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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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