BIOL 202 LAB 7: C-Fern® Investigations: Genetics in Action Mendelian Genetics

(Prepared with acknowledgment toDineh Judd and Sichangu Lee for supplying material for micrographs used in this document.)

Equipment, Supplies & Materials:

  • Genetics in Action: Mendelian Genetics C-Fern® Kit (Item #156708, Carolina Biological Supply Company), including:
  • Presterilized C-Fern® spores of an F1 hybrid, 5mg dry weight
  • Basic C-Fern® Medium, 480 mL (pre-made bottled containingnutrients in agar)
  • Sterile Petri dishes, 60 x 15 mm
  • Sterile distilled water, 200 mL
  • Sterile transfer graduated pipets (or graduated cylinder or volumetric flask) to measure 4 mL of sterile water
  • Sterile pipets
  • Spore spreaders (paper clips bent into a "T" shape, with alcohol prep pad to sterilize)
  • Culture domes (i.e., thoroughly clean plastic greenhouse trays covered with transparent humidity domes)
  • Marking pen
  • Fluorescent light source
  • Thermometer (to measure temperature inside culture dome)
  • Stereomicroscopes
  • C-Fern® culture instructions and background information (Hickok & Warne, 1998a)—instructor's copy
  • C-Fern® Investigations: Genetics in Action Mendelian Genetics Student Instructions (Hickok & Warne, 1998b)—one copy per student
  • Ceratopteris: A model plant for the 90s (Chasan, 1992)—one copy per student
  • Laminar flow hood
  • 70% isopropyl alcohol (IPA) to clean laminar flow hood and work surfaces
  • Microscope slides and coverslips

Activities:

  • Instructor presentations on pteridophytesand Ceratopteris richardii (PowerPoint and Prezi)
  • Overview of Lab 7: Visualization of basic principles of Mendelian inheritance in C-Fern® by following the segregation of a visible marker, polka dot, in both the F1gametophyte and F2 sporophyte generations. Students sow spores of an F1 hybrid (wild type x polka dot) to produce F1 gametophytes. Sporophytes also may be observed.
  • Follow procedure as described in lab instructions (and as indicated by instructor)

Assignment:

  • Lab Report #7 (final version to be submitted to instructor by next Friday at 5:00 pm, following editorial review by the Science Writing Mentor)

Procedure:

  1. Preparing Medium and Petri Dishes—Using a hot water bath, liquefy the Basic C-Fern® Medium. (Note: This step will be completed prior to the beginning of the lab.)
  2. Using the laminar flow hood that has been cleaned with 70% IPA, pour the liquefied medium into Petri dishes and allow to cool. (Ditto.)
  3. Preparing Spores—Preparean aqueous suspension of presterilized C-Fern® spores of an F1 hybrid as indicated in Table 1 and as follows:
  4. 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.
  5. Transfer the appropriate amount of sterile distilled water to the spore vial using a sterile transfer pipet.
  6. Do not return any water to the sterile water bottle.
  7. Wet spores completely by firmly attaching the cap and inverting the vial two or three times.
  8. With the cap on, check the bottom of the vial to be sure that all spores have been suspended. Allow the spores to soak for 15 minutes prior to sowing.

Note: The C15023 C-Fern® Polka Dot spore vial used in the 156708 kit is at half-density. The dry weight of the spores in the vial is 5mg and there are approximately 1,250 spores per mg dry weight. The 5mg vial is designed specifically for use of the kit. (See Table 1 for additional details.)

Table 1. Spore-sowing densities

FOR this density / USING 5-mg vial / ADD this much water (mL) to vial / SOW this many drops of spore suspension / MAKE about this many Petri dish cultures / WITH this number of spores per Petri dish
standard / 5 / 2 / 3 / 18 / 300+
  1. Sowing Spores—Use sterile pipets to sow (inoculate) spores into 60- x 15-mm Petri dishes containing Basic C-Fern®Medium (as per Table 1). Below are seven good habits for sowing C-Fern® cultures:
  2. Work very carefully when sowing spores. Secure the vial in a convenient holder.
  3. Suspend spores before every sowing by gently drawing the liquid along with the spores in and out of the pipet.
  4. Immediately sow three uniform drops (not squirts) onto the agar surface. Hold pipet at a constant angle.
  5. Do not touch the pipet to the agar surface.
  6. In most investigations, use a sterile spore spreader to distribute spores uniformly over the agar surface.
  7. Keep the Petri dish lid in place as much as possible. Tilt the lid of the Petri dish upward just enough to permit access of the pipet tip.
  8. Label dishes with name or initials, the date spores are sown, and the treatment code, if any. (Note: There is no treatment code for this investigation.)
  9. Spreading Spores—Using a sterile spore spreader (bend a paper clip into a "T" shape and sterilize with alcohol prep pad then let air dry), spread the spore suspension evenly over the surface of the culture medium in each Petri dish. Allow the spreader to rest on the agar surface without pressure and move it gently back and forth across the surface of the agar while rotating the dish slowly with the other hand. The goal is to uniformly distribute spores over the entire surface of the medium.
  10. Place Petri dishes in the culture dome (greenhouse tray, including the humidity dome).
  11. Place culture trays undercontinuous fluorescent illumination (e.g., 15-W fluorescent bulb) at 28°C (82°F). A screw-in 15-W fluorescent bulb with a simple fixture, such as a clamp or garage light, is long lasting, safe, and easy to use for light and temperature maintenance. Two 40-W cool-white fluorescent tubes at a distance of 45 cm from the cultures will accommodate two standard culture domes (54 x 27 cm). Smaller or larger setups may be used, and temperature is more important than light level, so the distance between the light source and culture dome should be adjusted to obtain a temperature near the optimum. Monitor the temperature with a thermometer to ensure best results.[1]
  12. Optional Observation of Spores—Prepare a wet mount using a small portion of the spore suspension to view the spores under compound microscope magnification.

Figures:

Compared to other ferns, C-Fern® spores(Figure 1, image reproduced from Hickok & Warne, 2009) are relatively large (ca. 120 μm in diameter)(Hickok & Warne, 2009). (Note: One μm—micrometer or micron—is one millionth of a meter.)

The images below show C-Fern® spores24 hours after having been spread on the surface of basic C-Fern® medium in Petri dishes. The spore at the center of Figure 4 appears to possibly be germinating, but compare Figures 5 and 6, which show spores actually germinating on Day 5.

Figure 1. C-Fern® spores, 80x magnificationFigure 2. C-Fern® spores, 150x magnification

Figure 3. C-Fern® spores, 250x magnificationFigure 4. C-Fern® spores, 350x magnification

Figure 5. C-Fern® spores, 400x magnificationFigure 6. C-Fern® spores, 1000x magnification

(showing primary rhizoids in two spores)(showing primary rhizoid in one spore)

References

Chasan, R. (1992). Ceratopteris: A model plant for the 90s. Plant Cell, 4, 113-115.

Hickok, L. G., & Warne, T. R. (1998a). C-Fern® culture instructions and background information [Pamphlet]. Burlington, NC: Carolina Biological
Supply Company.

Hickok, L. G., & Warne, T. R. (1998b). C-Fern® investigations: Genetics in action Mendelian genetics student instructions [Pamphlet]. Burlington, NC: Carolina Biological Supply Company.

Hickok, L. G., & Warne, T. R. (2009). C-Fern web manual. Retrieved from Sunnyside website:

Maxwell, D. (2004). C-Fern genetics. Retrieved from ThePingrySchool wiki website:

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[1] Optimal temperature may be obtained through the use of supplemental incandescent lighting.