Supplies found in your kitchen or the
local discount store:

You need to be aware of basic laboratory skills and lab safety including: the safe handling and disposal of alcohol and bleach solutions, disinfecting forceps and knives with alcohol (flame sterilization is not recommended), preparation of media (depending on student age, you may need to limit this activity), and the use of protective clothing such as vinyl gloves, goggles, plastic aprons, dusk masks, and leather or tennis shoes.

Supplies needed for one quart of medium (20 baby food jars) plus sterile water:

• 1 quart jar
• 20 baby food jars (4 or 6 oz)
• 2 pint jars (regular opening)
• 20 plastic or metal baby food jar caps‚
• 2 pint jar plastic caps‚ or regular metal rings and lids if using a pressure cooker (the white plastic caps will survive autoclaving though they might get a little soft)
• long handled spoon
• pH paper

Sterile water will be needed to rinse plant pieces after they are soaked in bleach solution. Fill pint jar 1/2 full with tap water or distilled water. Place cap on loosely. Set aside until media preparation is finished.

Sterile African violet medium is needed to grow the plant parts. The following is combined in a quart jar: 1 packet MS medium, 2 tablespoons table sugar, 1 mg BAP, 1 ml PPM, distilled water. Using pH paper, vinegar, and baking soda, adjust the pH to about 5.5 to 6.0.   Note that hormones solutions can be purchased in concentrations of 1 mg/ml and measured with a simple baby dropper hence you will not need an expensive balance to weigh them.

Add 3 tablespoons of liquid medium to each baby food jar using a plastic measuring tablespoon. Add one-half level "pink Baskin-Robbins" spoon OR one level "pinch" spoon of agar to each baby food jar. Place polypropylene baby food caps on the jars (if using a microwave) and press to tighten. Polypropylene caps or the original metal caps can be used in a pressure cooker.

The purpose of the clean area is to limit the number of particles that fall into your tissue culture jar. These airborne particles carry bacteria and fungi, and can kill your plant tissues because they grow faster than the plants. A clean area can be made from a plastic-lined cardboard box or a plastic storage box with a "window" cut out.

Pick up a leaf with forceps and dip into the 70% alcohol for a few seconds. This will remove some debris and wax. Place leaf in 10% bleach solution and allow to soak for 10 minutes. Stir occasionally. Move the bottle with leaves to the clean box. Transfer leaves to sterile water using the forceps, and allow to soak for 1-5 minutes.

Inside the clean box, wipe a small salad plate with 70% alcohol. Note that you could also use a paper towel laid directly on the table surface; spray it down with alcohol and you have a sterile surface.  Dip the forceps in 70% alcohol and transfer one leaf to the plate. Dip the kitchen knife in 70% alcohol. Holding the petiole end of the leaf with the forceps, cut the edges of the leaf away. Then cut the leaf into two pieces.

Loosen the caps on 2 baby food jars. Dip the forceps in 70% alcohol. Pick up one leaf piece. With your other hand, pick up the cover of the media jar and place the leaf piece in the jar. Quickly replace the cover. Wrap florists’ tape or surgical tape around the outside of the jar. This will help to minimize the debris that gets into the jar and causes contamination of the cultures.

                                       

Various containers can be used for culture:  Gladware (left-top), baby food jars and mason jars (right-top),
Combiness vessels (left-bottom) and "Quesa" jars (right-bottom)

 

Put the cultures in a bright room out of direct sunlight or set the cultures on shelves with cool-white fluorescent lights positioned about 9-12 inches from the shelf below. Lights should be on 16 hours per day. The leaves should start to swell in 2-4 weeks, and small bumps and then leaves will appear on the "mother" leaf’s surface.

The newly developing plantlets will grow better if they are transferred to fresh medium without growth regulators. The growth regulators can inhibit elongation of the shoots and the formation of roots.

After 4-6 weeks, make fresh medium using the "Home Style Medium" recipe below. Follow the same instructions as you did for the original medium using these ingredients:

"Home Style Medium"
In a quart jar filled with water, mix:

Mix well. The vitamin pill will not completely dissolve. It can be removed after a couple of minutes. Test pH and adjust as you did in the first batch of medium.

Measure 3 tablespoons medium into each baby food jar. Add two cotton balls, or 1/2 teaspoon gelatin, or agar (as previously described). Cap with polypropylene caps, or metal baby food jar caps if using a pressure cooker. Sterilize as described earlier.

When plants have developed shoots and roots, they are ready for transfer to sterile soil or soil-less medium (found at your local discount store). Gently remove the plants from the jar. Gloves should be worn when doing this in case your skin is sensitive to the culture medium. Rinse off all of the medium that is sticking to the stem and roots under lukewarm running water. Plant the tissue cultured plantlet in the moist soil. Water with a liquid fertilizer such as Peter’s or Miracle Gro.

Dirr, Michael A., and Charles W. Heuser, Jr. 1987. The Reference Manual of Woody Plant Propagation: From Seed to Tissue Culture. Varsity Press, Inc. 239 p.

Kyte, Lydiane, and John Kleyn. 1996. Plants from Test Tubes: An Introduction to Micropropagation (Third Edition). Timber Press, Inc. Portland, Oregon. 250 p.