An appropriate culture environment helps regulate temperature, humidity, evaporation, and helps give consistent results.
Culture Containers
Once cultures are inoculated with spores, place them into an appropriate culture container.
- Culture Dome – consisting of clean plastic greenhouse trays covered with transparent humidity domes. Culture Domes should be thoroughly clean to prevent contamination of cultures by soil-borne organisms. Standard greenhouse trays and clear domes are available commercially.
- Growth Pod – consist of an insulated ‘6-pack’ cooler fitted with an interior cardboard box lined with aluminum foil and covered with a ¼” clear acrylic lid. Growth Pods can be assembled from inexpensive and easily obtainable materials.
Culture Domes and Growth Pods serve to reduce the possibility of contamination, variations in temperature, fluctuations in humidity, reduce excessive evaporation, and permit easy handling of a larger number of dishes. Do not tightly seal the Petri dishes, for example with Parafilm®, as this results in poor growth presumably due to ethylene buildup.
Temperature
The temperature optimum for spore germination and gametophytic development is about 28 °C (82 °F). Relatively similar patterns of growth and development of gametophytes can be obtained at a temperature range of 26-30 °C (79-86 °F). However, lower temperatures will substantially delay developmental timing; for example, at 20 °C (68 °F) development times will be increased by about two-fold.
Temperature Stabilization
With constant temperature C-Fern growth and development of both gametophytes and sporophytes is reproducible and consistent. Place culture containers in an environment that is temperature stable, free of drafts, and traffic. It is a good idea to monitor and record the temperature inside Growth Pods or Culture Domes daily; use an inexpensive thermometer with a remote probe. Some control of the temperature inside the Growth Pods or Culture Domes can be achieved by adjusting the distance between the light source and C-Fern cultures. Once a suitable temperature is achieved, the height of the lights should remain constant during all phases of culture.
Humidity, Evaporation, Condensation
Culture containers also help successful gametophyte growth by buffering changes in humidity within the culture environment and reducing evaporation of water from the culture medium. In addition, stable temperatures help to reduce condensation of water on Petri dish lids. Condensation may occur if sufficient temperature variation occurs between the lid and agar surface. Moderate amounts of condensation, i.e. fogging on the lid, are not a problem. However, excessive condensation can result in free water falling onto the culture surface, i.e. “raining”, which may result in an uncontrolled release of spermatozoids from antheridia. If condensation continues to be a problem, cultures may be grown upside down once the water used for sowing spores has been absorbed into the agar medium.
