BIOLOGICAL TECHNIQUES

Microscopy
There are many different forms of microscopy but the one most commonly employed is
“brightfield” microscopy where the specimen is illuminated with a beam of light that
passes through it (as opposed to a beam of electrons as in electron microscopy). The
general requirements for a specimen to be successfully examined using brightfield
microscopy are:

 That the cells and other elements in the specimen are preserved in a “life-like”
state (this process is called “fixation”)
 That the specimen is transparent rather than opaque, so that light can pass
through it
 That the specimen is thin and flat so that only a single layer of cells is present
 That some components have been differentially coloured (stained) so that they
can be clearly distinguished.

Preparation options
Because of the microscopy requirements, options for preparing specimens are limited
to: 

 Whole-mounts, where an entire organism or structure is small enough or thin

enough to be placed directly onto a microscope slide (e.g. a small unicellular or
multicellular organism or a membrane that can be stretched thinly on to a slide)
 “Squash” preparations, where cells are intentionally squashed or crushed onto a
slide to reveal their contents (e.g. botanical specimens where cells are disrupted to
reveal chromosomes)
 Smears, where the specimen consists of cells suspended in a fluid (e.g. blood,
semen, cerebro-spinal fluid, or a culture of microorganisms), or where individual
cells have been scraped brushed or aspirated (sucked) from a surface or from within
an organ (exfoliative cytology). Smears are the basis of the well-known “Pap test”
that is used to screen for cervical cancer in women.
 Sections, where specimens are supported in some way so that very thin slices
can be cut from them, mounted on slides, and stained. Sections are prepared using
an instrument called a “microtome”.
Of these options only whole-mounts and sections preserve the structural relationships
between individual cells and extracellular components. Smears and squash
preparations provide detail about individual cells and relative cell numbers, but
structural relationships are lost. The preparation of sections is the most technically
complicated of these methods as it requires specialized equipment and considerable
expertise. The microscopic examination of sections by a pathologist forms the corner
stone of cancer diagnosis. Although the methodology for preparing sections from both
animal and plant material is similar, the following description relates to animal (human)
tissues.

Section preparation
Most fresh tissue is very delicate, easily distorted and damaged and it is thus impossible
to prepare thin sections (slices) from it unless it is supported in some way whilst it is
being cut. Usually the specimen also needs to be preserved or “fixed” before sections
are prepared. Broadly there are two strategies that can be employed to provide this
support.
1. The tissue can be rapidly frozen and kept frozen while sections are cut using
a cryostat microtome (a microtome in a freezing chamber). These are called “frozen
sections”. Frozen sections can be prepared very quickly and are therefore used when
an intra-operative diagnosis is required to guide a surgical procedure or where any type
of interference with the chemical makeup of the cells is to be avoided (as in some
histochemical investigations).
2. Alternatively specimens can be infiltrated with a liquid agent that can subsequently be
converted into a solid that has appropriate physical properties that will allow thin
sections to be cut from it. Various agents can be used for infiltrating and supporting
specimens including epoxy and methacrylate resins but paraffin wax-based histological
waxes are the most popular for routine light microscopy. This produces so-called
“paraffin sections”. These sections are usually prepared with a “rotary” microtome.
“Rotary” describes the cutting action of the instrument. In all histopathology laboratories
paraffin sections are routinely prepared from almost every specimen and used in
diagnosis.
The following paragraphs describe the major steps in preparing paraffin sections. These
steps generally dictate the layout and workflow in large, specialist histopathology
laboratories where hundreds of specimens are handled every day.

Figure 1: A diagnostic section being prepared with a cryostat microtome. The section, which has been cut from
 snap-frozen tissue, is being picked up onto a warm slide where it will be immediately fixed and stained.
.

Figure 2: A rotary microtome being used to cut paraffin sections. In the foreground a ribbon of sections is being
“floated out” ready for mounting on a microscope slide

Fixation:

As soon as tissues were taken from the specimen, a process of autolysis or self-
destruction starts. This process was initiated by the activity of intercellular enzymes
resulting in the disruption of proteins and ultimately liquefaction of the cells. Changes in
the tissues were also resulted because of bacterial decomposition brought about by the
bacterial production in the dead cells. After washing with water, the tissues were fixed in
a fixative. The fixatives were used to considerably harden and preserve the tissue
morphology possible nearest to living state and to minimize the cellular deterioration by
inactivating enzymes.

Numbers of chemicals were used as fixatives and are a mixture of two or more
chemical reagents mixed together. The fixatives preserved the structural accuracy of
tissues and cells. Normally there are following groups of fixatives:
 Picrates
 Aldehyde (formaldehyde)
 Mercuials
 Oxidizing agents
 Alcohols
Formalin:
 The most regularly used fixative is formalin because it permeates into tissues
swiftly. The fixative should be colorless and clear with no precipitates. Its pH should not
be below 6.5 to avoid brittle and unnecessary hardening of the tissue. As a result
natural tissue color is restored.

Chemical Composition:

For fixative normally 40% aqueous solution of formaldehyde is used as diluted

solution 10% formalin. 40% aqueous solution of formaldehyde normally exists as a gas.

Procedure of fixation:

 Specimen tissues collected were immediately processed.

 Cut the infected specimen 3-5 mm in length approximately.
 Later placed it in a refrigerator to store it in a cool state.
 Maintain a 10:1 ratio of fixative to tissue volume.
 Immediately submerged the specimen tissues in a fixative. To assure highest
contact between the fixative and tissues, stir the tissues in a fixative container.
 It is important to place the infected tissues in fresh fixative for 24h.
Embedding:
In this process impregnated specimen tissues were placed in a warm liquid
paraffin wax which solidified to a firm block on cooling down on room temperature. This
process is called as “blocking” or “casting”.

Procedure of Embedding:

The following steps were used for embedding process:

1. We took L-block layered with glycerin to avoid wax attachment to the surface.
2. Melted wax was poured at oven temperature into the block cavity.
3. The wax at the base of smooth glass piece was left to harden.
4. Then specimen tissue was transferred with the help of heated forceps to the L-
block bottom. It was placed vertically for transverse sectioning or horizontally for
longitudinal sectioning. Pour the remaining paraffin wax quickly and filled the L-
block up to the surface.
5. Avoid air bubbles and clear away the air bubbles with the help of needle or heated
forceps.
6. Allowed the wax to solidify at room temperature. Removed the L-block gently once
wax was solidified and labeled the blocks with tissue name and date of collection.
7. Store the wax blocks in cold place.
SECTIONING or MICROTOMY:

After embedding, the tissues were cut properly into sections suitable for the
slides. Cutting was done with the help of microtome. The microtome acts as a knife. It
worked on a mechanism of advancing a paraffin block at standard intervals crosswise
the knife. Paraffin block with tissue section was sliced in thin sections on a rotary
microtome.

Paraffin block with tissue sections was attached to tissue holder and placed on
microtome. In Paraffin technique, the average section thickness is 5 µm with section
thickness 3-7 µm. 4 µm is desirable in some cases.

The paraffin block with tissue section was moved closer to the knife with the help
of rotary handle of microtome. The sections were cut and came out in the form of a
continuous ribbon.

STAINING
There are different stains used as biological stains. Their use depends on availability
and their economic value. Staining result also plays important role in stain choice.

STAIN USE SPECIMEN COLOR

Acid Fuchsin Stains collagen fibers red and smooth muscle in contrast to Red/magenta/violet
collagen.

Congo Red Used to stain amyloid. (A starchlike substance. A hard, waxy Red
deposit consisting of protein and polysaccharides that results
from the degeneration of tissue.)

Crystal Violet The (positive) dye of choice in Grams stain. Bacteria Blue violet

Eosin B Interchangeable with Eosin Y. Ionization – acid (Color is red) Bluish cast

Eosin Y Stains alkaline cell parts (like cytoplasm). Use on plants, Pink
animals and blood.

Eosin Y ws Common counterstain to alum hematoxylin and Eosin method. Red

Gram Stain Positive – Staphylococcus, Streptococcus, Bacillus -subtilis Dark blue or purple
(crystal violet)
                                     
Negative – Neisseria, E. coli (Eosin Y ws) Red

Hematoxylin Stains nuclear chromatin using aluminum mordant. Acid Yellow/brown

resistant nuclear staining, muscle striations and some glial
fibers (The delicate network of branched cells and fibers that
supports the tissue of the central nervous system) with ferric
salts mordant.

Iodine Carbohydrates in plant and animal specimens Brown or Blue-Black

Glycogen (Animal starch.. A polysaccharide, (C6H10O5)n, that Red

is the main form of carbohydrate storage in animals.

Mercurochrome Marks tissue margins and faces for orientation. (Antiseptic) Red
 Similar to Eosin Y ws.

Methyl blue Stains collagen and connective tissue. Blue

Methylene blue Acidic cell parts (Use on animal, bacteria and blood specimens. Blue

Saffron Colors connective tissue yellow in contrast to the pink Yellow orange
cytoplasm given by phyloxine.

Safranin O Commonly used for counterstaining nuclei red. Cartilage is Red

stained yellowish.

Toluidene blue Stains acidic cell parts (like nucleus). Good to show mitosis in Dark Blue
plant cells. MITOSIS = The process in cell division by which the
nucleus divides, typically consisting of four stages, prophase,
metaphase, anaphase, and telophase, and normally resulting in
two new nuclei, each of which contains a complete copy of the
parental chromosomes. Also called karyokinesis.

H and E STAINING:
Different stains were applied to visible the differences in the tissue structure and
process is called as staining and counterstaining. A double staining technique including
hematoxylin and eosin was used and is most commonly used technique.
k) HARRIS HEMATOXYLIN:
Hematoxylin is used to stain acidic structure i.e. nucleus in purplish blue color
and is a basic dye. The nucleus showed strong likeness for this dye.
Chemical Composition:
The chemical composition of Harris hematoxylin is as follows:

 Ethyl alcohol 50ml

 Hematoxylin 50mg
 Distilled water 950ml
 Potassium or Ammonium alum 100gm
 Glacial acetic acid 40ml
 Sodium iodide 1gm
Method:
In an alcohol, dissolve the hematoxylin at water bath or gentle heat 56°C in an
oven and dissolved the alum in distilled water by using heat and frequently stir it.
Alcoholic hematoxylin solution was added in hot aqueous alum solution and boiled the
mixture. After boiling, kept the container in cold water and let it to cool down. After
cooling acetic acid was added to the mixture and filtered it. The solution was all set for
instant use.
L) 1% EOSIN:
Eosin is used to dye basic structure i.e. cytoplasm as it is acidic dye. It gives red
or pink color and so cytoplasm was stained in pinkish red or pink because of this dye.
Chemical Composition:
The chemical composition of 1% eosin is as follows:

 Distilled water 80ml

 Eosin Y (water soluble) 1gm
 Glacial acetic acid 0.4ml
 95% alcohol 320ml
Method:
Dissolved eosin in water and added it to 95% alcohol in a ratio of 1:4. In this
mixture few drops of glacial acetic acid (0.4ml) was added. The staining power of the
eosin was improved because of acetic acid. The stain should be cloudy in appearance
at the time of use but if not so add few drops of acetic acid.
OTHER REAGENTS:
 Dilute aqueous HCl (0.5%):
 Distilled water 500ml
 Conc. HCl 2.5ml
 Dilute ammonia water:
 Distilled water 500ml
 Strong ammonia (28%) 1.5ml
5.4 STAINING and DEHYDRATION PROCEDURE:
1. Slides were washed thoroughly under tap water and rinsed with distilled water.
2. Slides were then stained with Harris hematoxylin for 3-5 min. For each batch it is
important to determine time factor by trials.
3. Again washed the slides completely with tap water and bathed with distilled water.
4. Dipped the slide in and out of HCl 0.5% for few times. Slides should be checked
under microscope after every dip until clear differentiation was observed between
the nuclei (stained dark purple) and rest of the tissue appeared pale in color.
Repeat the staining process after enough rinsing in distilled water in case the
nucleus didn’t appear dark in color.
5. Washed with water for 50-60 seconds.
6. After dipping in dilute ammonia water several times, the sections changed to blue
in color.
7. Washed completely with water and bathed with distilled water.
8. Later rinsed with 95% alcohol.
9. Agitated for 10-60 seconds in eosin staining solution. The time duration depends
on the age and staining strength of the stain. After desirable staining, drained the
stain.
10.Dipped in 70% alcohol for 30-60 seconds.
11.Then dipped in 90% alcohol for 30-60 seconds.
12.Then dipped in absolute alcohol for 2 times each for 30-60 seconds.
13.Precautions:
14.Every 10 seconds comprised of 1 dip therefore timing can be noted by number of
dips.
15.When alcohol appeared to be of a color of stain change it immediately and xylene
was changed 2 times a week.
16.It took 15 minutes to stain with HARRIS hematoxylin for decalcified tissues.
17.It is necessary to check the intensity of routine stain with experienced plant
pathologist for standardized routine staining.
18.It is compulsory for technician to prepare best H &E staining. The slides should be
checked under microscope during staining with hematoxylin and detaining with
acid alcohol so that all slides should be microscopically differentiated.
MOUNTING:
Mounting or side preparation can be done by using different chemicals. Most common
and economical is mounting by using paraffin wax. In this process tissue sections or
specimen is placed in the centre of the clean glass slide in the middle of the glycerin
drop at the surface of the slide. Paraffin wax ribbons were added around the glycerin
drop having specimen and carefully covered with clean cover slip. Slide is then placed
on hotplate so the paraffin wax melted and evenly spread under the cover slip. Remove
the glass slide and let it to cool on room temperature. One can observe clearly
specimen or tissue in the centre under the microscope.
Another method of slide preparation is by mounting permanently in Canada balsam.
Reagents used:
 Xylene 100ml
 Canada balsam 55-65g
Method:
Prepare the solution for mounting by using above mentioned quantities. Greater
quantity of resin will result in viscous solution. Specimen is placed on the clean glass
slide and covers the slide with Canada balsam. Place the cover slip carefully and avoid
air bubbles. Place the glass slides in autoclave at temperature of 54 °C for next 48houts
or until Canada balsam get hard. Label the slides.
Slides prepared by both ways can be stored for longer time and future use.

Cryosections
Cryosections are rapidly and relatively easily prepared prior to fixation, and they provide
a good system for visualizing fine details of the cell. Although cryosections are
physically less stable than paraffin- or resin-embedded sections, they are generally
superior for the preservation of antigenicity and therefore the detection of antigens by
microscopy. The preparation of cryosections does not involve the dehydration steps
typical of other sectioning methods, and, furthermore, sectioning, labeling, and
observation of specimens can usually be carried out in one day. In general, the sample
is frozen quickly in either isopentane or liquid nitrogen. (Small samples such as cells
and small tissues may be mixed in a slurry of an inert support medium such as optimal
cutting temperature [OCT] compound before freezing). Rapid freezing reduces ice
crystal formation and minimizes morphological damage. Frozen sections may be used
for a variety of procedures, including immunochemistry, enzymatic detection, and in situ
hybridization.