Histo

20.What are mounting media and how are they used in histopathology?What are the different
type of mounting media that can be used to mount tissue sections ,what are their advantage and
disadvantage?

Ans. The mounting medium is the solution in which the specimen is embedded generally under a
cover glass.It may be liquid, gum or resinous, soluble in water alcohol or other solvents and be
sealed from the external atmosphere by non-soluble ringing media. The main purpose of mounting
media is to physically protect.

 Add the mountant on the coverslip in the center.

 Bring the slide down (invert) to the coverslip and let the surface tension pull the coverslip. Use
only enough mountant to fill the space on the coverslip/slide and not excess and this assessment
comes with experience.
 Too little mounting media will cause air bubble at the edges of coverslip and one will be tempted
to press down on the coverslip to ensure a tight seal.
 Too much mounting media will make it messy and move the samples around and it can make the
sample impossible to image at 1000x due to the very short working distance of high
magnification oil immersion objective lens.

different type of mounting media- There are two main types of mounting media: water based
(aqueous/hydrophilic/non-adhesive) and organic solvent based
(resinous/hydrophobic/adhesive).

organic solvent based (resinous/hydrophobic/adhesive)- These are natural or synthetic

resins dissolved in benzene, toluene or xylene and are used when a permanent mount is
required and frequently used in routine H and E staining procedures.

 Canada balsam-It is a natural resin that is dissolved in xylene or toluene to create

a mounting medium.(Advantages and disadvantages from pdf)
 Dammar balsam - are similar to Canada balsam and are rarely used as mountant
because of dirt and impurities usually present and difficulty of filtering prepared
mountant.
Advantage-
1. Dammar balsam is highly transparent, allowing light to pass through
easily.
2. Dammar balsam has a refractive index close to that of glass, which helps
reduce light refraction and scattering. This property enhances the clarity
and sharpness of the image
3. Dammar balsam provides excellent long-term preservation for mounted
samples.
4. It has a low viscosity, making it easy to apply and spread evenly on the
slide
Disadvantages
 1. Over time, Dammar balsam tends to yellow and become brittle. This
can impact the appearance and quality of the mounted specimen.
2. Dammar balsam is not compatible with certain staining techniques
and specific imaging modalities. For example, it may not be suitable
for fluorescence microscopy due to its auto fluorescence properties.
Other modern mounting media may offer better compatibility with a
wider range of applications.
3. Dammar balsam forms a strong bond with the slide, which can make
the removal of coverslips challenging.
 Euparal - is a semi-synthetic mountant. Euparal is commonly used to mount
histological specimens.
Advantage-
1. Euparal is highly transparent, allowing for clear visualization of the
sample under a microscope.
2. Euparal has a refractive index close to that of glass, reducing light
refraction and scattering. This property enhances image clarity and
sharpness.
3. Euparal provides excellent long-term preservation, protecting the
mounted specimen from degradation and damage.
4. Euparal is compatible with various staining techniques, allowing for
the retention of stained colors and patterns during the mounting
process.
Disadvantage-
1. Euparal has a relatively long drying time, which can be a
disadvantage if quick mounting is required
2. Euparal contains toxic substances, such as xylene and toluene,
which are used as solvents in its preparation
3. Euparal may not be compatible with certain modern imaging
techniques, such as fluorescence microscopy
 DPX- DPX is a synthetic non-aqueous mounting medium for microscopy. A
traditional resin-based slide mountant with xylene solvent.(from pdf advantage
and disadvantage)

 Permount - Permount Mounting Medium consists of a synthetic resin dissolved in

Toluene. Simply apply the medium directly to the sample on a microscope slide for
short-term mounting and long-term preservation. (from pdf)

Other organic solvents are-

Histomount
Cover bond
Gurr's neutral mounting medium
Histoclad
Pro-texx
Technicon Resin
Uv-inert
water based (aqueous/hydrophilic/non-adhesive)- Aqueous mounting medium are used for
mounting sections from distilled water when the stains would be decolorized or removed by alcohol
and xylene as would be the case with most of the fat stains (Sudan methods).Water based mounting
media are-
 Water
 Glycerine jelly
 Glycerine-Glycerol
 Apathy's medium .
 Farrant's medium
 Highman's medium
 Fructose syrup
 Polyvinyl alcohol.

 Water- ater serves as a convenient temporary mountant for some whole specimens
for examining certain microorganisms live (saline mount) and particularly when
checking sections during the staining procedures.(from pdf)
 Glycerine jelly- his is usually regarded as the standard mountant for fat stains.
Dissolve the gelatin in the distilled water in a conical flask in a water bath and add
glycerine and phenol mix well and store.
Advantage-
1. Glycerine jelly is highly transparent, allowing for clear visualization of the
specimen under a microscope.
2. Glycerine jelly is easy to work with and apply to slides. It has a gel-like
consistency, making it simple to spread evenly over the specimen.
3. Glycerine jelly provides good long-term preservation, helping to protect the
mounted specimen from degradation over time.
Disadvantage-
1. Glycerine jelly has a lower refractive index compared to other mounting
media, such as Permount. This can result in increased light refraction and
scattering, which may impact image quality and clarity.
2. Glycerine jelly has a longer drying time compared to some other mounting
media. It may take longer for the medium to dry completely, potentially
delaying further analysis or observation.
3. Glycerine jelly is soluble in water, which can be problematic if the slides
come into contact with water or undergo water-based processing steps. It
may cause the mounting medium to dissolve or be removed.
 Glycerine-Glycerol- This commonly utilized aqueous mountant is a mixture of
glycerol and gelatine. It should set quite hard but for long-term preservation
sections are best ringed and sealed. Various formulations are in use.
Advantage-
1. glycerol mounting media is highly transparent, allowing for clear
visualization of the specimen under a microscope. It provides good optical
clarity, enabling detailed observation of the sample.
2. Glycerine-glycerol has hygroscopic properties, meaning it can absorb and
retain moisture. This feature helps prevent dehydration and shrinkage of the
mounted specimen,
3. Glycerine-glycerol mounting media is compatible with a wide range of
staining techniques and is suitable for various microscopy applications.
Disadvantage-
 1. glycerol has a relatively longer drying time compared to some other
mounting media.
2. Glycerine-glycerol may not be suitable for certain advanced imaging
techniques, such as fluorescence microscopy, due to its autofluorescence
properties
3. glycerol mounting media may have stability issues over time.
 Polyvinyl alcohol-often used as a mountant in immunofluorescence microscopy, has
been recommended as an alternative for glycerine jelly.
Advantage-
1. PVA mounting medium is highly transparent, allowing for clear
visualization of the specimen under a microscope
2. It can be used with both water-soluble and organic-soluble dyes.
3. it forms a protective layer that helps prevent drying out and preserves
the structural integrity of the specimen.
4. It can be used for both routine and specialized microscopy applications.

Disadvantage-
1. Exposure to incompatible chemicals can cause the mounting media to
dissolve or degrade, leading to loss of specimen integrity.
2. PVA mounting media may undergo some shrinkage during the drying
process, which can affect the positioning and integrity of the specimen.
Care must be taken to minimize this effect.
3. PVA mounting media requires a longer drying time compared to some
other mounting media options.

19.What is the Hematoxylin and Eosin staining technique ? How it is used to visualize tissue
sections?What is counter staining and why it is important in the Hematoxylin and eosin staining
technique?
Ans.
 A common laboratory method that uses two dyes called hematoxylin and eosin that
make it easier to see different parts of the cell under a microscope.Steps of H&E
staining-
1. Deparaffinization: flame the slide on burner and place in the xylene. Repeat the treatment
to remove the wax.
2. Hydration: Drain xylene and hydrate the tissue section by passing through decreasing
concentration of alcohol baths (100%, 90%, 80%, 70%) and water.
3. Nuclear Staining: Stain in hematoxylin for 3-5 minutes.
4. Wash in running tap water until sections “blue” for 5 minutes or less.
5. Differentiation: selective removal of excess dye from the section). Dip in 1% acid alcohol (1%
HCl in 70% alcohol) for a few seconds.
6. Blueing: Rinse in running tap water. Dip in ammonia water until the sections become blue,
followed by tap water wash.
7. Counterstain: Stain in 1% Eosin Y for 10 minutes.
8. Wash in tap water for 1-5 minutes.
9. Dehydration: Dehydrate in increasing concentration of alcohols.
10. Clearing: Put slides in two xylene baths for clearing.
11. Mounting: Mount in DPX or other mounting media.
12. Observe under compund microscope.
  The principle behind H & E stain is the chemical attraction between tissue and
dye. Hematoxylin, a basic dye imparts blue-purple contrast on basophilic
structures, primarily those containing nuclei. An acidic eosin counterstains the
basic elements . Hematoxylin shows the ribosomes, chromatin (genetic material)
within the nucleus, and other structures as a deep blue-purple color. Eosin shows the
cytoplasm, collagen, connective tissue, and other structures that surround and
support the cell as an orange-pink-red color. Hematoxylin and eosin staining helps
identify different types of cells and tissues and provides important information about
the pattern, shape, and structure of cells in a tissue sample. It is used to help diagnose
diseases, such as cancer. Also called H and E staining.
 A counterstain is a stain with colour contrasting to the principal stain, making the
stained structure easily visible using a microscope.In this case Eosin is used to stain
tissues after the primary dye.

Importance of eosin staining-

1. Eosin is a fluorescent, xanthene dye or red dye formed with the action of
bromine and fluorescein which binds to salts with eosinophilic compounds
containing positive charges.
2. In order to understand the general histological architecture of tissues, eosin is the
most suitable stain combined with an alum hematoxylin. This stained solution can
examine cytoplasm, collagen, and muscle fibers under a microscope.
3. An acidic eosin counterstains the basic elements. Eosin shows the cytoplasm,
collagen, connective tissue, and other structures that surround and support the
cell as an orange-pink-red color.
4. Used to differentiate one component or cellular structure from another or to
differentiate an entity from another in a specimen.
5. It does so by coloring a portion of a specimen that remain uncoloured following
the 1st staining using hematoxilin.

18.Explain the principle and applications of supravital staining in histopathology and

cytopathology.What are the advantages and limitations of this technique compared to other
staining method.
Ans.
The principle of the supravital method is to stain with relatively non toxic dyes some of the
structures of living cells removed from the body so that these living cells can be examined directly
under the microscope.These structures are the mitochondria which are stainable by janus green and
certain granules and larger bodies which are stainable by neural red.

Application- .
1. To determine the cell viability: Evans and Schulman noted that when leukocytes suspended
in solution of Tryphan blue, readily took up the dye upon mechanical injury. This is by
supravital staining.

2. Chromoendoscopy: Is a procedure of using vital stains to identify abnormal mucosa. In patients

who are at increased risk for squamous cell carcinoma, vital staining with lugol's iodine is performed
at the time of upper endoscopy to aid in cancer detection, in such cases it is applied through spray
catheter. Here, the dye stains the glycogen in normal squamous epithelium as dark brown. Areas
that are unstained particularly those that are larger than 5 mm are likely to be dysplastic / malignant
and can be targeted for biopsy. This is an intravital staining, which is quick and easy to perform.
3.To obtain cytological details of Protozoa.

4. Used to stain various cell organelles - Janus Green stains cell mitochondria, DIOC (3,3 Dihexyloxa
carbocyamine lodide) is used stain endoplasmic reticulum as a fluroscent dye, Rhodamine 1,2,3
fluroscence for mitochondria.

5. Used to stain various inclusions within the cell - Nile red fluroscence stains lipid vesicles.

6. Nerve fibers - Using Methylene blue by Coers and Woolf which is a supravital staining.

Advantages –
1. Unlike other staining techniques,supravital staining does not required fixation, dehydration
or embedding of the tissue sample.
2. It is quick and easy to use.
3. This method can be performed on fresh tissue samples, allowing for the observation of the
living cells in their natural state.
4. It is inexpensive and can be used for screening oral cancers.
5. It provides more cytological detail of the cell where the specific organelles can be stained.

Limitation-
1. Most of these stains are dyes and are extremely toxic, which can result in death, so
it has to be diluted to a larger extent
2. Some of the dyes like flourochromes are carcinogenic and mutagenic. So one should
avoid breathing any of the powder or allowing it to come in contact with the skin
while preparing the solutions
3. Some vital dyes, like Tryphan blue stain clothes and skin for a long time. So care
should be taken to avoid direct contact with the dye.
4. Supravital staining has limited applications, as it can only stain living cells and cannot
be used for the detection of dead or fixed cell.
5. The dyes used in supravital staining have a limited range of specificity, and may bind
to multiple cell components, making it difficult to identify specific structures within
the cell.
6. supravital staining may alter the morphology or behavior of the living cells,
depending on the type of dye used or the concentration of the dye

17.Discuss the types of dyes and stains used in histology and cytopathology.What are the
difference between natural,acidic,basic,neutral,fluorescence and moderent dye?(5+5+5)

Ans.
1. Hematoxylin and eosin (H&E) is the most widely used stain in histology and allows
localization of nuclei and extracellular proteins. Hematoxylin, not a dye itself,
produces the blue Hematin via an oxidation reaction with nuclear histones causing
nuclei to show blue. Eosin, most often tetrabro-mofluorescein, disodium salt, is
employed as a counterstain that stains most tissues, especially acetic, with a red
(acetic cytoplasm) blue, pink (collagen or muscle) or purplish (basic cytoplasm)
2. Feulgen staining The Feulgen technique selectively stains DNA, and under controlled
conditions, can be used for the photometric determination of DNA content. Feulgen
reaction chemically converts the deoxyribose in DNA into a magenta
colored product.
 3. PAS staining- PAS helps to demonstrate glycogen, cellulose and starch. This is useful
to detect glycogen deposits in liver when glycogen storage disease is suspected.
Basement membrane of various tissues may also be visualized through the PAS stain.
The PAS is most commonly used to demonstrate the thickness of glomerular basement
membrane when renal disease is being assessed.

4. PAP Staining- t is a polychromatic stain that uses multiple dyes to differentially stain
various components of the cells. It is a histological and cytopathological staining
technique used to differentiate cells in a smear preparation. It is the most common
screening method for cervical cancer.

DYE-

Dyes used in staining Dyes are classified in various ways : 1. According to source- a.
Natural b. Synthetic
2. Affinity to tissues - a. Acidophilic b. Basophilic
3. Chemical composition-a. Thiazines b. Azo-dyes c. Rosailins
Synthetic dyes have greater staining capacity, much greater spectrum of colours.

Natural dyes These are very few in numbers. They are mainly two in common use.
1. Haematoxylin : This is the most popular dye used as a nuclear stain. It is derived
from the log tree mainly found in Mexico. It develops staining property after
oxidation. It is a weak dye and to make it give sharp stain a mordant is needed
2. Carmine : It is a scarlet dye made from the ground bodies of cochineal beetles.

Acid dyes are anionic. They form salts with cationic groups in cells and tissues,
particularly the ionized amino groups of proteins. Acidophilic or oxyphilic is applied
to parts, which show a greater affinity for acid dyes. The cytoplasm is usually
acidophilic. Eosinophilic components are cationic compounds that have an affinity
for that acid dye.

Basic dyes are cationic. They form salts with tissue anions (components that carry a
net negative charge), especially the phosphate groups of nucleic acids and the
sulfate groups of the glycosaminoglycans. Basophilic is the term used to designate
the components of a cell or tissue, which take up the basic stain rather than the acid
stain of a combination. Nuclei are basophilic.

Neutral dyes are produced by combining basic and acid dyes. Since the base dye
stains the nuclei whereas the acid dye stains the cytoplasm a neutral dye would therefore
stain both the nuclei and the cytoplasm. 1 Eosinate of methylene blue and Giesma stain are
examples of a neutral dye.
 Fluorescent dyes, also known as reactive dyes or fluorophores, have been used by
biologists for decades. Fluorescent dyes offer higher photostability and brightness
compared to fluorescent proteins and do not require a maturation time. However,
fluorescent dyes are usually targeted to proteins of interest by antibody conjugates or
peptide tags.

Mordant is a chemical that serves as a link between the dye and the substrate. The result is
an insoluble compound that helps adhere the dye to the cells.The most useful mordants for
hematoxylin are salts of aluminum, iron, tungsten, and occasionally lead.

16.Explain the principles of freeze drying and the applications of this technique in various
fields.Additionally discuss the advantages and limitations of freeze drying compared to
other drying method.(8+7)

Ans- Principle

The process of lyophilization involves a phenomenon known as sublimation. An object that

is liquid passes directly from one state to another, unlike an object that is solid (ice). It
involves removing the water from the frozen state material and then heating it (by
conduction or radiation or even both) to the point that the evaporated liquid leaves only the
solids or dry components of the original liquid. It is necessary to dry materials at lower
temperatures and pressures than the triple point.

The process of freeze drying is complex and requires an optimal balance between
equipment, product, and processing technique. After a product has been frozen, water is
removed from it and the product is put under a vacuum, causing the ice to turn directly into
vapour without passing through a liquid phase. Under these conditions, frozen material can
be sublimated at temperatures below the triple point of liquid. Temperatures and pressure
remain low throughout. lyophilized product is obtained after the sample is prepared, frozen,
dried, and then secondary dried. During lyophilization, water is removed from the sample
through a concentration gradient of water vapour. Water vapour pressure increases as
temperature rises during the primary drying process.Keeping the primary drying
temperature above the critical temperature will prevent the loss of cake structure.

Application

 Vaccines and other injectables are often freeze-dried by pharmaceutical companies

in order to increase their shelf life.
 Material can be stored, shipped, and later reconstituted into its original form by
removing the water and sealing the material in a vial under vacuum.
 By freeze-drying biologicals, it is possible to preserve them and make them
lightweight.
 The freeze-drie form of blood products is used for preservation.
 Often, freeze-dried products are used in chemical synthesis to stabilize them or
make them more soluble in water.
  By freeze-drying, solvents can be effectively removed that can be used in the final
steps of bio- separation.

•Furthermore, low molecular weight substances that are too small to be removed by a
filtration membrane can be concentrated using this technique.

Merits

 Vacuum conditions provide good protection for oxidizable substances.

 A long drying time is attributed to water removal of 95%-99.5%.
 There is uniformity in the content of loaded quantities.
 Aseptic process resulted in little contamination.
 Volatile chemicals and nutrient- and fragrance-sensitive components are minimized.
 Due to the fact that growth of bacteria and enzymes cannot occur at low
temperatures, there are very few changes in the properties.
 Under normal temperatures, thermostable products can be transported and stored
 Reconstitution is done rapidly, usually within 10 seconds.
 The dried material maintains a homogeneous distribution of constituents
 It is possible to achieve and maintain product stability.

Demerits

 It may be necessary to use a high vacuum to remove volatile compounds.

 Operation with the highest unit cost.
 The ability of an individual drug to cope with low temperatures is one of its primary
drawbacks.
 Aseptic loading of vials into the dryer chamber and sterilization and sterility
assurance of the chamber raises some concerns.