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organisms retaining the primary stain and Gram-negative organisms losing the primary stain
are the result of the chemistry and structure of the organism’s cell walls. The tissue elements
are stained as follow:
• Gram-positive organisms: blue
• Gram-negative organisms: red
• Background: varying shades of blue/green
Medical Laboratory Technology: Volume 3
Grocott’s Methenamine Silver (GMS) stain kit
Groco’s Methenamine Silver (GMS) stain kit is intended to identify fungal organisms and
Pneumocystis jiroveci (formerly known as carinii) in tissue sections on the ArtisanTM Link
and ArtisanTM Link Pro Staining Systems. Fungi and Pneumocystis jirovecii are stained black
while other tissue elements are stained green. This stain is not recommended for cytology
specimens.
Iron stain kit
Iron stain kit is used to identify iron pigment in tissue sections. Iron is an important component
of the human body, especially as a vital constituent of oxygen carrying haemoglobin. Iron is
stored in bone marrow, and a loss of iron stores is indicative of anaemia. An excess of iron
deposited in organs such as liver, spleen, and bone marrow may be a result of hemochromatosis.
• Ferric deposits: blue
• Nuclei: red
• Background tissue elements are stained pink. This stain is not recommended for bone
marrow smears.
Jones’ Basement Membrane (PAS-M) stain kit
Jones’ basement membrane stain kit is used to identify basement membranes, specically
glomerular and tubular basement membranes in renal tissue sections cut at 2 mm.
• The Bowman’s capsule: black
• Inner basement membrane: black to gray
• Nuclei: red
• Collagen: rose
• Cytoplasm and other tissue elements: pink
Masson’s trichrome stain kit
Masson’s trichrome stain kit is used to identify muscle, collagen bers, brin and erythrocytes
in tissue sections. Masson’s Trichrome is oen used to demonstrate increased collagen
deposition that is associated with replacement of functional tissue by scar tissue. This is
useful in the assessment of sclerosis of the liver in which thickened collagen replaces normal
tissue causing liver dysfunction.
• Muscle: red
• Collagen: blue
• Fibrin: pink
• Erythrocytes: red
• Nuclei: blue/black
Mucicarmine stain kit
Mucicarmine stain kit is used to identify epithelial mucins in tissue sections. These mucins
are a family of polysaccharides covalently linked to proteins in epithelial cells.
• Mucins: pink
Laboratory Techniques in Histology
• Nuclei: black
• Other tissue elements: yellow
1197
PAS-Green stain kit
PAS-Green stain kit is used to identify fungi in tissue sections on the ArtisanTM Link
and ArtisanTM Link Pro Staining Systems. The most commonly used application is for
demonstrating fungi in skin infections. Unlike GMS, the PAS-Green technique will not stain
all fungi and yeast. However, fungi typically found in dermatologic specimens are commonly
identied using PAS-Green.
• Positive fungi: magenta
• Remaining tissue elements: blue/green
Periodic acid-Schiff (PAS) stain kit
PAS stain kit is used to identify glycogen in tissue sections. PAS-positive entities and structures
are also numerous in tissue sections. The most common application is for demonstrating
glycogen in the liver. Duplicate sections are stained with or without a pre-treatment, such
as the ArtisanTM Alpha-Amylase, which is a glycogen-digesting enzyme. Comparing PAS
signal intensity in a digested tissue sample with one that has not been digested will give
an indication of the amount of glycogen present. A loss of glycogen may be indicative of a
metabolic disorder or damage to the liver.
• Positive glycogen: magenta
• Nuclei: blue
• Background: pink
Reticulin/No counterstain stain kit
Reticulin/no counterstain stain kit is used to identify a primitive form of connective tissue,
called in tissue sections on. An Ammoniacal Silver Nitrate solution is applied to
stain the reticulin bres in tissue. The silver is then reduced and toned to produce a black
coloration of the bres, which are visible by light microscopy. A counterstain may be applied
oine.
Reticulin/Nuclear fast red stain kit
Reticulin/nuclear fast stain kit is used to identify a primitive form of connective tissue, called
reticulin, in tissue sections. An Ammoniacal Silver Nitrate solution is applied to stain the
reticulin bers in tissue. The silver is then reduced and toned to produce a black coloration of
the bers, which are visible by light microscopy. Other tissue elements will be stained pink.
Warthin-Starry stain kit
Warthin-Starry stain kit is used to identify Helicobacter pylori, spirochetes and other
microorganisms in tissue sections. H. pylori and spirochetes are stained black while the
background is stained golden yellow.
Special Stains for the Evaluation of Mucins
Mucins are high molecular weight glycoproteins that are found dispersed throughout the
epithelia of the gastrointestinal, respiratory, and reproductive tract. They are composed of
a central protein core with multiple chains of carbohydrates (polysaccharides) aached.
Special stains that are used for the evaluation of mucins, mucin-like molecules and other
carbohydrate containing macromolecules are utilized frequently in the histology laboratory.
The typical mucin special stains contain cationic (positively charged) dye molecules in
solution at a specic pH. This is true in the case of mucicarmine, Alcian blue as well as the
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older metachromatic techniques that utilized such dyes as azure A or toluidine blue. The
cationic dye molecules bind via electrostatic forces to the anionic carboxylated or sulfated
polysaccharide chains of the mucin molecules.
Medical Laboratory Technology: Volume 3
Mucicarmine
Mucicarmine is one of the oldest techniques for the detection of mucins. Although not as
commonly used as it was, mucicarmine is still a valuable technique for the evaluation of
acid mucins particularly those of the gastrointestinal tract. In addition, this technique is also
useful for staining the capsule of the fungus Cryptococcus neoformans. It is found that tissue
sites that contain an abundance of neutral mucins demonstrate lile or no staining while
the sites of the gastrointestinal tract that are known to contain acid mucins typically stain
strongly with mucicarmine.
Alcian blue
The Alcian blue dye molecules are aracted to the anionic sites in mucin molecules. Like
mucicarmine, Alcian blue does not stain neutral mucins. Varying the pH of the Alcian blue
solution is a useful means for further characterization of the subtypes of acid mucins present
in a tissue specimen. The standard Alcian blue –pH 2.5 stains all acidic mucins. In contrast,
mucins that contain predominately carboxylated carbohydrates will stain strongly with
Alcian blue –pH 2.5 but not with Alcian blue at a pH of 1.0. The carboxyl groups do not ionize
at this lower pH and thus the mucins will display neutral characteristics.
Colloidal iron
The colloidal iron technique is based upon the araction of positively charged iron ions
(ferric cations) for the negatively charged carboxylate and sulfate group endings in acidic
mucins. The bound ferric ions are detected or visualized by subsequent treatment with potassium
ferrocyanide to form bright blue deposits of ferric ferrocyanide or Prussian blue. While the
colloidal iron technique is an extremely sensitive method for the detection of acid mucins, it is
not as frequently used as other techniques such as Alcian blue. However, like Alcian blue the
colloidal iron technique may be used in combination with the periodic acid–Schi technique.
The Periodic Acid–Schiff (PAS) technique
The PAS technique is perhaps the most versatile and widely used of the techniques for the
demonstration of glycoproteins, carbohydrates and mucins. Unlike the other techniques
described thus far, the PAS technique also recognizes neutral mucins as well as acid mucins
that contain signicant quantities of sialic acid. In addition to mucins, the PAS technique is
also widely used for the detection of glycogen and various glycoproteins. The PAS technique
is particularly valuable for the visualization of basement membranes due to the presence of
Schi reactive glyoproteins within these structures.
Alcian blue/PAS
The combination of the Alcian blue and the PAS techniques can be used as a means of
distinguishing neutral mucins from acid mucins. In most protocols, sections are stained with the
standard Alcian blue (pH 2.5) method followed by the PAS technique. The Alcian blue at a pH
of 2.5 will stain all acid mucins deep blue but will not color the neutral mucins. The subsequent
application of the PAS technique will stain the neutral mucins bright magenta. Tissues or cells
that contain both neutral and acidic mucins may demonstrate a dark blue or purple coloration.
The combined application of Alcian blue and PAS is useful for several reasons. Changes in
the distribution or paern of expression of neutral and acid mucins are indicative of certain
pathological conditions. In addition, the combined Alcian blue/PAS technique is perhaps the
most sensitive or comprehensive means for detection of mucins as all mucins should react
regardless of the charge nature of the mucin.
Laboratory Techniques in Histology
1199
Alcian Blue/PAS/Hematoxylin stain kit
Alcian Blue/PAS/Hematoxylin stain kit is intended to identify acidic and neutral mucins in
tissue. This procedure clearly separates the acidic and neutral mucins by color and can be
used to distinguish all mucins in tissue sections. Alcian Blue pH 2.5 stains the acid mucin blue
while the Schi's reagent stains the neutral mucins pink to red. Mixtures of the two mucins
will appear purple due to the positive reactions with both Alcian Blue and Schi's reagent. A
hematoxylin counterstain is then applied to impart a blue/black color to the nuclei.
Use of Special Stains in Commercial Autostainer
A commercial automatic stainer was successfully used to automate 2 versions of the
hematoxylin and eosin stain as well as 10 special stains frequently performed in a histology
laboratory. The special stains included van Gieson, diastase/periodic acid-Schi (PAS),
PAS, Alcian blue, Alcian blue/PAS, Gomori 1-step trichrome, Alcian yellow/toluidine blue,
Schmorl, Perl prussian blue, and luxol fast blue. The capabilities of the existing hardware and
soware of the Leica Autostainer XL were explored and some of the hardware was modied:
the staining rack adaptor was used to accommodate membrane and some opportunistic
organisms such as Pneumocystis carinii in tissue specimens. Periodic Acid-Schi (kidney).
PAS staining is mainly used for staining.
Elastic stain kit
As already discussed before.
Reticulin/No counterstain stain kit
As already discussed before.
Giemsa stain kit
As already discussed before.
Feulgen stain kit
As already discussed before.
H&E stain
The H&E stain uses two dyes, hematoxylin and This combination is used as the dyes
stain dierent tissue elements. Hematoxylin reacts like a dye with a purplish blue
colour. It stains acidic, or basophilic, structure including the cell nucleus (which contains
DNA and nucleoprotein), and organelles that contain RNA such as ribosomes and the rough
endoplasmic reticulum.
Eosin is an dye that is typically reddish or pink. It stains basic, or acidophilic,
structures which includes the cytoplasm, cell walls, and extracellular bres.
Hematoxylin is extracted from the logwood tree and puried. It is then oxidized and combined
with a mordant (typically aluminum) to allow it to bind to the cell structures. Of the many
hematoxylin preparations used in histology—Gill’s hematoxylin, Harris’s hematoxylin and
Mayer’s hematoxylin are the most popular. (Mordant is a substance, typically an inorganic
oxide, that combines with a dye or stain and thereby xes it in a material).
Eosin is formed by a reaction between bromine and uorescein. There are two eosin
variants typically used in histology—eosin Y which is slightly yellowish, and eosin B which
is slightly bluish. Eosin Y is most popular.
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Medical Laboratory Technology: Volume 3
Rapid Giemsa kit
The rapid Giemsa stain kit is a high quality stain kit specially formulated to provide rapid
turnaround Romanowsky type staining, on air-dried blood and bone marrow smears, as well
as non-gynaecological cytology samples such as: sputum, urine, ne needle aspirates and
tumour imprints. It can also be used to stain microorganisms such as H. pylori, protozoa and
spermatozoa. The easy to use kit consists of 3 × 500 mL boles, containing a xing solution
(Solution A), a red acid dye (Solution B) and a blue basic dye (Solution C). The kit also contains
5 × pH 6.8 buer tablets to produce a buered rinsing solution.
Acid fast bacillus special stain kit
Acid fast bacillus stain kit stains are used to detect and identify acid fast bacilli in tissue. Bacilli
are rod-shaped bacterial organisms. A primary function of this stain is to identify tuberculosis
in lung tissue, as well as other bacterial bacillus infected tissue.
Alcian blue special stain kit
Alcian blue is normally prepared at an acidic pH of 2.5 and is used to identify acid
mucopolysaccharides and acetic mucins. Excessive amounts of non-sulphated acidic mucosubstances are seen in mesotheliomas, certain amounts occur normally in blood vessel walls
but increase in early lesions of atherosclerosis.
Alcian blue and PAS special stain kit
This kit is a combination of the components of Alcian Blue and Periodic Acid–Schi (PAS)
stain kits. Alcian Blue is normally prepared at an acidic pH of 2.5 and is used to identify
acid mucopolysaccharides and acetic mucins. Excessive amounts of non-sulfated acidic
muco-substances are seen in mesotheliomas, certain amounts occur normally in blood vessel
walls but increase in early lesions of atherosclerosis. PAS staining is mainly used for staining
structures containing a high proportion of carbohydrates such as glycogen, glycoproteins,
and proteoglycans typically found in connective tissues, mucus and basement membranes.
Oen used to stain kidney biopsies, liver biopsies, certain glycogen storage diseases in
striated muscles and suspected fungal infections.
Congo red, amyloid special stain kit
The Congo red, amyloid stain kit is intended for use in histological detection of amyloid in
tissue sections. Examination of stained tissues with conventional microscopy reveals a dull
brick red coloration of amyloid. Examination utilizing polarizing microscopy reveals apple
green birefringence of amyloid.
Gomori’s trichrome special stain kit (Blue collagen)
Trichrome stains are used to stain and identify muscle bers, collagen and nuclei. They can be
used to contrast skeletal, cardiac or smooth muscle. The Gomori Trichrome is a simplication
of the more elaborate stains and combines the plasma stain (chromotrope 2R) and connective
tissue with Gomori’s Blue to provide a brilliant contrasting stain.
Gomori’s trichrome special stain kit (Green collagen)
Trichrome stains are used to stain and identify muscle bers, collagen and nuclei. They
can also be used to contrast skeletal, cardiac or smooth muscle. The Gomori Trichrome is a
simplication of the more elaborate stains and combines the plasma stain (chromotrope 2R)
and connective tissue with Gomori’s Green to provide a brilliant contrasting stain.
Laboratory Techniques in Histology
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Perls’ prussian blue iron special stain kit
Perls’ Prussian Blue Iron Kit stains are used to detect and identify ferric (Fe3+) iron in tissue
preparations, blood smears, or bone marrow smears. Minute amounts of ferric acid are
commonly found in bone marrow and in the spleen. Abnormal amounts of iron can indicate
hemochromatosis and hemosiderosis. Lack of iron identication can indicate decient anaemia.
Modified GMS silver special stain kit
The modied GMS silver stain kit is intended for use in histological observation of fungi,
basement membrane and some opportunistic organisms such as Pneumocystis carinii in tissue
specimens.
Schiff’s reagent
Schi reagent is produced in small batches, ensuring maximum consistency and shelf life.
It is used in the standard Periodic Acid–Schi (PAS) reaction for neutral muco-substances,
glycogen, basement membrane and fungal cell walls. A positive reaction produces a purplish
red/bright rose color.
Periodic acid and Schiff stain
PAS staining is mainly used for staining structures containing a high proportion of
carbohydrates such as glycogen, glycoproteins, proteoglycans typically found in connective
tissues, mucus and basement membranes. Oen used to stain kidney biopsies, liver biopsies,
certain glycogen storage diseases in striated muscles and suspected fungal infections.
Alcian blue and PAS stain
Leica Alcian blue and PAS special stain kit combines the components of the Alcian Blue and
Periodic Acid-Schi (PAS) stain kits. Alcian blue is normally prepared at an acidic pH of
2.5 and is used to identify acid mucopolysaccharides and acetic mucins. Excessive amounts
of non-sulfated acidic muco-substances are seen in mesotheliomas, certain amounts occur
normally in blood vessel walls.
Periodic acid stain
Periodic acid is a 0.5% aqueous solution used to oxidize reactive tissue elements to aldehydes
prior to treatment with Schi reagent. This solution is prepared immediately upon placement
of an order.
Fixative and Mordant
Bouin’s fixative/mordant
Used for testes biopsies and also as a mordant for staining procedures.
Saturated picric acid 1500 mL
Formaldehyde 500 mL
Glacial acetic acid 100 mL
Mix well. Stable for 1 year. Label, initial and date.
Caution Carcinogenic, contains formaldehyde, irritant, toxic.
• Work in a well-ventilated area.
• Wear gloves, lab coat and goggles.
• Avoid contact and inhalation.
• Picric acid can become explosive if allowed to dry out.
1202
• Toxic through skin exposure.
• Formaldehyde:severe eye and skin irritant.
• Sensitizer by skin and respiratory contact.
• Toxic by ingestion and inhalation.
• Target organ eects on respiratory system.
Fixationtime:Small biopsies x in 2 to 4 h, large specimens may remain in xative up to 3 days.
1. Fixed tissue may be retained in 10% formalin or 70% alcohol.
2. Remove the picric acid from the tissue prior to staining by:
(a) washing in tap water
(b) grades of alcohol (50%), or
(c) 70% alcohol saturated with lithium carbonate
Medical Laboratory Technology: Volume 3
frOzen SectiOn tecHnique
Frozen sections are used in emergency when results are needed fast. This is typical during
surgery when the surgeon needs to know the excision margin while removing a tumour.
Typically do not create the same quality as the The
process for frozen section preparation is as follows:
• Tissue is quickly frozen to preserve and harden it.
• The frozen tissue is sectioned in cryostat (a sectioning microtome in a freezing chamber)
and placed on a microscope slide for staining.
• The section is before it begins to decay and is then stained.
Frozen sections can be prepared for histological examination in a few minutes. The principle
involved is simple in comparison with paran wax embedding. The.water in the tissue is
frozen and as the ice acts as the embedding medium. The
consistency of frozen blocks is aected by the nature of the tissue and amount of water in the
tissue. Tissues that have a low water content section beer at colder temperatures; those with
a higher content, being harder due to more ice crystals, section beer at higher temperatures. It
is possible to improve sectioning of frozen blocks in the cryostat by adjusting the temperature
of the block tissue. For example, the optimum sectioning temperature of brain is –12°C while
the adipose tissue of breast may require –35°C or lower. Fixed tissue of any nature is sectioned
at –5°C to –10°C.
The rapid development of the cryostat over the last three decades has made it possible to
examine the ‘urgent frozen sections’. It is now an indispensable technique for rapid diagnosis
and intraoperative surgical consultation. In addition, many histochemical methods cannot be
done without freezing microtomy because the drastic steps involved in paran sectioning
destroy or lose the material sought.
Preparing frozen sections for histological studies has some disadvantages also. The structural
details are distorted because of the lack of embedding material during cuing and handling.
It is impossible to obtain serial sections in the case of cryostat sections and the staining is not
as satisfactory in the case of unxed frozen sections as seen in properly xed material. The
histopathologist will also nd many freezing artefacts in the cryostat sections. Moreover, some of
the ner details cannot be determined by this technique and certain special stains cannot be used.
We will try to describe here the general process of handling the freezing microtome (cryostat).
However, because of the wide variations found between various microtomes we will leave to
the reader the details of their operation available in the manufacturer’s instruction booklet.
A suitable block of fresh tissue is selected and trimmed with a sharp
scalpel so that its sides are parallel. The block should be about 5 mm thick. The tissue is
directly taken for cryostat sectioning.
Laboratory Techniques in Histology
Due to the water in the tissue, it is advisable to freeze the tissue slowly
to avoid tissue disruption. In most instances it will be wise to place the tissue on the freezing
stage of cryostat and allow it to freeze. When the tissue is frozen, place the tissue block rmly
on the microtome holder.
Pull the stage holding knife forward to allow about 5 mm clearance
between the subsequent down-travel of the block and the knife edge. Release the drive wheel
lock and line up the tissue block to the knife to within 1-2 mm of the tissue with the frozen
block parallel to the knife edge. When the face is almost in contact with the knife, release the
ratchet from the micrometer wheel. Turn the wheel with the hand, a fraction of a turn at a
time, to advance the tissue until the knife begins to cut the sections.
The cryostat cuts individual sections unlike the ribbons of sections with the paran
preparations. Sections should be cut with a slow and even motion. If the adjustments are
correct, the section will glide smoothly and at beneath the antiroll plate. Some technicians
use a camel-hair paint brush to start the section and to keep it at as it glides out on the knife
surface. Sectioning may be dicult in hot, humid weather.
The cuing of a frozen section requires experience and ‘touch’. Start sectioning and
continue until a complete section is obtained. Usually the block will have thawed to about the
right consistency by this time. Do not freeze the tissues too hard or the sections may shaer.
If this happens, allow the block to thaw slightly and try again. If it has become too so the
sections will also shaer or fracture. The technician will have to develop a proper feel for
the correct temperature. Sometimes rubbing a nger across the block will give it the right
consistency for good sections to be cut. Cut the sections slowly at about 5–10 mm thickness.
Mounting of the frozen section should be done carefully. The sections
are not rigid like the paran sections and should be handled dierently. The cryostat is provided
with an antiroll plate which is ipped back aer the section is cut. In practice, one edge of the glass
slide is rested on the knife surface about 1 inch beyond the section and the other end is lowered
gently until it is about 0.5–1.0 mm from the knife face, and the section will automatically transfer
from the cold knife to the relatively warm slide. Never press the slide down on the section. A frost
mark will remain where the section rested on the knife. This should be wiped away with so
gauze. The antiroll plate is re-positioned and another section may be cut.
No adhesive is needed to stick the section of unxed tissue on the slide; air drying for
about 30–60 s will be good enough to hold the section.
1203
Staining of Frozen Sections
The staining technique for frozen sections is basically the same as for paran sections. Since
the major use of the cryostat is for rapid surgical diagnosis, time may be saved by using
unxed tissue. The laer will also adhere easily to the glass slide without the need for any
adhesive mixture. Most oen the sections are handled singly and they receive the top priority
in the laboratory. In addition to the rapidity of diagnosis, there is another advantage of using
unxed frozen sections for histological studies. Freezing preserves the cellular enzymes and
other substances that may be studied by histochemical techniques. If the tissues are xed,
they should be washed thoroughly before staining.
There are numerous methods of staining frozen sections, but for rapid surgical diagnosis
two methods are widely used: H&E and polychrome methylene blue. Many workers also use
toluidine blue with carbol fuchsin counterstain. There are many modications of these but
only the commonly used ones will be described here:
Haematoxylin–eosin for fresh cryostat sections
Although xation prior to staining is not necessary, a brief 1–2 min immersion of the slide
in 10–15% formalin will enhance the nuclear basophilia. Sections of certain material, for
example, mucoid tumours may li o the slides when placed in the formalin and this step
should, therefore, be avoided and a dry section celloidinized instead.
1204
The routine H&E staining method can be applied. It is a lile more time-consuming, and
in some laboratories a special staining row is set up just for frozen sections.
Prepare the reagents as described earlier with routine staining.
• Harris’s haematoxylin (pre-ltered): Described earlier.
• Acid alcohol (1%, v/v)
• Sodium bicarbonate solution (2%, w/v, aqueous)
• Eosin yellow water soluble (1%, w/v, aqueous)
1. Wash section in water for 10–20 s.
2. Stain with haematoxylin for 1 min.
3. Wash briey in water; dierentiate in acid-alcohol for 1–2 s.
4. Wash briey in water and ‘blue’ in sodium bicarbonate for 10 s or so.
5. Wash in water 10–20 s and, if time permits, check nuclear staining microscopically.
6. Stain with eosin for 30 s.
7. Wash briey, dehydrate, clear and mount as desired.
Medical Laboratory Technology: Volume 3
Polychrome methylene blue staining
The method of polychrome methylene blue staining is recommended for rapid diagnosis with
frozen sections. Its use is, however, limited. Many laboratories use this as a preceding stain
so that the pathologist can study the methylene blue-stained section whilst the comparatively
slower H&E-stained section is being completed. Touch or imprint smears of unxed tumours,
breast lumps or lymph nodes may also be stained to advantage with polychrome methylene
blue. Polychroming of methylene blue is achieved by mixing with potassium carbonate when
various azures are formed. The process is accentuated by ageing of the solution.
• Staining solution:
Methylene blue 1 g
Potassium carbonate 1 g
Glacial acetic acid 3 mL
Distilled water 300 mL
Place the distilled water in a litre ask and add, with mixing, methylene blue and
potassium carbonate. Boil for 10–15 min. While still hot add glacial acetic acid dropby-drop, shaking vigorously until the formed precipitate is dissolved. Boil until the
volume of uid is reduced to 100 mL. Cool and lter. Allow to stand 4 weeks prior to
use.
1. Rinse frozen sections and imprint smears in water.
2. Stain with the polychrome methylene blue for about 30–60 s.
3. Wash in water and blot dry.
4. Dehydrate in tertiary butyl alcohol; clear in xylene and mount in a DPX-type mountant
(conventional ethanol dehydration will diminish the polychromasia).
• Nuclei—blue
• Background—various shades of red-purple
Laboratory Techniques in Histology
1205
HanDLing anD embeDDing SmaLL tiSSue fragmentS
Minute fragments of tissue (e.g. biopsy materials, bone marrow aspirate, endoscopy
fragments and others) may require special handling to prevent loss during processing. One
of the recommended ways is to wrap the fragments of tissues in lens paper for the processing
through various reagents.
In the case of bone marrow aspirate, as soon as the smears have been made, whatever
remains should be placed in xative for 30–60 min. Allow the fragments to sele down, pour
o the supernatant and transfer the marrow fragments into a Petri dish to x in formalin for
about 30 min and then process the marrow particles by wrapping in the lens paper.
review queStiOnS
1. What is the role of histology laboratory in clinical diagnosis?
2. What are the responsibilities of a histotechnician?
3. How is the specimen prepared for cuing sections?
4. Which xative is considered ideal in the laboratories of developing countries? What are
the problems of using formaldehyde as a xative?
5. How would one remove the mercuric chloride deposits in sections?
6. Explain the steps of tissue processing? What are the advantages and disadvantages of
using automated tissue processor? How is it dierent from automated stainers?
7. What is the purpose of clearing? Which is the commonly used clearing agent?
8. Explain the process of ‘take to water’. Why is it necessary?
9. Explain the process of dehydration.
10. Describe how you will prepare a biopsy specimen for microscopic examination.
11. Why is Canada-balsam no longer used in histotechnology laboratory? Which are the
popular mounting media currently used?
12. Why fat is treated dierently in the staining process than other constituents of the body?
State its clinical signicance.
13. What are the stains used for amyloids?
14. Describe the way the frozen sections are handled in the laboratory.
15. What is the use of a cryostat?
16. How is rotary microtome dierent from sliding microtome?
17. State the principle of uorescent microscope.
18. List the stains used in identifying bacteria, fungi and protozoan.