OBJECTIVES:
To produce agar media for the culture of Leptosphaeria maculans, Pyrenepeziza brassicae and other fungi
OPERATORS:
Staff after reading SOP who know how to use an autoclave and microwave safely
PROCEDURE:
Warning: it is essential that a face mask, gloves and labcoat be worn when dealing with fine powders to avoid nasal, sinus, skin irritation or future allergy problems. I find that goggles are also advisable.
General Method
1. Weigh/ measure out the required ingredients to make up 400 ml of agar media.
2. Transfer to 500 ml Duran bottle or large conical flask (twice the final media volume).
3. Add 200 to 300 ml distilled or deionised water. Loosely apply lid to bottle or bung to flask.
4. Melt the agar in a microwave (3 to 4 min on maximum power 650 watts)
Warning: Do not place any metallic materials into the microwave. Check frequently after 3 min that media is not boiling over.
5. Wait 30 seconds. Using heat resistant gloves, swirl agar/media round the bottle/ flask until completely dissolved.
6. Adjust volume to 400ml with distilled H 2 O.
7. For Durans: ensure plastic ring is present round the neck of each bottle (for easy pouring) and screw on the lid loosely to avoid explosion in the autoclave. For conical flasks: place a sponge bung into the neck and a cap of silver foil over that – secured round the neck by a ring of autoclave tape.
8. Attach a piece of autoclave tape to each flask or bottle and label with [ PDA/MA/V8A; your initials; date ]. Media is best used within six months stored at 12 - 16°C.
9. Autoclave at 121°C for 15 minutes – check autoclave tape has turned colour appropriately
10. Once autoclaved the media can either be poured after 30 min or left to solidify until required. When required the media can be melted by microwaving at full power for about 1 minute per 100 ml
11. Store, prepare slopes or pour plates.