Virtual Plant  Appendices - Techniques

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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.

 

1Technique 1: the use of the microscope

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

  • Arrange the microscope so that you can use it comfortably. If you have a monocular microscope before you  then try to use your left eye to view the specimen If you have a binocular microscope before you then adjust the eyepieces so that both eyes present a n in-focus image. This will prevent eye strain.

  • You should arrange your seating position such that you do not have to lean across the bench, or sit in a strained, or uncomfortable position. Try to find a position in which you have to move the microscope or your seat during your microscopic observations. Your laboratory book should lie close to the microscope on the left or right hand side to suite yourself.

  • Make sure the L.P. (x10) objective is in position, turn on the lamp, open the diaphragm to its widest aperture and, looking through the eyepiece, adjust the light which is transmitted so that it is evenly distributed within the field of view.

  • By means of the sub stage adjustment, bring the condenser to its highest position, then lower it very slightly so that the whole field of view is brightly and evenly illuminated.

  • Partially close the diaphragm and adjust the lamp brightness to cut out glare.

  • Use of the higher power objectives. The microscope which you have will normally be equipped with not more than three objectives; a 3 to 4X, a 10X, and a 40X. The 40X objective is thus referred to as the high power objective. Some exercises may require the use of oil immersion objectives. Special precautions necessary for their use will be given to you, when the time arises. When the nosepiece is rotated to bring the HP. objective into position, the specimen will still be visible, but may not sharply in focus (most student microscopes are not completely parfocal). Re-focus by means of the fine adjustment, until the image is sharply focused.

  • Remember that adjustment of the condenser is just as essential for critical resolution, as is focusing.

  • When you have finished with the HP, rotate the nosepiece, to bring the low power objective into position and re-focus with the fine adjustment. This practice will prevent the fine adjustment from being run to the end of its traverse.

  • Never insert a slide directly under the HP. objective! Always focus using the L.P. first. Apart from obviating risk of damage to the HP lens, time will be saved because no two specimens will be of exactly the same thickness.

  • 7.Care of the microscope. The microscope is an expensive precision instrument; it is your responsibility to use it properly. Please observe the following simple guidelines.

  • If the microscope develops any faults or becomes damaged, report it to the instructor immediately. Do not on any account unscrew any of the components, particularly of the lens system.

  • Always carry the instrument in an upright position and set it down gently.

  • If water or reagents are spilt on the stage, wipe them off immediately.

  • All optical parts must be kept clean and dry. They should not be touched with the fingers or wiped with dusters, etc. Any cleaning should only be done with lens tissue , which is available on the instructor's bench).

  • The specimen must always be enclosed by a cover glass when examined with the HP objective.

  • After use make sure the LP objective is in position and the instrument quite clean. Do not leave any slides on the microscope. If you are right handed, place your workbook to the right of the microscope and make sure your workspace is uncluttered. Remove any extraneous books, papers and other items not needed during the exercise. return

     

    click here to see arrangement of workspace for right handed use

 

2Technique 2: mounting of freehand sections

    Whenever you make mounts for microscope examination - 

    Make sure the slide and cover slip is quite clean.

    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

     

    Click here to see an illustration for the above technique

 

3Technique 3: drawing cell detail 

  1. The drawing must be large, with clearly defined, non-fuzzy lines.

  2. One particular cell must be represented, i.e. not a generalized impression.

  3. The parts must be represented in their true proportions. Thus when making a large drawing of a single cell, the cell wall must be indicated by double lines, faithfully following any variation in wall thickness. (In later exercises, when cells are drawn to a smaller scale, single lines will normally be used).

  4. Indicate the cytoplasm by stippling and try to show the approximate density of cytoplasm granules.

  5. Pay particular attention to the "corners" of the cell and show how the walls are connected to adjacent cells.

 

4Technique 4: the use of stains

The purpose of using a stain in microscopy, is firstly to render transparent objects more opaque and secondly, to find out something about their chemical composition. The affinity for a stain is largely dependant on the composition of the various parts of the cell and a single cell will differentially stain by virtue of its heterogeneous composition. For example, the protoplasts of the Zebrina guard cells were stained brown and the starch grains black by iodine solution. The cell wall became a yellowish colour, but was not very efficiently differentiated from the cytoplasm. It would however have been possible to stain the cell wall with another reagent which had a specific affinity for cellulose. Thus whilst it is possible to differentiate some of the cell components by using a single stain or reagent, this can be done more effectively by using two (or more) stains, each more or less specific for a certain component. The two stains may be applied separately or may be combined in a single solution. Fabil stain. In this image of a section of Gloriosa superba stem shows a typical Fabil staining result. Plant material, either fresh or preserved, can be mounted directly in Fabil. It may be examined immediately, but the staining will be found to be better after about 5 to 10 minutes immersion, and continues to improve for some time. The main colour reactions are listed in the table below:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cytoplasm and nuclei

deep blue

Cellulose and hemicellulose walls

pale or silvery blue

Lignin-impregnated walls

shiny yellow, orange or pink

Suberin-impregnated walls

crimson

Starch grains

black

 

 

 

5Technique 5: section cutting

Study the diagrams and make sure you understand the different planes of sectioning, viz. transverse, longitudinal radial and longitudinal tangential.

Reference points, lines and planes

Click here to see illustrations of reference points, lines, and planes

 

CUTTING SECTIONS

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.

 

Click her to see the illustration of  section cutting 

 

 

6Technique 6: handling and staining of sections.

(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.

 

7Technique 7: illustrating organ structure. The low power drawing

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).

 

 

The LP Diagram: Shows the distribution of the zones of tissue within the structure.

 

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.

 

8Technique 8: illustrating organ structure, the high power diagram

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:

 

  • chloroplasts -- (can be diagrammatic)

  • starch in starch sheath -- (diagrammatic)

  • crystal inclusions -- (can be diagrammatic)

  • sieve plates -- (diagrammatic)

  • cytoplasm of companion cells -- --(stipple)

  •  

(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.

 

 

9Technique 9 interpretation of prepared slides

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:

  • Cellulose : bluish-green

  • Cytoplasm : bluish-green

  • Lignin : red

  • Suberin : red-orange

  • Cutin : red-orange

  • Tannins : red-brown

  • Chromatin : red-purple.

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.

Stock Solutions.

 

 

A

Aniline Blue 0.5% in lactophenol

B

Basic Fuchsin 0.5% in lactophenol

C

Iodine 3g; potassium iodide 0.6g in lactophenol

 

 

 

 

 

 

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) 

Lactic Acid

50 ml

Phenol Crystals

50 gm

Glycerol

50 ml

Dist H2O

50 ml

Dissolve phenol in H2O without heat. Then add glycerine and lactic acid.

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INDEX to techniques

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Click on the headings, to go to the technique


Technique 1: the use of the microscope

 

Technique 2: mounting of freehand sections

Technique 3: drawing cell detail

 

 


Technique 4: the use of stains

Technique 5: section cutting

 

 

Technique 6: handling and staining of sections.

 

 

Technique 7: illustrating organ structure. The low power drawing

 

 

Technique 8: illustrating organ structure, the high power diagram

 

 

Technique 9 interpretation of prepared slides

 

 

Technique 10: Making Fabil Stain

 



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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

 

 

 

 

 

 

 

Shading Convention

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.