Exercise 2 — Preparation &
Staining
Your name:
___________________
Date: ______________
Objectives:
By completing this laboratory session you will be able to:
1. Gain experience in freehand sectioning to prepare plant specimens for light
microscopy.
2. Learn the appropriate use of stains to enhance observations of specimens.
3. Understand the use of selective staining for botanical specimens.
4. Make effective use of polarizing filters and understand the concept of birefringence.
Materials:
Letter “e” prepared slides
Polarizers (1 large & 1 small)
65% H2SO4
Ruthenium Red
Baby Carrots
Potato
Celery
Yellow Apples
Pine needles & branches
Razor blades
Coleus slides
Phloroglucinol
IKI
Ferric sulfate
Syringa (lilac) slides
Laboratory Exercises:
Freehand Sectioning & Staining –
Starch: Based on the handouts entitled “Freehand Sectioning” and “Some Histochemical
Tests for Fresh Tissue Slices.” Either slice or scrape off starch grains from a potato.
Very thin sections should allow for better resolution of cells. Stain lightly with IKI
solution and observe with 10, 20 and 40× objectives. A drop or two of water added to the
IKI solution may dilute the staining enough to prevent over-staining.
What structures are stained? _____________________________________________
In the space below, draw one or two cells and their contents. Label structures.
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Pectic substances:
Make a thin section of apple from the surface inwards approx. 5-10 mm to obtain tissue.
Stain with ruthenium red and observe (allow 1-2 min. for stain to maximize). Also,
make a thin slice of potato through the “skin.” Mount on a slide and stain with ruthenium
red.
What structures are stained? ________________________________________________
Cellulose:
Make thin sections of celery stalk and pine stem. Stain with the IKI/H2SO4 method.
(Refer to handout sheet on Histochemical Tests.) Caution: Use care when handling
strong acids as H2SO4 so as not to damage yourself, clothing or the microscopes!
Describe the staining reaction as a function of cell types: _________________________
_______________________________________________________________________.
Lignin:
Prepare thin cross-sections of a pine branch and stain with toluidine blue-O solution. Do
you observe metachromasia? Draw a cross-section of your sample and label structures
that show selective staining.
Repeat the above procedure with phloroglucinol. How differently do the stains appear?
(Caution: Use care as phloroglucinol contains a strong acid!)
Tannins:
Look for tannins in pine needle cross-sections. The baby carrots will help in obtaining
thin slices. Stain with ferric sulfate (works similar to ferric chloride).
What color do the tannins appear? ___________________________________________
Where are they found? ____________________________________________________
Polarizing Filters
A polarizing filter screens light in all but one plane to obtain ordered light. As you rotate
one of the polarizers, those parts of the specimen, which appear to change in color and
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intensity, are said to possess birefringence and are usually crystalline or para-crystalline
at the molecular level.
Observe birefringence of several specimens available in lab (e.g. letter “e,” potato, celery,
pine). Experiment both with and without stain. Draw one example of birefringence and
name the source/structure.
Permanent stains
Observe prepared slides of Syringa (lilac) leaves and Coleus leaf abscission to illustrate
staining using the permanent stains, Safranin (red) and Fast Green. What structures are
stained with safranin (red)?
________________________________________________________________________
What structures are stained with fast green? ____________________________________
________________________________________________________________________