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Fungal Media recipes used in the laboratory

SOLID MEDIA

Malt Extract Agar (MEA)

Note: For 1 L of MEA (Gutz and Doe 1973). Sterilize by autoclaving at 10 psi for 30 min. Plates should be stored at 4°C.

Ingredient Amount
malt extract 30 grams
agar 20 grams
distilled water 1 Liter

Malt Extract Agar + chloramphenicol

For general isolation of fungi (standard medium for isolating fungi from leaves)

Ingredient Amount
Malt Extract 20 grams
Agar 15 grams
chloramphenicol 0.200 grams
deionized water 1 Liter
  1. Add all ingredients into water, mix, and heat gently to dissolve.
  2. Autoclave. Chloramphenicol can be autoclaved.
  3. Cool and pour plates.

Carnation Leaf Agar (CLA)

Notes for Fusarium: Used for getting good macroconidia formation for identification.

Ingredient Amount
Carnation Leaves
Agar 20 grams
  1. Collect carnation leaves and cut into 3-5 mm2 pieces.
  2. Microwave ~30 seconds to sterilize and dry.
  3. Make 2% water agar: a. Add 20 g agar (Fisher # BP1423-500) to 1 L of H2O. b. Autoclave for 20 minutes on the fluid setting. c. Cool in a water bath to ~50°C.
  4. Add 10-15 pieces of carnation to each plate.
  5. Pour ~30 mL water agar into each plate.

Lactic acid PDA (APDA)

Good for Fusarium graminearum and allies. Used for isolation and for inoculating corn and MB broth. Lactic acid inhibits bacterial growth.

Ingredient Amount
Potato Dextrose Agar 39 grams
85% Lactic Acid 1 mL
Distilled H20
  1. Add 39 g PDA to 1 L distilled H2O.
  2. Autoclave for 20 minutes on the fluid setting.
  3. Cool in a water bath to approximately 50°C.
  4. Add 1 mL 85% Lactic acid and swirl to mix.
  5. Pour approximately 30 mL per plate.

PDA odered before was Fisher #DF0013176

Mung Bean Agar (MBA)

Used for Fusarium spore production on culture plates. Protocol developed by the lab of Dr. Ruth Dill-Macky.

Ingredient Amount
  1. Boil water in 2 L beaker.
  2. Place beans in water and boil for 25 min. NOTE: Time from when the water has returned to the boil. The beans will have started to crack open by 25 min (this may happen as early as 15 min). Good spore production is dependent on a cooking time of 25 minutes.
  3. Remove bean broth from heat.
  4. Mark 1 liter volume on the side of a 2-liter Erlenmeyer flask.
  5. Place a large Buchner funnel on the flask.
  6. Line the funnel using two gauze pads (4-6 layers of cheesecloth may be substituted). Ensure that the gauze/cheesecloth covers the funnel holes.
  7. Pour the bean broth slowly into the flask through the funnel
  8. Discard the beans (and gauze) from the funnel
  9. Soak the funnel and beaker in water NOTE: The residue is difficult to remove once dried onto surfaces!
  10. Adjust the total volume of broth up to 1 liter by adding distilled water to the mark on the flask
  11. Add 15 grams of agar (Fisher # BP1423-500) and mix.
  12. Autoclave at 115 oC for 20 minutes, fluid setting
  13. Cool in a water bath to ~50°C.
  14. Pour ~30 mL per plate.

V8 Juice Agar

Used for sporulation of some fungi and oomycetes

Ingredient Amount
V8 Juice 163 ml (1 can?)
CaCO3 1.8 grams
Agar 15 g
deionized water add up to 905 ml (should be 742ml diH20)
  1. Mix and heat to dissolve all ingredients;
  2. Autoclave, cool, and pour

Malt Extract Agar + Benlate + chloramphenicol

For isolation of wood decay fungi

Ingredient Amount

20g Malt Extract 15 g Agar 0.200 g chloramphenicol 1 L deionized water Mix and heat to dissolve all ingredients. Autoclave, and cool to pouring temperature. After autoclaving, just before pouring add: Benlate (10 mg/L) as 1mL of a 10 mg/mL stock suspension (0.200g Benlate in 20mL 70% EtOH stock solution)

MAG

Malt Extract Agar (1 L) To 960 mL of ddH2O add: Dextrose 20 g Malt Extract 20 g Peptone 2 g Vitamin mix 1 mL Trace elements 1 mL Add 15 g of agar for solid media Autoclave on P2, or the second longest liquid autoclave time (30 mins vacuum) * For MEA, leave out the vitamin mix and the trace elements *

YPD

Yeast Peptone Dextrose Agar To 960 mL of ddH2O add: Bacto yeast extract 10 g Bacto Peptone 20 g Dextrose 20 g Agar (for solid media) 20 g * It is suggested that when adding dextrose to media that you autoclave the dextrose separately from the media, in this case you would add the 20 g of Dextrose to 50 mL of ddH2O and remove 50 mL of ddH2O to the media recipe. After autoclaving the dextrose and the rest of the media just pour the dextrose into the larger flask*

LIQUID MEDIA

Hillman Broth Media

Note: Eddie's Media for growing fungi in liquid culture

Ingredient Amount
yeast extract 2%
dextrose 5%
sucrose 10%
malt 4%
distilled water 1 Liter

Mung Bean Broth (MBB)

Good for Fusarium spore production in liquid culture.

Ingredient Amount
  1. Bring 1 L distilled H2O to a boil.
  2. Remove from heat and allow to sit 30 seconds.
  3. Add 40 g of mung beans and allow to steep 10 minutes.
  4. Fix a Buchner funnel to a 2 L flask and cover the holes with cheesecloth. Filter the beans from the broth.
  5. Divide the broth as needed into flasks and put a sponge cork in the top.
  6. Autoclave 20 minutes on the fluid setting.
  7. Allow broth to cool overnight before inoculating.

VITAMINS & TRACE ELEMENT ADDITIONS

Vitamin Mix

To 100 mL of ddH2O, add 10 mg of each of the following: Biotin Pyridoxin Thiamine Riboflavin P-aminobenzoic acid (PABA) Nicotinic acid Store in the dark (wrapped in aluminum foil) at 4°C

Trace Elements

To 80 mL of ddH2O, add these components in order: ZnSO4 – 7H2O 2.2 g H3BO3 1.1 g MnCl2 – 4H2O 0.5 g FeSO4 – 7H2O 0.5 g CoCL2 – 6H2O 0.17 g CuSO4 – 5H2O 0.16 g Na2MoO4 – 2H2O 0.15 g EDTA (Na4) 5.0 g Boil; cool to 60° C; adjust pH to 6.5; bring up to 100 mL with ddH2O Or Autoclave for 20 mins?