A Novel Method for Surface-Sterilizing and Sowing Fern Spores

Abstract

Numerous methods are available for the sterilization of fern spores (see Dyer, 1979). Many of these methods are inefficient in terms of time and spore loss and require bulky equipment such as a centrifuge or vacuum filtration units. We present an easy and effective method for spore sterilization that requires no specialized tools or equipment, other than a sterile area. We have used the method presented here routinely to sterilize Ceratopteris richardii Brongn. spores. In addition, this technique has worked with spores of Lygodium, Pteridium, and other species of Ceratopteris (unpublished data). The basic innovation in this method involves the removal of liquid from a spore suspension in a conical centrifuge tube by means of a Pasteur pipet while leaving the spores in place. This method requires the following supplies: 1) sterile Pasteur pipets (Fisherbrand? borosilicate glass, 53/4 inches) with square-cut ends that are not cracked or chipped; 2) sterile pipet bulbs (natural rubber dropper bulb); and 3) sterile conical centrifuge tubes (Pyrex? No. 8060, 15 ml). To accomplish spore removal, insert a pipet with attached bulb into a conical centrifuge tube that contains a liquid and spores and suspend the spores in solution by bubbling air into the liquid. While continuing to bubble air, gently, but securely, seat the pipet tip onto the base of centrifuge tube. A slight rotation of the pipet may assist seating, but excess downward or lateral force will crush the pipet tip. Squeeze the pipet bulb to force additional air out of pipet, then slowly release the bulb to aspirate liquid into the pipet; most spores should collect at the base of the centrifuge tube and not be drawn into the pipet. This procedure has been incorporated into the following generalized spore sterilization and sowing method. Spore preparation.-These procedures can be performed outside of a sterile area. Transfer spores into a conical centrifuge tube and presoak them with distilled water for 16 hours. We routinely dry-sterilize (120?C, 2 hours) centrifuge tubes to eliminate cross-contamination of spore stocks and minimize contamination from the tube. If the spore source is exceptionally dirty or contaminated, wetted spores may be transferred with a Pasteur pipet to a clean sterile centrifuge tube immediately prior to sterilization. This significantly reduces the amount of coarse debris that may harbor and shield contaminants during sterilization. We prepare and sterilize from 2 to 250 mg of Ceratopteris spores per tube, depending on the desired sowing density and number of cultures to be sown. Spore sterilization.-All procedures for spore sterilization and spore sowing should be performed in a sterile area using standard sterile technique. All materials that come into contact with spores should be sterilized. To avoid contamination, use a clean sterile pipet and pipet bulb for each step. To sterilize spores, remove the presoak solution using the method described