You are on page 1of 2

A typical fungal life cycle features formation of threadlike vegetative hyphae which form a

mycelium, a three-dimensional structure of hyphae capable of effective assimilation of


nutrients and aggressive growth. Hyphae emerge from germinating spores (conidia) that may
be uni- or multinucleate, haploid or diploid. Fungi are typically isolated by plating a sample
(e.g., soil, organic matter, liquids) on a Petri dish containing a rich medium such as malt
extract agar and potato dextrose agar (PDA) supporting the growth of a variety of fungi. In
addition to the nutrients available, the main external factors affecting the fungal growth
include pH, temperature, humidity, and light. The type and concentration of carbon and
nitrogen source and the cultivation temperature are amongst the most important physical
factors having an effect on the type of reproduction. (e.g., vegetative vs. sexual) in fungi that
possess these life cycles. Sporulation can be induced by the selection of the growth medium,
humidity of the cultivation environment, and, in some cases, light. It should be noted that
especially in the soil environment, fungal species capable of aggressive sporulation are easily
overrepresented in the samples; therefore, dilution of the sample before plating is
recommended to expose the less abundant species.
Fungi can be grown in liquid cultures for various purposes such as enrichment of a type of
fungus of interest, production of fungal biomass, and production of enzymes and antibiotics.
For industrial purposes, filamentous fungi are grown in fermenters (up to 100,000 L in
volume) where the cultivation parameters can be controlled and the process automated. The
so-called solid-state fermentation can be performed in vessels especially designed for this
type of culture. One typical application of solid culture is production of fungal mycelia and
spores to be applied for biological control. Solid culture is also seen as a way to modify the
enzyme profiles produced by fungi as the profiles may differ from those produced in liquid
culture.
Materials

Plate Cultures

Plate cultures usually contain agar as a solidifying agent. Some agars, such as PDA, are
enriched with nutrients and can be purchased as “ready-made,” whereas minimal agars
require addition of relevant nutrients such as a carbon and nitrogen source. In addition to
nutrients essential for growth, various other components can be introduced into the agar
media. For example, antibiotics may be added for the selection of fungal transformants or to
prohibit bacterial contamination. The surfactant Triton-X100 is typically applied to restrict
the growth of fungal colonies on the plates.

Components for PDA Plates


PDA, Triton-X100, Hygromycin B, Cellophane discs cut to fit into the 9 cm Petri dishes,
Whatman filter paper discs, Glass 9 cm Petri dish to autoclave and store the cellophane discs

Componentsfor Minimal Agar Plates


Minimal agar, KH2PO4 , (NH4)2SO4 , MgSO4, CaCl2 , 5M KOH, 100× mineral stock (100 mg
FeSO4 × 7H2O; 20 mg MnSO4 × 4H2O; 20 mg ZnSO4 × 7H2O; 40 mg CoSO4 × 7H2O to 200
mL of distilled H2O, Birch wood xylan 0.5% (w/v), NaCl 1M solution made in purified
water, Congo Red 1 % (w/v)

Liquid Cultures

Liquid cultures are typically carried out in conical (Erlenmeyer) flasks placed on a shaker.
This type of culturing is usually performed with a view of testing particular properties of the
fungal strains of interest such as production of an enzyme or a metabolite. Fungi are excellent
protein secretors; thus, high amounts of proteins can be found in the culture supernatants.
Composition of the growth medium and cultivation conditions depend on the goal of the
experiment. The procedure involves choosing the carbon or the nitrogen source and setting
the pH and the shaker speed. A fungal shake culture in a laboratory is typically carried out in
50 mL of medium placed in 250 mL Erlenmeyer flasks, but cultivations can also be carried
out on a smaller or a larger scale such as 15 mL test tubes, 11 mL “Duetz System” deep well
plates, and laboratory fermenters ranging from 0.5 to 20 L.
Methods

Carry out all procedures at room temperature unless otherwise specified. Take care for not
dusting the environment with agar or other easily spreadable medium components.
Autoclaving is carried out at 121 °C for 20 min if not stated otherwise. Cultivation media can
also be sterilized in a pressure cooker for 30 min at 121 °C (at 15 psi).

Fungal Sporulation

1. Prepare PDA plates


2. Thaw out fungal spore suspension kept in cryogenic storage solution at −80 °C or use fresh
spore suspension prepared. Spread 50–100 μL of spore suspension onto dry PDA plate (s)
and spread with a sterile spreader.
4. Incubate at 28 °C for 7–10 days or until the surface of the plate is fully covered with
spores.

Inoculation and Incubation of Plate Cultures


1. Inoculation with fungal spores: Prepare a spore suspension by pouring 5–7 mL of sterile
0.9 % NaCl (w/v) + 0.01 % Tween 80 on a PDA plate containing a premade fungal culture,
and scrape the spores into solution. Filter the spore solution into a sterile 10 mL test tube
through a sterile funnel containing a cotton wool plug to remove hyphae. Take 1 mL of the
spore solution and dilute further if required for obtaining a viable count or separate colonies,
using aseptic techniques. Plate out 100μL aliquots and spread aseptically using a sterile
spreader. A glass spreader can be sterilized by flaming in 70 % (v/v) ethanol.
2. Inoculation with a soil sample: Measure 1 g of the soil sample in a test tube containing 10
mL of sterilized 0.9 % NaCl (w/v) + 0.01 % (v/v) Tween 80, and prepare a tenfold dilution
series until 10 −6 . You may need to use pipette tips of which the mouth has been widened by
cutting off the end of tips. Mix well between every transfer.
3. Inoculation from colonies: Plates can be inoculated from colonies growing on older plate
cultures. Transfer is carried out by lightly touching the growing colony by a sterile rod or
toothpick and making a 1–2 mm streak onto the new plate. Sometimes a piece of agar
containing fungal growth is cut out aseptically and placed onto a fresh plate.
4. A general incubation temperature for mesophilic fungi is +28 °C. Incubation times usually
vary from 3 to 7 days depending on the fungal species. Plates will be incubated bottoms up to
avoid condensation of water onto the cultures

You might also like