Hello Hagos,
(To make 1L of Rye A agar)
- Surface-sterilize 60g of rye seeds with 10% bleach solution for 30-60 sec in a beaker.
- Wash with flowing tap water in a sieve until no bleach smell is left.
- Put seeds in a box, spread them out and add tap water, not submerged. Soak rye grains for 16-24 hrs in dark at 25oC.
- Rye should germinate-should see germination on ≥ 50% of seeds.
- Put the soaked rye seeds and approx. 200ml tap water in a blender and macerate for 3x20 seconds. Check after 20 seconds that the seeds don´t stick to the wall-put them down with a spoon. Seeds should be halved, not totally mashed.
- Incubate at 50°C for 3 hrs. (water bath or incubator)
- Transfer to a sieve, press slightly with a spoon to get the nutrients out. Add approx. 200mL tap water, mix and press through sieve again. Repeat (3 times) until 1 L is used.
- Mix and divide into 2 one litre Duran bottles. (500mL in each)
- Add 3.75g of bacto-agar and 10g sucrose into each of 2x 500ml Duran bottles.
- Autoclave at 120°C/25min,
- Cool it down (55oC) and pour onto petri dishes immediately!
- Store plates 1-2 month (I prefer to use fresh).
To make some changes you can refer to http://www.eucablight.org/EucaBlight.asp and http://www.plantpath.cornell.edu/Fry/Protocols-RyeA-Agar.html.
Hello Hagos,
(To make 1L of Rye A agar)
- Surface-sterilize 60g of rye seeds with 10% bleach solution for 30-60 sec in a beaker.
- Wash with flowing tap water in a sieve until no bleach smell is left.
- Put seeds in a box, spread them out and add tap water, not submerged. Soak rye grains for 16-24 hrs in dark at 25oC.
- Rye should germinate-should see germination on ≥ 50% of seeds.
- Put the soaked rye seeds and approx. 200ml tap water in a blender and macerate for 3x20 seconds. Check after 20 seconds that the seeds don´t stick to the wall-put them down with a spoon. Seeds should be halved, not totally mashed.
- Incubate at 50°C for 3 hrs. (water bath or incubator)
- Transfer to a sieve, press slightly with a spoon to get the nutrients out. Add approx. 200mL tap water, mix and press through sieve again. Repeat (3 times) until 1 L is used.
- Mix and divide into 2 one litre Duran bottles. (500mL in each)
- Add 3.75g of bacto-agar and 10g sucrose into each of 2x 500ml Duran bottles.
- Autoclave at 120°C/25min,
- Cool it down (55oC) and pour onto petri dishes immediately!
- Store plates 1-2 month (I prefer to use fresh).
To make some changes you can refer to http://www.eucablight.org/EucaBlight.asp and http://www.plantpath.cornell.edu/Fry/Protocols-RyeA-Agar.html.
V8 juice agar Media
V8 Juice...................200.0 ml
CaCO3 ........................3.0 g
Agar........................15.0 g
Tap water to.................800 ml
Adjust pH to 7.2.
Autoclave at 121C for 15 minutes.
pea-agar media
The preparation is the same as Potato's dextrose agar
but the ingredients are 140g of fresh pea+ 20g d’agar-agar+ 1000ml d’eau distillée
Incubation at 18 to 22°C, one week for Mycelia growth and 2 to 3 weeks for a good sporulation. Good luck
Sorry for this delay in sending an answer.
1. Isolation
Surface sterilize infected plants in 96% ethanol for 10 minutes and place on a sterile slicing board for about 1 minute to allow the ethanol to evaporate.
Dip a dissecting knife in 96% ethanol and then heat sterilise in a spirit flame. Using the sterilised knife, the infected fruits are cut into 5 mm thick slices. The fruit discs are put in 94mm diameter Petri dishes and incubated at room temperature (25°C) for 5 days.
2. Culturing of isolates
Hyphal tips of the fungus are aseptically transferred onto Rye A agar and cultured. Further purification in subsequent subculturing is done by adding pimaricine, rifampicine and oxytetracycline antibiotics, plus a fungicide carbendazim into Rye A agar.
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