C O N T E N T S |
C-Fern Home |
Web Manual |
Introduction C-Fern Nutrient Medium Culture Conditions Gametophyte Culture Gametophyte Observations Sporophyte Culture Spore Mutagenesis Bibliography |
Light Stand Growth Pod DNA Extraction C-Fern T-shirt Who's Who Selection and mutants Media formulation error Image gallery |
Web Journal |
Educational Resources |
National Science Standards Gametophyte development Student research questions Research in the classroom Research with C-Fern Brief description of C-Fern C-Fern manipulation Root tropism? |
Frequently Asked Questions |
Workshop and Exhibit Schedule |
Educational Materials |
Investigations Mutant & wild type spores Manual & Culture Instructions Supplies - Domes, media |
Development
of C-Fern is supported by the National Science Foundation (NSF-DUE) |
Copyright
© 1997-2000
Thomas R. Warne and Leslie G. Hickok. All rights reserved. |
C-Fern
Web Manual - Spore Sterilization
Surface Sterilization of C-Fern Spores
Spore sterilization
and sowing should be performed on a sterile bench or laminar flow hood.
Materials:
Procedure: 1. WEIGH SPORES. Weigh out spores onto glassine weigh paper and transfer them to a sterile centrifuge tube with conical bottom.
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.
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..
|