Assignment Topic:Preparation Of Various Reagents, Buffer And Stain Used In

Necropsy And Clinical Pathology Laboratory

REAGENTS:

A reagent is a compound or mixture added to a system to start or test a chemical reaction. A

reagent can be used to determine the presence or absence of a specific chemical substance
as certain reactions are triggered by the binding of reagents to the substance or other
related substances

Reagent Examples:
More reagents are inorganic compounds or small organic molecules. Some commonly used
reagents are:
1.Grignard reagent,
2. Tollens’ reagent,
3.Fehling’s reagent,
4.Millon’s reagent,
5.Collins reagent, and
6. Fenton’s reagent
These are named reagents. However, not all reagents are named with the word “reagent.”
Reagents also include solvents, enzymes, and catalysts.

A:Preparation Of Grignard Reagent:

The process of preparing Grignard reagents is described in the points provided below. It can
be noted that many of these reagents can also be purchased commercially.

A Grignard reagent is an organomagnesium compound which can be described by the

chemical formula ‘R-Mg-X’ where R refers to an alkyl or aryl group and X refers to a halogen.

They are generally produced by reacting an aryl halide or an alkyl halide with magnesium

B:Preparation OF TOLLEN`S Reagent:

It is prepared by using a two-step procedure.

Step 1: Aqueous silver nitrate is mixed with aqueous sodium hydroxide.

AgNO3+NaOH2AgOH→AgOH+NaHO3→Ag2O+H2O

Step 2: Aqueous ammonia is added drop-wise until the precipitated silver oxide completely
dissolves.

Ag2O+4NH3+H2O→2Ag(NH3)+2+2OH−

C:Preparation OF Fehlings Reagent:

Fehling's solution is prepared by combining two separate solutions:
Fehling's A, which is a deep blue aqueous solution of copper(II) sulfate, and
Fehling's B, which is a colorless solution of aqueous potassium sodium tartrate (also known
as Rochelle salt) made strongly alkali with sodium hydroxide.

These two solutions, stable separately, are combined when needed for the test because the
copper(II) complex formed by their combination is not stable: it slowly decomposes into
copper hydroxide in the alkaline conditions. The active reagent is a tartrate complex of Cu2+,
which serves as an oxidizing agent. The tartrate serves as a ligand. However, the
coordination chemistry is complex and various species with different metal to ligand ratio
have been determined

D:Preparation Of Millons reagent :

Millon’s reagent is prepared by dissolving mercuric nitrate in nitric acid, and then adding
water to dilute it.

The reagent is used in Millon reaction test wherein few drops of the reagent are added to the
test solution, and then heated gently to boiling. When a red-coloured solution or red
precipitate forms, this means that tyrosine is present (+ result)

E:Preparation Of Collins reagent:

Collins reagent or dipyridine chromium (VI) oxide is prepared by the addition of one
equivalent chromium trioxide to a stirred solution of two equivalents of pyridine in methylene.
It precipitates in a yellow microcrystalline form, and on continuous stirring at 15°C, it reverts
to a deep red macrocrystalline form, which can be isolated, dried, and stored. This complex
is both challenging and dangerous to make. The reaction of chromium trioxide with pyridine
is very hygroscopic, exothermic, and can inflame during its preparation. The preparation
should be conducted in a hood, observing the precautions

F:Preparation Of Fentons reagent :

The Fe2+ catalyst for the Fenton's Reagent was prepared by dissolving 69.50 g of
analytical-grade ferrous sulfate (FeSO4. 7H2O) in 500 mL of sterilized deionized water (DI)
containing 5 mL of concentrated H2SO4 (Weast, 1980

BUFFER SOLUTIONS:

Buffers are solutions that resist a change in pH on dilution or on addition of small amounts of
acids or alkali.

A lot of biological and chemical reactions need a constant pH for the reaction to proceed.
Buffers are extremely useful in these systems to maintain the pH at a constant value. This
does not mean that the pH of buffers does not change. It only means that the change in pH
is not as much as it would be with a solution that is not a buffer.
 TYPES OF BUFFER:

Buffers are broadly divided into two types –

1:acidic and
2: alkaline buffer solutions.
Acidic buffers are solutions that have a pH below 7 and contain a weak acid and one of its
salts. For example, a mixture of acetic acid and sodium acetate acts as a buffer solution with
a pH of about 4.75.

Alkaline buffers, on the other hand, have a pH above 7 and contain a weak base and one of
its salts. For example, a mixture of ammonium chloride and ammonium hydroxide acts as a
buffer solution with a pH of about 9.25

Preparation Of Different Buffer Solution:

A:Preparation Of Bicarbonate -Carbonate buffer (pH 9.2-10.8)

Required components:
Component Amount Concentration
Sodium
bicarbonate(
mw:84.01
gm/mol) 1.05 g 0.0125 M
2.Sodium
carbonate
anhydrous
(mw: 105.99
gm/mol) 9.274 g 0.0875 M

1.Prepare 800 mL of distilled water in a suitable container.

2.Add 1.05 g of Sodium bicarbonate to the solution.
3.Add 9.274 g of Sodium carbonate (anhydrous) to the solution.
4.Add distilled water until the volume is 1L.

B:Preparation Of Citrate Buffer (pH 3.0–6.2)

Required components:

Component Amount Concentration

1.Sodium Citrate
dihydrate
(mw: 294.10
g/mole) 16.34 g 0.05556 M
2.Citric Acid
(mw: 192.12
g/mol) 4.503 g 0.02344 M

1.Prepare 800 mL of distilled water in a suitable container.

2.Add 16.34 g of Sodium Citrate dihydrate to the solution.
3.Add 4.503 g of Citric Acid to the solution.
4.Adjust solution to final desired pH using HCl or NaOH
5.Add distilled water until the volume is 1L.

C.Preparation Of phosphate buffer Saline Sokution(pH 5.8–8.0 at 25°C)

Preparation
For 1 litre of 10x PBS:

1.Begin with 800ml of distilled water in a Duran bottle.

2.Measure out and add 80.1g of NaCl.
3.Add 14.4g of Na2HPO4.
4.Add 2g of KCl.
5.Add 2.7g of KH2PO4
6.Bring the pH to 7.4 or 7.2 (depending on the application) using HCl.
7.Add distilled water until the total volume reaches 1 litre.
8.Sterilise in an autoclave on a liquid cycle.
9.Store at room temperature.

For 1 litre of 1x PBS:

1.Begin with 800ml of distilled water in a Duran bottle.

2.Measure out and add 8g of NaCl.
3.Add 1.44g of Na2HPO4.
4.Add 0.2g of KCl.
5.Add 0.24g of KH2PO4
6.Bring the pH to 7.4 or 7.2 (depending on the application) using HCl.
7.Add distilled water until the total volume reaches 1 litre.
8.Sterilise in an autoclave on a liquid cycle.
9.Store at room temperature

D:Preparation of Tris- Buffered Saline(TBS 10X Stock Solution)

Tris-buffered saline (TBS) is an excellent wash buffer for many types of immunoassays.

To make 1 L of 10X TBS stock solution, 1.dissolve 24 g Tris and 88 g NaCl in 900 mL of
water
2. adjust the pH to 7.6 and final volume to 1L.

E:PBS (Mg2+- and Ca2+-free)

137mM NaCl
2.7mM KCl
4.3mM Na2HPO4
1.4mM KH2PO4
The final pH should be 7.4

STAINS:

Staining:

Staining is widely used in histopathology and diagnosis, as it allows for the identification of
abnormalities in cell count and structure under the microscope. A huge range of stains is
used in histology, from dyes and metals to labeled antibodies. For staining, paraffin sections
of tissue are normally used. They are rehydrated and then made translucent (cleared) using
a clearing substance such as xylene, before being stained.

The most common stains used in histology and pathology are:

1.Hematoxylin and Eosin Stain
2.Special Stain
a)Van Gieson
b)Giemsa
c)Nissl
d)PAS
e)Wright And Wright Giemsa Stain
f) Aldehyde Fuschin stain.

Hematoxylin&Eosin Stain:
Hematoxylin and Eosin (H&E) staining is the most widely used staining technique in
histopathology. As its name suggests, H&E stain makes use of a combination of two dyes,
namely hematoxylin and eosin.

Principle
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 nucleic acid moeties such as chromtatin, ribosomes and cytoplasmic
regions rich in RNA. An acidic eosin counterstains the basic elements such as RBCs,
cytoplasm, muscle and collagen in varying intensities of pink, orange and red.

Preparation:
Requirements:
1.Harri’s Hematoxylin stain
A = 1 gm hematoxylin in 10 ml ethanol
B = 20 gm ammonium alum in hot distilled water
2.Mix A & B, boil and add 0.5 gm of mercuric oxide and filter.
3.Eosin solution
Yellow eosin = 1 gm
Distilled water = 80 ml
Ethanol = 320 ml
Glacial Acetic Acid = 2 drops
4.0.5% HCl
5.Dilute ammonia water

Special Stains:

Special stains are used to identify and demonstrate particular structures and tissues which
are not visualized by H&E stains. There are variety of special stains, a brief detail of some of
them is given below.

Van Gieson

The van Gieson stain is a very common stain used to highlight the difference between
collagen and other connective tissue such as muscle tissues. It is often used to identify the
characteristic arrangement of fibers in different types of tumours. The stain uses a mixture of
picric acid and acid fuchsin to penetrate the tissue sample, causing collagen to become red.
Surrounding muscle tissues and blood cells are stained yellow.

Preparation:
100 mL of Saturated Aqueous solution of Picric Acid added to 5 mL of 1% Aqueous solution
of Acid Fuchsin.

Giemsa Stain:

This is a blood stain that can be used histopathologically. It was designed primarily to
demonstrate malarial parasites. It was later used in histology because of its high quality
staining capabilities of chromatin and nuclear membranes. It is also used to stain blood cells,
so that their composition and structure may be observed

Nuclei are stained purple, and cytoplasm is stained blue to pale pink, depending on cell
type.Basophils show dark blue granules and eosinophils show orange granules.

Preparation of the Giemsa Stain Stock solution:

1.Add 3.8 g of Giemsa powder and dissolve into 250 ml of methanol,
2.heat the solution up to 60°C.
3. Then, add 250 ml of glycerin to the solution slowly.
4.Filter the solution and leave it to stand for about 1-2 months before use.

Nissl stain:

Nissl staining is used to visualise Nissl substance (clumps of rough endoplasmic reticulum
and free polyribosomes), which is found in neurons. This stain distinguishes neurons from
glia and the cytoarchitecture of neurons can be well studied with the help of this stain. A loss
of Nissl substance can signify abnormalities such as cell injury or degeneration, which in turn
can indicate disease.

PAS (Periodic Acid Schiff)

This stain colours glycogen, and is therefore used to look at membranes, mucosubstances
as well as the presence of fungus. The process of PAS staining usually involves two steps,
1.the first one is the oxidation reaction with periodic acid leading to the formation of
aldehydes,
2 the second step is the demonstration of these aldehydes with the help of Schiff’s reagent.

Preparation:

Schiff's reagent: Pour 10 ml of 10% formalin to a beaker, add a few drops of the Schiff's
reagent to be tested. Good Schiff's reagent will rapidly turn a red purple color

Wright And Wright Giemsa Stain:

The Wright and Wright Giemsa stains are polychromatic stains because they contain eosin
and methylene blue.Different blood cell types become stain differently allowing them to be
differentiated by the observer.

These changes are listed below:

1.Neutrophils have purple nuclei, light pink cytoplasm, and reddish-purple granules
2.Basophils have dark blue or purple nuclei, and very dark purple granules.
3.Eosinophils have blue nuclei, light pink cytoplasm, and red granules.
4.Red blood cells become red or pink
Platelets appear purple

The process for staining blood with Wright stain is as follows:

1.A drop of blood is placed between glass slides and air dried in a thin layer to be stained
2 The smear is then placed in Wright stain for 1 to 3 minutes
3.A phosphate buffer is added for double this time
4.It is then rinsed with water, and dried by blotting

Preparation:
A. 15 to 17 parts of Wright staining powder in weight,
B. 3 to 4 parts of Giemsa staining powder in weight,
C.33 to 35 parts of glycerinum in volume and D.5000 parts of methanol in volume.

Aldehyde Fuchsin Stain:

This stain, sometimes known as Gomori's aldehyde fuchsin stain, was originally created to
stain elastic fibers. However, it can also be used to stain other tissues, particularly the beta
cell granules in the pancreas. It is also highly selective with few other high affinity basophilic
sites, like mast cell granules and cartilage matrix.

Preparation :
Aldehyde fuchsin solution
a.Basic fuchsion - 1 g
b.70% ethanol
c.200 ml
Concentrated hydrochloric acid (HCl)
d.Paraldehyde -2 ml

THANK YOU