These experiments are to be performed individually by each student and your diagrams must be handed-in at the end of the class. Do not forget to write your name and number on your work. If you use more than one sheet of paper please staple your sheets together. A stapler is provided on the front bench for this purpose.

1. Cytoplasmic streaming in pollen tubes (Zingiber officinale)

1. Transfer some pollen to a drop of germination medium on a microscope slide..

2. Gently add a coverslip and a leave to germinate.

3. Observe (x40), it may take some time for the tubes to appear (about 20 min).

4. How many different types of streaming can you identify?

5. Add a drop of ethanol near the edge of the coverslip.

6. Observe the structural changes in the pollen tube.

7. Record your observations.

2. Cytoplasmic streaming in Hydrilla leaves

1. Place a drop of water on the top of a slide.

2. Carefully place a Hydrilla leaf into the drop of water, be sure to choose a young thin leaf from near the tip of a shoot.

3. Gently place a coverslip on the top of the leaf.

4. Record the rate of cytoplasmic streaming. (Hint, calibrate your field of view with a ruler)

5. How many different types of streaming can you recognize?

6. Streaming is activated by light and heat and as the Hydrilla leaf is on the illuminated stage streaming will increase. If      you cannot immediately observe streaming leave the slide under a light and look at it later.

3. Freehand Sectioning of Stem Tissue

Purpose: To review basic procedures of freehand sectioning, staining, and temporary mounting of plant materials; to continue examining different types of cells seen in transverse sections (T.S.) of stem. Each student will be assigned a plant. You must investigate its anatomy by cutting, staining, and mounting T.S. sections of its stem. You should submit the results of your investigations as labelled drawing of the stem T.S. which must be handed in at the end of the class.

Materials: Razor blades which must be clean and sharp, preferably new. Single-edged blades are preferred to double-edged ones because the former are safer to use and stiffer, and so will cut flatter, more even sections. Without a good razor blade you cannot hope to cut good sections. Always have several new blades on hand. Keep some water in a petri dish, to keep the specimen and blade wet while sectioning and in which to float cut sections. Sections can be manipulated using the small brush.

Procedure (for right-handed people):

1. Hold the specimen in your left hand between thumb and the first finger, with only a few mm of the specimen     projecting above your fingers.

2. Hold the blade in your right hand, resting it flat on the first finger of your left hand.

3. Cut sections by drawing the blade above your finger and through the specimen. Use the whole length of the blade in    one continuous motion (do not saw back and forth); cut quickly; regulate the position of the blade with the first finger  of your left hand.

4. After several sections accumulate on the blade, transfer them to a dish of water.

5. Re-wet blade and specimen; continue cutting as needed.

Precautions:

1. Keep blade and specimen constantly wet.

2. Keep thumb out of the way of the blade.

3. Cuts should be made at right angles to the axis of the specimen.

4. If your cuts begin to slant, trim specimen back to a right angle.

5. Cut more sections than you think you will need.

Assessment:

The best sections are one-cell thick, cut perpendicular to the axis of the specimen. THINNESS is more important than completeness; that is, a very thin partial section is better than a thick complete section. Furthermore, because of their thinness, the best sections are the most difficult to see in a petri dish of water, and they are the most delicate: Handle with care, always with a soft brush.

Staining:

The purpose of staining is to make sections or specimens easier to see, and to differentiate tissues based on their chemistry; that is, because the chemical constituents of tissues differ, the tissues turn different colors when exposed to a given stain.

TOLUIDINE BLUE O: An aqueous stain which is used alone, on fresh or fixed material, on handcut sections or embedded microtome preparations. Traditionally used for zoological material, equally good for botanical specimens. Simple and quick, giving multicoloured (i.e., polychromatic) results.

Formula: Aqueous 0.05% Toluidine blue O in 0.1 M phosphate buffer at pH 6.8.

Procedure for temporary mount; single stain (Toluidine blue O)

1. Put 2 drops of stain in watchglass.

2. Using a brush, transfer the sections (soaked in water) to stain.

3. Leave for approximately 1 minute: time will vary with different plant materials and according to thickness of sections.     You must learn by experience what is best for your sections.

4. Transfer sections to clean water in another watchglass. Leave for 2-3 minutes, agitating them occasionally.

5. Mount in a drop of dilute glycerine.

6. Examine. Tissues should be distinct and clearly stained. If understained or overstained, prepare another slide,     adjusting time in stain as appropriate.

STAINING REACTIONS

Tissue Element                           Toluidine Blue O

Lignified walls                         Green or bluish green

Non-lignified walls*:

Collenchyma                                    Purple

Parenchyma                                     Purple

Sieve tubes                                        red

Companion cells                                red

Middle Lamellae                    Reddish-purple or Red

Callose, starch                               Unstained

Nuclei, chromosomes              If stained,blue-purple

* The colour of the non-lignified walls is due to pectin and polyuronides present in the wall and not due to cellulose which is unstained.