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Submit your questions to be posted (and hopefully answered!) on this page. Send your contributions to cfern@utk.edu.

• How much medium should I pour into the culture dishes?
  
  
• What can I do about my culture dishes drying out?
  
  
• What type of agar can I use for C-Fern cultures?
  
  
• My students are having difficulty visualizing swimming sperm under the dissecting scopes. Are there any tricks that can be used to enhance visibility?
  
  
• Why do some of my C-Fern gametophyte cultures show very long rhizoids?
  
  
• Will contaminants such as fungi and algae affect my C-Fern cultures?
  
  
• Why do my students sometimes get genetic ratios that are quite different from the expected?
  
  
• How can I grow sporophytes to maturity?
  
  
• Swimming sperm are typically easily observed shortly after adding water to the cultures, but in a few moments they seem to be all gone. Why is this?
  
  
• The Chemotaxis Kit says to use 12-18 day old cultures. Does this mean I should sow the spores 12-18 days in advance, or does the 12-18 day age indicate the number of days after the gametophytes germinate?
  
  
• We got the C-Fern to grow in soil and for a month or so they were doing really well. Then, after returning from vacation, the ferns were wilted in their pots. What happened?
  
  
• What kind of soil is needed to grow sporophytes? Does soil pH need to be adjusted?
  
  
  
  
• How much medium should I pour into the culture dishes? TOP
  For 60-mm diameter Petri dishes approximately 18 milliliters (ml) of nutrient medium should be poured into each dish. One liter (l) of nutrient medium will pour approximately 55 60-mm dishes. Dishes will be approximately 3/4 full.
  
  
• What can I do about my culture dishes drying out? TOP
  It is important to use Petri dishes that are filled properly. Because C-Fern cultures are maintained for a prolonged period of time it is necessary to fill each dish three-quarters full with medium. (See How much medium should I pour into the culture dishes?). Another factor may be the environment in which the dishes are kept. Environmental growth chambers may have a constant air flow which can cause premature drying out of cultures. In this situation, to reduce evaporation of media, the cultures can be kept within culture domes that are firmly secured. Newly developed C-Fern Growth Pods maybe a solution to culture drying as well as maintenance and regulation of optimal temperature for C-Fern growth. C-Fern Growth Pods can be assembled from inexpensive and easily obtainable materials.
  
  
• What type of agar can I use for C-Fern cultures? TOP
  We recommend using Difco-Bacto agar. This is a relatively purified and inexpensive brand that has consistently given excellent results. Other agars, for example, those recommended for tissue culture uses or more highly purified types, sometimes yield poor or unusual growth of gametophytes.
  
  
• My students are having difficulty visualizing swimming sperm under the dissecting scopes. Are there any tricks that can be used to enhance visibility? TOP
  Magnification is typically not the problem, since sperm can be adequately visualized even at a total magnification of 25 X. Usually difficulties observing sperm are associated with improper or deficient illumination. Bottom (transmitted) illumination is preferred. If adjustments are possible, try increasing or decreasing the intensity of the light source. Also if a mirror is present, try a different positions so that the highly refractive for sperm appear in a pseudo-dark field context. If a separate light source is available, light from the side can sometimes help. In some student-level scopes, there is little or no adjustment concerning intensity. Thus, if the light source is too bright the largely transparent sperm will be difficult to visualize. If there is no adjustment, a simple device can be made from the top lid of a Petri dish. With a marker, make a series of straight lines in a grid-like pattern covering approximately one square inch of the Petri dish lid. Spaces between the lines should be much smaller than the thickness of the lines. By placing this lid on underneath the specimen (gametophytes of a slide in water or a culture-dish) and moving it around enough of a contrast can be created to enhance sperm visualizations.
  
  
• Why do some of my C-Fern gametophyte cultures show very long rhizoids? TOP
  Certain types of fungal contaminants can cause excessively long rhizoid growth. In fact, this can be starting point for very interesting student research projects.
  
  
• Will contaminants such as fungi and algae affect my C-Fern cultures? TOP
  C-Fern cultures are largely resistant to many fungal contaminants, although some fungi can be pathogenic. For routine observations, the presence of some contamination will not seriously interfere with gametophyte growth or development. Contamination by certain types of algae can be much more destructive since contamination can spread rapidly and infect other cultures. If algal contamination is observed it is best to isolate or destroy the cultures.
  
  
• Why do my students sometimes get genetic ratios that are quite different from the expected? TOP
  "Unexpected" genetic ratios are typically associated with sampling error and an unconscious tendency to bias counts towards one of the phenotypes. Students should be carefully instructed in the proper ways of data collection, including a very throrough explanation of appropriate random sampling. One common problem is the tendency to 'select' individuals for counting rather than counting ALL of the individuals within a given (randomly selected) area. Other factors, of course, can also come into play, such as incomplete fertilization is due to underwatering the cultures and/or less than ideal growing conditions.
  
  
• How can I grow sporophytes to maturity? TOP
  The easiest way for most users to cultures sporophytes is to use a mini-terrarium constructed from a plastic drink bottle. Full instruction are available on the mini-terrarium web page. Bottles as small as 8-ounces or as large as 2-liters can be used. The commercially available potting soil (i.e. ProMix®) works very well. Fertilization using time-release pellets (i.e., Osmocote) are effective and easy to use.
  
  
• Swimming sperm are typically easily observed shortly after adding water to the cultures, but in a few moments they seem to be all gone. Why is this? TOP
  If water is added to a C-Fern culture, sperm are typically released within a few minutes. Because there are many hermaphroditic gametophytes in the culture, sperm are immediately attracted to them and cluster tightly around the area of mature archegonia. Once attracted to the archegonial region, sperm do not appear to swim away in search of other adventures! Consequently, in a culture with many mature archegonia, the sperm swim freely only for a short period of time. If it is desired to visualize freely swimming sperm for longer periods of time, male gametophytes should be removed from the culture and suspended in a drop of water or Sperm Release Buffer (SRB - see Chemical Attraction - C-Fern Sperm Chemotaxis kit). If there are no hermaphroditic gamtophytes are present, sperm may swim for a very long period of time. Under optimum conditions (e.g. low temperature) sperm can swim for as long as two hours in SRB.
  
  
• The Chemotaxis Kit says to use 12-18 day old cultures. Does this mean I should sow the spores 12-18 days in advance, or does the 12-18 day age indicate the number of days after the gametophytes germinate? TOP
  The timing refers to the age of the cultures, day 0 being the day the spores are sown. Under the recommended conditions the spores should germinate begining at day 3 and then gametophyte development will be quite rapid. At 28-30 C and under constant light the gametophytes will be sexually mature by day 12 and the chemotaxis can be done any time from then until about day 18. Be sure to note that when the cultures are removed from warm culture conditions to room temperature, the sperm will release from the antheridia even in the absence of liquid, so students should have all the preparations made prior to obtaining the cultures.
  
  
• We got the C-Fern to grow in soil and for a month or so they were doing really well. Then, after returning from vacation, the ferns were wilted in their pots. What happened? TOP
  If sporophytes are badly wilted, it may be too late to save them. If they get too stressed the apical cells in the shoots will die and even if they seem to recover some, they may not grow new leaves. However, the small buds on the leaves will begin to grow and can be used to clone the plants (see the C-Fern Manual or Greenhouse Culture of Sporophytes in the C-Fern Web Manual for details). Several factors may be responsible for the wilting of sporpphytes. Could they have not gotten watered over a weekend? Did environmental conditions change drastically (i.e., lower humidity, cold weather)?
  
  
• What kind of soil is needed to grow sporophytes? Does soil pH need to be adjusted? TOP
  The soil we routinely use now is readily available commercially as ProMix® potting mix. ProMix works very well for both greenhouse or terrarium culture (see mini-terrarium instructions) and has some starter fertilizer in it to get things going. We do not adjust pH with this potting material. Sporophytes grown in mini-terraria typically need no additional fertilization to grow to maturity. For prolonged greenhouse culture, follow the fertilization instructions in the Fertilization of Sporophytes section of the C-Fern Web Manual or in the C-Fern Manual.