Other Resources

Materials

• Surface sterilized C-Fern spores in a graduated spore vial. Wild type (RNWT1) and mutant spores are available as presterilized class size and bulk unsterilized units. Bulk unsterilized spores and spores collected directly from sporophytes and surface sterilized.
• Sterile distilled water
• Sterile Transfer pipet (plastic) OR equivalent
• Sterile spore spreader
• Prepared Petri dishes containing C-Fern Medium (prepared in advance)

	C-Fern spores at bottom of spore vial

Procedures

The instructor or students may sow spores. Inoculate spores in an area that is clean and relatively free from drafts. A spore vial holder provides a stable container that help prevents spillage and provides a place to hold the pipet. Instruct and monitor students on proper and careful technique for sowing and spreading spores, handling and labeling dishes.

PREPARING SPORES Before opening any spore vial, be sure that all spores are at the bottom of the vial by tapping the bottom of the vial on a hard surface. If using presterilized spores, loosen the cap. Transfer the appropriate amount of sterile distilled water to the spore vial using a sterile transfer pipet. Do not return any water to the sterile water bottle.

	For each inoculation, thoroughly suspend the spores in water. Aspirate the spore suspension into a pipet. Grasp the lid of a petri dish
	Elevate the petri dish lid to about a 45 degree angle. Carefully dispense 3 drops - not squirts - of spore suspension onto the agar surface.
	Drops of spore suspension should be evenly distributed across the agar surface. After dispension spores, replace petri dish lid.
	Use a sterile spore spreader to distribute spores randomly on the agar surface.
	To spread spores, elevate the petri dish lid to a 45 degree angle and move the spore spreader rapidly but gently across the agar surface. Rotate the petri dish to help in distributing the spores randomly.

The standard presterilized spore unit will sow ca. 35 dishes at a standard density of 300+ spores per plate when 4 ml sterile distilled water is added. Allow spores to wet completely by firmly attaching the cap and inverting the vial 2-3 times. With the cap on, check the bottom of the vial to be sure that all the spores have been suspended. Return vial to holder and loosen cap. If using unsterilized spores, determine the appropriate amount of spores for inoculation and surface sterilize spores.

SOWING SPORES To achieve consistent sowings, spores must be thoroughly suspended between each sowing. Suspend spores gently by drawing the liquid along with the spores in and out of the pipet. To sow, withdraw a small amount of the spore suspension into the pipet and immediately dispense the appropriate number of drops - not squirts! - onto the agar surface. A standard sowing density of 300+ spores per Petri dish requires three drops of spore suspension. When sowing, tilt the lid of the Petri dish upward only just enough to permit access of the pipet tip. To insure a consistent drop size, hold the pipet at a constant angle (45°) throughout the sowing. Do not touch the agar surface with the tip of the pipet. Resuspend spores between each sowing by gently squeezing and releasing the pipet bulb.

SPREADING SPORES If you are using a plastic spore spreader, it may be effectively sterilized by wiping or dipping in alcohol, e.g. 70% isopropanol or ethanol and allowing to completely air dry. Metal wire spore spreaders may be sterilized by heating in a flame and allowing to cool. When distributing spores, allow just the tip of the L-shape (about 5 mm) of the spreader to just rest on the surface without pressure. Move the spore spreader rapidly but gently back and forth across the surface of the agar while rotating the Petri slowly with the other hand. The goal is to distribute spores over the surface of the medium in a somewhat 'random' arrangement.