Media
A large number of media have been developed for fungi. Many of these are general purpose media, suited to the growth of most fungi, while others are selective media for the isolation of particular fungi from plants or soil. All media have in common a gelling agent, usually agar, and a supply of the nutrients essential for fungal growth. Media may be synthetic, that is made up from defined chemical constituents, or may be made from natural materials, usually plant extracts.
Many mycological media have high carbohydrate levels. These give rapid growth of fungi, with abundant aerial mycelium. However, repeated subculturing on rich media frequently leads to cultural degeneration and loss of virulence. Spores and other structures used in identification are often highly variable on rich media. For these reasons, low-nutrient media are to be preferred for maintenance of cultures and for identification.
Comments on some components of media
Tap water is suitable for use for most media, as it contains trace elements which may be missing from distilled water. However in some areas tap water may contain substances which are toxic to fungi. One of the most significant is copper, which is inhibitory to many Phytophthora species among others. In these cases distilled water is preferred.
Agar is an extract from algae, and its quality can vary depending on its source. It is available as a powder, or in a block or flake form. Many powdered agars dissolve readily during autoclaving; the recipes given below are for agar of this type. Some powdered agars and all solid forms of agar need to be dissolved by boiling in water before adding to the media. To test whether this is the case, make up a 1.5% water agar medium and autoclave for the standard time. When poured into Petri plates, this should give a firm, clear gel. If the medium will not set properly, or is cloudy, the agar will need to be dissolved first. To do this, suspend the required amount of agar in half of the volume of water specified in the recipe, and boil for two hours. Add water to bring it back to the initial volume, then add the agar solution to the rest of the ingredients, made up in the other half of the water, before autoclaving.
Antibiotics may be added to isolation media to prevent the growth of bacteria. Most antibiotics (except chloramphenicol; see below) are unstable if heated and need to be added to the medium after autoclaving. These antibiotics are dissolved in a small quantity of sterile distilled water, according to the recipe. For most purposes this may be added directly to the medium, but for critical work the antibiotic solution should be filter-sterilised before use. The most commonly used antibiotics are:
- Penicillins, active mostly against Gram-positive bacteria, water soluble;
- Streptomycin, active mostly against Gram-negative bacteria, water soluble;
- Neomycin, active mostly against Gram-positive bacteria, water soluble;
- Chloramphenicol, active against both Gram-negative and Gram-positive bacteria, ethanol soluble.
Chloramphenicol may be added to the medium before autoclaving. Chloramphenicol is a suspected carcinogen, and all antibiotics must be handled with care.
Fungicides are frequently used in selective media. For example, Fusarium species are relatively tolerant to pentachloronitrobenzene (PCNB; Terrachlor® or Quintozene®) and dichloronitroaniline (DCNA; Allisan®) and these fungicides are added to media selective for Fusarium.
Rose Bengal is added to some media used for isolation from soil. It inhibits the growth of all fungi, and is added to prevent fast-growing species from overgrowing colonies of slow-growing fungi. Rose Bengal becomes more toxic on exposure to light. Plates of Rose Bengal media should be stored and incubated in the dark.
General Purpose Media
1. Water Agar (WA)
Water Agar (2%) consists of 20g agar in 1L of water and is recommended as the substrate for the germination of conidia used to initiate single spore cultures.
Hyphal growth is sparse on this medium so it is suitable for cultures from which single hyphal tips are to be taken for the initiation of new colonies. Sparse growth on Water Agar also facilitates the isolation of fungi from plant material, particularly roots.
Water Agar (0.05%), 0.5g agar in 1L of water, is used in the preparation of soil dilution series. The small quantity of agar slightly retards sedimentation rates of fungal propagules. The agar is dissolved in water before being dispensed into McCartney bottles. Bottles are capped loosely during sterilisation and caps are tightened when sterilisation is complete.
2. Carnation Leaf-Piece Agar (CLA)
CLA is a natural substrate medium (Snyder & Hansen, 1947) prepared by placing sterile carnation leaf pieces (approximately 1 piece per 2ml agar) in a Petri plate and then adding sterile 2% Water Agar.
The carnation leaf pieces are prepared from fresh carnation leaves free from fungicide or insecticide residue. Immediately after collection the leaves are cut into 5-8mm pieces and dried in a forced-air oven at approximately 70°C for 3-4 hours until brittle. Leaf pieces can also be dried in a microwave oven. The dried leaf pieces are packaged in aluminium or polycarbonate containers and sterilized by gamma irradiation (2.5 megarads). Sterilized leaf pieces can be stored at 2-5°C for up to 12 months before use.
Many species sporulate on CLA in 6-10 days. Using this medium conidial shapes are more uniform than when using carbohydrate rich media such as PDA. Macroconidia are formed mainly in sporodochia which usually develop on the leaf pieces. Macroconidia formed in sporodochia are preferred in identification as they are more consistent in shape and length than macroconidia formed from solitary monophialides on hyphae on the agar.
Microconidia are more common on hyphae growing on the agar, often away from the leaf pieces. The mode of formation of microconidia, the presence of chains of microconidia, and the presence of chlamydospores can be determined by direct examination with a compound microscope when small plates of CLA (5cm diameter) are used for routine identification of Fusarium cultures. CLA is also suitable for the production of large numbers of conidia for experimental work, and can be prepared for this purpose in large flat bottles or large Petri plates.
A number of fungi form perithecia readily on CLA if incubated under light.
3. KCl Agar
The addition of 4-8gL-1 KCl to WA or CLA enhances the number and length of chains in cultures with this characteristic. Chains are also easier to observe as there is less moisture on the surface of the agar and fewer droplets of moisture in the aerial mycelium. Species of Cylindrocladium will more readily form stipes on this medium.
4. V8-Juice Agar
A 30% (v/v) suspension of V8 Juice® (Campbell’s Soups Pty. Ltd.) is made up with 2% Water Agar. Before addition of the agar the pH of the juice should be adjusted to 5.5-6.5 using 1.0M NaOH.
The medium has been used to promote the production of perithecia by Haemanectria haematococca. It can also be used to encourage pseudothecia in Pyrenophora tritici-repentis.
5. Potato Dextrose Agar (PDA)
PDA is a carbohydrate rich medium which contains 20g dextrose, 20g agar and the broth from 250g white potatoes made up to 1L with tap water. The potatoes are unpeeled but washed and diced before boiling until just soft. The boiled potatoes are filtered through cheesecloth leaving some sediment in the broth.
Conidia formed on PDA are usually variable in shape and size and so are less reliable for use in identification. However colony morphology, pigmentation and growth rates of Fusarium species on PDA are reasonably consistent if the medium is prepared carefully and if the cultures are initiated from standard inocula and incubated under standard conditions. These colony characteristics are useful secondary criteria for identification.
Although PDA is useful for the isolation of many fungal species many saprophytic fungi and bacteria also grow on PDA and may inhibit the recovery of the pathogen. It is recommended that the concentration of potato and dextrose be reduced by 50-75% when the medium is used for the isolation of Fusarium species and other fungi from plant material.
6. Carrot Agar
Used in fertility studies with Gibberella species and for the growth of Thielaviopsis basicola.
400 grams of carrots are washed and cut up into small pieces. Autoclave for 10 minutes in 400mL of water. Puree (blend) the carrots and liquid till it forms an even mixture and add an extra 500mL of water. Add 20g agar and autoclave.
7. Soil Agar (SA)
Chlamydospore formation is enhanced on Soil Agar (Klotz et al., 1988) and so this medium is a critical supplement in the identification of some species of Fusarium.
SA is prepared by adding 500g sieved dry soil and 15g agar to 1L water. The amount of soil used can be varied with soil type. Abundant chlamydospore formation by various species has been observed on SA prepared with 250g black clay soil. The autoclaved medium should be regularly agitated while plates are being poured to ensure the even distribution of the solids to all plates.
Selective Media
Peptone PCNB Agar / Selective Fusarium Agar / Dichloran Chloramphenicol Peptone Agar / Spezieller Nährstoffarmer Agar / Modified potato Dextrose Agar / Komada’s Medium / Phytophthora selective medium / Grass-blade Medium for Oomycetes / Selective medium for the recovery of Thielaviopsis
1. Peptone PCNB Agar (PPA / Nash - Snyder Medium)
PPA is comprised of a basal medium to which antibiotics and fungicides are added and enables the selective isolation of Fusarium species from soil dilutions (Nash & Snyder, 1962). It is highly inhibitory to most other fungi and bacteria but allows slow growth of Fusarium which form small colonies of 5-10mm diameter after 5-7 days.
Basal medium in 1L water:
Agar 20.0g
Peptone 15.0g
KH2PO4 1.0g
MgSO4.7H20 0.5g
Terrachlor® 1.0g (contains PCNB 75% w/w)
The basal medium is autoclaved and cooled to 55°C before adding, in 10ml sterile water:
Streptomycin sulfate* 1.0g
Neomycin sulfate * 0.12g
The prepared plates should be allowed to 'dry' in a cool dark place before use so that the water in the soil suspension is rapidly absorbed. Most species of Fusarium do not form distinctive colonies on PPA, sporulation is poor and conidial morphology abnormal. Colonies must be subcultured for identification. Fusarium cultures should not be maintained on PPA because the metabolism of peptone leads to the accumulation of toxic ammonia.
* Streptomycin is effective against Gram negative bacteria, Neomycin against Gram positive bacteria.Typical isolation of Fusarium species from soil on PPA
2. Selective Fusarium Agar (SFA)
Developed for the selective isolation of Fusarium species from soil debris, SFA is a modified Czapeck-Dox medium containing antimicrobial agents (Tio et al., 1977).
Basal medium in 1L water:
Agar 20.0g
Dextrose 20.0g
KH2PO4 0.5g
NaNO3 2.0g
MgSO4.7H2O 0.5g
Yeast extract 1.0g
1%FeSO4.7H2O soln 1ml
The basal medium is autoclaved and allowed to cool to 55°C before antimicrobial agents are added in 10ml sterile water:
Streptomycin sulfate 0.1g
Dichloran,*added as Allisan® 0.013g
Neomycin sulfate 0.01g
SFA permits the slow growth of Fusarium species from plant roots and soil debris, and is less inhibitory than PPA to most fungi. Colonies of different species developing from a single root fragment or piece of debris can be differentiated more efficiently than on PPA. SFA is not suitable for the isolation of Fusarium species from soil dilutions.
*Dichloran (2,6-dichloro-4-nitroanaline) 0.0065g may be substituted by Allisan® 0.013g or by the fungicide PCNB (pentachloronitrobenzene) at a concentration of 0.75gL-1, as 1g Terrachlor®.
3. Dichloran Chloramphenicol Peptone Agar (DCPA)
DCPA was developed for the selective isolation of Fusarium species and dematiaceous hyphomycetes from cereal grains (Andrews & Pitt, 1986).
The basal medium, made up with 1L distilled water, contains:
Agar 20.0g
Peptone 15.0g
K2HPO4 1.0g
MgSO4.7H2O 0.5g
Chloramphenicol* 0.2g
After autoclaving add, in 10ml ethanol:
Dichloran 0.002g
Fusarium species produce well formed colonies on DCPA. It is an alternative medium for use in identification of Fusarium species as macroconidia are uniform and similar to those formed on CLA (Hocking & Andrews, 1987). Aerial mycelium is more sparse on DCPA than on CLA so microconidia are generally produced less abundantly. Chlamydospores are also less abundant on DCPA than CLA. DCPA should not be used as a maintenance medium because the metabolism of peptone leads to accumulation of ammonia to toxic levels. Mucoraceous fungi are suppressed by dichloran, and the absence of a carbohydrate source is selective against Aspergillus and Penicillium species.
* Chloramphenicol is a broad spectrum antobiotic that is able to be autoclaved.
4. Spezieller Nährstoffarmer Agar (SNA)
SNA is a weak nutrient agar which can be used for the identification and maintenance of Fusarium and Cylindrocarpon isolates (Nirenberg, 1976). In addition to limiting cultural degeneration, this medium promotes uniform sporulation and good conidiogeneous cell development.
SNA is prepared by autoclaving, in 1L distilled water:
KH2PO4 1.0g
KNO3 1.0g
MgSO4.7H2O 0.5g
KCl 0.5g
Glucose 0.2g
Sucrose 0.2g
Agar 20g
Two pieces of sterile filter paper (1 cm square) placed on the agar surface when set assist in stimulating sporulation.
As SNA is transparent, cultures can be viewed by direct examination under the microscope or small blocks can be mounted on a slide with a drop of water and cover slip for observation. A liquid broth made from this medium but with no addition of agar is often used for the preparation of mycelium for extraction of DNA.
5. Modified Potato Dextrose Agar (MPDA)
MPDA was developed for the selective isolation of fungal pathogens from the crown region of wheat plants. Specifically targeted are the fungi causing crown rot, Fusarium pseudograminearum and common root rot, Bipolaris sorokiniana.
The basal medium is prepared as for PDA but the concentrations of potato broth and dextrose are halved. 10g dextrose and the broth from 125g potato is made up to 1L. The concentration of agar can also be reduced to 15g when large numbers of wheat crowns are to be plated in stainless steel trays.
After the basal medium has been autoclaved and cooled to 55°C antibiotics are added:
in 10ml sterile water: Streptomycin sulfate 0.16g
Dichloran (added as Allisan®) 0.013g
Neomycin sulfate 0.06g
MPDA allows the formation of distinctive colonies of F. pseudograminearum, and suppresses the growth of mucoraceous fungi and Trichoderma species.
6. Komada’s Medium
Komada’s medium is recommended for the selective isolation of F. oxysporum from soil (Komada, 1975). F. oxysporum colonies are pigmented. Other Fusarium species are suppressed.
The basal medium contains the following constituents in 1L distilled water and is autoclaved (121°C for 15m) and cooled to 55°C before antimicrobial agents are added.
K2HPO4 1.0g
KCl 0.5g
MgSO4.7H2O 0.5g
F3Na EDTA 0.01g
L Asparagine 2.0g
D Galactose 20.0g
Agar 15.0g
To the basal medium is added in 10ml sterile distilled water:
PCNB as Terrachlor® 1.0g
Oxgall 0.5g
Na2B4O7.10H2O 1.0g
Streptomycin sulfate 0.3g
The pH is adjusted to 3.8±0.2 with 10% phosphoric acid.
7. Phytophthora selective medium (PSM)
20 mL carrot puree
80 mL potato puree (recipes below)
8 g agar
Make up to 1 L with distilled water, autoclave and when cooled to 60°C, add:
3.7 mL Hymexazol in water
400 microlitres Pimaricin
200 mgms Penicillin
Wrap the plates in plastic wrap and store them in the fridge out of the light. Discard after a month. To make a medium selective for Phytophthora and Pythium do not add:
A. CARROT PUREE - 400 gms carrots washed and diced, autoclaved 10 mins in 400 mL distilled water. Puree the mix, then add an additional 500 mL water. This can be measured out and frozen in plastic containers until needed.
B. POTATO PUREE - Dice 200 g potato and boil in 500 mL tap water until tender. Puree and make up to a total of 800 mL with additional water. Store as above.
C. HYMEXAZOL STOCK SOLUTION - Add 0.3 g pure Hymexazol to 20 mL sterile water.
D. PIMARICIN - Pimaricin can be added directly to the molten agar. Shake well before dispensing. Store wrapped in foil in the fridge.
Baiting of Soils
Make a sludge of soil at about 100 mL of soil to 400 mL distilled water. Stir and add cubes of PSM cut from a plate. Leave 24 hours. Put the sludge into a fridge for about 2 hours for a cold shock. Remove cubes by carefully pouring sludge through a sieve. Gently and quickly pat them dry on absorbent paper then place them on a plate of fresh PSM. Allow about 48 hours for growth to occur. Phytophthora cinnamomi can be identified under a compound microscope as very thick, knobbly hyphae.
8. Grass-blade Medium for Oomycetes
Sporangia and oogonia of Pythium and Phytophthora can be observed best in mycelium growing in water. This can be prepared by culturing on pieces of grass blade floating in Petri plates of water. Cut pieces of grass blades, or maize or sorghum leaves, into pieces about 0.5-1 cm square. Boil in water for 10 minutes, then place 4-5 pieces into Petri plates containing 15-20 mL sterile water. Inoculate with a plug from an agar culture. The fungus will colonise the grass pieces, and mycelium will grow over the surface of the water. To mount for observation, place a coverslip under the surface of the water. Carefully tease off some of the mycelium, and draw it onto the coverslip. Remove the coverslip from the culture, invert, and place onto a drop of water on a microscope slide.
9. Selective medium for the recovery of Thielaviopsis - TB-CEN
To water agar that has been cooled to 45-50°C add the following ingredients:
Carrot juice 40-60 mL
Etridiazol 400 mg
Nystatin 250,000 units
Chlorotetracycline HCl 500 mg
CaCO3 1 g
Medium for Natural Inoculum - Chaff Grain Medium
Inoculum suitable for addition to soil in pathogenicity tests can be prepared using colonised grain -chaff as a substrate. It is particularly appropriate for species which do not form chalmydospores but which normally persist in soil as hyphae in plant residues.
Cereal chaff and ground cereal grain (ratio 5:1) are used together. The chaff-grain mixture is soaked in water overnight at 5°C to leach phenolic compounds, then drained thoroughly before distributing to glass jars or polyester oven bags. The containers are sealed with a large cotton wool plug and autoclaved for 15m each on two successive days. Containers are inoculated with a conidial or mycelial suspension (2ml per 250ml chaff-grain mixture) which is thoroughly shaken through the medium then incubated under standard conditions. The mixture should be shaken regularly to encourage colonisation. When the medium has been thoroughly colonised it is air dried and crushed to the required size for addition to soil or may be stored for up to 12 months at 2-5°C. |
