This booklet introduces a range of simple techniques for the beginner with the Lensman microscope. Many specimens can be examined without much by way of preparation, and many more can be mounted on a slide for examination without your needing special training. Among the methods outlined here are some new techniques which bring microscopy within the reach of the complete novice. In the laboratory, microscopy is a complex business. The simplest preparations are smears. Here, a fluid specimen is wiped across the surface of a glass slide. After drying the smear is fixed, in order to make its biochemical components easier to stain and to stick the specimen firmly to the slide surface. After fixation the slide is stained, usually with a combination of two or more coloured dyes that single out different parts of the specimen. The specimen is then differentiated (so that the dyes stain the correct parts of the smear), dried, possibly cleared with agents that are solvents for the mountant, and then mounted beneath a cover-slip. Solid biological materials are often prepared as sections. In the laboratory, the specimen is passed through a series of increasing concentrations of a dehydrating liquid until all water has gone. The dehydrated material is subsequently cleared in a second series of increasing concentrations of a solvent until the dehydrating agent is replaced. After this, the material is embedded in a molten wax (or sometimes in a cold curing resin) so that it is firmly held in a solid block at the end of the process. This block is mounted on a microtome, a machine capable of cutting very fine sections across the embedded specimen. These sections are then transferred to a slide where they are fixed to the surface before being stained, differentiated, cleared of all unwanted solvents and finally mounted ready for storage. The entire process takes many days.
Here are presented techniques which avoid the need to stain or permanently mount specimens. These methods, many of them specifically devised for users of the Lensman microscope, are rapid and simple substitutes for more advanced techniques. Anyone can use them. Solid specimens are often ground to form fine slices which are thin enough to transmit light. This is how rocks, for example, are often prepared. The surface topographv of many examples can be examined direct. These include leaf surfaces, composite rocks, such as granite, or tiny fossils. Cloth specimens are ideal for this form of microscopy. Here a metallurgical microscope fitted with a reflected light system would be utilized. The Lensman has reflected light built in, enabling such surface examinations to be made without any costly apparatus. Small living life-forms are studied in the laboratory using phase- contrast microscopy. This shows up the differences in optical property of transparent organisms and makes them more easily seen. A less widely used method of illumination is dark ground microscopy, in which light is shone onto the specimens but does not otherwise fill the field of view. In this mode the organisms shine out brightly against a dark background. The level of contrast is higher than in phase contrast. The Lensman microscope offers dark ground by shining light obliquely across the field of view; no special attachment is necessary.
Many of the techniques of microscopical examination can be carried out simply and effectively. In addition, the Micropage reference system provides back-up information that is ideal for use out and about, when working in the field. Enthusiasts may find it helpful to photograph their specimens. The Lensman microscope can be adapted for direct photography using a purpose built T-mount. This device, complete with a descriptive booklet that introduces the amateur microscopist to the field of photomicrography, is under preparation with details obtainable from: Vector Services Ltd., 13 Denington Road, Wellingborough, Northants., NN8 2RL.
1. Basic Methods
Smears may be made from soft specimens and suspensions in liquids. A small droplet of the material is placed near one end of the slide and it is then literally smeared along to the opposite end. In the process a fine film of specimen material is left on the surface. When dry, this can be examined direct. It is important that the smear is not too thick; if so it is hard to make out individual cells within the specimen. The ideal smear looks, to the naked eye, like a slight area of dulling along the slide. If it looks like a great grey swathe running from one end to the other it is too thick by far. There are three ways to make a smear, and the method depends on the nature of the specimen:
The specimen is cut open to present a moist surface. This is laid against the slide, and a sliding movement wipes a thin smear along the surface. In this way a layer containing well-spread particles remains on the slide. This method is applicable to the observation of starch grains in fresh potato.
In this technique a small dropletof the material is put on a slide and it is then spread out using a loop. The standard laboratory implement is a loop made of platinum. For amateur use, an opened-out paper clip should suffice. See: fig 1. Cells from the lining of your cheek are observed through this form of smear.
A droplet of blood is placed at one end of a clean microscope slide. A second slide is then brought up to the drop, and the end of this slide is used to draw the droplet along the length of the slide; the second slide acts rather like a wall-paper stripping knife, as it were. It has the effect of spreading the blood thinly along the surface, leaving the blood cells well separated. See: fig 2. Though this method is intended for blood, it can also be used for thick suspensions of starch grains or concentrated colonies of aquatic microbes. The smears may be examined wet or dry. A wet smear is usually made by the direct or loop method and a coverslip is added before the specimen dries out. Starch grains can be observed best by this method, and living preparations of pond microbes can make good wet smears too. Dry smears are obtained by leaving the smear for a moment or two, during which time the excess moisture will evaporate. Outlines of small organisms and grains show clearly in a dry smear, though the internal details are largely lost. However this is a simple method for observing blood cells without the need to stain them.
Making Droplet Mounts
This is the simplest mounting method using a slide and a coverslip. A small drop of liquid containing the specimen(s) is placed at the centre of the slide. A clean coverslip is then slowly lowered onto the droplet until it spreads out to the edge of the coverslip. The easiest way to do this is to rest one side of the coverslip on the slide, immediately alongside the specimen droplet. Hold it in position with a probe which you can make by straightening out one of those paper clips. Then lower the cover-slip onto the slide, so that the droplet spreads out (see fig 3). The preparation is then ready for observation. Live organisms may best be spotted using dark ground illumination. Although high power may give you the most close-up view, the movement of organisms usually makes the low-power lens the best choice. This technique applies to specimens suspended in fluids, such as pond microbes. Water from a stagnant gutter can provide a source of many intriguing microbes which can be studied using this method.
This is the technique for examining specimens which can be easily observed in their unaltered state. A small portion of a specimen, or an entire object if it is small enough, is put onto a slide and restrained beneath a coverslip. Some are mounted as they are; others require suspension in a liquid mountant.
Small solid structures, such as small floral parts and insect eggs, are best examined this way. The specimen is laid on the slide and examined as it is. For small specimens it is best to add a coverslip.
Some specimens are seen more clearly if a drop of water is added to the slide before the coverslip is carefully lowered onto the surface. This prevents small objects from being blown away and provides a good optical surface for the transmission of light. The refractive index of water will improve visibility of some small specimens. Methods like this are used for pollen grains and dust samples.
Whole Specimen Studies
The reflected light capacity enables whole specimens to be observed in situ. The microscope itself is laid onto the edge of a rocky outcrop for mineral studies; it can be offered up to a sturdy plant leaf to study the living leaf surface; mosses and lichens can also be observed where they grow. Insects need to be restrained under a coverslip. This is best for minerals, plant surfaces and lichens. A microchip circuit laid directly on the stage can be closely examined by this means too.
2. Advanced Methods
By experimenting with different mounting media, it is possible to prepare materials for microscopy using adaptations of standard laboratory methods.
Thin layers of cells can be examined using methods anyone can master. The easiest specimen to use as an example is an onion. Remove one of the fleshy layers of an onion and cut a small piece out of it. The piece should be about two centimetres square, for ease of handling. Snap it by bending it sharply, and a portion should break almost through. The layer that does not break, but which merely bends, is the surface layer of cells. If the broken portion of the onion is now carefully pulled back, this fine layer - finer than tissue-paper - is separated from the main body of cells. It consists of a single layer of onion cells. This fine tissue can then be spread with finger pressure onto a slide. This is how many early microscopists made their material. However, a better view, and a more long-lasting mount, is obtained if you mount the tissue in a droplet of water before adding the cover-slip (see fig 4). Many fresh tissues can be prepared this way, including the epidermis of leaves and the surface cells of flowers.
Sections of bulk tissues
To observe cells in plant tissues usually necessitates cutting sections. Here we are concerned with roots (such as carrots), tubers (like potatoes) and stems (from asparagus and lilies to apple and begonia).
Sections cannot: be cut with a scalpel or a knife, as the finest cutting edge is necessary. For amateur purposes, a razor blade is best. Care is needed in handling these, for their edge is exceedingly sharp and they can section fingers if carelessly handled. The type recommended has a safety backing on one side, and a cutting edge on the other. A cut is made to obtain a flat and smooth surface. Then a second is made below the first, removing a section of tissue. This is laid on the slide, a drop of water added, and a coverslip lowered into position. It is often easiest to float the section from the blade onto a saucer of water, and then pick it up with, for example, a half-straightened paper clip (see fig 5). Roots, stems, buds, fruits and tubers can be sectioned in this way. Cell structures are clearly visible under the low power magnification of your microscope.
Sections Of Fine Tissues
To examine stamens, leaves or other fine tissues the basic sectioning technique has to be supplemented. Even the sharpest razor blade cannot be used to cut hand sections of such tenuous structures. The solution here is to support the tissue inside something builder. Carrot is an ideal cutting medium. Cut a rod and make a small incision in one end; into this slit the leaf, or whatever, is carefully placed. Sections are now cut of carrot tissue, with which the leaf is sectioned at the same time. Float the sections onto a saucer of water and the fine leaf sections can be transferred with a paper clip loop onto a slide. This method is ideal for leaves, petals and other thin plant structures.
Water is usually used to mount preparations of these materials. But there are other mountants which allow slides to be prepared that will not dry out so soon. The main mounting fluids are listed here, with some of the specimens to which they could be applied:
For many plant subjects as wet mounts there is often enough tissue fluid to act as a mountant. This may apply to onion and petal preparations. Specimens of fluids such as saliva or semen can be mounted direct, too, using the suspending fluids as mountant.
A drop of water is often helpful to display wet mounts of plant tissues. This is in many ways the 'universal mountant' for fresh tissues, including sections of leaves.
This viscous liquid, chemically known as glycerol, provides a suitable mountant that lasts longer. Many stem sections and tissue films can be successfully mounted in this familiar product.
A straightened paper clip can be used to add a small droplet of washing-up liquid to many preparations prior to adding the coverslip. This is ideal for fibres (hairs, etc.), pollen grains, and some other small entire specimens.
Clear Nail Varnish
Permanent slides may be made by inserting specimens including artificial fibres and some other small dust-like particles into a drop of nail varnish on a slide. If the drop is encouraged to spread to the edge of the coverslip by gentle pressure on its surface with the paper clip, and it is then left for a week to dry, a form of permanent mount is produced for addition to your collection. Animal tissues are soft and difficult to examine without careful preparation. However, some insight into the way we are made up can be gained from an examination of smear preparations.
3. Human Specimens
A droplet of blood is positioned near one end of a slide, and a second slide used to make a smear. Use the smallest amount of blood possible; a large drop will prevent the cells from separating. Under the low power lens the dry smear will show millions of circular cells. These are the red cells, or erythrocytes, which transport oxygen. The high power lens may enable occasional cells which are less evenly rounded, and are also slightly larger, to be observed. These are the white cells which are concerned with immunity. [NOTE: A cubic millimetre of blood contains five million cells].
Wipe your finger across the inside of your cheek and transfer the saliva to a slide before preparing a smear and drying it. The irregular outlines of cheek cells can be seen. In each cell a small rounded body will be observed; this is the cell nucleus. The body is made up of cells based on this essential pattern. There is a nucleus inside each cell, apart from the red cells of the blood stream.
Cut a few short hairs and add them either to washing-up liquid or nail varnish on a slide. This will provide a handy preparation for observations. Fine scales may be seen to mark the hair surface; white hairs have a central core of air bubbles which reflect light; black ones contain dark pigment granules.
4. Plant Specimens
The under-surface of a mature fern leaf shows dumps of sporangia (often shielded by an umbrella tissue). When they ripen each sporangium slowly uncurls and then shoots the spores out tike a catapult. This can be observed under low magnification.
Small plant specimens can be examined entire, whilst others are small particles that need to be mounted in glycerine or washing-up liquid.
Dust some pollen onto a drop of washing-up liquid, water, or nail varnish, on a slide before adding a coverslip. Note the fine surface sculpturing. Of great interest is pollen from the pine tree. Each grain has paired air-sacs, like waterwings, which give them buoyancy in the air.
Many plant hairs are fascinating to study. Note the blue hairs from the flowers of Tradescantia virginiana. They grow between the stamens. Each cell is large and has a pronounced nucleus. Threads suspend the nucleus inside the cell, and the cytoplasm inside the cell flows along these threads.
The above section of a plant stem has been cut at a slight angle. Sections need to be made at exactly right-angles to the axis of the stem (see figs 8 and 9). There are some clear starch-grains visible on the left, but the remaining tissues have a somewhat 'blurred' appearance, even though they are in focus (as the starch grains show).
The underside of a stinging-nettle leaf is covered with spines made from silica, the same chemical from which sand is made. Put a specimen (carefully!) on the microscope stage, held in place with the stage magnet, and use reflected light. Note the poison sac at the base of each spine: these contain acid sap. When the tip of the spine is broken off it leaves a sharp and hollow needle which injects the poison into the skin.
Look at the feathery flowering head of a grass plant and detach one of the single flowers. This, mounted in washing-up liquid or examined dry by reflected light, shows the sac-like stamens and the feathery pistil in each flower.
The surface hairs and round glands can be studied on the undersurface of a leaf portion mounted directly on the stage with the magnet. Note that the glands are less frequent on the upper surface.
The illustration shows a section exactly at right-angles to the stem axis, but the use of a somewhat blunted razor blade means that parts have been torn away; in particular, the central xylern vessels have cauoht on the blade and are now missing. See also figs 7 and 9.
Use the razor edge with care to cut very fine sections of plant stem and root structures. Sections of potato merely show starch grains; use carrot as supporting medium - the starch grains in potato obscure details in the section. Young stems and roots are easier to cut. The best mountant here is washing-up liquid; nail varnish cannot be used.
Note the large xylem vessels; these are tubes of cells in which the walls are thickened with cellulose and lignin. Sap is transported from roots to leaves through this system of vessels. The smaller, delicate cells associated with these are the phloem through which downward transport takes place. This is how the plant feeds its lower parts with the foods made in the leaves. Most of the stem is made of rounded cells of the cortex, which give bulk to the structure
The above illustration shows a section through a fern root as in fig 7 but a correctly sectioned specimen, and at higher magnification. In the centre are the thick-watled xylem vessels, which transport sap from the roots, with the thinner phloem tissue surrounding it. Much of the rest of the root is made of larger cortex cells, most of which contain starch grains. There is a darker layer of cells here, stained with tannin.
Structures are much the same as in stem sections. Small roots will show root hairs. These are only a single cell in thickness and gather soil water and nutriment during life. Good specimens of root hairs are found in window-sill cultures of mustard and cress.
Thin leaves like sunflower or fuchsia show an upper layer of cells which stand side by side like columns. These are the palisade cells which collect most of the sunlight. Sunflower has two layers of palisade cells; fuchsia only one. The xylem and phloem that carry sap are found in the veins where they form vascular bundles. Fuchsia leaf contains crystals of calcium oxalate. Thick leaves, like rhododendron and holly, have a cuticle that covers the top of the leaf. This is clearly seen in a section. Most evergreen leaves have five or six layers of palisade cells, though holly manages with two. Crystals of calcium oxalate might be observed in both kinds of leaf; these are a waste material.
These claws are a feature of some of the minute spider-like creatures which live in moss and leaves. To the naked eye they look like small grubs, but the microscope shows the fearsome appearance they present to their prey.
Small branched outgrowths in rock pools look like moss to the naked eye; however, under the microscope can be seen small polyps with tentacles, tike miniaturized sea-anemones. This example is called Sertularia; a related genus, with alternating polyps rather than pairs is Obelia.
5. Pond Life
A drop of pond water reveals a host of living microbes. They were discovered more than three centuries ago in Holland, and the first sight of pond life on a microscopic scale is one of the most memorable sights you will see. What looks like stagnant water to the naked eye becomes a beautiful world of its own when magnified. Even water from a damp gutter will provide intriguing life forms (see fig 10).
The green growth known as witches' hair or slime is made up of fine, bright green threads. Each one is a chain of cells joined end-to-end. These are green algae. The leaf-green structure inside each cell is its chloroplast. You will have seen small, round chloroplasts inside each leaf cell in the previous paragraphs. They capture sunlight and put the energy to work. A spiral cholorplast is found in Spirogyra, see: fig 12; two star-shaped ones occur in Zygnema.
Here we see seven filaments of fresh-water witches' hair, mounted in glycerine. The central filament is made up of ten or eleven loaf-shaped cells. Inside each one is a flat spiral ribbon, the chloroplast; this contains the chlorophyll with which the alga captures solar energy. Other algae swim around, their single cells gliding and twisting across the field of view. Little round ones are likely to be Chlamydomonas, longer tapering ones, often with a translucent portion to the cell, are Euglena.
Many organisms are greyish and glide slowly along. These are the ciliate protozoa, which swim with a surface layer of beating cilia that move like a wheatfield in the wind; Paramecium is one familiar example. Euplotes scurries like a mouse along the green algal filaments. Some ciliates are normally anchored down in colonies, like
Vorticella which jerks shut into a tiny ball when disturbed. Amoeba is also a protozoan. Pour a little mud into a saucer and next day minute white specks may be seen on the surface; these are amoebae, which can be collected with an eye-dropper, and mounted in water for observation.
Some of the creatures you will see seem to have paired wheels at the front. They look like gear-wheels. In fact these are discs of cilia, beating together. The organisms are called rotifers, and make beautiful objects of study.
The rounded, jerking water fleas are probably Daphnia: inside the back may be observed a beating heart. The brown, curving line that runs through the body is the gut from mouth to anus; note also the delicate eye muscles. Swifter in movement is Cyclops, shaped like a fat road-drill with antennae for handles. The young of Daphnia are formed in brood pouches inside the body; in Cyclops the egg cases hang outside the body as a pair of projections. The new-born young are called nauplius larvae and are quite unlike the adults.
The above diagrams are drawn at very high magnification (1000x) but the essential structures can be seen with the Lensman in high magnification mode.
A trace of mould on stale food or neglected
bread provides a source of fungi for study. Otherwise, leave
some moist bread under an up-turned jam jar for a week and you
can grow your own. The threads of the fungus are its hyphae,
and show clearly in a wet mount. Note the oval spores that form
inside the round spore cases in Mucor (see fig 13a), the
classical pin-mould. In some other species, including the blue
Penicillium (see fig 13b), in which the spores form in
a brush-shaped array at the end of a hypha.
7. Other Suggestions
Look at the underside of a fern, using reflected light, to study its spore cases. Then mount some in washing-up liquid and look in more detail. Lay the microscope on textiles to see how cloth is made. Examine printed colour photographs in a magazine. Separate dots of the three colours which go to make up the coloured image. Starch grains develop beautiful maltese cross patterns under polarized light, if you have one piece of Polaroid underneath the slide and another above it. Rotate the upper filter to get the best effect. Now study different kinds of starch - rice, wheat, maize - and see how they look. Can you spot starch grains in cheap curry powder, put there to bulk it out? Out in the field, watch for the glistening components of igneous rocks, or the tiny shells in chalk; study wood grain in detail; observe the blood circulation in midge larvae from a water-butt. Learn how to distinguish between cotton and rayon, between acrylic and nylon fibres. By using a combination of the right techniques, you can put much of your immediate world under the Lensman microscope.
You will never look at the world in quite the same way again.