UNIVERSITY OF MALTA
DEPARTMENT OF BIOLOGY
BIO1010 Cellular and Biochemical Basis of Life
PRACTICAL WORK IN CELL BIOLOGY
Contents:
I.
Setting up a microscope
For Critical illumination
For Kohler illumination
Use of Immersion Objectives
Selecting an area for observation
Light and illumination in practice
Observations
Care of the stand and cleaning of optical components
II.
Microscopic Preparations
Notes on the various methodologies
Professor V. Axiak
MICROSCOPIC OBSERVATIONS
Setting up a Microscope
For Critical Illumination
1. Adjust seat height (and inclination of the tube, when possible) so that the eye can be
brought easily before the eyepiece; the draw-tube, if fitted, should be adjusted to the
correct tube-length.
2. Turn on a low-power objective, e.g. l0x, with the revolving nosepiece; bring the
condenser in its highest position.
3. Switch on the lamp and manipulate the (flat!) mirror until the front lens of the condenser
lights up.
4. Move a stained object in the light beam from the condenser, using the mechanical stage
(object lights up).
5. Look in eyepiece and bring the object into focus with the coarse adjustment so that an
image appears.
6. Remove the eyepiece from the tube and manipulate the mirror and the height adjustment
on the condenser so that a disc with even illumination is seen when looking down the
tube. Close the substage diaphragm so that the outer border (about ¼-¾ of the diameter)
of the disc, actually the entrance pupil of the microscope, is screened off. This is no more
than a rule-of-thumb to obtain a tentative adaptation of the numerical aperture of the
condenser to that of the objective, so that with an average stained specimen not too much
glare is generated and no appreciable loss in aperture occurs.
7. Re-insert the eyepiece and adjust the condenser so that maximal intensity of illumination
is obtained. When the image of the surface of the opal bulb is found disturbing, the
condenser may be moved slightly up or down.
For Kohler-Illumination
In setting up a microscope with a built-in Kohler-illumination, the steps 1-5 are the same; the
subsequent steps are:
6. Close the field diaphragm and focus the border of the illuminated disc sharply in the
specimen.
7. Bring the illuminated disc to the centre of the field using the centring screws of the
condenser, which should be present with any microscope with Kohler-illumination.
8. Open the field diaphragm until its image fills the field of view, so that the border is no
longer visible.
9. Adjust the aperture of the illumination cone with the substage diaphragm, as described in
Crit. III. 6). As the Kohler illumination in itself already reduces glare, an excessive
aperture of the illumination becomes less easily manifest than with a critical illumination;
this adaptation is required nevertheless in order to obtain an optimal result.
10. In changing an objective, both field diaphragm and aperture diaphragm should be
adjusted, while the condenser has not infrequently to be realigned with the objective.
Use of Immersion Objectives
The main practical point to consider here is not so much the introduction of an oil film
between the cover glass and the front lens, but the combination of a very short working
distance with a very thin depth of field. Missing the image plan in focusing is a far from
imaginary event; in the assumption that the image still has to appear, the objective can be
forced without noticeable resistance against the object with the advancement of the fine
adjustment. This can have serious consequences, as the front lens of the objective might get
damaged or become loose in its mounting because of the pressure (which often leads to a
crushing of the cover-glass too): due to the high costs of individual manufacture the
reparation of a damaged front lens is often more expensive than a complete new objective of
the same type.
A few technical devices exist for this very old problem. The most universally applied
guarding device is the so-called spring-mount; this consists of a telescoping system in which
the objective proper can be pushed against the pressure of a spring in an outer tube-mount.
This type of mount is not only applied with oil immersion-objectives - for which it has
become more or less the standard practice - but also for some high-power dry objectives.
When the correct image plane has been missed, the telescoping of the spring-mount, when the
objective is forced down onto the slide with the fine adjustment, becomes evident only when
the lens is inspected from the side, for there is no image! When the spring- mount has reached
the end of its movement, it has no more effect and thus the system is far from fool-proof. The
same is the case with other guarding devices, such as a slipping of the fine adjustment when a
certain pressure is exceeded, or an adjustable mechanical stop to the fine adjustment. These
latter devices can even give a false feeling security; with any appreciable variation of the
thickness of slide + mounting medium + coverslip they fail completely.
The following general rules can be recommended for maximal safety, although caution
should always be exercised.
I
First select the area to be studied with a low-power dry objective.
II
Apply one, and not more than one, medium drop of oil on the cover glass exactly where
the specimen is illuminated by the condenser, e.g. with a plastic bottle with a long spout.
It is important to let any air with escape first from the spout, as air bubbles in oil on the
cover glass are very difficult to remove.
III In the case of parafocally adjusted objectives the immersion objective can be rotated with
the nosepiece into position in the oil without changing the height adjustment of coarse or
fine adjustment.
IV When the immersion objective has snapped into position, further adjustment should be
made with the micrometer until the image comes into focus. When the same low-power
objective is always used first, the necessary correction will be identical each time (e.g.
half a turn forward with the fine adjustment).
With objectives which are not parafocally adjusted such as may be the case with an older
microscope type, or a set of objectives of various makes, the focusing of an immersion
objective cannot be performed in the way just described. When a suitable area has been
selected with a low-power lens, the oil immersion objective is rotated into position and
racked up 8-10 mm above the object. When the oil has been applied as described previously,
the eye level should be placed near the top of the object slide, so that the objective can be
inspected from the side. The oil-immersion objective should be lowered carefully with the
coarse adjustment until it touches the oil. At the moment of contact, the oil spreads with a
clearly visible jerking movement in the slit-like space between the lower surface of the
objective mount and the coverslip. The objective is now lowered towards the object with the
fine adjustment, while looking in the eyepiece until the image appears. This is a very
important point, which may also apply when an oil-immersion lens can be set up in the easier
way discussed at the beginning of this section, with a parafocally adjusted low-power
objective.
Selecting area for observation
In selecting an area to be focused with oil-immersion objective, one has to make sure that
under any circumstances an image can be expected to appear. Often the rather small object
field of a high-power objective is overestimated, so that one may try to focus on an emptyfield. Moreover, the centring of the objectives is seldom perfect. It is important, therefore, to
select a homogeneous area with contrast-rich details for setting up the high-power objective,
if necessary even outside of the area to be studied. When the correct level of focus has been
found, it is relatively easy to pass a few “empty” areas in the specimen with the mechanical
stage. Uncertainty whether one is under or over the level of the focus is a situation which
should be avoided; when this state of affairs has been reached (and movements with the
mechanical stage do not yield anything like an image either) it is best to stage anew with a
low-power objective. In some situations, e.g. a smear of scattered cells of low contrast, it can
be difficult to find the correct level of focus with the oil immersion lens. In some
circumstances it is even recommended to mount a very small piece of cigarette piece or thin
metal foil which is visible to the naked eye with the specimen under the coverslip, so than an
approximate level of focus can be found at once at the border of this object in the specimen.
With parafocally adjusted objectives, it is easy to turn back eventually to a low-power
objective without removing the oil. In contrast to what is often thought, the film of oil does
not interfere very much with the formation of an image at low power, as long as the front lens
does not touch the oil (which will not easily occur with e.g. a l0x objective with its long free
working distance). The oil film on the cover glass will cause, of course, an exaggeration of
the cover-glass effect.
Even in using a non-resinifying oil both in oil immersion objective and the slide should
always be cleaned carefully after use. When a condenser immersion has been applied, the
condenser should be cleaned, moreover, and the slide on both sides. As the use of a
condenser immersion is seldom justified, even from a theoretical point of view, the rather
tedious immersion of the condenser and the cleaning it entails (often also of the stage) is kept
for a few selected situations with a highly corrected condenser and a contrast object, so that
the full condenser aperture can effectively be used.
Light and illumination in practice
In describing the standard-procedure for setting up a microscope, a position of the condenser
diaphragm has been indicated which, in the case of a stained specimen with average contrast,
leads to a good adaptation of the N.A. of objective and condenser. In many cases the
illumination cannot be considered as optimal under these conditions, however: the aperture of
the condenser may need further adjusting, or the intensity of illumination may be too high or
too low. These two factors have to be dealt with independently; the use of the condenser
diaphragm for tempering the brightness of the image is one of the most common mistakes of
beginners in microscopy. It is true that in closing the substage aperture diaphragm the amount
of light passed is reduced, but this is accompanied by a change in the aperture cone of the
illumination, which quickly brings about a loss in resolving power and an increase in
diffraction phenomena in the specimen. The best way to change the brightness of the image is
a variable resistance on the lamp tension, as can be easily installed with a low voltage lamp.
When such a control is not feasible (e.g. with a simple high voltage illumination) or the lamp
current should be kept constant for certain purposes (e.g. in photometry or
photomicrography), an eventual change in image brightness should be brought about with
filters. In view of the low luminance of the high voltage bulb poverty of image brightness at
high power observation will be a more probable event than a too intense illumination,
although this may occur with low-power work. With high-power observation (e.g. using a
l00x, N.A. 1.25-1.35 objective) the high-voltage bulb will certainly fall short when a
binocular tube is used.
The uninitiated observer generally has a tendency to use a too low level of image brightness
with a stained specimen, underestimating the consequences of this. A dimly illuminated
image reduces the actual resolution (not the resolving power) considerably, however, certain
details can be made clear merely by increasing the image brightness. Local differences in
light absorption which determine the contrasts in the image of a stained specimen, come out
only with a certain degree of lighting intensity. With monocular observation, a too low
brightness of the image will increase moreover the habit of tightly closing the eye which is
not in use. The tense attitude which this brings about will promote fatigue and headache,
which phenomena are often ascribed too easily to monocular observation. A somewhat more
dim lighting of the image, on the other hand, is sometimes preferable with unstained objects
which are poor in absorbing details and in which the contrasts are determined mainly by
diffraction at boundaries. The condenser diaphragm should be in a much more closed position
than with a stained specimen, in view of the fact that a higher degree of coherence in the
illumination beam, in combination with a minimum of glare in the specimen is desirable in
these situations.
Observations
The study of a microscopic specimen starts - after an inspection with the unaided eye - by
focusing a low-power objective on it as already described. With some experience this can be
performed with a few quick actions. When the microscope has been set up and the object
focused with e.g. a l0x objective, the observation proper can start. This will differ so much
from case to case, that it is virtually impossible to make any sensible general remark in this
respect. Problems which will often recur, however, will be those of a systematical search for
certain details and marking and re-locating of a particular field, once such a detail has been
found.
In searching a microscopic specimen, a comparatively arbitrary way can be followed when a
structure to be studied with high-power can be found easily. The situation is quite different,
however, when a specimen has to be examined systematically, e.g. in making a differential
count of various components or in finding (or excluding the occurrence of) a certain rarely
occurring detail. It is often the best in these situations to search the specimen meander- like,
in which use is made of the fact that it is possible to pursue straight tracks through the
specimen with the mechanical stage. The track is followed in a given direction up to the
border of the preparation after which the position of the object is shifted with the other
control until a detail which could just be observed at one side still remains visible at the other
side. When the track is followed back after this shift, the strips of object-field just overlap, so
that a complete inventory of the specimen is possible if the same shift (or slightly less than
the diameter of the object field) is always made when the border of the preparation is
reached.
In surveying quickly large series of preparations in the search for certain details, it is
sometimes easier to move the specimen by hand (depending on the magnification, entirely
free on the stage or with one side under a spring clip) than with a mechanical stage. In many
cases the mechanical stage is detachable; a built-in mechanical stage can be moved often to
an extreme position so that the object can be put on the stage outside of its object holder.
When quick screening of larger specimens is often required, the mounting of a gliding stage
can be considered.
Care of the stand and the cleaning of optical components
A microscope stand of good make hardly needs any maintenance for long periods; the
moving parts, such as the rack-work, only need cleaning and new grease at very long
intervals. Care should be taken never to use thin oil on rack-work, as this may cause the
spontaneous sinking of a tube or condenser.
A microscope should be kept as much as possible free from dust in a case or under a plastic
cover; dust do not only adhere to the stand and its moving parts; but accumulate especially on
lenses and other glass surfaces (cover plates of built-in illumination, mirrors). The stage
should be cleaned regularly; if anything is spilled on the stage it should be wiped off.
Especially mounting material (Canada balsam and the like) should be removed at once (when
necessary with some xylol) as it can form hard cakes and be difficult to remove afterwards.
The following general rules can be given for the cleaning of optical surfaces, they hold true
especially for objectives. It should be kept in mind that external surfaces of optical parts,
treated with anti-reflection coating are relatively hard, but that those coverings are very thin.
Some of the internal coatings are soft and easily damaged.
1. Non-adhering dust can be removed best with a dry soft brush (if shortly warmed against
the surface of a bulb it picks up dust particles more easily) or lens paper. Dust should
never be removed by fingers from optical surfaces; the skin which is moist and greasy
will usually take up the dust, but leaves a greasy substance instead. Fingermarks or other
adhering grease or dirt should be removed with soft cloth or lens paper barely moistened
with xylol, or petrol if available. Never soak or immerse a lens in xylol; avoid under any
circumstance the use of methylated spirits or other alcohols, ether or acetone in view of
the cementing material. When water is used aqua dest. should always be taken instead of
tap-water, which leaves a deposit of salts on evaporation.
2. Always take lens paper, linen, etc. double over the finger to prevent infiltration by
moisture or grease from the skin. When the border of the mount is protruding over the
lens and/or the front lens is concave (as is often the case with plan-objectives) it can be
difficult to reach the entire lens surface with lens paper or a cloth. The cleaning should be
performed then with a piece of cotton wool impregnated with some xylene around a
match stick.
3. Oil-immersion objectives should always be cleaned after use, even with use of nonhardening oils, as these also alter slowly with time. Preferably first use dry lens paper,
which is mostly sufficient, finish when necessary with a piece of linen slightly moistened
in xylene. When too much of a solvent is on the cloth, touch it with a dry part; the lens
should never be soaked in any cleaning fluid. Some very old objectives have lens
cements which can be weakened by xylene; cleaning of older objectives should
preferably be done entirely dry.
4. Never attempt to take an objective lens to pieces for cleaning; even when everything has
been brought back into place correctly, mutual distances can be changed. This does not
hold true for eye-pieces with their more simple construction; they can be unscrewed for
cleaning without any danger. With most modern objectives unscrewing has become
impossible because the mount is made in one piece. Condensers, on the other hand often
have a front lens which can be unscrewed to lower aperture of the illumination apparatus;
even when this device is not used, dirt can accumulate between both lens systems and
should be removed from time to time. Condensers with a swing-put front lens quickly
become dusty on the uncovered top side of the large lens. When they cannot be cleaned
sufficiently with an air current from a blower brush (a soft brush at the end of the spout
of a rubber blower, also useful for cleaning the top cover of a built-in illumination) it
should be taken out and cleaned in the manner already described.
Recording observations from the microscope:
Well annotated drawings are extremely useful in recording microscopical studies. Please
follow the following rules in making drawings which are to be presented to the tutor for
assessment:
•
•
Draw only what you see, not what is indicated in the textbook or suggested by your
imagination.
Remember that you are examining a three-dimensional object. Therefore in some cases, a
more accurate impression is obtained by racking the focus back and forth, to bring
different levels into focus. If possible serial section in T.S. and in L.S. should be
examined.
•
•
•
•
•
Use the microscope with BOTH eyes open and have your notebook on the right of the
instrument.
Drawings under L.P. should show the outlines and the main features drawn to the same
scale as seen under the microscope. Magnification should be noted and recorded.
Drawings under H.P. should show only representative areas of the field of view, never
the whole of it, and different cells should be properly labelled. Drawings of cells should
not represent holes or brackets or fish scales! They should represent individual cells as
seen under YOUR microscope. Careful observation will indicate that not only have the
individual cells their individual boundaries but that adjacent boundaries are
interconnected in some quite specific manner according to the nature of the cells.
Drawings should be in pencil, while shading or other markings should be kept to the
essential minimum and then in such a way that the shading is clearly differentiated from
the detail on the drawing - each dot or line on a drawing represents a dot or line on the
original and vice versa.
Each drawing is to be properly titled: i.e. name of specimen in full, magnification,
objective used, and when possible the stains utilised, together with the orientation (L.S.
or T.S.).
MICROSCOPIC PREPARATIONS
Microscopic preparations may either be temporary or permanent. Both involve a series of
micro technical processes to which a specimen or tissue must be subjected. These processes
include fixing the tissue to kill it, hardening, sectioning, staining, dehydration clearing and
mounting. These processes all have some effects on the cellular structure of the specimen.
This may result in artifacts or distortions of the actual structure of the tissue. The presence of
these artifacts must be borne in mind when interpreting a microscopic preparation.
TEMPORARY PREPARATIONS
Temporary preparations are well suited for the examination of living or fresh material. One
main advantage is that the specimen is subjected to as little preparation as possible, so that it
is more or less in a natural state. However the microscopic study of living material presents
several problems. The isolated cells of larger organisms, eggs embryo or unicellular
organisms must be kept alive, or at least the changes occurring after death delayed. The cells
are mostly colourless and transparent, so that their examination presents some difficulties.
The latter have been overcome by the use of special techniques for illuminating and
observing the specimens, for example phase contrast microscopy, interference microscopy,
etc.
Mounting in water
This is the familiar method of studying small organisms in a drop of water. It is often
necessary to support the coverslip using a glass ring or else use a cavity slide. Marine
microorganisms should be examined in sea water.
Mounting in isotonic saline solutions
This technique is used to examine cells from small pieces of tissue. The fluids used are
solutions whose composition resembles the plasma normally surrounding the tissue. They are
usually dilute solutions of sodium chloride and other salts.
Vital staining
Certain dyes, known as vital dyes, are taken up by living cells where they colour elements
within the cell, e.g. mitochondria. Dye particles can also be taken into the cell by
phagocytosis. Intravital staining refers to differentiation of cellular constituents by injecting
dyes into a living animal. Supravital staining indicates staining of isolated cells, small pieces
of tissues, or small organisms by placing them in isotonic saline solutions containing dyes.
PERMANENT PREPARATIONS
The techniques mentioned in the previous section will only last a few hours at most: to keep a
permanent record they must be photographed or accurately drawn. Moreover certain cellular
components are only poorly defined in such preparations. The following sections illustrate
the processes involved in making permanent preparations.
FIXATION
The first step is to fix the tissue or specimen. Fixation is intended to terminate life processes
quickly, preventing decay by bacteria and autolysis by the enzymes present in the cells
themselves. Fixation, therefore, preserves the cellular components as faithfully as possible
and stabilizes their condition during the steps to follow. Fixatives (or fixing agents) also
harden the tissue so that it can be embedded and sectioned, and also improves the effect of
certain stains. The following are a few of the fixatives in use:
Desiccation
This often brings about distortion in most kinds of cells, but is used for smear preparation of
blood, lymph, pus, spermatozoa etc. Fixation is often completed by heat or by treatment with
alcohol.
Heat
Heat brings about denaturation of enzymes, but also causes extreme shrinkage and distortion.
It is now used in conjunction with other methods.
Formaldehyde
This is a gas which is soluble in water, to give formalin. It is normally employed as a 10%
formalin solution. It is a strong fixing agent, rendering tissues tough and elastic. It can be
used on its own or in combination with other chemicals (compound fixatives).
Alcohol
70% ethanol precipitates cell proteins such as albumen and globulin in an insoluble and
denatured form while it renders glycogen and nucleic acids water soluble. It tends to shrink
the tissues.
Compound Fixatives
Choosing a fixative depends on what parts of a tissue are to be investigated. If the
organisation of the tissue is important, then a micro-anatomical fixing agent is to be used. If
intracellular components are to be demonstrated, then use a cytological fixing agent.
Zenker’s Fluid
This is a microanatomical fixative used for routine fixation. It penetrates rapidly and evenly.
Fixation is usually complete after 12 hours. Zenker’s fluid consists of a solution of mercuric
chloride (5g), potassium dichromate (2.5g) and sodium sulphate (1g) in distilled water
(l00ml). 5mls of glacial acetic acid are added just before use.
Formal-saline
This is the most popular and useful of all fixatives. Tissues left in it for long periods suffer
very little from its effect. Fixation time is about 24 hours. It consists of l0mls of formalin and
0.9g of sodium chloride in l00ml of distilled water.
Carnoy’s Fluid
Intended as a cytological fixative, it penetrates rapidly and gives excellent nuclear fixation. It
can be made mixing absolute alcohol (60ml) chloroform (30ml) and glacial acetic acid
(l0ml).
APPLYING FIXATIVES
It is not only necessary to use a suitable fixative, but also to apply it in the most effective
method, so that it would be able to penetrate as rapidly as possible, without excessive
distortion. As with other reagents, fixatives are to be treated carefully as many contain
poisonous chemicals.
Fixing by Immersion
This is the usual method of fixation. With larger specimens it may be necessary to inject the
fixative as well. Always use about 20 times the volume of fixative as that of the tissue to be
fixed. The penetrating power determines the size of the tissue to be fixed. For example use
only small pieces of tissue with low penetrating fixatives. Hot fixatives usually penetrate
faster; if heat is to be used, the fixative is to be warmed before the specimen is added.
Fixing by injection (Perfusion)
A fixative penetrates dense or bulky tissues rapidly when it is injected into the blood cavities
of body cavities. The fixative is introduced using a cannula.
Washing
It is often necessary to wash the fixative out of the tissues before proceeding with the
preparation. For example, mercury components, unless removed, tend to form precipitates
which are very difficult to remove. Washing is carried out with water or with alcohol,
depending on the fixative. Small specimens, must be washed using repeated changes of water
or alcohol. It is best to use running water with larger specimens.
Bleaching
Bleaching is carried out on specimens which contain dark pigments, in order to render the
rest of the material more visible. Mayer’s Chlorine bleach is suitable for bleaching sections of
skin.
Decalcification
It is impossible to section material which contains calcium salts, unless these are first
removed. An acid is always incorporated in the decalcifying solution, together with other
substances to prevent distortion. Some fixatives also contain sufficient acid to decalcify small
objects during the course of fixation. Recently, chelating agents have been used to decalcify
tissues. EDTA (ethylene-diamine-tetraacetic acid) is useful for this purpose.
EMBEDDING AND SECTIONING
The cutting of sections is the most important way in which organisms or tissues are separated
into thin portions for microscopic study. Sections must be thin enough for light to pass
through them. Generally, sections are one or two cells thick (about 8- 12µm).
Embedding Methods
Using Pith or Cork
This was the earliest aid to hand sectioning, using pieces of pith or cork to support the tissues.
These methods are no longer in much use.
Paraffin Wax Method
The tissue is impregnated with wax and so it can be cut into extremely thin sections. This
method consists of the following steps:
1. fixation (and washing).
2. dehydration - because water is immiscible with paraffin wax, the fixed tissue must be
dehydrated before impregnating with wax. This is carried out by transferring the tissue
from 30% to absolute alcohol, passing through 50%, 70% and 90% grades. The final bath
in absolute alcohol should have at least two changes.
3. “Clearing” - because alcohol itself is immiscible with paraffin wax, the dehydrated tissue
must next be transferred to a “clearing agent” which are solvents of paraffin wax. They
are so called because they render the tissue transparent. Chloroform, xylene and
cedarwood oil are the commonest clearing agents.
4. Impregnation with wax - this is carried out in molten wax at a temperature some 2-3°C
higher than the melting point of the wax (usually about 54°C). All the wax must be
filtered before use, to keep the crystals as small as possible. The tissue is first transferred
from the clearing agent to a solution of wax in the cleaning agent, before being actually
transferred to successive baths of molten wax.
5. Block making - “blocking” is carried out by transferring the impregnated tissue to a
mould containing molten wax (this may be a plastic container, paper boxes two L-shaped
pieces of metal resting on a glass). The tissue is oriented in the mould and a slip of paper
bearing the necessary details inserted in one corner. The wax is then cooled rapidly by
blowing gently and then by playing a jet of water it. When cold, the wax block is
removed from the mould.
6. Trimming the block - before sectioning, the block must be trimmed. This should be done
with a safety razor blade. Trim each side straight and make the opposite sides parallel;
leaving about 2 mm from the widest part of the specimen. The face of the block should
be a perfect rectangle. The trimmed block should now be attached to the chuck of the
microtome. The chuck should have a layer of wax on its surface. Melt this wax with a hot
scalpel handle and press the block firmly on and let the wax harden. The long side of the
rectangle should be parallel to the edge of the microtome knife.
Sectioning
Microtomes are designed to cut paraffin sections in the form of continuous ribbons. Before
using the microtome, the feed mechanism should be turned back as far as it will go. The knife
is then inserted and secured. It is important to tilt the knife at the correct angle. This is
normally very acute (about 5°) so that the knife edge passes freely through the material rather
than acting as a scraper. Take great care as microtome knives are extremely sharp. The
section thickness gauge is then set to the required position (about 8-12µm). The microtome is
operated until a ribbon of sections is formed. The ribbon should be lifted from the knife by
means of a metal seeker, and the support maintained until the ribbon is about 30 cm long. The
ribbons should be stored in a draught-free place.
Attaching the sections to the slides
Mount the sections on a clean glass slide using egg albumin as an adhesive. Put 1 drop on the
slide and spread it evenly with a clean finger. If the film is too thick, it may subsequently take
up stain and make the preparation untidy. Flood the surface of the albumenized slide with
distilled water. Single or short ribbons of sections are carefully lifted with a moistened paint
brush and laid on to the water, taking care not to trap any air bubbles. The slides are
transferred to a hot plate (at about 50°C) and left until the wax expands. When the sections
are flat, drain the water and orientate the section in the correct position. The sections are left
in an incubator at 37°C to dry completely (12 hrs) before using them. Care should be taken
during the process not to melt the wax. The prepared slides may be stored indefinitely in a
dust free container.
Other methods
One disadvantage of the paraffin wax method is that relatively high temperatures are required
during preparation. Therefore other methods can be used to avoid heat denaturation. One
solution is to use polyester wax, which has a lower melting point than the paraffin wax (about
38°C). The Celloidin method does not require any heat, but impregnation is slow. One further
method which deserves mention are frozen sections. Tissues may be frozen and sections of
the frozen tissues, which are embedded in ice, cut on a special freezing microtome (cryostat).
This method has the advantages of rapid preparation of sections with little fixation being
necessary, so the sections can be used for histochemical studies. Frozen sections are not as
satisfactory as paraffin or celloidin sections.
NON-SECTIONAL METHODS FOR THE PREPARATION OF TISSUES
Spreading
This method is used for the preparation of membranous tissue, to prevent it from shrinking
and wrinkling. For example, the membrane may be stretched between two plastic rings or a
coverslip. It is then fixed before proceeding with the preparation.
Smears
The preparation of smears is used in the study of blood, lymph, spermatic fluid etc. as well as
for the study of soft tissues such as testes and lymph glands. Smears are classified as wet or
dry. Wet smears are those which are fixed when they are still moist.
Dissociation of Cells
The technique of dissociation or maceration of cells is of great value for the study of isolated
cells. There are many methods, some of which are listed below.
1. Teasing: This is the process of separating tissue using sharp needles. This is used for
fibrous tissues such as nerves and tendons, which are not extremely fragile or held
together firmly.
2. Dissociation using Chemicals: The tissue is soaked in a suitable solution for a
considerable length of time; this loosens the connections between the cells so that they
can be easily shaken or teased apart.
3. The use of digestive enzymes: This brings about the removal of certain structures while
leaving the rest of the tissue intact. For example pepsin dissolves albumens, collagen,
mucin and elasten but has no effect on fats, carbohydrates, chilin and keraten.
4. Corrosion: This is the process by means of which hard structures such as sponge spicules
or fish scales are separated from the soft, surrounding tissue. A 10% solution of
potassium hydroxide may be employed for this purpose.
STAINING
The purpose of staining is to enhance natural contrast and make more evident various cell and
tissue components. Stains are coloured organic substances capable of being held by the tissue
components. They are in fact similar to textile dyes in their chemistry. The chemical structure
which confers colour to the stain is known as a chromophore. For example, the nitro group
(NO2) in trinitrobenzene gives this compound its yellow colour. Compounds containing
chromophores are known as chromogens. Chromogens, though coloured, are not dyes in
themselves as they have no affinity to bind to tissue constituents. To be a dye a compound
must contain in addition to a chromophore another group which enables the compound as a
whole to combine with the tissue, such groups are known as axochromes.
Classification of Stains
Stains may be classified according to the chromnophoric group present: Basic stains are those
having a base as a chromophore combined with acetate, chloride or sulphate groups. Basic
stains usually stain acid compounds, of cells (e.g. nuclei). Acidic stains have an acidic
organic chromophore and stain basic components of cells (e.g. cytoplasm). Neutral stains
contain both types of chromophores.
Mordants
Certain stains such as haematoxylin, will not stain tissues directly, unless a third compound is
present. This compound is known as a mordant and forms a link between the tissue and the
stain. The commonest mordants are the salts and alums of aluminium and iron (Fe3+).
Accentuators
These are substances which increase the selectivity or staining power of certain dyes, which
actually combine chemically in any way.
Methods of Staining
1. Progressive staining: the tissue is left in the stain until it reaches the required depth of
colour, this is determined by viewing the preparation under a microscope.
2. Regressive staining: the tissue is overstained and then decolourised (differentiated) to the
required depth of colour. Nuclear stains are frequently used regressively.
3. Counter staining: first part (or all) of the cell is stained with a suitable dye, then another
component of the cell is stained with a contrasting dye (or the second dye displaces the
first).
4. Double staining: a mixture of two dyes is used. Tissues may be stained in bulk or as
sections.
PROCEDURE FOR STAINING AND MOUNTING PARAFFIN SECTION
The basic procedure is as follows:
1. Dewaxing: this is the removal of the paraffin wax with xylene. The slide with the
attached sections is first placed on a hotplate for about 2 minutes, and then placed in the
xylene. The wax should be entirely removed and the tissue becomes transparent.
2. Hydration: this is the reverse of dehydration. Most solutions of stains contain water and
this is immiscible with xylene. Hence the section must be hydrated gradually by passing
it through successive solutions of alcohol of decreasing concentration (absolute alcohol,
90%, 70%, distilled water). Unless hydration is carried out gradually, the cells absorb
water too fast and become distorted.
3. Staining: the staining process to be carried out depends on the stain(s) to be used.
4. Dehydration: the slide is passed from distilled water to absolute alcohol through the
graded alcohols.
5. Clearing: The absolute alcohol is replaced by the cleaning agent, which is miscible with
the mounting medium. Xylene, benzene, oil of cloves or cedarwood oil may be used as
the clearing agent.
6. Mounting: the choice of a mounting medium determines the permanency of the
preparation. The mounting medium should be stable, and have no effects on the stains.
The refractive index of the medium is a very important consideration. The medium
should render the specimens transparent enough so that light will pass readily through
them, but not to such an extent that important structures become difficult to see. Media
having refractive indices close to that of crown glass are suitable. Canada balsam and
D.P.X. are two mounting media in common use. A drop of the mounting medium is
placed on the centre of the slide, over the section. Great care should be exercised not to
trap any air bubbles. Cover with a coverslip by resting the coverslip against a finger and
lowering it gently down using a mounted needle. The coverslip should settle by itself, to
prevent entrance of air bubbles. Label the slide properly with the name of the tissue,
method of fixation and staining and the date. Leave it to dry for at least a week.