Introduction
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
Making Smears
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:
Direct Smear
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.
Loop Smear
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.
Blood 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.
Fresh Preparations
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.
Dry Mounts
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.
Wet Mounts
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.
Tissue Peels
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.
Mounting Methods
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:
Tissue Fluid
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.
Water
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.
Glycerine
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.
Washing-Up Liquid
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
Blood Cells
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].
Cheek 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.
Human Hair
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.
Entire
Small plant specimens can be examined entire, whilst others are small particles that need to be mounted in glycerine or washing-up liquid.
Pollen Grains
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.
Stamen Hairs
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).
Nettle Leaf
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.
Grass Flowers
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.
Sage Leaf
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.
Sections
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.
Stem
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.
Root
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.
Leaves
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).
Algae
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.
Protozoa
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.
Rotifers
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.
Water fleas
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.
6. Fungi
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.
