Lettuce preparation

1. Cut the lettuce leaf into pieces roughly 5 mm x 5 mm.

2. Add 15~20 lettuce pieces to 9.5 cm³ 13% sorbitol solution in a test tube.

3. Incubate the tube in a water bath at 37^oC for 5 minutes. During this time the lettuce tissue
    will equilibriate to the osmotic potential of the sorbitol solution.
 

1. Gently stir 0.5 cm³ of the carbohydrase enzyme mixture into the sorbitol and lettuce
    preparation.

2. Return the tube to the water bath for another 20 minutes. Gently agitate the tube from time
    to time while it is incubating. The carbohydrase mixture contains pectinases, cellulases and
    other cell wall-degrading enzymes.
 
 

Recovery of protoplasts

1. Tightly pack the spout of the filter funnel with the nylon gauze. This will produce a fine
    filter of roughly the correct pore size (able to trap cell debris greater than 50 mm in
    diameter).

2. Pour the digested lettuce suspension into the filter funnel.

3. Wash any trapped protoplasts through the filter using 10 cm³ of 13% sorbitol solution.
    Collect all the filtrates in a centrifuge tube. The protoplasts will pass through the filter while
    the majority of the undigested cell debris will remain on top of the gauze.

4. Ensure that the centrifuge is correctly balanced then spin the filtrate for roughly 5 minutes at
    2,000 rpm (you may need to adjust this according to the type of centrifuge available).

5. Carefully discard the supernatant, leaving the pellet of protoplasts at the bottom of the tube.
    Use a tissue to wipe away excess liquid from the inside wall of the tube.

6. Resuspend the pellet in approximately 0.1 cm³ of 21% sucrose solution. This is an
    osmotically balanced (isotonic) medium in which the delicate protoplasts will not burst.
 
 

Examination of protoplasts

The protoplasts will remain stable in the sucrose solution for several hours and can easily be seen without staining using a microscope with a x40 objective.
 
 

Safety

Care should be exercised when loading and operating the centrifuge. Students using the centrifuge must be closely supervised. Spills of enzyme should be wiped up promptly using plenty of water.