Preparation
Given
certain basics there are many options for procedure, equipment and
supplies
for plant
tissue culture. Some decisions will be based upon the amount of time and
money available, others are merely a matter of personal preference.
Catalogs, such as Sigma, Carolina Biological, or Edmund
Scientific for
are essential
for
reference
or purchasing are very important resources.
OPTIONS
The following discussion lists some of the various options you have as
you
begin the
tissue culture of plants. Options used in
in the
sample protocol are identified with an
asterisk (*).
Which plant?
Perhaps the easiest decision you need to make is which plant you would
like
to place
in culture. To begin with I would suggest one of the following:
- Boston fern (runner tip)
- Rex Begonia {petiole segment}
- Kalenchoe (stem tip)
- *Strawberry (runner tip)
- African violet
, leaf part
- Arrowhead plant
, stem tip
Work Space
If your school already has, or can afford to buy, a transfer chamber
with a
HEPA (high
efficiency particulate air) filter then there is no decision, you will
use
the transfer
chamber. For now we shall rule out this unlikely option. An open bench
or
desk-top in the school room will work but you may have as much as 95%
contamination which
would be very discouraging. Other options include the following:
- Find a small room with still air
- Build a still air box of wood; with a slanted front of glass (or
Plexiglass)
- *Use a large cardboard carton; tape clear plastic over the top and
front
(cut
holes in the sides or front for access by hands and forearms)
- Use a large, clear plastic bag
- Purchase a small HEPA filter and blower and build a little
hood.
Medium (plural media)
Next you will need to decide on the nutrient medium. The options are
limited:
- *Buy premixed medium in powder form ready to mix with water, heat,
dispense and
sterilize.
- Buy media already in test tubes ready to use (expensive).
- Buy the individual chemicals (expensive for limited use and requires
a
balance
which can weigh milligram quantities).
- Buy "off the shelf" ingredients from local stores.
Equipment for Media Preparation
You will need
a vessel in which to mix and heat the medium:
- A 2 or 4 liter Erlenmeyer flask (if using a hot plate or hot
plate/magnetic
stirrer}
- *A stainless steel pan (if using a stove or burner)
You will need
a means of stirring while heating
:
- *A long handled spoon
- A hand mixer
- A hot plate/magnetic stirrer
You should have one to ten test tubes per student.
- If you are really desperate use baby food jars.
- *Test tubes, "disposable" glass 25 X 100 mm is standard
(80
for $54.60 1995
Sigma Plant Culture Catalog)
- *Test tube caps
You will need
racks to hold the test tubes vertical
vertically?
(see the catalogs)
- Buy one or more racks
- *Build one or more from wood or wire
You will need
a means of dispensing the medium
into test tubes:
- Buy an automatic dispenser (expensive and fragile)
- *Use a glass (Pyrex) quart pitcher
The
test tubes containing the medium should be sterilized
in:
- *A household pressure cooker
- An autoclave (very expensive)
You will also need a clean cupboard in which you can store the
sterilized
tubes of
media.
Equipment and Materials for Use in the Transfer Chamber
- *Forceps (2 or more) 8 inch
- Tweezers (large, o.k. for baby food jars)
- *Kitchen paring knife
- Scalpel
- *Rubber gloves
- Spray bottle with 70% isopropyl alcohol (flammable)
- *Household bleach
- *Plastic dish to hold 10% bleach solution (1 part bleach and 9 parts
sterile water)
for sterilizing instruments and gloved hands.
- *Plastic dish to hold 1% rinse solution (1 part bleach and 99 parts
sterile
water)
for rinsing instruments and gloved hands.
Other options for sterilizing instruments but too dangerous in a
cardboard
transfer
chamber:
- Bunsen burner
- Bacti-Cinerator
- Glass bead sterilizer
- Lead melting pot containing sand
Device to hold sterilized instruments as they cool or
dry:
- *Test tube laid horizontally to rest instruments on
- Build a rack of hardware cloth (wire)
- Up-end a metal test tube rack
Sterile surface on which to cut cultures:
- A 12 inch square of plate glass (spray with alcohol)
- *Single sheet paper towels (which have been wrapped in foil and
sterilized
in pressure
cooker for one hour at 15 lbs pressure. Don't forget to have sufficient
water in
the base of the cooker and place pack of towels on rack or other holder
to
keep them
out of the water.)
Growing Space
*Shelves lighted with fluorescent lights
(cool white or equivalent)
Test tube racks
*A seedling tray 11 X 22, 78 holes, to hold test tubes on lighted
shelves
background | laboratory protocol
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