Dissection and microscopy of plant vascular tissueTechnical & Teaching Notes

Introduction and context

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

Toluidine blue made up as 1% (w/v) or less in water is a very low hazard. Eye protection should be used when handling this stain. Care should be taken when handling and transporting blades – teacher should demonstrate safe use. Sharp

scalpels can produce usable sections and are the simplest blades to use safely. Single edged razor blades can provide good sections but require more care when transporting and handling. Double edged razor blades produce the thinnest sections most easily but are considerably harder to use safely. Your choice of blade may be determined by the characteristics of the class and the particular safety precautions you put in place.

Coverslips and slides can sometimes be broken by students. Appropriate methods for handling breakages and facilities for the disposal of these are necessary.

Some plant material may need to be avoided if any students are allergic to them. Always perform your own risk assessment for practicals.

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 Pipettes

Science & Plants for Schools: www.saps.org.ukDissection and microscopy of plant vascular tissue – Teaching and Technical notes: p. 1

Tap water Eyepiece graticules Watch glasses (can stain on the slide and rinse into a sink/beaker)

Other apparatus for extension activities

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, lriso Dicots: Carnation, Thyme, Mint, Broccoli

Information about Toluidine Blue and using it as a stain

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).

General practical tips Thin, perpendicular sections are the key to good images – they don’t need to be whole sections so

concentrating on thin rather than whole is better. Sectioning and staining is very quick so multiple attempts are suggested to then observe the best specimens.

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Over-staining leads to lack of differentiation between the different colours that Toluidine blue produces – if this is the case then dilution and/or reduced time in the stain is necessary. Modifications to the procedure can even be made in the lesson.

Viewing under low and medium power is sufficient – high power is not needed and would only be useful for the very best sections and when using microscopes that are in very good condition.

Photos can be taken down the eyepiece with a mobile phone camera with a little care and persistence, and can be kept by students as evidence of their work.

Fresh celery from a supermarket is good and many different plant stems can be found around the fruit and veg., section as well as from the cut flowers and pot plants section. We advise against using plant material with very tough stems as sectioning may become more hazardous. For best results keep plant material from wilting by storing in containers containing water or moist tissue.

Colours Toluidine blue produces

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.

Tissue Element or Structure ColourXylem Green or Blue-greenPhloem RedSclerenchyma Blue-green, sometimes GreenCollenchyma Red-PurpleParenchyma Red-Purple

(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)

Students may need to be taught or reminded about

best and safe use of a microscope carrying and handling blades safely wearing eye protection when using the stain what to do if coverslips or slides break or if there is a stain spillage the process of producing a good scientific drawing

Notes on plant material choice

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. The potted herb Thyme (Thymus sp.) allows for comparison of newer stems (less woody) and slightly older

stems (more woody). Mint (Mentha sp.) is interesting as it has a square cross-section with strengthened corners.

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Notes on student tasks

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.

Possible questions for students1. Follow the protocol and answer the following questions

What is the eye-piece graticule for?What is a stage micrometer for?What species did you investigate (and did you look at transverse and/or longitudinal sections)?What difficulties are there in reliably measuring the diameter of cells or the thickness of cell walls?How can these difficulties be overcome?Why is it important to produce very thin slices of plant tissue?Why is it important that the sections are truly transverse (or longitudinal) and not cut at an angle?What did you find was the best way to get sections as thin as possible?Why are stains useful in microscopy?Why is Toluidine blue useful for this protocol?What did you find was the best way to modify the staining protocol to produce the most informative staining?What adjustments did you make to the microscope to optimise the image?What challenges were there in taking photographs of what you could see down the microscope?What things did you try and how did you overcome these challenges?What did you end up taking good photomicrographs of?

2. Produce drawings of the specimens you 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.

3. Do calculations to learn about your specimens

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 drawingsWhat is the magnification of this photomicrograph? (given the cell diameter from x to y)What is the actual size of the cell in this photomicrograph? (given the magnification of the image)Draw a table to show the average diameter of each cell type, the average cell wall thickness and the average percentage of the cell diameter that is cell wall.Draw a table to show the percentage of the cell diameter that is cell wall for 10 xylem vessels and 10 parenchyma cells.

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Do a statistical test to see if there is a significant difference between the relative cell wall thickness of xylem vessels and parenchyma cells.Plot a histogram to show the distribution of diameters for a variety of cell types.How can you increase the reliability of your measurements of each cell diameter?How could you get an unbiased selection of cells for your analysis of cell diameters and cell wall thickness?

4. Answer these questions about the biology of what you see

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?

How can you tell if what you are looking at is phloem? Can you identify the difference between sieve tube elements and companion cells? If so how? If not what is

the difference between the two and how might you be able to identify one from the other? Where are the phloem in a stem compared to the xylem? What adaptations does phloem have and which ones can you see in your preparations? How are the features of phloem linked to their function?

How can you tell if what you are looking at is sclerenchyma? Is sclerenchyma present is all species you looked at? What adaptations do sclerenchyma fibres have and which ones can you see in your preparation? In which two ways are sclerenchyma fibres walls adapted? What evidence you do have that this is the case in

your preparation? Is this true for all species you’ve looked at? What are the similarities and differences between the ways xylem and sclerenchyma cell walls are

specialised? Can you quantify any differences? How are the features of sclerenchyma linked to their function?

Identify xylem, phloem and sclerenchyma (and any other cell types) in photomicrographs.

Getting learning value from the practical

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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).

PRACTICAL SKILL DEVELOPMENTYou should be able to: use a light microscope safely and proficiently at low, medium and high power use an eye-piece graticule and stage micrometer to calibrate the graticule use a calibrated eye-piece graticule to measure the size of objects viewed under a light microscope dissect plant tissue to obtain usable, thin slices (both transverse and longitudinal) for microscopy produce an annotated biological drawing for a specimen viewed under a light microscope use a staining procedure to highlight different structures within a biological specimen explain why specimens for viewing under a slide microscope need to be thin explain why stains are a valuable tool in microscopy

EXTENSION TO PRACTICAL SKILL DEVELOPMENTYou should be able to: explore the stem structure of a variety of specimens to identify similarities and differences between species locate xylem, phloem, sclerenchyma, parenchyma and collenchyma in the variety of specimens adapt a staining procedure to optimise contrast and identification of different structures within a biological specimen adjust the illumination of a light microscope to optimise contrast and identification of different structures within a

biological specimen learn from experience and subtly adapt your method to cut even thinner slices take simple photomicrographs

MATHS SKILLS DEVELOPMENTYou should be able to: perform calculations to calibrate an eye-piece graticule calculate the magnification of your drawing calculate the magnification or actual size of a cell using information given about a photomicrograph or diagram calculate an average diameter for a variety of cell types

EXTENSION TO MATHS SKILL DEVELOPMENT plot a histogram of cell diameters for variety of cell types analyse data to investigate whether there is a significant difference in cell diameter in different cell types

ASSOCIATED SUBJECT KNOWLEDGE DEVELOPMENTYou should be able to:

1. list the types of cells found in plant stems2. state the function of xylem vessels, phloem and sclerenchyma fibres in plant stems3. list the two components of phloem and explain why both are needed4. describe the structure of xylem vessels, phloem and sclerenchyma fibres5. identify xylem vessels, phloem and sclerenchyma fibres in preparations, photos and diagrams of plant stems6. describe how the structure of xylem vessels, sieve tube elements, companion cells and sclerenchyma fibres

are adapted for their specific function7. identify similarities and differences between xylem vessels, phloem and sclerenchyma fibres

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RECORDING EVIDENCE OF STUDENTS WORK

Students can use the worksheet provided to produce evidence of their work in this practical that can contribute to their practical skills accreditation.

MEETING ASPECTS OF THE USE OF APPARATUS AND TECHNIQUES

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 responsesb. Physiological functions

2. Use microbiological aseptic techniques, including the use of agar plates and broth3. 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)

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MEETING ASPECTS OF THE COMMON PRACTICAL ASSESSMENT CRITERIA (CPAC)

Competency Practical Mastery Evidence from this practical / or opportunity to train students in this skill using this practical

Follows written procedures

Correctly follows instructions to carry out the experimental techniques or procedures.

Observational evidence from the teacher.Positive outcome for the student’s preparation.Being able to answer questions about the procedure.

Applies investigative approaches and methods when using instruments and equipment

Correctly uses appropriate instrumentation, apparatus and materials (including ICT) to carry out investigative activities, experimental techniques and procedures with minimal assistance or prompting.

Use of microscope, graticule, stain, slide and coverslip, plant material and blade (statistical package).

Caries out techniques or procedures methodically, in sequence and in combination, identifying practical issues and making adjustments when necessary.

Follow procedure and modify to get thinnest sections and best staining and viewing conditions.

Identifies and controls significant quantitative variables where applicable, and plans approaches to take account of variables that cannot readily be controlled.

When investigating best staining procedure.

Selects appropriate equipment and measurement strategies in order to ensure suitably accurate results.

Measuring and drawing at best magnification. Accurate use of graticule including movement of slide into appropriate place. Appropriate method for selecting cells to measure.

Safely uses a range of practical equipment and materials

Identifies hazards and assesses risks associated with these hazards when carrying out experimental techniques and procedures in the lab or field.

Use of blade. Use of stain.

Uses appropriate safety equipment and approaches to minimise risks with minimal prompting.Identifies safety issues and makes adjustments when necessary.

Use of blade. Use of stain.

Makes and records observations

Makes accurate observations relevant to the experimental or investigative procedure.

Differential staining observation and drawing.Diameter of cell measurements.

Obtains accurate, precise and sufficient data for experimental and investigative procedures and record this methodically using appropriate units and conventions.

Diameter of cell measurements.

Researches, references and reports

Uses appropriate software and/or tools to process data, carry out research and report findings.

Graph drawing packages. Statistical analysis packages. Presentation packages. Report writing packages (e.g. Word). Internet search engines.

Sources of information are cited demonstrating that research has taken place, supporting planning and conclusions.

Referencing in final report or presentation.

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Background information

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

Comparisons between different species of plant

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 19th Workshop/Conference of the Association for Biology Laboratory Education (ABLE).

The animation on the SAPS website (http://www.saps.org.uk/secondary/teaching-resources/1299) is directly relevant to the study of this topic showing transport of water and sugar as well as growth of a stem.

Acknowledgements

Protocol and support materials developed by Dr Chris Graham, Long Road Sixth Form College, Cambridge and Gail Webdell, Faculty of Education, University of Cambridge.

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