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	We recommend that C-Fern spores be surface sterilized in 6 ml plastic tubes with conical bottom and screw caps and handled with sterile plastic pipets. The photographs below use glass conical centrifuge to allow easy visualization of spores.

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Surface Sterilization of C-Fern Spores

Spore sterilization and sowing should be performed on a sterile bench or laminar flow hood.

Materials:

• Sterile tubes with conical bottom
• Sterile plastic Pasteur OR sterile glass pipets
• Sterile distilled water
• Sterile pipet bulbs
• Clorox® bleach OR equivalent 5.25% sodium hypochlorite solution
• Waste container

Procedure:

1. WEIGH SPORES. Weigh out spores onto glassine weigh paper and transfer them to a sterile centrifuge tube with conical bottom.

	Dry C-Fern spores in conical tube. A pipet full of liquid, either sterilization solution (dilute bleach) or sterile distilled water
	Liquid added to C-Fern spores

2. PRESOAK SPORES. Cover spores with glass distilled water (approximately 2 mL) and let stand for 5 min to 24 hours. Spores may be soaked for just enough time to accomplish wetting before proceeding. Water for soaking spores does not need to be sterile.

3. REMOVE FLOATING SPORE DEBRIS AND PRESOAK WATER. Remove floating spore debris, if any, by placing pipet tip at water meniscus and slowly drawing off water. The debris will be sucked into the pipet along with the water. If the spores are exceptionally dirty or contaminated, wetted suspended spores should be transferred to a clean sterile centrifuge tube and care should be taken not to touch the internal sides of the tube. Remove remaining water from the spores using a sterile pipet (spores will have settled to the bottom of the tube) using the technique described below. This procedure for removing liquids from tubes will be used also in spore sterilization and rinsing.

 

	Insert pipet into liquid while expelling air from the pipet
	Continue expelling air from pipet and move pipet towards bottom of conical portion.
	Seat pipet onto conical portion of tube. Expel sufficient air to allow for complete aspiration of liquid.
		With pipet seated, release pipet bulb. Liquid aspirates and C-Fern spores are retained at base of pipet.
		When all of the liquid is aspirated a 'pellet' of C-Fern spores is left in the tube.

PROCEDURE FOR REMOVING LIQUIDS Select a sterile Pasteur pipet. (For glass pipets choose one with a tip that is not cracked or chipped and attach a rubber bulb on the pipet.) Insert pipet into tube and suspend spores by bubbling a small amount of air into the water. While air is slowly bubbling out of the pipet, gently but securely seat the pipet onto the base of tube, but don't press too hard on the pipet or you will crush the tip. Sometimes it helps to gently rotate the pipet tip. Squeeze the bulb to force additional air out of the pipet. When the bulb is released, water should enter the pipet and the spores should collect around the outside of the base of the pipet tip, provided the pipet is securely seated on tube bottom. If you can't remove liquid without bringing the spores along, try another pipet. Remember that time is critical when the sterilizing solution (sodium hypochlorite) is in the tube! Practice this technique with water prior to sterilizing the spores. With practice you should be able to remove liquids free of spores, in less than ten seconds. When the presoak water is successfully removed, discard the pipet.

4. SURFACE STERILIZE SPORES. To sterilize, suspend spores in one full Pasteur pipet of 0.875% sodium hypochlorite (1 part Clorox (5.25% sodium hypochlorite) to 5 parts distilled water). Rinse down lip and sides of tube with sodium hypochlorite solution. To insure that the spore mass becomes evenly suspended it may be necessary to bubble air through a clean, sterile pipet (discard this pipet after use). Sterilize spores for three minutes. Remove sodium hypochlorite solution with clean, sterile pipet using technique described above. For precise timing of surface sterilization, time needed to remove sodium hypochlorite must be factored in. Discard this pipet.

5. RINSE SPORES. To rinse spores, add one full pipet of sterile glass distilled water. Pipet used to add sterile distilled water may be used repeatedly as long as care is taken to prevent contamination with foreign spores. Remove rinse water with clean, sterile pipet. Repeat rinse one or two more times. Pipet used to remove sterile distilled water may be left in tube for use in sowing spores.

6. SOW SPORES. Add the appropriate amount of sterile glass distilled water to sow a given number of plates. Dispense spores to plates (refer to information on SPORE SOWING DENSITY AND CALCULATIONS). Spores may be uniformly distributed across the agar surface by gently spreading with a sterile wire bent in the shape of a small coat hanger or other appropriate spreader..