xercise 1. Microscopy and the interpretation of cell structures

  

Suggested specimens                             

 

   

 

 

 

 1.       Pollen  of Hibiscus

 

 2.      Stage Micrometer                 

 3.       Potato  Tuber

 4.       LS Onion root 

 5.     Grass seedlings

  

      Core Objective                                          

 

 

 

 

 

To demonstrate appropriate use of the microscope, through the observation of simple microscopic objects.

 

 

 

    Introduction                                            

 

 

 

 

The microscope is perhaps one of the most fundamentally important pieces of equipment that you will use in the laboratory environment. The compound microscope allows us to visualize objects that are too small to see with the eye. Using a microscope correctly is a fundamental requirement in any laboratory environment, where sections or microscopic particles have to be examined. This exercise concentrates on the correct use of a compound microscope, as this is the more difficult to use correctly, than is a dissecting microscope.  There are a number of issues related to the use of a compound microscope, that most students will be either unaware of, or will tend to ignore completely either through their own lack of understanding, or perhaps, they feel the student does not have to go through certain setup procedures.

 

 

 

     Techniques                                                           

 

 

 

 

This session will concentrate on the use a typical student compound microscope. It is imperative that correct procedures are adopted when using a microscope,  otherwise you may become frustrated by inability to see small objects and details. Pay particular attention to focusing and the use of the sub stage diaphragm, which aligns and focuses the beam of light through the objective,  thus clarifying structural detail including fine cellular details.

The simple tasks that are presented here, have been included to help develop basic skills needed to use a compound microscope more effectively. Correct use of the microscope will lead to less frustration during practical sessions. This should result in a more stimulating experience, as you use the microscope to discover the microscopic structure of plant cells and their interrelationships with each other.

Limitations to resolving power are governed optics, light wavelength and the resolving power of a good light microscope, is approximately 0.2µm using white light.

 

 

         Procedure                                                         

 

 

 

You should always be very careful when removing your microscope from its case/cupboard. Place it in front of you, and take care not to have it too close to the edge of the work surface. Have a close look at the microscope in the image to the right. Does yours look like this? Move your mouse over the image, and 'hotspots' will appear. Clicking on these will present you with more information and a detailed image of the region of the microscope. Identify the major parts; objectives which magnify the object that you are looking at; eyepieces, the specimen stage, the substage condenser which is beneath the stage and in the light path. The condenser focuses the light through the object. Have a look at what happens to the object you are looking at when you open or close the condenser diaphragm, or raise, or lower its position. Move the mouse pointer over the stage, the eyepieces and the focusing system, and you will find 'hot spots' - either with text associated with the region when using Internet Explorer. If you are using Firefox or Safari (for the Mac) information about the new URL will appear in window at the bottom of your active screen. Click on the objects and learn more. Look carefully at the objectives on your microscope. A series of numbers are engraved onto each lens. For example Plan 40/0.65 means that the lens is a planachromatic objective, with a 40X magnification, and that it has a numerical aperture of 0.65.

The web page at:- http://micro.magnet.fsu.edu/primer/index.html  contains some very useful information about the compound microscope. Take time to view it.

Eyepieces - maginfy object 8 - 12.5X  Integrated focussing system; very low gearing on fine focus to allow small focus movementsNosepiece and objectives - usually four objectives; including a 6.6X; 10X;40X and 100X immersion objectiveMechanical stage; geared, with a integrated slideholderSubstage condenser, usually two position, one for low power and one for high power observationBuilt-in illuminationThe eyepieces create a magnified virtual image of the specimenThe condensor is optimized to focus the light beam optimally.

 

 

A.      Simple microscope exercises                    

 

 

 

 

 

One should make an outline drawing to show the main parts of the microscope . There may be different instruments in use in the laboratory environment. The Zeiss student microscope illustrated here is an example of a reasonably modern instrument, and is easy to use effectively. The condenser system is relatively simple in the example that we have chosen to illustrate here and is fixed in an optimum position. Please familiarize yourself with the condenser on your microscope. It may be quite different to this one. Examine any wall charts that your instructor may have put up, and acquaint yourself with the mechanical and optical workings of the instrument, as well as its light path.

 

 

 

 

 

 

 

 Pollen grains                                                           

 

 

 

 

 

Angiosperm pollen grains are quite unusual, in that they contain sperm cells. These sperm cells are encased in a cell wall which is demarcated internally by a plasmamembrane. If you take some pollen, and sprinkle a little (the Hibiscus  is useful, as it has large pollen grains) on a microscope slide, in a drop of water. Carefully lower the coverslip over the drop of water containing the pollen. Wipe off excess liquid round the coverslip, and ensure that the bottom of the microscope slide is dry as well. Put the slide on the microscope stage, after making sure that the low power objective is in position. Focus using the coarse focusing control, until the pollen grains are visible through the eyepiece. Change to a higher power objective (say 20 to 40X). You should see something like the image of pollen (below, right).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

Assignment:  As you can see from the micrograph to the right, the  surface structure of the pollen grain is sculpted and spiky. Why do you think this is so? Consult reference books, and the internet, to try to find out more information about pollen sculpturing, and the role of the spikes or protrusions from its surface.

Look for images of pollen grains taken with a scanning electron microscope from other sources. Can you use the surface features of pollen grains to help identify the source of the pollen?

 

 

      Using a stage micrometer      

 

 

 

 

 

To make measurements of length or area with a microscope, means that you will have to be able to calibrate the microscope image, using a micrometer slide.  This is used co calculate the diameter of the field of view which is the visible illuminated circle that is visible through the eyepieces. If you know this value, you can extrapolate and determine (at least) the approximate length of a specimen viewed with the microscope. However, micrometer slides are expensive and because of this, are thus not commonly available for use in introductory practical classes.

 

A simple and inexpensive micrometer slide may be made by mounting a 200 mesh electron microscope grid on a microscope slide under a coverslip. Making a permanent preparation is easy and convenient and will mean that you have a micrometer slide available, should you forget the calibration data, or use a different microscope, which means that you would have to recalculate the diameter of the field of view of your microscope again anyway.

 

 

      Magnification and size                                    

 

 

 

 

 

 

If you use a 200 mesh grid, then each aperture in the grid (the grid squares) will measure approximately 200x200 microns and the width of the copper wires or bars which make up the grid, will be approximately  40 microns wide. Note these values and write them down, as you will need them when you make the calculations that are required to determine field of view.

 

Place the micrometer on the microscope. Focus the micrometer slide until you can resolve the bars and spaces. Carefully line up one row of the grid so that it passes across the approximate center of the field of view. Count the number of bars and the number of grid squares  that you can see. Add in any estimated fractions of bars or grid squares that you can see.

 

Write these numbers down.

 

 

Example calculation: return

After you have placed the grid on the microscope stage and have focused the image, let us assume that you can see 10.5 grids and 11 bars when you look through the eyepiece. Look at the image below, and use it as a guide. Click here for an illustration and more information.

 

Calculation of the diameter of the field of view

Calculations of the diameter of the field of view are shown in the example below.

Remember: You need to count the number of grid squares (in microns)  = (10 X 200 = 2000), plus 0.5 = 100, = 2100 microns, and then add total width of the bars  = 11 X 40 = 440 microns.

 

Total diameter of field of view1 = total grid width + total bar width = 2540 microns

 

Calculate the diameter and area of the field of view for each objective on the microscope. Remember, the higher the magnification the smaller the diameter of the field of view becomes.

Complete the table below. These values will be very helpful, when you look at objects of unknown size in future.

 

 Objective Magnification

 Diameter of field of view (mm)

Area of field of view (mm2)

10X

 

 

20X

 

 

40X

 

 

other

 

 

 

 Measure your drawing and calculate the scale of magnification i.e., how much larger is your drawing than the actual size of the object itself?

Get into the habit of indicating the scale of magnification2 when you make drawings with the aid of the microscope in future.

 

Note that whatever the nominal magnification of the optical system may be, you will be able to use this table in two very useful (descriptive) ways:

·         To estimate the size of the microscopic objects that you look at during these and other practicals;

·         To estimate the scale of magnification of your drawings.

Look again at the mount that you made of the pollen grains an estimate the size of the pollen grains. Are they very similar in size? Measure a few, and calculate the mean diameter.

You can make your drawing any size you wish. Only by measuring the drawing and comparing this with the actual size of the object or specimen, using the data that you calculated in the table above, can you indicate the actual scale of magnification. You should perform the above operation until you can accomplish it easily.

 

 

       water droplets on a slide                                

 

 

 

 

 

Place a small drop of water on a slide and carefully drop a coverslip onto it. Examine the trapped air bubbles under a low power (10X) and a high power (40X) objective. Note the difference in appearance when the bubbles are seen in surface view and in optical section, as the level of focus is altered. Note particularly, the apparent “thick black wall” which is due to refraction of the light at the edge of the bubble. It is important to recognize bubbles for what they are and not to become confused, thinking they are strange-looking cells!

 

 

B. Observation of specimens                         

 

 

 

 

 

      starch grains                                                      

 

 

 

 

 

Cut through a potato and wipe the exposed surface across the centre of a slide. Quickly add a small droplet of water to the smeared potato, and place a coverslip over it. Examine your preparation under a 10X and a 40X objective and observe the starch  grains. Draw the outline of a few starch grains as seen under HP (at least 5cm in diameter). Experiment with different diaphragm apertures, as well as with different levels of focus and change the condenser lens setting (if your microscope can do this). Try to make the concentric lamellae, as well as the small, central dot, or hilum visible. Add these features to your drawing and complete it by labeling fully, and include a scale of magnification. These starch grains have been contrasted using a very dilute concentration of Toluidine blue stain.

 

Remember that when you have to examine colourless or transparent objects, adjustment of the diaphragm (by partially closing the diaphragm) as well as the condenser lens position (lowering it slightly) will greatly improve resolution and will help you see structures that would otherwise be invisible.

 

 

  Irrigating wet preparations with stains            

 

 

 

 

 

Irrigating the specimen is another simple technique. It is an important microscopic technique, as the process is used to introduce a dye or reactant to a section, which is already mounted under a coverslip. For example; place a small drop of iodine at one edge of the coverslip of the slide containing starch grains. Use a small piece of filter paper and draw the iodine solution through the mount from the opposite side. Replace any 'lost' liquid with more iodine.

Practice doing this several times, introducing more or less iodine Take care not to move the coverslip, else you may damage the specimen. Examine the starch grains at the edge of the solution and make a note of their appearance.

You may use the irrigation technique with any histochemical stain which may be available to you. The diagrams below, illustrate the irrigation technique

 

 

 

 

 

 

Figure 1. Illustrates the technique required to irrigate a section. (a) centre the wet specimen on the slide; (b) gently lower a coverslip over the specimen; (c) introduce dye at edge of coverslip, (d) draw excess water and stain through coverslip across the specimen. Allow to differentiate, add more stain as necessary.

 

 

 

 

 

 

 

       Using stains                                                     

 

 

 

 

 

 

Cutting freehand sections and then staining them, using  stains such at Toluidine blue or Fabil (see the appendix for details) allows us to identify cells more easily than by looking at unstained material. The production of wall colours depends on the chemical composition of the cell walls. The two images to the right, are from near-identical stem sections cut from Gloriosa superba stem. Toluidine blue is a metachromatic stain, which is most useful, as it helps distinguish many of the cell and tissue types. Fabil (rightmost section) is also metachromatic. E= epidermis; PVF = perivascular fibers,  P = phloem; PX = protoxylem.

 

 

 

Draw up a Table in which you contrast the colours which are produced when using these two stains. Which do you think is the most useful of the two?

 

 

 

        seedling structure                                          

 

 

 

You should look at a young grass seedlings, or an onion roots as an alternative, which have been germinated on moist filter paper. Carefully lift off one of the largest seedlings and mount it in water on a slide. Examine this with the aid of a hand lens, or with a stereo dissecting microscope and make a reasonably large drawing and outline the root.

 

 

Note and label:-

a)  The primary root, or radicle, which is short-lived in grasses and most monocotyledons. Transfer your mount to the microscope and examine the radicle under low power. Add to your drawing:

b)  The root hairs. Note that these are absent from the tip of the root. Those nearest the tip, are young and very short, those a little further back are longer and straight, whilst those nearest the “seed" are probably dying, and consequently, are rather shrunken and irregularly shaped. If the root is long enough, you should find a region, close to the “seed, which is bare of root hairs. It is important that you realize that root hairs develop just behind the growing and advancing root tip. Immediately behind this, is a zone containing mature and active root hairs. Behind this, is the zone where the root hairs begin to be shed and disintegrate.

c)  You should look for an irregular mass of tissue at the extreme tip of the root, that is surrounded by relatively loose cells. This is the root cap. The root cap is an important part of the root, as it affords protection from mechanical damage and is also the site of perception of gravity (geotropism) and of the secretion of mucilage, which probably acts as a lubricant and regulates the water content of the root cap cells. The surface cells of the root cap are continuously shed during the growth process and the lost cells are continually being replaced by the growing region behind.

d)  Immediately behind the root cap, and in front of the first root hairs, is the meristem or growing region of the root. This is a site of active cell division and is responsible for the formation of all the new tissues of the downward‑growing root.

e)  Note the vascular cylinder or stele running up through the centre of the root. You should see this as a darker, central core.

f)   Between the meristem and the root hair zone is a zone of active cell vacuolation and elongation. Note how the surface cells differ in shape from those at the meristem. Why are the root hairs borne (a) beyond the meristem (b) beyond the zone of cell elongation?

g)  Irrigate your mount with iodine solution  you will now be able to distinguish the root cap, and the sub‑apical meristem  more clearly. Note that the living root cap cells contain starch granules, which are probably associated with graviperception. Why does the meristem stain so darkly? Lift the coverslip, and detach the “seed”. Replace the coverslip and, using the end of a pencil, gently squash the root flat. Examine under the high power lens.

h)  Note the difference in shape, size and content of the cells of the root, and in particular,

(i)       Root cap cells of two kinds: loose ones without starch grains, and deeper‑seated, living cells, containing starch grains.

(ii)     Thin‑walled, cubical meristem cells, with very dense cytoplasm, and large nuclei.

(iii)   Behind the meristem, elongated cells with relatively small nuclei and large vacuoles.

(iv)    Cells of the stele: very elongated and narrow. Some, the protoxylem  vessels, have rings or spirals of wall thickening.

(v)      Epidermal cells, some with outgrowths or root hairs. Study the root carefully, and make a series of drawings which show stages in root hair development.

 

 

 

 

       onion root, longitudinal  section                  

 

 

 

 

 

look at cell division in more detail

Examine the micrograph to the left. It is shows part of a longitudinal section through the tip of the root, near the root apex of onion. Make a drawing of it, identify and make notes on:

(a) the root cap,

(b) the root meristem, many cells of which show stages of mitosis,

(c) the zone of cell vacuolation and elongation.

 

 

 

Questions:

1. What is meant by the term numerical aperture?

2. How does adjusting the substage condenser affect the virtual image that you see through the eyepiece?

3. What is the theoretical limit of resolution of a light microscope using "white" light?

4. How does shorter wavelength influence resolution?

 

 

        Section planes                                                

 

 

 

 

 

The majority of sections that you will be given to look at in The Virtual Plant exercises, will have been cut in the transverse plane (cut in cross section). Whilst it is accepted practice to provide only transverse sections in the practical environment, it is important that you realize that one cannot get a clear understanding of plant structure, simply by looking at cross sections the whole time. Cross sections will not be very useful if, say you need to distinguish between narrow-diameter vessels and tracheids within xylem for example. To achieve this, we need to examine material in two other planes -- radial and tangential. Radial sections are cut longitudinally, along a radius that intersects the central axis of the root or stem. Tangential sections are cut at right angles to the radius.

 

 

 Footnotes:

[1] Field of view: This is defined as the area that you can see when you look through the eyepiece. As magnification is increased, the field of view becomes smaller. You effectively see fewer cells, but at higher magnification.

[2] Scale of magnification:  Is based on the size that the object is drawn on paper (i.e., your drawing) divided by the actual size of the object, calculated from what you can measure, using the microscope.

 

 

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