LIST OF EXPERIMENTS:

1. Preparation of solutions: 1) percentage solutions, 2) molar solutions, 3) normal solutions

2. Standardization of pH meter, preparation of buffers, emulsions.

3. Spectroscopy: Determination of absorption maxima (λmax) of a given solution

4. General tests for carbohydrates, proteins and lipids.

5. Identification of Blood Collection Tubes and Phlebotomy equipment

6. Preparation of serum and plasma from blood

7. Estimation of Haemoglobin and blood glucose

8. Estimation of creatinine, urea and Uric acid

9. Separation of proteins by SDS electrophoresis (Demo) and amino acids by thin layer
chromatography (Demo).

10. Urine physical and chemical examination (protein, reducing substances, ketones, bilirubin
and blood)

11. Basic staining – Hematoxylin and eosin staining.

12. Special stains – cresyl fast Blue (CFV)- Trichrome – oil red O – PAS

13. Types of Staining : Simple stain, Gram stain

14. Study of parts of compound microscope

15. Study of Histopathological slides of benign and malignant tumours.

16. Study of Haematology slides of anemia and leukemia.

1
CONTENT

Ex. Page Faculty

Date Name of the Experiment Marks
No. No. Sign.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

2
12.

13.

14

15.

16.

17.

18.

19.

20.

21.

3
Expt. No.:_______ Date:____________

PREPARATION OF SOLUTIONS

AIM:

To prepare the percentage solutions, molar solutions and normal solutions.

REQUIREMENTS:

Weighing balance, sodium chloride, ethylene glycol, sulphuric acid, beakers.

PROCEDURE:

Percentage solution:

Percentage by weight (w/v) = [Mass of solute (g) / Volume of solution (ml)] x 100

For preparing 10% NaCl solution, weigh 10g of sodium chloride. Pour it into a graduated
cylinder or volumetric flask containing about 80ml of water. Once the sodium chloride has
dissolved completely (swirl the flask gently if necessary), add water to bring the volume up to
the final 100 ml.

Percentage by volume (v/v) = [Volume of solute (ml) / Volume of solution (ml)] x 100

For preparing 1000ml of a 5% by volume solution of ethylene glycol in water. First,

express the percent of solute as a decimal: 5% = 0.05. Multiply this decimal by the total volume:
0.05 x 1000ml = 50ml (ethylene glycol needed). Subtract the volume of solute (ethylene glycol)
from the total solution volume: 1000ml (total solution volume) - 50ml (ethylene glycol volume) =
950ml (water needed). Dissolve 50ml ethylene glycol in a little less than 950ml of water. Now
bring final volume of solution up to 1000ml with the addition of more water.

So, 50ml ethylene glycol / 1000ml solution x100 = 5% (v/v) ethylene glycol solution.

4
Molar solution:

Molar solutions are the most useful in chemical reaction calculations because they directly
relate the moles of solute to the volume of solution.

Molarity (M) is defined as moles of solute / 1 liter of solution or gram-molecular masses

of solute / 1 liter of solution.

The molecular weight of NaCl is 58.44, so one gram-molecular mass (=1 mole) is 58.44g.
The atomic mass (or weight) of Na is 22.99, the atomic mass of Cl is 35.45, so 22.99 + 35.45=
58.44.

Dissolve 58.44g of NaCl in a final volume of 1 liter, to prepare 1M NaCl solution.

To make molar NaCl solutions of other concentrations dilute the mass of salt to 1000ml
of solution as follows:

0.1M NaCl solution requires 0.1 x 58.44 g of NaCl = 5.844g

0.5M NaCl solution requires 0.5 x 58.44 g of NaCl = 29.22g

2M NaCl solution requires 2.0 x 58.44 g of NaCl = 116.88g

Normal solution:

Normality (N) is defined as the gram-equivalent weight of solute per liter of solution.

The normality of a 1.0 liter NaCl solution that contains 1.0 gram-equivalent weight will be
the GMW of NaCl divided by the valence of NaCl.

GMW of NaCl = 22.99 + 35.45 = 58.44 g

N = GMW/valence (the valence for NaCl is 1.0)

58.44 g/1.0 = 58.44 g = 1.0 gram-equivalent weight of NaCl = 1N solution of NaCl

In this situation, because NaCl has a valence of one, the molarity and normality of the
solution are the same.

5
 Some compounds, however, will not have the same normality as molarity, as in the case
of H2SO4:

The normality of a 1.0 liter solution of H 2SO4 containing 1.0 gram-equivalent weight will
be the molecular weight of H2SO4 divided by the valence of H2SO4:

GMW of H2SO4 = 1(2) + 32.06 + 16(4) = 98 g

N = GMW/valence (the valence for H2SO4 is 2, as there are 2 H ions that could be
displaced)

98 g/2 = 49 g = 1.0 gram-equivalent weight of H2SO4 = 1N solution of H2SO4

The molarity of this 1N solution of H2SO4 would be 0.5 (M = g /GMW per liter or 49g/98g=
0.5)

RESULT:

The procedure to prepare percentage solutions, molar solutions and normal solutions
were studied and the solutions were prepared according to the given values.

Expt. No.:_______ Date:____________

6
 STANDARDIZATION OF pH METERS, PREPARATION OF BUFFERS
AND EMULSIONS
AIM:

To study about the standardization of pH meters and the

preparation of Trisbuffer, potassium phosphate buffer and emulsions.

REQUIREMENTS:

Potassium dihydrogen phosphate, potassium hydrogen phosphate, Tris buffer powder,

alkali or acid to adjust the pH, pH meters, distilled water and weighing balance.

THEORY:

pH METER:

pH is the unit to measure the degree of acidity or alkalinity of a solution. It is defined as

the negative logarithm of hydrogen ion concentration i.e. pH = -log [H+]

Water molecules dissociate into H+ and OH- ions. In the state of equilibrium, the molar
concentration of H+ and OH-ions is 10-7 M each, at 25 °C. i.e. [H+][OH-] = 10-14 at 25 °C.

The pH value is therefore, measured on a scale of 0 to 14, where pH 7.0 is neutral, pH<
7.0 is acidic and pH > 7.0 is basic. An acidic solution has H+ concentration higher than OH- ions
whereas a basic solution has higher OH -concentration than H+ ions. Since pH scale is a
logarithmic scale, an increase in 1 pH unit corresponds to a reduction of H+ : OH- ratio by a factor
of 10.

A standard pH meter consists of an electrode for measuring pH. This electrode has a
glass membrane which is sensitive to H + ion concentration. Saturated KCl solution is used as
the reference solution as its output does not vary with H+ ion activity. Also, a high input meter
measures the difference between the voltages of the two electrodes which then translates the
voltage difference into pH and displays it on the screen.

7
BUFFER:

A buffer solution is a solution that resists changes in pH either when diluted or when
limited amount of acid or base are added to it. Such a solution can be prepared by combining a
weak acid and its salts with a strong base or analogously, a weak base and its salt with a strong
acid.

PHOSPHATE BUFFER:

The phosphates are among the most widely used buffers. These solutions have high
buffering capacity and are very useful in the pH range 6.5 to 7.5. Because phosphate is a natural
constituent of cells and biological fluids, its presence affords a more “natural” environment than
many buffers. Sodium or potassium phosphate solutions of all concentrations are easy to
prepare.

The major disadvantages of phosphate solutions are

i. Precipitation or binding of common biological cations (Ca2+ and Mg2+) and

ii. Inhibition of some biological processes, including some enzymes.

KH2PO4 + K2HPO4

[Weak acid] [salt, conjugated base]

The Henderson-Hasselbalch equation describes the behavior of such a buffer and for the
mixture of weak acid and its salts with the strong base has the form:

8
 pH = pKa + log Cs/Cac

where

pKa is the negative logarithm of the dissociation constant for the weak acid

Cs is the substrate concentration of the salt

Cac is the substrate concentration of weak acid

The equation of a weak base and its salt with a strong acid has the form:

pH = pKw - pKb+ log Cb/Cs

where

pKb is the negative logarithm dissociation constant for weak base

Cb is the substrate concentration of the base

Cs is the substrate concentration of the salt

pKw = 14 = -log10 -14 [Ionic product of water]

TRIS BUFFER:

The use of the synthetic buffer Tris [tris(hydroxymethyl)aminomethane] is now probably

greater than that of phosphate. It is useful in the pH range 7.3 to 9.3. Tris is available in a basic
form as highly purified crystals, which makes buffer preparation especially convenient. Although
Tris is a primary amine; it causes minimal interference with biochemical processes and does not
precipitate calcium ions. However, Tris has several disadvantages, including,

i. pH dependence on concentration, since the pH decreases 0.1 pH unit for each 10 fold dilution:

ii. Interference with some pH electrodes.

9
iii. A large ΔpKa/ °C dependence of pH on temperature.

Most of these drawbacks can be minimized by:

i. Adjusting the pH after dilution to the appropriate concentration,

ii. Purchasing electrodes that are compatible with Tris,

iii. Preparing the buffer at the temperature at which it will be used.

PROCEDURE:

Standardization of pH meter:

• Before measuring the pH of the test solution, calibrate the pH meter using the standard
buffer solutions.

• Rinse the probe thoroughly with distilled water. Immerse the probe in standard buffer
solution pH 7.0, allow the display to stabilize and set the display to read 7.0 by adjusting
calibration button.

• Rinse the probe with distilled water and again follow the previous step for standard buffer
solutions pH 4.0 or 9.4.

• To measure the pH of the test solution, rinse the probe and gently blot it using a tissue
paper. Place the probe into the test solution, the corresponding pH is displayed on the
screen.

• Rinse the probe and store in 4.0 M KCl solution.

Preparation of Tris buffer:

• Tris buffer (6.05 g) was weighed.

• The weighed tris buffer was dissolved in 30 ml of distilled water.

• The pH of the solution was measured and noted.

• Concentrated HCl was added dropwise and stirred continuously till the pH reaches 7.8.

10
 • The volume of the solution was made upto 50 ml with the distilled water.

• The resulting solution was 100M Tris-HCl buffer. Preparation of 1M potassium dihydrogen
phosphate:

Dissolve 13.6 g of potassium dihydrogen phosphate in 100ml distilled water.

Preparation of 1M Dipotassium hydrogen phosphate: Dissolve 17.4 g of K 2HPO4 from 100ml

of distilled water.

From this, the corresponding volumes of 1M potassium dihydrogen phosphate and 1M

dipotassium hydrogen phosphate as given in the table were mixed together to prepare the
required potassium phosphate buffer.

11
TABULATION:

pH Volume of KH2PO4 (ml) Volume of K2HPO4(ml)

5.8 8.5 91.5

6.0 13.2 86.8

6.2 19.2 80.8

6.4 27.8 72.2

6.6 38.1 61.9

6.8 49.7 50.3

7.0 61.5 38.5

7.2 71.7 28.3

7.4 80.2 19.8

7.6 86.6 13.4

7.8 90.8 9.2

8.0 94.0 6.0

10 times dilution of the resulting solution will yield 100mM potassium phosphate buffer

RESULT:

The procedure for standardization was studied and buffer solution were prepared at the required
pH values

12
Expt. No.:_______ Date:____________

SPECTROSCOPY: DETERMINATION OF ABSORPTION MAXIMA OF A

GIVEN SOLUTION
AIM:

To determine the absorption maxima (λmax) of a given solution by using a

spectrophotometer.

REQUIREMENTS:

UV-Vis Spectrophotometer, Cuvettes, test tubes, distilled water, given solution for
determination of λmax

THEORY:

The quantitative measurement of the absorbance or transmission properties of a material

as a function of wavelength is referred to spectrophotometry. This technique involves
electromagnetic radiations in the visible, near UV and near infrared regions. These radiations are
absorbed / transmitted differently by different material. Spectrophotometer is the instrument used
to measure the fraction of incident light transmitted through a solution. It is designed to
transmitlight of narrow wavelength ranges and the intensity of the absorption is measured as a
function of frequency and/or concentration of the test material.
The relationship of absorbance and concentration can be explained by Lambert- Beer law,
absorbance of a light absorbing material is proportional to its concentration in solution and path
length of the incident light.
A= ɛlc
Where:
ɛ = the extinction coefficient of the substance, has units of M-1 cm-1 (unique for
each substance)
l = the sample path length measured in centimetres (i.e. the width of the cuvette— almost
always 1 cm)
c = the molar concentration of the solution

13
 A colorimeter uses only visible wavelength of light (400 – 700 nm) and the
monochromatic light is obtained using a color filter. A color filter can only give a band of
wavelengths (generally a band width of 10 nm) and therefore, the light is not truly
monochromatic. In a UV-visible spectrophotometer, both ultra violet and visible
wavelengths are used. There are two light sources, a deuterium (D2) lamp for UV rays
and a tungsten lamp for visible light. Instead of color filters, a dispersion grating is used
to obtain monochromatic light as narrow as 01nm bandwidth. This not only improved
the accuracy of measurements but also the sensitivity of the measurement. Unlike the
colorimeter, spectrophotometer uses square cuvettes made of quartz which is UV
transparent. The incident light is usually split into two beams and both the blank and
sample are simultaneously compared in a double beam spectrophotometer. This further
improves the accuracy and measurements are faster allowing scanning of the sample
for the entire length of the spectrum (generally, 200 – 800 nm).
PROCEDURE:
1. Switch on the spectrophotometer and set it in scanning mode.
2. Select the range from 200 nm to 800 nm.
3. Take two paired cuvettes.

14
 4. Clean both the cuvettes with distilled water and blot it with a tissue paper.
5. Add 2 mL of distilled water in both the cuvettes.
6. Place one of the cuvettes in the reference position and the other in the sample
position (both positions will now have blank solutions (distilled water) only).
7. Carry out “Auto Zero” / “Baseline correction” for the spectrophotometer. The
spectrophotometer will now adjust DDW for 100% transmittance for all
wavelengths from 200 nm to 800 nm.
8. Remove the cuvette from the sample position.
9. Drain the distilled water and replace it with 2 mL of the given solution.
10. Place the cuvette back in the sample position (Reference position will now have
distilled water and the sample position will have given solution).
11. Start the scanning procedure. The spectrophotometer will now scan (record the
absorbance from 200 nm till 800 nm) given solution for the entire range of spectrum
specified.
12. From the data obtained / the spectrum obtained, note the wavelengths at which the
given solution shows maximum absorbance (λmax).

RESULT:
The maximum absorbance (λmax) of the given solution was found to be_______nm.

Expt. No.:_______ Date:____________

15
 General Test for Carbohydrates, Proteins and Lipids

Aim:

Apparatus required:

Test tubes, Reagents, Water bath, Test tube holders, Beakers, Pipettes, etc.

S. No Experiment Observation Inference

Test for Carbohydrates

Molish’s Test:

To 5ml of given solution added 2

drops of Molisch’s reagent and 3ml A violet colour ring was It indicates the
1 of Conc. Sulphuric acid along the got at the junction of two presence of

sides of the test tube with out liquids carbohydrate.

shaking so as to form an acid layer

beneath the sugar

solution

Preparation of Molisch’s reagent 5% solution of α-naphthol in alcohol

Iodine test:
It indicates the
2 To a small amount of given solution A blue colour solution was presence of
added a few drops of dilute iodine obtained
polysaccharide
solution

16
 Benedict’s test:

To 2ml of given solution added 8

First greenish yellow and It indicates the
drops of Benedict’s reagent and
3 then a reddish orange presence of
heated in a boiling water bath for 3
precipitate was obtained. reducing sugar.
minutes and then allowed to cool

spontaneously

Preparation of Benedict’s reagent

Dissolved 173gms of sodium citrate and 100gms of sodium carbonate in 800ml of

distilled water by warming, filtered through a filter paper and made up the volume of
the filtrate to 850ml with distilled water. Dissolved 173gms of copper sulphate in 100
ml of distilled water. Poured the copper sulphate solution with stirring in the
carbonate-

citrate solution and made up the volume with distilled water to 1 litre.

Barfoed’s test:

To 5ml of given solution added 5ml A brick red precipitate It indicates the
4 of Barfoed’s reagent and heated in was got at the bottom and presence of
a the sides of test tube monosaccharide.

boiling water bath for 3 minutes

Preparation of Barfoed’s Reagent

Dissolved about 13.3gms of neutral crystalline copper acetate in 200ml of distilled

water. Filtered, it necessary and then added 1,8ml of glacial acetic acid.

Selivanoff’s test: A cherry red This shows the

5 To 5ml of given solution added 5ml of colour solution was presence of keto
Selivanoff’s reagent and heated in a obtained. sugar
direct flame for few minutes

17
 Preparation of Selivanoff’s reagent

To 0.5 gms of Resorcinol in 100ml of 10% hydrochloric acid

Test for Proteins

Nitric acid test:

Layered the given solution carefully A white precipitate was This Indicates the
6 to a few ml of conc. Nitric acid in a formed at the junction presence of

test tube so as to get a sharp line of of two layers protein.

demarcation

Coagulation or Boiling test:

Fill the test tube with 2/3 full of
given solution and gently heated
the upper half of the fluid to boiling This indicates the
7 and being carefully mixed with A turbidity was noticed presence of
lower half which serves as control ( protein
acidify the given solution slightly by
the addition of 3-5
drops of dilute acetic acid)

Biuret’s Test:
A purplish violet This Indicates the
8 To 2ml of the given solution added
colouration was got presence of
2ml of the Biuret’s reagent
protein
Preparation of Biuret’s reagent
Added 1% copper sulphate drop by drop with constant stirring to 40% sodium
hydroxide solution till the mixture assumes a light blue colour

18
 Xantho Protein Test:
To 5ml of the given solution,
A white precipitate which
1ml of Conc. Nitric acid is
on heating turned yellow on
added and boiled for about 10
the addition of sodium This indicates the
9 minutes in a water bath. Allow
hydroxide yellow presence of protein
it to cool and add 40 % sodium
colouration deepens to
hydroxide in
orange
drops to make the solution
strong alkaline
Ninhydrin test:
To 5ml of given solution added A purple colouration was This indicates the
10
0.5ml of 0.1% ninhydrin and got presence of protein
heated in a boiling water bath.
Test for lipids
Grease Spot Test:
This indicates the
11 A drop of sample placed over a A translucent spot visible
presence of lipids
piece of ordinary paper.
Emulsification Test:
2 ml of water is taken in one
test tube and 2 ml of diluted
Note the stability
bile salt solution in another This indicates the
12 of emulsification
test tube. Add 3 drops of the presence of lipids
formed
given sample to
each test tubes and shake
vigorously .

19
13 Saponification Test:
Take 3 ml of the given sample in a test tube. Add 20 drops of 40% sodium hydroxide
and 2 ml of glycerol to it. Gently boil for about 3 minutes until complete saponification
occurs. If the oil globules are visible, boiling must be continued. Divide the solution
into 3 parts to carry the following experiments.

i) To test tube No.1 add The soap separates out This indicates the
saturated solution of sodium and floats to the surface presence of lipids
chloride.

ii) To test tube No.2 add a few An oily layer of the fatty This indicates the
drops of Conc. Hydrochloric acids rises to the surface presence of lipids
acid

iii) To test tube No.3 add a few The insoluble calcium This indicates the
drops of calcium chloride soap precipitated. presence of lipids
solution

Result:

From the above tests, it is confirmed that the given solution contains --------------

20
Expt. No.:_______ Date:____________

IDENTIFICATION OF BLOOD COLLECTION TUBES AND PHLEBOTOMY

EQUIPMENTS

AIM:

To identify the various blood collection tubes and phlebotomy equipments used in
hospitals.

THEORY:

Blood collection tubes:

Blood collection tubes are sterile glass or plastic tubes with a vacuum inside facilitating
the draw of a predetermined volume of liquid. Most commonly used to collect blood samples
in vein puncture, they are designed for the collection, transport, and processing of skin
puncture blood. The insides of these tubes may be coated with anticoagulants to keep blood
from clotting after being drawn and stored inside the tube. Tight seals preserve sample
integrity.

Types:

1. Veterinary and Laboratory Blood Collection Tubes:

Blood collection tubes for use in veterinary and laboratory environments. Choose glass or
plastic tubes.

21
 2. Venous blood collection tubes:

Sterile vacuum blood collection tubes are for serum determination. Tubes with a closure
incorporate a plastic shield that protects lab personnel from contact with blood on the stopper
or around the outer rim of the tube, and from potential splattering when tube is opened. The
closure's rubber stopper is recessed inside the plastic shield to isolate blood drops left by the
needle.

3. Blood collection tubes with clot activators:

They contain thrombin-based clot activator and polymer gel for serum separation. Samples
processed in these tubes are used for serum determinations in chemistry. A five-minute
clotting time makes this tube ideal for STAT testing in the emergency department as well as
clinical laboratories striving to improve test turnaround and workflow efficiencies. This
sterile, disposable plastic rapid serum tubes have paper label with orange closure to use for
blood collection. Latex free.

22
Phlebotomy equipments:

1. Micro-collection tubes:

Micro-collection tubes used to collect blood from a skin puncture (heel or finger).
Collecting this type of sample should be considered for patients who are difficult draws such
as elderly patients with fragile veins, infants, small children and other patients with difficult
veins. This can only be done for tests which can be performed on small quantities of blood.

2. Vacuum blood collection system:

It is composed of a multi-sample vacuum collection needle and a disposable tube holder.

Note the needle safety device is directly attached to the needle. When the phlebotomy
procedure is complete, the safety device is activated with the thumb.

3. Multi-sample blood collection needle:

This device has TWO needles. The needle is screwed into the holder. One needle is inserted
into the vein, the vacuum blood collection tube is inserted into the holder and is punctured
by a needle on the opposite end. The rubber sheath allows multiple tubes of blood to be
collected and covers the needle in between tube changes so blood does not collect in the
holder.

23
 4. Vein puncture safety device:

This is a needle safety device used with the vacuum tube blood collection system. Note,
this blood collection device uses a standard multi-sample collection needle. The needle
safety device is activated by pressing it against a hard surface to permanently cover the
needle.

5. Microcapillary tubes:

Microcapillary tubes are used to collect small volumes of blood. The tubes have a ring /
tip color to indicate any additive.

Color coding is as follows:

red tip = sodium heparin,

green tip = ammonium heparin, and

blue tip = plain/no additive.

The most common test using the microcapillary tube is the hematocrit which is routinely
collected in the red tipped tube.

24
 6. Microhematocrit centrifuge:

The microhematocrit centrifuge is used to spin down microhematocrit tubes.

7. Syringe with needle:

A syringe with a syringe needle is sometimes used to collect blood from patients with
difficult, small or fragile veins. Collecting with a syringe and needle is especially useful when
the phlebotomist needs to control the amount of vacuum being applied to prevent or reduce
the chance of the vein collapsing.

8. Urine dipsticks:

Urine dipsticks are used for the chemical examination of urine.

25
 9. Butterfly needles:

Butterfly (Winged Collection Set) needles are used to draw blood from patients with small or
fragile veins.

10. Blood culture bottles:

Blood culture bottles - used to collect blood from patients suspected of having
septicemia or bacteremia. Proper preparation of the vein puncture site is crucial in
preventing contamination with skin bacteria (causing a false positive). The blood collection
site must be aseptically cleansed to produce a sterile site with a product such as a
Chlorascrub. To aid in preventing false positives, the phlebotomist cannot repalpate or
touch the vein puncture site after it has been prepared. Not putting a sufficient amount of
blood into the bottle(s) and/or not drawing the blood at the correct time could result in a
false negative.

26
 11. Erythrocyte Sedimentation rate (ESR) tubes:

Blood drawn into the purple-top EDTA tube can be used, but will require mixing the
blood with a diluent prior to testing. This mixing process occurs when the blood is dispensed
into the special reservoir prior to being loaded into the ESR tube. It may be more convenient
to draw the blood specimen into the black-top ESR vacuum blood collection tube which
contains a 3.8% buffered sodium citrate solution. The advantage of using this tube is that the
required pre-testing dilution will already have been made; the disadvantage is that the ESR
is the only test that can be performed on blood collected in this tube. This non-specific test is
used to monitor patients with inflammation and/or necrosis, and is not diagnostic of any one
specific disease or illness.

12. Lancets:

Lancets are used for capillary blood sampling and come in a wide variety of shapes
and sizes. Modern blood lancets are safety engineered, self-contained single use
automated puncture devices designed to produce a uniform puncture of a specified depth.
Punctures are routinely made on the side and near the tip of the 3rd or 4th finger of adult
and pediatric patients. Capillary blood is collected from the sides of the heel of non-walking

27
 infants. It is critically important the heel stick is performed in the correct area of the heel to
prevent nerve or bone damage.

RESULT:

The blood collection tubes and Phlebotomy equipments were studied and identified.

28
Expt. No.:_______ Date:____________

Preparation of Serum and Plasma from Blood

Aim:

To separate the serum and plasma from the blood sample.

Apparatus required:

Centrifuge, centrifuge tube, anticoagulants, blood sample.

Separation of Serum:

The blood sample is collected into a container without anticoagulant and kept
undisturbed. Blood clots, as there is no coagulant. The sample is then centrifuged and the
supernatant serum can be collected.

Separation of Plasma:

The blood sample is collected into a test tube containing anticoagulant. The sample is
centrifuged and the supernatant plasma is collected.

Anticoagulants:

These are chemical substances that prevent clot formation. Several anticoagulants
are available for use in the clinical lab. Each one is used for a specific purpose. These are
broadly categorized as in vitro and in vivo anticoagulants.

In vitro coagulants:

a) Double oxalate mixture

b) Ethylene diamine tetra acetic acid

c) Trisodium citrate

d) Sodium Fluoride
29
 e) ACD and CPD

In vivo:

a) Heparin

b) Dicumarol

Result:

By using the above procedure the serum and plasma were prepared from the
given blood sample.

30
Expt. No.: _______ Date: ____________

Estimation of Haemoglobin

Aim:

To estimate the amount of haemoglobin present in the given blood sample.

Principle:

When blood is added to 0.1N hydrochloric acid the haemoglobin is converted to

brown colored acid hematin. The resulting color after dilution is compared with standard
brown glass reference blocks of a Sahli’s Haemoglobinometer.
Specimen:

Capillary blood or thoroughly mixed anticoagulated (EDTA or double oxalate) venous

blood the specimen need not be a fasting.

Apparatus required:

❖ Sahli’s Haemoglobinometer.
❖ Sahli’s pipette graduated to 0.02ml (20mm 3 or 20 μl )
❖ Dropping pipette
❖ Filter paper
❖ 0.1N Hcl
A Sahli’s Haemoglobinometer consists of
1. A haemoglobin pipette
2. A stirrer
3. The standard haemoglobin comparator
4. The haemoglobin tube.
Procedure:

th
1. Fill the graduated haemoglobin tube to the 2 mark (for the mark 3g/100 ml
with 0.1N Hcl)

31
2. Draw capillary (or venous) blood to the 0.02ml mark of the Sahli’s pipette. Do not allow
air bubble to enter (do not take the first drop of the blood from the finger) with the
venous blood ensure that it is well mixed by inverting the
bottle containing it and the anticoagulant repeatedly for about one minute immediately
before pipetting.

3. Wipe the blood from the pipette with the filter paper. Check that the bottle is still in the
mark.
4. Blow the blood from the pipette into the graduated tube of the acid solution. Rinse the
pipette by drawing in and blowing out the acid solution 3 times. The mixture of blood
and acid gives a brownish color. Allow it to stand for 5 minutes.
5. Place the graduated tube in the Haemoglobinometer stand facing a window. Compare
the color of the tube containing dilute blood with the color of the reference tube. If the
color is the same or lighter than that of the reference tube, the haemoglobin value is
40gm/L or less.
6. If the color darker than that of the reference tube, continue to dilute by adding 0.1N
Hcl drop by drop. Stir with a glass rod and compare the color match. Distilled water can
also be used in this step instead of 0.1N Hcl to continue the dilution of the blood.
7. Note the mark reached. Depending on the type of Haemoglobinometer, this givesthe
Haemoglobin concentration in g/100 ml or percentage.

Result:
Amount of haemoglobin present in 100 ml of blood is _________ Gms.

32
Expt. No.: _______ Date: ____________

Estimation of Glucose by O- Toluidine method

Aim:

To estimate the amount of Glucose present in 100ml of blood.

Principle:

Glucose reacts with various aromatic amines in hot acetic acid solution to produce
coloured derivatives. O- Toluidine is an aromatic amine which condenses with the aldehyde
group of glucose to form an equilibrium mixture of a glycosyl-amine and corresponding sciff’s
base (bluish green colour).The bluish green colour developed was read in a colorimeter/
spectrophotometer at 660 nm (millimicron) against a reagent blank.

Apparatus required:

Test tubes, beakers, water bath, Colorimeter/ Spectrophotometer, Pipettes.

Reagents:

(i) O-toluidine reagent:

Transferred 3.75 gms of thiourea to 3litres Erlenmeyer flask and added 2375ml of glacial
acetic acid and 125ml of O-toluidine. Mixed well until the thiourea is dissolved and store in a
brown bottle at room temperature. Contact with skin should be avoided. The reagent should
be dispended from a burette.

(ii) Stock Standard Glucose Solution (100mg/ml)

Dissolved 200mg of glucose in distilled water and made up to 100ml in standard flask.

(iii) Working Standard Glucose Solution (200µg/ml)

Diluted 10ml of stock standard solution to 100ml with distilled water. 1ml of the working
standard solution contains 200µg of glucose.

33
Procedure:

Into a series of test tubes added 0.5, 1.0, 1.5, 2.0 and 2.5 working standard glucose
solution corresponding to 100, 200,300,400 and 500 µg values. The volumes of the standards
were made up to 3.0ml with distilled water. A blank was also prepared by taking 3.0ml of
distilled water and 1ml of the diluted serum sample was taken for the experiments.

To all the test tubes added 5ml of O-toluidine reagent. Mixed well and heated in a
water bath at 100۫ C for about 10 minutes. Then the test tubes were cooled in a water bath
for 2 to 3 minutes. The bluish green colour developed was read in a colorimeter/
spectrophotometer at 660nm against reagent blank.

A standard graph was drawn by plotting the concentration on X-axis and optical
density on the Y-axis. The amount of glucose present in 100ml of the given solution was
calculated from the standard graph.

Tabulation:

Volume of Volume of Volume of O- Optical

S. No working Distilled water Conc. in μg Incubation
density
standard (ml) (ml) Toludine (ml)

1 Blank 3.0 0 5.0 heated in a

water bath
2 0.5 2.5 40 5.0
at 100۫˚ C

3 1.0 2.0 80 5.0 for about

10minutes&
4 1.5 1.5 120 5.0 cooled
bluish green
5 2.0 1.0 160 5.0
colour
6 2.5 0.5 200 5.0 developed
was read at
7 Sample 1.0 2.0 - 5.0 660nm

34
Calculation

Spectrophotometer reading corresponds to µg of glucose.

1.0 ml of the given sample contains = µg of glucose .

100 ml of given sample contains = x100 mg of glucose 1.0 x 1000

Result:

The amount of glucose present in 100ml of the given blood is ---------

35
Expt. No.:_______ Date:____________

Estimation of Creatinine in Urine by Jaff’s method

Aim:

To estimate the amount of creatinine present in the 24 hours urine sample.

Principle:

Creatinine is estimated by making use of Jaff’s method in which it gives an orange

yellow colour with alkaline picrate solution whose intensity is measured at 540 nm.

Apparatus required:

Test tubes, beakers, water bath, Colorimeter/ Spectrophotometer, Pipettes.

Reagents:

1. Stock solution: 100 mg of creatinine is dissolved in 100ml of 0.1N hydrochloric

acid. Conc. 1mg/ml

2. Working standard: 4 ml of stock solution is made up to 100ml with distilled water.

Conc. 40μg/ml

3. 10% sodium hydroxide

4. 1% picric acid solution

5. Preparation of urine sample:1 ml of urine is diluted to 100 ml with distilled water.

Procedure:

0.4 to 2.0 ml of working standards concentration range 16 to 80μg are pipetted into
five test tubes. The volume is made up to 8 ml with distilled water. 1.0 ml of 10%sodium
hydroxide and 1.0 ml of 1% picric acid solution are added to all the test tubes and mixed well.

36
 A blank and the unknown urine sample 2.0 ml are also treated as above. The colour
developed is read after 15 minutes at 540 nm. The standard graph is drawn by taking
concentration of creatinine in the X axis and OD in the Y axis. From the standard graph the
concentration of creatinine in urine sample is calculated.

Tabulation

Volume of Volume
Volume of Volume Optical
working Conc. of 1 %
S. No Distilled of 10% Incubation density at
standard in μg picric
water ml NaOH 540nm
ml acid ml

Incubate
room
temperature
1 Blank 8.0 0 1.0 1.0 for 15
minutes
yellow colour
developed

2 0.4 7.6 16 1.0 1.0

3 0.8 7.2 32 1.0 1.0

4 1.2 6.8 48 1.0 1.0

5 1.6 6.4 64 1.0 1.0

6 2.0 6.0 80 10 1.0

Sample
7 6.0 - 10 1.0
2.0

37
2 ml of diluted urine contains X μg creatinine

100 ml of dilute urine contains Y= X x 100/1000 x 2 mg of creatinine

100 ml of dilute urine contains = 1.0 ml of urine

1.0 ml of urine contains = Y mg of creatinine 1200 ml of urine contains = Yx1200 mg of

creatinine Normal value is 1-2 grams/day.

Result:

The amount of Creatinine present in 1200ml (24 hrs.) of the given urine is .

Expt. No.:_______ Date:____________

38
 Estimation of urea by DAM-TSC method

Aim:

To estimate the amount of urea in 100ml of blood.

Principle:

Urea reacts with diacetyl monoxime in the presence of thiosemicarbazide to form a

red colour which is measured colorimetric ally at 540nm against the reagent blank.

Reagents:

1. Acid Reagent: Water-100ml, concentrated H2SO4 – 8ml, Orthophophoric acid -

20ml, 5%of Ferric chloride-1ml.

2. Colouring Reagent: Acid Reagent-30ml, Water-20ml.Diacetyl monoxime 2.5%-

1ml, Thiosemicarbazide 0.25%-0.25ml Colour reagent should be prepared just
before use. This solution is not stable for more than one hour.

3. Stock standard urea solution (1000µg/ml): 100mg of urea was dissolved in a 100ml
of distilled water in a 100ml of standard flask. This solution was dissolved in
benzoic acid for long use.

4. Working standard urea solution (20µg/ml): 2ml of the stock standard solution was
diluted to 100ml with water. 1ml of this solution contains 20µg of Urea.

Procedure:

Took 1.8ml of 3% tricholoro acetic acid solution and 0.2ml of blood, after 10 minutes,
it is centrifuged and 1.0ml of the supernatant was taken for the experiment. Into a series of
test tubes taken 0.5 to 2.5 ml of working standard urea solution corresponding to 10 µg to 50
µg values respectively. The volume of each tube was made up to 3 ml with water. Added 5ml
of the colour reagent to all the test tubes and mixed well. Cooled and heated in a vigorously
boiling water bath for 20 minutes. Along with these, a blank was also conducted removed the

39
tubes and cooled. The red colour developed was read in a colorimeter against a regent blank
at 520 nm.

A standard graph was drawn by plotting the concentration of urea in µg on X-axis and
the colorimeter reading on Y-axis. From the graph, the concentration of urea in 100ml of
blood was calculated.

Tabulation:

Standard Solution Optical Density

Volume in Concentration in Volume of Volume of

ml µg water in ml colouring
reagent in ml

Blank - 3.0 5.0

Standard

0.5 10 2.5 5.0

1.0 20 2.0 5.0

1.5 30 1.5 5.0

2.0 40 1.0 5.0

2.5 50 0.5 5.0

Serum

1.0 - 2.0 5.0

40
Calculation

Colourimeter reading ------------ corresponds to µg of Urea.

1.0 ml of serum supernatant contains = µg of Urea.

2.0ml of serum supernatant contains = µg of Urea.

0.2ml of blood was made up to 2.0ml

1. ml of the blood contains = µg of Urea.

100ml of the blood contains = µg of Urea.

Normal amount of urea present in blood is

Result:

The amount of urea present in 100ml of the blood was found to be ______ .

41
Expt. No.:_______ Date:____________

Separation of proteins by SDS-PAGE electrophoresis

Aim:

To separate the protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE Electrophoresis

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a

technique used separate proteins according to their electrophoretic mobility. The solution of
proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures
secondary and non–disulfide–linked tertiary structures and dithiothreitol (DTT) or 2-
mercaptoethanol (beta-ercaptoethanol/BME) which reduces the disulfide bonds, and applies
a negative charge to each protein in proportion to its mass.

Materials:

1. 30% Acrylamide/bis (29:1) solution;

2. 4 x Resolving gel buffer: 1.5 M Tris-HCl (pH 8.8), 0.4% SDS;
3. 4x Stacking gel buffer: 0.5 M Tris-HCl (pH 6.8), 0.4% SDS;
4. 1x Elecrtophoresis buffer: 25 mM Tris-HCl (pH 8.3), 250 mM glycine, 0.1% SDS.
5. 3xprotein loading buffer: 0.19 M Tris-HCl (pH 8.0), 3% glycerol, 6% SDS, 300 mM
1. DTT, and 0.25% bromophhenol blue;
6. 10% Ammonium persulfate (APS);
7. TEMED;
8. Gel staining buffer: 1 g of Coomassie Brilliant Blue dissolved in 400 ml of solution
containing 45% methanol, 45% H2O, and 10% acetic acid;
9. Destaining buffer: 25 % methanol , 10% acetic acid, and 65% H2O;
10. Gel Drying Buffer: 40% ethanol , 4% glycerol, and 56% H2O.
11. Protein samples.
12. .Protein standards.

42
Experimental Procedure:

1. Prepare 40 ml of 15% resolving gel solution as following: 9.68 ml of H2O 20 ml of

30% Acrylamide/bis solution 10 ml of 4x resolving gel buffer 300μl of 10% APS 20μl
of TEMED

First assemble the gel caster, and add the 15% gel mix to gel caster. Overlay the
solution gently with water or 1-butanol saturated with H2O, and allow to be
polymerized. You should see the gel-H2O interface disappear and then reappear
when the gel is polymerized;

2. Prepare 20 ml of 5% stacking gel solution as following: 11.5 ml of H2O 3.3 ml of

30% Acrylamide/bis solution 5 ml of 4x stacking gel buffer 200 μl of 10% APS 10
μl of TEMED

Add the gel mix to the gel caster, and insert the comb to the gel solution; you should
wait until gel is fully polymerized. Once the gel is polymerized, the comb can be
removed gently. Then assemble the gel into the electrophoresis apparatus.

3. Load your samples: Mix two volumes of your samples with 1 volume of 3x sample
loading buffer, and heat the samples over 90 0C for 5 min, and load 20 μl of your
samples to each well of the gel. Also add the protein standards in two lanes on
each gel.

4. Run the gels: Apply power at 100-150 V until the dye has reached the top of the
resolving gel, and then increase the power into 200 V. When the dye reaches the
bottom of the resolving gel, turn off the power supplier. Disassemble the gel
apparatus, and stain the gel for about one hour at Gel staining buffer. Destain your
gel at the destaining buffer until the protein bands are clearly seen in the gel, and dry
the gel if it is possible.

43
Result:

The given mixture contains ____________

44
Expt. No.:_______ Date:____________

Separation of amino acids by thin layer chromatography

Aim

To separate and identify the amino acids in a mixture by thin layer chromatography.

Chromatography:

Chromatography is the most useful technique available for the separation of closely
related compounds in a mixture. Here the separation is effected by differences in the
equilibrium distribution of the components between two immiscible phases, viz., the
stationary and the mobile phases. These differences in the equilibrium distribution are a result
of nature and degree of interaction of the components with these two phases. The stationary
phase is a porous medium like silica or alumina, through which the sample mixture percolates
under the influence of a moving solvent (the mobile phase).

Thin layer chromatography [TLC]:

Thin layer chromatography is a technique used to separate and identify compounds

of interest. A TLC plate is made up of a thin layer of silica adhered to glass or aluminum for
support. The silica gel acts as the stationary phase and the solvent mixture acts as the mobile
phase. In the ideal solvent system the compounds of interest are soluble to different degrees.
Separation results from the partition equilibrium of the components in the mixture.

The separation depends on several factors; (a) solubility: the more soluble a
compound is in a solvent, the faster it will move up the plate. (b) attractions between the
compound and the silica, the more the compound interacts with silica, the lesser it moves,
(c) size of the compound, the larger the compound the slower it moves up the plate.

An important characteristic used in thin layer chromatography is Rf value.

45
Chromatographic Separation of Amino acids:

All 20 of the common amino acids [standard amino acids] are a-amino acids. They
have a carboxyl group and an amino group bonded to the same carbon atom (the α- carbon).
They differ from each other in their side chains, or R groups, which vary in structure, size,
and electric charge. The interaction of the amino acids with the stationary phase like silica
varies depending on their 'R' groups. The amino acid that interacts strongly with silica will be
carried by the solvent to a small distance, whereas the one with less interaction will be moved
further. By running controls [known compounds] alongside, it is possible to identify the
components of the mixture.

Materials Required:

1. 2% solution of individual amino acids.

2. Solvent mixture of normal butanol, acetic acid and water in the ratio 12:3:5 by
volume.

3. Ninhydrin reagent.

4. TLC plate.

5. TLC chamber.

6. Capillary tubes.

7. Reagent spray bottle.

46
 8. Conical flasks.

9. Beakers.

Procedure:

1. Pour the solvent mixture in to the TLC chamber and close the chamber.

2. The chamber should not be disturbed for about 30 minutes so that the
atmosphere in the jar becomes saturated with the solvent.

3. Cut the plate to the correct size and using a pencil (never ever use a pen)
gently draws a straight line across the plate approximately 2 cm from the
bottom.

4. Using a capillary tube, a minute drop of amino acid is spotted on the line.

5. Allow the spot to dry.

6. Spot the second amino acid on the plate [enough space should be provided
between the spots].

7. Repeat the above step for spotting the unknown acid.

8. Place the plate in the TLC chamber as evenly as possible and lean it against
the side(immerse the plate such that the line is above the solvent). Allow
capillary action to draw the solvent up the plate until it is approximately 1 cm
from the end.

9. Remove the plate and immediately draw a pencil line across the solvent top.

10. Under a hood dry the plate with the aid of a blow dryer.

11. Spray the dry plate with ninhydrin reagent.

12. Dry the plates in hot air oven at 105°C for 5 min. [Ninhydrin will react with the
faded spots of amino acids and make them visible as purple coloured spots.]

47
 13. After some time, mark the center of the spots, then measure the distance of
the center of the spots from the origin and calculate the Rf values.

14. Rf value can be calculated using the formula:

Result:

The given mixture contains

48
Expt. No.:_______ Date:____________

URINE PHYSICAL AND CHEMICAL EXAMINATION (PROTEIN, REDUCING

SUBSTANCES, KETONES, BILIRUBIN AND BLOOD)

AIM:
To perform physical and chemical (protein, reducing substances, ketones, bilirubin and
blood) analysis of the given urine sample.

PRINCIPLE:

URINARY pH

Fresh urine specimens can have pH values ranging from acidic to alkaline. Upon
standing the decomposition of urea into ammonia causes the urine to become more
alkaline. Lower pH values are observed in cases of diabetes and in patients with fever.
Urine retention by some patients can result in more alkaline urine. The standard method
for pH measurements uses glass electrodes. Urinary pH measured with indicator paper is
more than accurate enough for clinical purposes, since small changes in urinary pH are of
little clinical significance. There is no confirmatory testing for urine pH.

Normal—The urinary pH range is usually 4.5 to 8. Extremely acidic or alkaline urine

usually indicates a poorly collected specimen.

PROTEIN

Based upon a phenomenon called the "protein error of indicators," this test uses a
pH indicator, such as tetrabromphenol blue, that changes color (at constant pH) when
albumin is present in the urine. Albumin is important in determining the presence of
glomerular damage. The glomerulus is the network of capillaries in the kidneys that filters
low molecular weight solutes such as urea, glucose, and salts, but normally prevents
passage of protein or cells from blood into filtrate. Albuminuria occurs when the glomerular
membrane is damaged, a condition called glomerulonephritis.

49
 pH 3

Tetrabromphenol blue Positive results (green-blue)

Protein

pH 3

Tetrabromphenol blue Negative results (yellow)

No protein

Normal—A healthy person will excrete up to approximately 100 mg/day, a very small
fraction of the plasma protein that is filtered at the glomerulus.

GLUCOSE:

The presence of significant amounts of glucose in the urine is called glycosuria (or
glucosuria). The quantity of glucose that appears in the urine is dependent upon the blood
glucose level, the rate of glomerular filtration, and the degree of tubular reabsorption.
Usually, glucose will not be present in the urine until the blood level exceeds 160–180
mg/dL, which is the normal renal threshold for glucose. When the blood glucose exceeds
the renal threshold, the tubules cannot reabsorb all of the filtered glucose, and so
glycosuria occurs. Normally, this level is not exceeded even after the ingestion of a large
quantity of carbohydrate.

50
Copper reduction (Clinitest, Benedict's test)

Heat

Cupric ions + Glucose Cuprous Cuprous

+
(or other reducing substances) oxide hydroxide

Alkali (red) (yellow)

Normal—Health individuals normally will have no detectable sugars in their urine.

KETONES:

Ketones are compounds resulting from the breakdown of fatty acids in the body.
These ketones are produced in excess in disorders of carbohydrate metabolism,
especially Type 1 diabetes mellitus. In diabetes, excess ketoacids in the blood may cause
life-threatening acidosis and coma. These ketoacids and their salts spill into the urine,
causing ketonuria. Ketones are also found in the urine in several other conditions,
including fever; pregnancy; glycogen storage diseases; and weight loss produced by a
carbohydrate-restricted diet.

Alkaline pH

Acetoacetic acid + Sodium nitroprusside + Glycine Purple color

Normal—Health individuals normally will have no detectable ketones in their urine.

BLOOD:

Hematuria is the presence of blood or intact RBCs in the urine. A urine that is highly
alkaline or has a very low specific gravity (_1.007) can cause the red cells to lyse, thus
releasing their hemoglobin into the urine. The presence of this type of hemoglobin is still

51
considered to be hematuria as far as the origin is concerned, but it is very difficult to
distinguish from true hemoglobinuria. When lysing occurs, the microscopic examination
may show the empty red cell membranes which are often referred to as “ghost” cells. In
microhematuria there is such a small amount of blood in the urine that the color of the
specimen is unaffected and the hematuria can only be detected chemically or
microscopically. On the other hand, gross hematuria alters the color of the urine and is
easily visible macroscopically.

Alkaline pH

Oxidized chromogen (blue)

Hydrogen peroxide (H2O2) + Chromogen
+ H 2O

Normal—Health individuals normally will have no detectable blood or myoglobin in their

urine.

BILIRUBIN:

Bilirubin is a breakdown product of hemoglobin. Most of the bilirubin produced in

humans is conjugated by the liver and excreted into the bile, but a very small amount of
conjugated bilirubin is reabsorbed and reaches the general circulation to be excreted in
the urine. The normal level of urinary bilirubin is below the detection limit of the test.
Bilirubin in the urine is derived from the liver, and a positive test indicates hepatic disease
or hepatobiliary obstruction.

Acid

Bilirubin glucuronide + Diazonium salt Azobilirubin (brown)

Normal—Urine from healthy individuals does not contain detectable bilirubin.

52
REQUIREMENTS:

1) 2% acetic acid

2) conc. Nitric acid

3) saturated benzidine

4) 3% Hydrogen perroxide

5) A and B (Fehling’s)

6) Benedict’s reagent

7) liquid ammonia

8) % freshly prepared sodium nitro prusside

9) ammonium sulphate crystals

10) sulphur powder.

11) tincher of iodine

12) Ehrelich reagent

13) HCl

PROCEDURE:

Collection of specimen and its preservation

Like all biological specimens, urine has to be collected and adequately preserved
to prevent contamination and bacterial overgrowth since it is a very good culture medium.
The type of urine specimen to be collected is determined by the test to be performed:

A clean, early morning, fasting specimen is generally the most concentrated

specimen and preferred for microscopic examination and for detection of abnormal
amounts of constituents e.g. protein.

53
 A clean, timed specimen is one obtained at specific times of the day or during
certain phases of the act of micturition .

-First 10ml of urine voided is most appropriate to detect urethritis.

-Midstream specimen is best for bacteriological study.

Catheter specimens are used for microbiological examination in critically ill patients
or in urinary tract obstruction, only.

Preservatives used

The most satisfactory form of preservation is refrigeration at 40C combined with

chemical preservation.

Commonly used forms of preservation used earlier were formalin (2 drops of 40%
in 30 ml of urine) or Thymol (0.1mg per 100ml of urine sample). Nowadays tablets
containing a mixture of chemicals are widely used. They act by lowering the pH and by
releasing formaldehyde.

For Ketone bodies: Investigation is to be done immediately or within 2 hours of

collection or it should be refrigerated with adequate preservative.

54
 QUALITATIVE ANALYSIS OF URINE

EXPERIMENT OBSERVATION INFERENCE

APPEARANCE: Clear Normal

The appearance of the given sample was

noted

COLOUR Amber Yellow Normal

The colour of the sample was noted

ODOUR Aromatic Normal

The odour of the sample was noted

pH Blue litmus turns red Acetic in nature

pH of the sample was checked

TEST FOR PROTEIN: Coagulated Presence of protein

HEAT COAGULATION TEST:

To 0.5ml of urine added 2% acetic acid

mixed and heated. The upper portion is
compared with lower portion

TEST FOR ALBUMIN: (HELLER’S TEST) White precipitate Presence of albumin

was formed
2 to 3 ml of Urine taken in a test tube 3
ml of conc. Nitric acid was added gently
over it

TEST FOR BLOOD: 3 ml of saturated Blue or green colour Presence of blood

benzidine followed by 1 ml of 3% Hydrogen formed
perroxide. Add 2ml of Urine

TEST FOR REDUCING SUGAR: Reddish brown Presence of reducing

precipitate is formed sugar
BENEDICT’S TEST:

To 5ml of Benedict’s reagent add 3

drops of urine and heated in a test tube

55
FEHLING’S TEST: Orange Red Presence of reducing
Precipitate is formed sugar
To equal volumes of A and B (Fehling’s)
added 2 drops of urine and heated in a
boiling water bath.

TEST FOR KETONE BODIES: Pink colour is Presence of Ketone

(ROTHRA’S TEST): obtained bodies

About 5ml of urine is saturated with

ammonium sulphate crystals and added 5
drops of 2% freshly prepared sodium nitro
prusside. It is mixed well and 10 drops of
liquid ammonia is added

TEST FOR BILE SALTS: HALF – Sulphur floats in the Absence of bile salts
SULPHUR TEST: tube

Half the test tube urine was taken and

gently sprinkle the sulphur powder.

TEST FOR BILIRUBIN: IODINE TEST: No characteristic Absence of bilirubin

change
Dilute and some tincher of iodine 1 – 2
volume of water and layer it on some urine
in the test tube.

TEST FOR UROBILINOGEN: EHRLICH’S Red colour was Presence of Urobilinogen

TEST: observed

1 ml of urine added Ehrelich reagent and

acidified with HCl

56
Result:

COLOUR:

CLARITY

(i) Sample ‘A’ contains presence of sugar and Ketone bodies

(ii) Sample ‘B’ contains presence of proteins

(iii) Sample ‘C’ contains of blood, reducing sugar, Ketone bodies, bile salts,
Bilirubin.

APPLICATION:

✓ Urine is an excretory product of the body and presence of certain substances

in the urine reflects the metabolic state of the body.

✓ Since it can be easily collected and examined, routine and microscopic

examination of urine are preliminary and important in diagnosis of various
pathological conditions.

57
Expt. No.:_______ Date:____________

BASIC STAINING – HEMATOXYLIN AND EOSIN STAINING

AIM:

To perform basic staining – Hematoxylin and eosin staining for the given tissue sample.

PRINCIPLE:

The most commonly used staining system is called H&E (Haemotoxylin andEosin).
H&E contains the two dyes haemotoxylin and eosin.Eosin is an acidic dye: it is negatively
charged (general formula for acidic dyes is: Na+dye-). It stains basic (or acidophilic)
structures red or pink. This is also sometimes termed 'eosinophilic'. Thus the cytoplasm is
stained pink in the picture below, by H&E staining. Haematoxylin can be considered as
a basic dye (general formula for basic dyes is: dye+ Cl-). Haemotoxylin is actually a dye
called hematein (obtained from the log-wood tree) used in combination with aluminium
ions (Al3+). It is used to stain acidic (or basophilic) structures a purplish blue. (Haematoxylin
is not strictly a basic dye, but it is used with a 'mordant' that makes this stain act as a basic
dye. The mordant (aluminium salts) binds to the tissue, and then haematoxylin binds to
the mordant, forming a tissue-mordant-haematoxylin linkage.)Thus the nucleus is stained
purple by H&E staining. This means that the nucleus, and parts of the cytoplasm that
contain RNA stain up in one colour (purple), and the rest of the cytoplasm stains up a
different colour (pink).DNA (heterochromatin and the nucleolus) in the nucleus, and RNA
in ribosomes and in the rough endoplasmic reticulum are both acidic, and so haemotoxylin
binds to them and stains them purple. Some extracellular materials (i.e. carbohydrates in
cartilage) are also basophilic.Most proteins in the cytoplasm are basic, and so eosin binds
to these proteins and stains them pink. This includes cytoplasmic filaments in muscle cells,
intracellular membranes, and extracellular fibres.

DIAGRAMMATIC REPRESENTATION:

58
REAGENTS:

Reagent alochol - HPLC Fisher A995-4 or histological A962, FLAMMABLE store at room
temp. in a flammable cabinet

Eosin Y, disodium salt

Harris Hematoxylin Stain, acidified

Permount -

Xylenes

Solutions:

1. Eosin Y, 1 % aqueous

Eosin Y dye 1 g

Deionized water 100 ml

2. Harris Hematoxylin, acidified

Filter before use

59
3. Alcohol 50, 70, 80, 95%

Reagent alcohol 50, 70, 80, 95ml

Deionized water 50, 30, 20, 5ml

PROCEDURE:

1. Immerse sections in the filtered Harris Hematoxylin for 1 minute.

2. Rrinse with tap water.

3. Exchange tap water until the water is clear.

4. Immerse sections in EOSIN stain for 1-2 minutes.

5. Rinse with tap water.

6. Exchange tap water until the water is clear.

7. Dehydrate in ascending alcohol solutions (50%,70%,80%,95%x2, 100%x2).

8. Clear with xylene (2X).

9. Mount coverslip onto a labeled glass slide with Permount.

OBSERVATION:

60
Sl.No Structures Stained in Colour

RESULTS:

Nuclei and other basophilic structures are purple.

Cytoplasm and acidophilic structures are light to pink.

APPLICATION:

✓ Hematoxylin and eosin stain (H&E stain or HE stain) is one of the principal stains
in histology. It is the most widely used stain in medical diagnosis and is often the
gold standard; for example when a pathologist looks at a biopsy of a suspected
cancer, the histological section is likely to be stained with H&E and termed "H&E
section", "H+E section", or "HE section".

61
Expt. No.:_______ Date:____________

BRIGHT FIELD: CRESYL VIOLET STAINING (NISSL STAINING)

AIM:

To perform Cresyl Violet Staining (Nissl staining) for the given tissue sample.

PRINCIPLE:

Cresyl Violet Acetate solution is used to stain Nissl substance in the cytoplasm of
neurons in para-formaldehyde or formalin-fixed tissue. The neurophil will be stained a
granular purple-blue. This stain is commonly used to identify the neuronal structure in brain
and spinal cord tissue. The Cresyl Violet method uses basic aniline dye to stain RNA blue,
and is used to highlight important structural features of neurons. The Nissl substance
(rough endoplasmic reticulum) appears dark blue due to the staining of ribosomal RNA,
giving the cytoplasm a mottled appearance. Individual granules of extra-nuclear RNA are
named Nissl granules (ribosomes). DNA present in the nucleus stains a similar color.

REAGENTS:

- 95% Ethanol

- 70% Ethanol

- Differentiation solution: 2 drops glacial acetic acid in 95% ethanol

- Cresyl Violet Acetate 0.2% in Acetate Buffer (Fronine, Cat No:HH155)-Filtered

PROCEDURE:

Method (for paraffin embedded sections):

1. De-wax sections in xylene (2 or 3 changes of 3min each)

2. Rehydrate in alcohol (100% x2), 3min each.

62
3. Stain in 0.1% Cresyl Violet 4-15min.

4. Quick rinse in tap water to remove excess stain

5. Wash in 70% ethanol (the stain will be removed by this method).

6. If required immerse sections for 2min in Differentiation solution - check staining on

microscope.

7. Dehydrate through 2x3min changes of absolute ethanol.

8. Clear in xylene x2 and mount in DePeX. Allow dry in the fume hood.

Method (for free floating sections, mounted and air-dried)

1. Wash slides briefly in tap water to remove any residual salts.

2. Immerse slides through 2x3min changes of 100% ethanol

3. Defat the tissue: 15min in 100% xylene (2-3 changes as directed), then 10min in 100%

ethanol.

4. Rehydrate through alcohol (100% x2) 3min each.

5. Wash in tap water.

6. Follow the protocol for paraffin sections above from step 3.

The thicker sections require exposure to each solution for longer to allow the diffusion of
reagents.

OBSERVATION:

63
RESULT:

• Nissl substance-Purple/dark blue

• Neurones and cell nuclei - Purple/blue

APPLICATION:

✓ Cresyl Violet Acetate solution is used to stain Nissl substance in the cytoplasm of
neurons in paraformaldehyde or formalin-fixed tissue. The neuropil will be stained
a granular purple-blue. This stain is commonly used to identify the neuronal
structure in brain and spinal cord tissue.

Expt. No.:_______ Date:____________

64
 COLLAGEN - MASSON'S TRICHROME STAIN(TRI)

AIM:

To perform Collagen - Masson's Trichrome Stain(Tri) staining for the given tissue
sample.

PRINCIPLE:

As the name implies, three dyes are employed selectively staining muscle, collagen
fibers, fibrin, and erythrocytes. The general rulein trichrome staining is that the less porous
tissues are colored by the smallest dye molecule; whenever a dye of large molecular size
is able to penetrate, it will always do so at the expense of the smaller molecule.Others
suggest that the tissue is stained first with the acid dye, BiebrichScarlet, which binds with
the acidophilic tissue components. Then when treated with the phospho acids, the less
permeable components retain thered, while the red is pulled out of the collagen. At the
same time causing a link with the collagen to bind with the aniline blue.

FIXATIVE: Bouin's is preferred, 10% formalin.

TECHNIQUE: Cut paraffin sections 4m

EQUIPMENT: Rinse glassware in DI water. Coplin jars, 60°C oven or waterbath,

microwave

REAGENTS:

Bouin’s Fixative: Saturated picric acid 1500.0 ml Formaldehyde 500.0 ml Glacial acetic
acid 100.0 ml

Mix well, label, date and initial. Stable for 2 years.

Biebrich Scarlet:Biebrich scarlet 2.7 gm Acid fuchsin 0.3 gm Distilled water 300.0 ml
Glacial acetic acid 3.0 ml

Mix well, label with initial and date. Solution is stable for 6months.

Weigert's Iron Hematoxylin

65
Stock Solution A: Hematoxylin 5.0 gm95% alcohol 500.0 ml

Mix well, label with initial and date. Stable for 1 year.

Stock Solution B: 29% ferric chloride 20.0 ml Distilled water 475.0 ml

Hydrochloric acid 5.0 ml

Mix well, label with initial and date. Stable for 1 year.

Weigert's Hematoxylin

Working Solution: Solution A 25.0 ml Solution B 25.0 ml

Mix well, solution will remain stable for 3 - 4 days.

Phosphotungstic/Phosphomolybdic Acid Solution: Phosphotungstic acid 25.0 gm

Phosphomolybdic acid 25.0 gm

Distilledwater 1000.0 ml

Mix well, label with initial and date. Solution is stable for 6 months.

Aniline Blue: Aniline blue 2.5 gm Distilled water 100.0 ml Glacial acetic acid 1.0 ml

Mix well, label with initial and date. Solution is stable for 6months.

1% Acetic Acid: Glacial acetic acid 10.0 ml Distilled water 1000.0 ml

Mix well, label with initial and date. Solution is stable for 1 year.

PROCEDURE:

1. *Mordant in Bouin's solution, microwave 1 minute, allow to stand 15 minutes.

2. Wash in running tap water to remove the picric acid, 5 minutes.

3. Weigert's working hematoxylin, 10 minutes.

66
4. Blue in running tap water for 5 minutes, rinse in distilled water.

5. Biebrich scarlet for 5 minutes.

6. Rinse in distilled water.

7. Phosphotungstic/phosphomolybdic acid for 10 minutes, discard solution.

8. Transfer directly into Aniline blue for 5 minutes.

9. Rinse in distilled water.

10. 1% Acetic acid for 1 minute, discard solution, rinse in distilled water.

11. Dehydrate, clear, and coverslip.

*Conventional method: Mordant in Bouin's solution, 60°C for 1 hour.

OBSERVATION:

Sl.No Structures Stained in Colour

67
 1 Nuclei

2 Cytoplasm, muscle,
erythrocytes

3 Collagen

RESULTS:

Nuclei – black

Cytoplasm, muscle, erythrocytes – red

Collagen – blue

NOTES:

1. Light green may be substituted for Aniline blue.

2. 5% phosphotungstic acid for 5 minutes, must be substituted whenusing Light green.

3. When staining liver biopsies, the collagen is better light blue thandark blue.

APPLICATION:

✓ Used to differentiate between collagen and smooth muscle in tumors, and the
increase of collagen in diseases such as cirrhosis. Routine stain for liver and kidney
biopsies.

Expt. No.:_______ Date:____________

68
 OIL RED O - PROPYLENE GLYCOL – FAT

AIM:

To perform Oil Red O - Propylene Glycol – Fat staining for the given tissue sample.

PRINCIPLE:

Staining with oil-soluble dyes is based on the greater solubility of the dye in the
lipoid substances than in the usual hydroalcoholic dye solvents. Oil red O is a lysochrome
(fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen
sections and some lipoproteins on paraffin sections. In histology, a supersaturated solution
of oil red O in isopropanol may be used to stain fat in tissue.

CONTROL:

Use a positive control of a fat smeared slide, and a negative control slide of a
paraffin processed tissue, such as lung.

FIXATIVE: 10% formalin.

TECHNIQUE: Cut frozen tissue sections 10µ

EQUIPMENT: Cryostat, coplin jars. (Making stain, stir plate, filter paper, fritted glass filter,
and vacuum) and a 60°C oven. Rinse all glassware in DI water.

REAGENTS:

Propylene Glycol:

Place in two coplin jars, label #1and #2, can be reused.

85% Propylene Glycol: Propylene glycol 85.0 ml Distilled water 15.0 ml

Hematoxylin: Commercial Gill-3

Glycerin Jelly

69
Oil Red O Solution: Oil red O 0.7 gm Propylene glycol 100.0 ml

Dissolve oil red O in propyleneglycol, slowly, while stirring. Heat to 100°C, but not over
110°C. For a few minutes stirring constantly. Filter through Whatman #2 filterpaper. Cool,
and filter again through a frittered glass filter of medium porosity with suction.

Store in a 60°C oven. Solution stable for 1 year.

PROCEDURE:

1. Pick-up frozen sections on clean glass slides if fresh, albuminized slides if fixed.

2. Fix slides in 10% formalin if fresh.

3. Wash well it tap, rinse in distilled, drain off excess water.

4. Propylene glycol, two changes, 5 minutes each.

5. Oil red O, 7 minutes, agitate.

6. 85% Propylene glycol, 3 minutes.

7. Rinse in distilled water.

8. Hematoxylin, 1 minute.

9. Wash in water.

10. Bluing solution, 20 dips, or running tap water.

11. Wash in tap water, rinse in distilled.

12. Mount with aqueous mounting media, Glycerin Jelly.

OBSERVATION:

Sl.No Structures Stained in Colour

70
 1 fat

2 Nuclei

RESULTS:

Fat – red

Nuclei - blue

PURPOSE:

✓ To demonstrate fat or lipids in fresh tissue sections. Fat occurring in an abnormal

place, such as fatty emboli that may develop after either a bone fracture or an injury
that crushes a fatty body area. Tumors arising from fat cells (liposarcomas) can be
differentiated from other types of tumors.

Expt. No.:_______ Date:____________

71
 SIMPLE STAIN

AIM:

To perform Simple stain for the given tissue sample.

PRINCIPLE:

A simple stain consists of a solution of a single dye. Some of the most commonly
used dyes are methylene blue, basic fuchsin, and crystal violet. Simple stains allow one
to distinguish the shape (morphology) of the bacteria. For example, E. coli and Bacillus
subtillus are bacilli or rod-shaped bacteria. Many bacilli occur singularly, but chains may
also be observed. Bacilli very greatly in length and diameter. Staphylococcus
aureus and Streptococcus pneumoniae are cocci or spherical bacteria. Cocci may occur
singularly, in pairs (as in Streptococcus pneumoniae), or in clusters (as in Staphylococcus
aureus). R. rubrum is a spirillum or curved bacterium, Spirilla always occur singularly.

DIFFERENTIAL STAINS:

Differential stains are more complex than simple ones and use more than one stain
to differentiate cellular components. They are used to examine structural differences
between bacterial groups or to provide contrast to different structures within the same
organism.

GRAM STAIN

The Gram stain procedure uses 3 different stains. These are crystal violet, Gram’s
iodine, and safranin. The cells are first stained with crystal violet, then Gram’s iodine.
Following a rinse in alcohol, to de-colorize the cells, the cells are then stained with safranin.

MATERIALS NEEDED

• Bacillis subtillis liquid culture and various other bacterial cultures

• Sterile loops

72
 • Microscops slides

• Coverslips

• Compound microscope

• Sterile water or sterile 0.9% NaCl (sterile saline)

• methylene blue stain (Ward’s)

• gram stain kit (Wards): crystal violet, Gram’s iodine; safranin

• 95% ethanol

EXPERIMENTAL PROCEDURE

A. WET MOUNT

The wet mount is a preparation of a culture to observe motility (movement) or structure of

microorganisms.

Use a sterile inoculating loop to place a loopful of a motile bacillus culture on a slide. Cover
immediately with a coverslip. Do not allow the preparation to dry out. Observe under the
microscope. Draw a picture of what you see.

B. SIMPLE STAIN

73
1. Place a loopful of bacillus culture into a test tube of sterile distilled water to make a
suspension of bacterial cells in the water. Place a loopful of this bacterial
suspension on a clean slide. Allow the bacteria on the slide to air dry.

2. Heat fix the cells by passing the slide quickly through the flame of a Bunsen burner
two or three times, with the glass surface exposed to the flame. Each pass should
only be a second or two. The slide should not be so hot as to be uncomfortable to
touch. (NOTE: your instructor will demonstrate this for you!)

3. Flood the slide with methylene blue stain for 30 seconds.

4. Rinse the slide with distilled water, blot it dry, and examine it under the microscope.

5. Draw what you observe.

74
Observation and result:

Draw the results from your stains.

1. Magnification

2. Cell shape

3. Cell color

4. Acid-Fast

APPLICATION:

✓ Simple stains are single dyes used to stain the organism and it has limited clinical
application. The dye is negative and the bacterium is positively charged and they
will get stained due to the interaction of the opposite charges. It doesn’t provide a
lot of detail on structure though.

Expt. No.:_______ Date:____________

75
 GRAM STAIN

AIM:

To perform Gram stain for the given tissue sample.

PRINCIPLE:

The Gram stain procedure separates almost all bacteria into two large groups:
the Gram-positive bacteria that stain blue and the Gram-negative bacteria that stain pink.
Bacteria take up the Gram stain differently because they differ in cell wall composition.
Gram-positive bacteria have a thick cell wall layer. Alcohol does not readily penetrate to
decolorize the cell wall of the previously applied crystal violet stain. Gram-negative cells
have a thinner cell wall through which the alcohol readily penetrates. The crystal violet is
removed from these cell walls that are then stained with the safranin counter stain

MATERIALS NEEDED

Cultures to select from (18-24 hour broth or agar):

Bacillus cereus (Gram-Positive rod)

Enterobacter aerogenes(Gram-Negative rod)

Enterococcus faecalis(Gram-Positive coccus)

Escherichia coli (Gram-Negative rod)

Proteus vulgaris (Gram-Negative rod)

Pseudomonas aeruginosa (Gram-Negative rod)

Staphylococcus epidermidis (Gram-Positive coccus)

• Sterile loops

• Microscops slides

76
• Coverslips

• Compound microscope

• Sterile water or sterile 0.9% NaCl (sterile saline)

• methylene blue stain (Ward’s)

• gram stain kit (Wards): crystal violet, Gram’s iodine; safranin

• 95% ethanol

77
PROCEDURE

78
 1. Transfer a loopful of the bacterial suspension to the surface of a clean glass slide,
and spread it over a small area. Allow the slide to air dry. Fix the cells by passing
the slide briefly through the Bunsen burner flame.

2. Flood the slide for one minute with a crystal violet solution. Wash off briefly with tap
water (not longer than 5 seconds).

3. Flood slide with Gram’s iodine solution and allow to sit for one minute. Wash off
with tap water and drain off excess liquid.

4. Flood slide with 95% alcohol and pour off immediately. Re-flood with 95% alcohol
for 10 seconds and wash off with tap water. Drain off excess liquid. (NOTE: The
first flooding with alcohol removes the excess water from the slide, so that alcohol
used for decolorization is not diluted)

5. Flood the slide with safranin solution and allow it to stain for at least one minute.
Wash off with tap water. Drain the slide and blot it dry with bibulous paper. DO NOT
RUB.

6. Observe the slide under the microscope. Record what you see. Compare the size,
shape and color with each of the 3 different techniques you have used (wet mount,
simple stain, Gram stain).

7. Perform wet mounts, simple stains, and Gram stains on the additional
microorganisms (including, for example, bacillus, staphylococcus, and spirillum)
that will be provided for you in the lab.

GRAM STAIN (Steps): Heat fix the stain with the following steps

a) Apply crystal violet - 1 min

b) Rinse for 5 seconds with water

c) Cover with Gram’s iodine for 1 min

79
d) Rinse for 5 seconds with water

e) Decolorize with 95% ethanol for 15-30seconds

f) Rinse for 5 seconds with water

g) Counterstain with safranin for 1 minute

h) Rinse for 5seconds with water

i) Blot with filter paper

OBSERVATION:

Sl. No. Gram +ve/-ve Name of the Bacterium

80
RESULT

Streptococcus pneumoniae, Staphylococcus aureus, and Bacillus subtillis are Gram-

positive and stain blue.

E. coli and R. rubrum are Gram-negative and stain pink.

APPLICATION:

✓ Gram staining is a bacteriological laboratory technique used to differentiate

bacterial species into two large groups (gram-positive and gram-negative) based
on the physical properties of their cell walls.

✓ The Gram stain is almost always the first step in the identification of a bacterial
organism. While Gram staining is a valuable diagnostic tool in both clinical and
research settings, not all bacteria can be definitively classified by this technique.
This gives rise to gram-variable and gram-indeterminate groups as well.

81
Expt. No.:_______ Date:____________

PARTS OF COMPOUND MICROSCOPE

Aim:

To study the different parts and their uses in a microscope. A microscope is

an instrument used to study the structural detail of an object, which cannot be observed
by a naked eye. A monocular light compound microscope is used in the laboratory.

Requirements:

Compound microscope, stained blood film, source of light.

Parts of a microscope:

A compound microscope has the following parts:

1. Base

2. Body

3. Stage

1. Base: It is made up of a heavy metal for providing stability to the microscope.

2. Body: It consists of:

a) A ‘C’ shaped metallic structure.

b) A body tube.

c) Revolving nose piece with objective pieces.

d) Eye piece.

82
 a) A ‘C’ shaped heavy metallic structure: This connects the base with the
body tube.

a) Body tube: It is a metallic tube having a fixed length of 16 cm. The body
tube is fixed to the upper end of the ‘C’ shaped metallic structure either
vertical or in slanting position. It has a revolving nose at its lower end and
an eye piece at its upper end.
b) Revolving nosepiece: The revolving nosepiece is located at the lower
endof the body tube with three or four objective pieces. The resolving power
of each objective piece is indicated on it. Low power objective 10X High
power objective 40X Oil immersion objective 100X The correct position
of an objective piece is indicated by click sound while the revolving
nosepiece is rotated.
b) Eye piece: Eyepiece is located at the upper end of the body tube. They vary
in their resolving power. Eyepieces are 5X, 8X, 10X, 15X. Certain eyepieces
are provided with a pointer, which is useful to point a specific structure, E.g.
Neutrophil, Eosinophil & etc. Some other eye pieces are provided with small
windows, which restrict the size of the field. This facilitates the counting of
cells in a smaller field especially when there are large number of cells in a
single field, E.g. RBC and Reticulocytes.

The stage of compound microscope consists of:

a) Mechanical stage.

b) Sub stage with the condenser and iris diaphragm.

These are fixed to the lower end of the ‘C’ shaped structure.

Mechanical Stage:

It is meant for placing the slide and has a central aperture for focusing light on to the slide.
A slide moving arrangement is attached to the stage for moving the slide forward,
backward and side to side.

83
Sub stage:

It carries a condenser for focusing light on to the slide. The condenser is adjustable with
the help of knob. While studying finer details, the condenser is kept at the highest level
and at the lowest level for studying the gross detail of an object. An iris diaphragm present
in the sub stage controls the quantity of the light that enters the condenser. Just below the
sub stage a small ring meant for placing a filter is fixed. The filter helps in selecting a
particular wavelength of light.

Adjustable Knobs:

There are two adjustable knobs, one for coarse and another for fine adjustment. The
coarse knob is used for gross movement where as fine knob is used for fine adjustment.

Setting up of microscope:

• Place the compound microscope on a flat surface of the table and put
light source on.

• Put the low power objective piece into (10X ) focus with the concave
mirror in position reflecting the light into the condenser.

• Take a glass slide with stained blood smear and place on the stage
and fix it between the clips.

84
• Look through the eyepiece while adjusting the coarse and fine knobs till you come
into clear focus of the blood cells, under low power objective. Note the appearance
of the blood cells.

85
 • Now bring the high power objective into focus by rotating the revolving nose.

• Bring the blood cells into clear focus by adjusting the adjustable knobs and note
down the appearance of the blood cells.

• Now try to bring the oil immersion lens into the focus in the following steps to obtain
greater resolution of the object

• Place a drop of oil on the slide and open the iris to allow maximum illumination.

• Raise the condenser to the maximum and change the mirror to plane side

• Turn the oil immersion lens into the position.

• Looking from the side lower the objective by using coarse knob till it touches the oil
drop.

• Now looking through the eyepiece carefully adjust the coarse knob till the cells
appear in the field.

• Bring it to the final focus by adjusting the fine knob and note down the appearance
of the blood cells. The oil immersion lens is used for maximum resolution and for
studying finer details of fixed and stained tissue preparations.

Magnification:

The image of an object is magnified twice in a compound microscope

Mirror:

It is fixed below the sub stage and carries plane mirror on one side and concave
mirror on the other side. Plane mirror reflects the parallel light rays to the condenser and
is used for studying finer details when oil immersion lens is used. For other purpose
concave mirror is used.

86
Oil:

Cedar wood oil or liquid paraffin or castor oil can be used while the oil immersion lens is
at focus. The oil has the same refractive index as that of the glass slide and so prevents
refraction of light rays. This helps in the better resolution of the lens system.

Light Source:

It can be either natural light or an incandescent light. For studying gross detail natural light
is used and for studying finer details of an object a light source of constant intensity is
necessary.

First it is magnified by the objective lens so that an inverted, enlarged real image is formed
in the upper part of the tube. The second magnification is brought about by the eyepiece
lens.

Precautions:

• Keep the microscope covered when not in use to prevent deposition

of dust on the lenses.

• Wipe out the remnants of oil on the lens with a clean dry soft cloth or
tissue paper.

• Manipulate adjustable knobs gently.

• While using place the microscope on a stable plat form.

• Replace the microscope in its position after use.

• Provide adequate illumination by using external constant light source.

87
 Ray diagram of light microscope

RESULT:

Thus the different parts of microscope and their uses were studied.

88
Expt. No.:_______ Date:____________

STUDY SLIDES OF BENIGN AND MALIGNANT TUMORS

BENIGN TUMORS:

Benign tumors are generally spherically or Ovid. They are encapturated or well
circumscribed. Freely movable, firm and uniform. The cells of benign cancer are well
differentiated and the structure may be difficult of tissue or organ. The rate of growth is
usually progressive and slow. They may come to a standstill or regress. The mitotic figure
are rare and normal. The local invasion is usually cohesive well demarked masses that do
not in way or infiltration the surrounding normal tissue. Metastasis is absent

MALIGNANT TUMORS:

Malignant cells are irregular in shape poorly circumscribed the cells of malignant cancer
cells lag differentiation with anaplasia and the structure is often a typical. The rate of growth
may be slow to rapid and the mitotic figures may be slow to rapid and the mitotic figures
may be numerous and abnormal. It is locally invasive infiltrating the surrounding normal
tissue. Sometimes they may be cohesive and expensile. Metastasis is frequently present.
The larger and more undifferentiated the primary the more likely are the metastasis.

89
90
Expt. No.:_______ Date:____________

STUDY SLIDES OF ANEMIA

Anemia (from Greek word “anemia’ meaning ‘lack of blood’) is a decrease in the normal
number of red blood cells (RBC) or less than the normal quantity of hemoglobin in the
blood. However it can also include decreased oxygen winding ability of each hemoglobin
molecules due to deformity or lack of development as in some types of hemoglobin
deficiency.

Sine hemoglobin found inside the RBC’s normally carried oxygen from the lungs to
the tissues anemia leads to hypoxia. Hypoxia or lack of oxygen in the organs. Since all
human beings depend on oxygen for survival, varying degrees of anemia can have a wide
range of clinical consequences.

The three main classes of anemia include excessive blood loss such as hemorrhage
or excessive blood cell destruction. (ie) Hemolysis or deficient red blood cell production
which is due to ineffective hematopoisis. Anemia is the most common disorder of the blood
can cause pallor some patients experience other symptoms like fevers chills, night sweat
and other flue like symptoms are feeling fatigue. Some patients experience nausea or a
feeling of fullness due to enlarged a liver or spleen .This can result in unintentional weight
loss.

91
92
Expt. No.:_______ Date:____________

STUDY SLIDES OF LEUKEMIA

Leukemia (fromeek ‘leukos’ meaning white ‘amia’ meaning blood) is the cancer of blood
or bone marrow and it is characterized by an abnormal proliferation(Production by
multiplication) of blood cells. Usually white blood cells are leukocytes. Leukemia is
clinically and pathologically subdivided into two parts.

i) the acute forms and

ii) Chronic forms.

Acute leukemia is characterized by the rapid increase of immature blood cells. This
crowding makes the blood cells. Immediate treatment is required in acute leukemia due to
the rapid progression and accumulation of malignant cells which then spill over into the
blood stream and spread to other organs of blood. Acute forms of leukemia are the most
common form of leukemia in children.

Chronic leukemia is distinguished by the excessive build up of relatively matured but

still abnormal white blood cells. Typically taking months or years to progress the cells are
produced are a much higher rate than normal cells resulting in many abnormal white blood
cells in the blood where as acute leukemia must be treated immediately chronic forms are
sometimes monitored for sometime before treatment to ensure maximum effectiveness of
therapy. Chronic leukemia mostly occurs in order people but can theoretically occur in any
age group. Additionally the diseases are subdivided according to the kind of blood cell
affected. This divides leukemia’s into lymphoblastic or lymphocytic leukemia and myeloid
or myelogeneous leukemia.

The myeloid or myelogenous leukemia the cancerous change takes place in a type of
marrow cell that normally goes on to form red blood cells. Some type of white cells and
platelets.

93
 Damage to the bone marrow by way of displacing the normal bone marrow cells with
higher numbers of immature white blood cells results in a lack of blood platelets which are
important in blood clotting process. This means people with leukemia may easily bruisede
bleed excessively or develop pin bleeds. The WBC’s which are involved in fighting
pathogens may be suppressed or dysfunctional. This could the patients immume system
to be unable to fight of simple infection or to start attacking other body cells. Because
leukemia prevents the immune system working normally. Some patients experienced
frequent infection ranging from infected tonsils. Soars is mouth or diarrhea to life
threatening pneumonia or opportunistic infections.

The high number of WBC’s appearances when a blood sample is viewed under a
microscope. These extra WBC’s are immature are dysfunctional. The excessive number
of cells can also interfere the levels of other cells causing a harmful imbalance in blood
count.

OBSERVATION:

94