Virtual Plant Appendices - Techniques
Useful laboratory techniques
WARNING: MANY OF THE CHEMICALS THAT ARE RECOMMENDED FOR USE IN THESE APPENDICES MAY BE HARMFUL TO YOUR HEALTH. PLEASE EXERCISE CAUTION WHEN USING THEM AND FOLLOW SAFETY INSTRUCTIONS WHERE GIVEN.
Read these notes carefully and whatever procedure you may have followed in the past, we strongly recommended that you follow the advice given here. Please refer also to the proper use of a microscope in Virtual exercise 1
Ensure that the specimen does not project beyond the edge of the cover slip - else this will act as a 'wick' and you will loose mounting medium over time..
Add just sufficient mounting medium to fill the area of the cover slip. If excess is present, mop up with absorbent paper. If insufficient is present, add a further drop at the side of the cover slip.
Lower the cover slip gently (use a needle to help lower the cover slip), to avoid the inclusion of air bubbles.
Place the coverslip squarely in the centre of the slide.
Take some time to make a neat neat slide. In an exam, you may be required to label it with your name, and the specimen number.
If you think carefully you will appreciate that there is at least one scientific reason for each of the requirements
Study the diagrams and make sure you understand the different planes of sectioning, viz. transverse, longitudinal radial and longitudinal tangential.
a) Whilst a single edge blade is preferable to a double edged one, not only for ease of holding but because they are usually made of thicker metal and therefore not so likely to bend during sectioning, one can cut very thin sections, with a double edged blade with practice.
b) At commencement of sectioning, trim the surface of the material level and truly orientated. Then cut several sections in rapid succession, transferring them to a watch glass as cut. It will be advantageous to rotate the material at intervals to counteract any tendency towards obliqueness in the sections.
c) Keep the blade and material wet whilst sectioning; use water for fresh material and 70% alcohol for preserved material. If the section dried at this or any subsequent stage, it would become full of air bubbles. NOTE: Air bubbles often look like "cells" in that the edge of the bubbles approximate the average cell wall in thickness.
d) It is often supposed that the thinner the section, the better. However, cells vary greatly in size and two equally thin sections might consist of only one, or of several layers of cells respectively. In the thinnest hand section of, for example, a stem, the very large cells of the pith may be cut in half, whilst in other parts of the section, the smallest cells may be two deep. The reason for attempting to cut the thinnest section possible, is to allow as much light to pass through the specimen, and to allow one layer of cells to be sharply focused. Within limits, it is as desirable for the section to be truly transverse as well as being thin.
e) The area of the section is determined by the variety of cell types and tissues present. The section must therefore include all cell and tissue types present within the specimen. The only exception to this, is when a complete section of an organ is required, in order that a plan may be drawn, to show the distribution of the cell and tissue types within the organ. In such cases, the section need not be particularly thin.
(a) As cut, transfer the sections to a watch glass by means of a fine paint brush. For fresh material the watch glass should contain water, for preserved material, use 70% alcohol.
(b) Examine the sections: if thin enough, they will be almost transparent, but if too thick, they will be white and opaque.
(c) Using a fine paint brush and not a needle, transfer two or three sections only to a clean slide.
(d) Add a drop of Fabil stain, just sufficient to fill the cover slip when it is lowered. (You will learn by experience, just how little is needed). Make sure that the stain covers the sections, before adding the cover slip, otherwise the cover slip may make a seal over the section and the stain will not penetrate.
(e) Do not stain sections in the watch glass: You won't be able to find them if they are immersed in a pool of Fabil.
(f) Use filter paper to mop up excess stain. Do not spill stain on clothes or books, as it cannot be removed. Fabil is poisonous and caustic - wash off with water if it should get on your hands.
It is customary to illustrate the internal structure of organs by means of a diagrammatic plan, and a detailed drawing (hereafter referred to as the LP diagram and the HP drawing).
It is advisable to have a "B" and an "HB" pencil for drawing.
(a) In the case of TS of stems or roots, draw half of the section. For TS of leaves , and all LS, draw the complete organ. A complete, but not necessarily thin section will be required for the latter.
(b) Commence by drawing an outline of the zones. It is very important to maintain the correct proportions - Note for example, the relative width of the cortex, and stele in stems and roots. Indicate accurately, the position, size and number of vascular bundles.
(c) The LP diagram should occupy at least half a page in your practical book.
(d) Now mark the position of the tissues. Cell structure is on no account to be shown in the LP diagram. Use the conventional shading, as illustrated below.
(e) Label your drawing as fully as possible (i.e.., zones and tissues). Arrange the labels neatly and systematically, preferably going from the outer, to the innermost tissues as one reads down the page. You will find it convenient to arrange the labels on the right hand side of the page, with the plan of the organ on the left.
(f) Lines should be clear and decisively drawn, not fuzzy.
(g) All labelling should be be in pencil. This means you can erase your mistakes easily. Try to be neat and ensure that the lettering runs horizontally. Labels may be either printed, or written, according to what you can do most neatly. Rule indicator lines (without arrowheads) and terminate these unambiguously. Indicator lines should not cross one another and should go as directly as possible from the label, to the feature indicated.
(h) Although the plan is called an "LP" diagram, it is not intended that it should be based solely on the examination of the structure using the low power objective alone.
The conventional shading given below is suggested for use in the LP diagram. The accompanying text book, shows the shading convention used in Metcalfe and Chalk, Anatomy of the Dicotyledons, 1979.
HP Diagram: To show the structure of the tissues and their component cell types.
(a) Draw sufficient of the specimen to enable you to illustrate a representative sample of all the tissues and cell types present. A thin and truly orientated section is necessary, though it need not be complete.
(b) Commence by selecting the best part of your section and examine it under LP. Taking a full page, map out, with faint lines, the limits of the different tissues.
(c) The drawing should represents a continuous narrow sector, say 6 to 10 cells wide, and should not contain isolated or disconnected patches of tissues. There is one exception to this however, in the case of very wide zones of homogeneous tissues, for example, as found in the parenchymatous cortex of roots. It is permissible here to rule two parallel lines and writing between the lines "6 rows of cells omitted" as has been done in the illustration on the preceding page for example.
(d) The detailed study of cell and tissue structure must consist of an accurate drawing i.e., it should not be diagrammatic. It must show the cells present in your preparation and must not in any way, be a generalized representation.
(e) To make a good HP drawing will at first be a tedious process. The epidermal and cortical cells are generally large and relatively easy to draw accurately. The smaller cells are more difficult e.g., phloem, tracheids fibers etc. Do not draw the epidermal / outer cortical cells too large-you may run out of paper.
(f) Work out a suitable scale before you start to draw. The smallest cells, e.g. in the phloem, cannot be represented with any degree of clarity at less than about 4mm diameter. This will mean that the largest cells (cortex / epidermis) may have to be about 2cm in diameter, to retain the proportions.
(g) Do not show any cell contents. Do not use any shading except:
(h) Use a single line for unthickened (e.g. primary) cell walls, and a double line for a thickened (e.g. secondary) wall. It is quite effective to use an "H" pencil for all primary walls, and an "HB" pencil for all secondary walls the secondary wall is usually drawn a little thicker that the primary wall-see the example on the preceding page.
(i) Draw the cell walls, and the cell cavities will draw themselves. This is particularly important in cell types such as collenchyma, sclerenchyma, and tracheary elements. Construct the walls by first drawing the primary wall. Then add the secondary wall. What you are doing is merely following the development process in your drawing.
(j) Pay particular attention to the shape of the cells and the way in which they fit together. Extend the walls at the edges of your drawing, to show how the cells join one another. Show the junctions between different cell and tissue types clearly and accurately.
(k) All drawings should be fully labeled. Observe the instructions (e) to (g) in the notes on the LP diagram.
The following general statements refer to permanent preparations made to show features of general anatomy, but do not apply to slides processed specifically to show the cytoplasm, nucleus, or chromosomes. Slides are made by killing and fixing small pieces of tissue in a special solution, commonly a mixture of formaldehyde, acetic acid and ethyl alcohol. The tissue is then dehydrated by means of alcohol, transferred to a wax solvent and then embedded in wax. It is next sectioned on a precision slicing machine called a microtome. The thin (usually 15-20 mm) sections are attached to a glass slide, the wax dissolved out and the sections then stained, dehydrated and mounted in a resin of low refractive index. A common stain combination for plant tissue is the red dye, SAFRANIN and the green dye, FAST GREEN. If these dyes have been used, certain chemical substances within the cells or their walls will be coloured as indicated below:
The above list is given only as a general guide, as the exact shade of colour developed is determined by a great number of factors, some owing to variability between one plant and another and some to variations in the staining technique. In order that you may make a correct interpretation of structures seen in prepared slides, you must be aware of certain artifacts; changes which occur when the slides are made, which do not exist in the living cells.
a) Shrinkage of the protoplast away from the cell wall. This results either from plasmolysis before death or from dehydration and shrinkage of the protoplast during processing. In all healthy living cells the protoplast is in contact with the cell wall.
b) Shrinkage and buckling of cell walls during dehydration. Many cell walls are thicker in living tissues than they appear to be in fixed and stained material, and are in a highly hydrated condition.
c) Tearing of cell walls, separation of cells, displacement of nuclei, etc., which occurs to some extent event in the best slides (see Greulach & Adams, Fig. 5..13)
d) Remember that cells are three dimensional structures, but that when they are sectioned the top and bottom walls may be removed, resulting in a ring-like open structure. The top or bottom wall of an occasional cell will remain in the section; in this event it will be stained probably somewhat lighter shade than the side walls.
e) Gross enlargement of vacuoles, owing to destruction or shrinkage of cytoplasm.
f) Loss of resolution, due to non-stainability or transparency. Certain features, such as chloroplasts, are much clearer in living cells; starch grains are best detected when stained with iodine and for chromosomes, mitochondria, etc. special staining methods have to be used which are not suitable for general anatomy. The cytoplasm is much more difficult to preserve in a lifelike condition than the cell wall. It is because the cytoplasm in an ordinary anatomical preparation is so distorted, that the cell contents are not drawn from such slides.
Technique 10: Making and using FABIL stain
FABIL is used extensively in our laboratories, for staining freehand sections. It is necessary to make up three stock solutions first before mixing these proportionally to make the stain itself. Please note: FABIL ripens and improves with age. It is also advisable to filter the stain to remove aggregated precipitate from time to time.
The stain is made up by mixing the stock solutions in the following proportions:
A=4: B=6: C=5
Allow the mixture to stand overnight and filter the next day. The stain ripens and improves with age. Periodic filtering is highly recommended.
LACTOPHENOL (take care not to get any of this on your clothes or skin. Wash immediately under cold running water)
Dissolve phenol in H2O without heat. Then add glycerine and lactic acid.
INDEX to techniques
Click on the headings, to go to the technique
Need to know how to mount that perfect freehand section? Look here for information!
Correct use of stains will make identification of the specimen under observation, so much easier!
A simple guide to the colours you can expect to see in sections stained with Safranin and Fast Green, or as in the image on the left, in Fabil
Get help with drawing the specimen here
Cutting sections is not easy. But, once mastered, will allow you to have a quick look at the specimen of interest, and will allow you to explore the cell and tissue types it contains
You will need sharp pencils and an eraser
Before you draw, work out the proportions & the location of the tissues. Make some simple measurements to ensure you get the proportions reasonably correct!
Be decisive about your drawing - there should be no fuzzy lines!
Struggling with high power drawings? Then read this and study the diagrams that go with this simple guide
Colours mean something - they indicate that the walls of the plant cells are composed of different molecules, and these will react differently, according to the nature of the histochemical stain used
This is an easy recipe to make a fairly complex stain, which works very well with freehand sections. The lactophenol will help preserve the section for a few days.