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This experiment allows students to go from the plant on the desk, to observing a stained specimen under the
microscope in less than 4 minutes. The viewed specimen clearly shows the location of vascular bundles and
the xylem, phloem and sclerenchyma or collenchyma. The use of the stain toluidine blue provides a colour
difference between lignified and non-lignified cell walls, clearly highlighting specialised cells and one
adaptation they have.
This experiment provides a quick and eye-catching way to teach about the vascular tissue in plants and the
structure of plant stems. It provides students with the opportunity to develop (and demonstrate) their
scientific drawing skills as well as their use of a light microscope and eye-piece graticule.
Simple extensions to the basic protocol would allow students to collect data in cell diameters of different
specialised cells, or from different plant species very quickly. Student could also explore modifications to the
protocol to try to get clearer images of the vascular tissue, thereby developing their skills of experimental
design.
Safety Notes
Check CLEAPSS guidelines for use of blades and Toluidine blue (both the use and the making up of the stain). Safety
precautions are very dependent on the solvent used.
Apparatus
Microscopes
Microscope slides
Coverslips
Fresh head of celery
0.5% Toluidine blue (unstained specimens also have value)
Scalpels or single edged razor blades (NB Scalpel blades must be sharp)
White tiles
Forceps
Stage micrometer (can use a table for looking up sizes of small divisions of the eyepiece graticule instead)
Glassware and pipettes for diluting Toluidine blue (if students are modifying staining protocol)
Suggestions of other species of plant for comparison
o Monocots: Asparagus, lris
o Dicots: Carnation, Thyme, Mint, Broccoli
Toluidine blue can be bought as Toluidine blue or Toluidine blue O. These are the same stain but toluidine blue O
(often the more expensive one) is certified for histological use as the stain content (of the powder purchased) is very
high. Toluidine blue is sufficient for the requirements of this practical.
Toluidine blue can be made up using water or in a buffer (e.g. McIlvaine (citrate and disodium hydrogen phosphate)
buffer (pH 6.8)). The use of the buffer enhances the staining but isn’t a necessity for this practical.
Staining protocols vary in stain concentration, staining duration, pre-soaking in water, rinsing duration, whether
toluidine blue or Toluidine blue O is used and whether the stain is made up using water or buffer. Some
experimentation prior to teaching may be useful to modify the suggested protocol to get the best results for any
particular teaching episode but obtaining functional results is very robust.
Suppliers
Toluidine blue is available through standard school suppliers, often sold as 5g of powder (at between
£10-£15) which would provide enough stain for many classes.
Plant material can be obtained from a local supermarket.
Stage micrometers are expensive so if you don’t have them students can look up an estimated length
between gradations on the student sheet provided.
Teaching Notes
Use of blades
Obtaining thin sections of tissue requires the use of sharp blades, there are 2 potential choices of blades that can be
used for obtaining sections: scalpels, single edged razor blades. It is easiest to get very thin slices using single sided
razor blades but care and appropriate guidance should be given by the teacher. It is up to the teacher to decide if
these blades can be used with their class and the appropriate precautions taken. Single edged razor blades produce
very usable sections but scalpels are the most easily available in schools (their blades should be as sharp [new] as
possible).
The table below show the colours that you should expect to see in your preparation. Generally non-lignified tissue
should be pink/purple and lignified tissue should be green/blue. Both colours tend towards dark blue when over
stained.
(From Parker, A. J., Haskins, E. F. and Deyrup-Olsen, I. (1982) Toluidine Blue: A simple, Effective Stain for Plant
Tissues. The American Biology Teacher., Vol. 44, No. 8, pp. 487-489)
Celery (Apium graveolens var dulce) is easily obtainable all year round and has large vascular bundles. The
celery stalks are technically leaf petioles rather than plant stems. The vascular bundles look the same as they
do in a stem but their arrangement within the petiole is not the same as the usual arrangement in
dicotyledonous stems.
Common species used, but more difficult to obtain all year round, are Buttercup (Ranunculus sp.) and
Sunflower (Helianthus annuus), sunflower flowers are periodically available in florists/supermarkets.
Asparagus and/or Iris are interesting as they are monocots and so have vascular bundles randomly arranged
throughout the stem – an interesting comparison.
Carnations (Dianthus caryophyllus) have a ring of vascular tissue rather than vascular bundles.
As a minimum students should prepare a slide and produce a scientific drawing from it.
Eye-piece graticules allow for magnification to be calculated and scale bars to be drawn.
To get students really thinking about what they have done and what they are seeing they can answer the
questions in the Students’ sheet document or completing the student worksheet. You can extend either of
these using some of the questions in the Possible questions for students list below.
The Possible questions for students section below has some suggestions that require students to look more
carefully at what they see and measure cell diameters of different cell types – this would be a useful extension
for those students who work quickly and could lead to some data being collected that could be shared with the
class (e.g. bar charts comparing average cell diameter of different cell types or of different plants, histograms
of size distribution, and/or statistical analysis of size difference).
A stage micrometer could be used to allow students to calibrate their own eye-piece graticules.
Students could modify the staining procedure (timing and stain concentration) to get the best colour contrast
as well as adjusting the light level or iris aperture diameter of the microscope if possible.
Students can use cameras on mobile phones to get photographs of what they have produced.
Give the drawings titles to describe the specimen, the staining procedure and the magnification of the eyepiece and
objective lens used.
Label the drawings to show different cell types and cell structures.
Annotate the drawings to provide more information about what you have labelled.
Provide a scale bar and magnification for the drawing you have produced.
Write out the calculations used to calibrate the eye-piece graticule at each magnification.
Write out the calculation used to work out the magnification of your drawing(s)
Explain how you produced a suitable scale bar for your drawings
What is the magnification of this photomicrograph? (given the cell diameter from x to y)
How can you tell if what you are looking at is a xylem vessel?
What adaptations do xylem vessels have and which ones can you see in your preparations?
In which two ways are xylem vessel walls adapted? What evidence you do have that this is the case in your
preparation? Is it true for all species you looked at?
How are the features of xylem vessels linked to their function?
Identify xylem, phloem and sclerenchyma (and any other cell types) in photomicrographs.
It is important to consider the purpose of practical work in Biology lessons and the following information provides
suggested areas where this practical can be used to help students develop their practical, mathematical and subject-
knowledge-based skills and understanding. The latter part of this section shows how this practical (or an extension of
it) can also be used to meet some of the A-level specification requirements for the Use of apparatus and techniques
as well as the Common practical assessment criteria (CPAC).
Students can use the worksheet provided to produce evidence of their work in this practical that can contribute to their
practical skills accreditation.
The full practical can allow students to meet various components of Appendix 5c of the GCE AS and A level subject
content for biology, chemistry, physics and psychology (DfE, April, 2014) in bold (with clarification where needed):
1. Use appropriate apparatus to record a range of quantitative measurements (to include mass, time,
volume, temperature, length and pH).
2. Use appropriate instrumentation to record quantitative measurements, such as a colorimeter or potometer.
3. Use laboratory glassware apparatus for a variety of experimental techniques to include serial
dilutions. (for experimenting with stain concentrations)
4. Use of light microscope at high power and low power, including use of a graticule.
5. Produce scientific drawing from observation with annotations.
6. Use qualitative reagents to identify biological molecules (identifying lignin)
7. Separate biological compounds using thin layer/paper chromatography or electrophoresis.
8. Safely and ethically use organisms to measure:
a. Plant or animal responses
b. Physiological functions
2. Use microbiological aseptic techniques, including the use of agar plates and broth
3. Safely use instruments for dissection of an animal organ, or plant organ.
4. Use sampling techniques in fieldwork.
5. Use ICT such as computer modelling, or data logger to collect data, or use software to process data.
(tabulating results of cell measurements, doing calculations, presenting results graphically, statistical analysis
of results)
The learning outcomes document for this practical summarises the key background information relating to vascular
bundles and their specialised cells.
Sample data
The two photographs below are examples of a transverse and longitudinal section of a celery vascular bundle
using this technique.
Further Investigations
As mentioned within this document there is scope to develop this practical in three directions:
Careful, logical, precise modification of the staining method to get the best differential colour staining.
Measurement of cell diameters of different specialised cells and analysis of the data
References
O’Brien, T. P., Feder, N. and McCully, M. E. (1964) Polychromatic staining of plant cell walls by toluidine blue O.
Protoplasma. Vol. 59, pp. 367-373
Parker, A. J., Haskins, E. F. and Deyrup-Olsen, I. (1982) Toluidine Blue: A simple, Effective Stain for Plant Tissues.
The American Biology Teacher. Vol. 44, No. 8, pp. 487-489
Yeung, E. (1998) A beginners guide to the study of plant structure. pp. 125-142, in Tested studies for laboratory
teaching, Volume 19 (S. J. Karcher, Editor). Proceedings of the 19 th Workshop/Conference of the Association for
Biology Laboratory Education (ABLE).
Acknowledgements