSO4 7H20 (0.3g/L) or MgSO4 anhyd. (0.146 g/L)
1.5% Agar (15 g/L) for solid OM only
Autoclave: 20 min sterile, 25 min slow exhaust

V8 Agar
100 ml V8 juice
10 g Agarose
1.5 g CaCO3
dH2O to 500 ml total

Keeping spores in Silica-Gel

  • sterilize silica gel in small capped glass vials in 180(C oven for 1 day
  • sterilize 5% (w/v in H2O) powdered milk (autoclave)
  • add approx. 4ml of milk to each petrie-plate containing sporulating fungal culture
  • if there is a lot of mycelium, filter the suspension through gauze
  • add 0.2ml of spore suspension to each vial containing silica gel
  • let air-dry for one week in vials with loosely screwed caps
  • tighten caps for permanent storage
  • to revive culture, inoculate plates with 3-4 pieces of silica gel

Protein Isolation from Frozen Ground Tissue

  1. Protein Isolation Buffer PIB)
    10 mM Tris-HCl pH 8.0, 1mM EDTA, 2% PVPP. Shake buffer before use. Add proteases inhibitors (50ul each in 10ml): chymostatin, aprotinin, leupeptin plus 500 ul PMSF in methanol
  2. Use 5ml of PIB per 2 g frozen tissue. Pour over frozen tissue and allow it to thaw on ice.
  3. Split into centrifuge tubes.
  4. Centrifuge at 8000 rpm for 30 min at 4 degrees C.
  5. Collect supernatant and add equal volume of acetone (freeze O/N).
  6. Centrifuge for 30 min at 6000-7000 rpm (4 degrees C).
  7. Pour off supernatant and allow pellet to air dry.
  8. Resuspend pellet in 1 ml TE (10 mM Tris-HCl, 1mM EDTA, and PMSF) and split into (5) eppendorfs (crude extract).
  9. To run samples on a gel, I typically mix 100 ul of crude extract with 100 ul of loading buffer, and then load some volume into the wells (e.g. anywhere from 2ul to 40ul, depending on the protein concentration).

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