Ward's World Working with Guides

Page 11 Media Preparation for Fungi Cultures (Pouring Plates from Bottled Media) Materials needed for plates: Sterile Petri plates, bottled media, large beaker, hot plate, thermometer, inoculating needle or loop, Bunsen or similar flame source Plates: Use sterile technique as outlined above. To make plates using bottled solid media: 1. Place the bottle of media in a large beaker. Add water to the beaker until the water level is just above the level of the medium in the bottle. 2. Loosen the cap on the bottle. 3. Put a thermometer in the beaker; set it on a hot plate and heat the water to boiling. 4. Meanwhile, set the Petri plates (each bottle of media will supply 5–7 plates, depending on the thickness poured) on the work surface. 5. Boil the water gently for several minutes until the medium is completely liquefied. Swirl the bottle gently to be sure all is melted. 6. Turn off the heat. Allow the water temperature to cool to between 45°C and 50°C. 7. Using an insulated glove, pick up the bottle, remove the cap, and flame the bottle mouth. 8. Lift the lid of a Petri plate just enough to admit the neck of the bottle, and pour the first plate, using just enough medium to slightly more than cover the bottom of the plate. Replace the lid. 9. Swirl the plate gently to distribute the medium. 10. Proceed to pour the rest of the plates. To transfer cultures to plates: 1. Follow aseptic technique as described for slants 2. Lift the lid of the Petri dish just enough to introduce the inoculating loop. Gently wipe the needle or loop across the culture (you only need a small amount ). 3. Replace the lid, and then lift the lid of the sterile petri dish containing fresh medium just enough to introduce the needle or loop. Gently wipe the needle or loop across the surface of the media, then replace the lid and sterilize the needle or loop as before. You have now transferred the fungal culture to the plate. Special Techniques for Some Fungi Cultures General: The techniques previously outlined apply to most fungal cultures. Some special techniques apply to specific fungi: Saprolegnia ferax, (water mold). This species is best grown on cornmeal agar. It can be grown in liquid medium by adding sterile rice grains or cucumber seed to distilled water and inoculating it with Saprolegnia. Demonstration of sexual reproduction in fungi can easily be done using Rhizopus stolonifer, Phycomyces blakesleeanus, or Mucor hiemalis. Inoculate plus and minus strains on opposite sides of a Petri plate containing sabouraud dextrose agar or potato dextrose agar. Be careful not to mix the strains when plating them. Fungi

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