Submitted to

VIDYASAGAR UNIVERSITY

For fulfilment of Bachelor degree in Medical Laboratory

Technology
Submitted by:
Mobile: 9547879900
 E-mail:- [email protected]

VIDYASAGAR INSTITUTE OF HEALTH

A Constituent of
Centre for Advancement of Higher Education, Midnapore
Rangamati Midnapore Paschim Medinipur – 721 102
(Affiliated: W.B.U.H.S. & Vidyasagar University for Para-Medical courses)

Date: 26.07.2021

TO WHOM IT MAY CONCERN

This is to certify that Subhadeep Dandapat, a regular student of Vidyasagar Institute of Health,

has been studying Bachelor of Medical Laboratory Technology (BMLT), a 3 and ½ years

regular degree course under the Vidyasagar University. His/her University Registration No. is

0000718 of the year 2017 – 18. He/She has completed the course with the 6moths mandatory

rotator internship at Midnapore Medical College and Hospital form 16.12.2020 to 15.06.2021.

He/she is a very diligent and disciplined student.

I wish him success in life.

Principal
(Dr. Harihar Bhowmick)

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 ACKNOWLEDGEMENT

It gives us an immense pleasure to express my profound gratitude to all those respective

personalities who helped me to complete my internship successfully.

My first and foremost expression of deepest and sincere gratitude goes to Prof. Dr. Tapan
Ghosh(Prof. & Head of Blood Bank dept, Bankura Sammilani Medical College and Hospital),
Prof. Dr. Swapan Pathak (Prof.& Head of Central Laboratory & Pathology dept, Bankura
Sammilani Medical College and Hospital), Prof. Dr. Jayanta Bikash Dey (Prof. & Head of
Microbiology dept, Bankura Sammilani Medical College and Hospital) and Dr. Falguni
Mukherjee(Head of Biochemistry dept, Bankura Sammilani Medical College and Hospital) for
his constant encouragement, tremendous support, patients guidance and suggestion throughout
the internship period. I would also like to thank Mr. Rajib Sengupta (MT. Lab), Mr. Apurba
(MT. Lab), Mr. Suman (MT lab), Mr. Moloy (MT lab), Mr. Lakkhan (MT lab), Mr. Subhankar
(MT. Lab), and Mr.Suvasish (MT. Lab) for kind cooperation during the entire internship period.
I would equally like to thank my friends and the lab. Technicians of the departments I
worked.

Finally, I would like to express special thanks to my family members for their constant
encouragement and endless support throughout my student life and during the project work.

………………………….
Amit Singha Mahapatra
Date. ROLL.1547766 NO.180053

REG.NO.0000689 of 2018-2019

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 INDEX

Sl.n0 contents Page no.

1. INTRODUCTION 6

2. AIMS AND OBJECTIVES 8

3. PATHOLOGY 9-48
a) Histopathology
b) Clinical pathology
c) Hematology
d) cytology
4. CLINICAL MICROBIOLOGY 49-76
a)media
b)bacteriology
c)serology
d)mycology
e)protozoology
5. CLINICAL BIOCHEMISTRY 77-103

6. BLOOD BANK & BLOOD 104-110

TRANSFUSION
7. RESULTS 111-121

8. OUTPUT OF INTERNSHIP 122

TRAINING
9. CONCLUSION 123

10. ABBERVIATION 124

11. REFERENCE 125

5
 1.INTRODUCTION

In response to the letter of the Principal of Vidyasagar Institute of Health , Midnapore, and
permission received from the Director of Bankura Medical Collage & Hospital, Bankiura, I
was allowed to carry out six months internship training program at Bankura Medical Collage
& Hospital, Bankura. As per the schedule decided by the Head of the Deaprtments, I
imparted the training in Blood Bank 13-09-2021 to 12-11-2021 ,Central Laboratory from 13-
11-2021 to 12-12-2021, Pathology from 13-12-2021 to 12-01-2022, Microbiology from 13-
01-2021 to 12-02-2022. I carried out work in Biochemistry from 13-02-2022 to 12-03-2022 .

6
7
 2.AIMS AND OBJECTIVES
The basic purpose of internship training is expanding our knowledge about the subject along
with other ancillary matters like.

 To learn the advance techniques of independent approach, manners as well as

development of personal skill.
 To learn the principle operation techniques to use different modern and sophisticated
instruments used for human health and health related disorders.
 To gain knowledge about the patient handling and how to give instructions to the
patient before the collection of sample.
 To gain knowledge about collection, preservation, testing process and handling
individual sample according to the disease condition of the patient.
 To learn at the time of test how to handle different hazardous and non hazardous
sample and after how to dispose such type of samples.

1. Role of Laboratory Technologist:

a. Maintaining the cleanliness and safety of the Laboratory.
b. Ensure that the equipment and glassware are kept safe and clean.
c. Handled & Maintained the Microscope.
d. Sterilize the equipment‟s as per required.
e. Dispose of specimens and infected materials in safe manner.
f. Maintain the necessary reports of investigations done and submit the report of the
superintend.
g. They will make indent for the materials and for the laboratory through the
superintend and ensure the safe storage of materials and supplies received.
h. Prepared “Periodical reports” regarding his work and authenticate for
transmission.
i. Practice the Thumb rules of bio medical waste disposal.

2. Laboratory investigations:
a. Carry out the examination of blood.
b. Carry out Biochemical examination.
c. Carry out the examination of Urine (RE, ME).
d. Carry out the examination of stool (OPC).

3. Carry out for Serological and Microbiological Investigation.

4. Carry out examination for Malaria.

5. Performed Blood grouping, Rh Typing and Compatibility testing.

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 3. PATHOLOGY
a) HISTOPATHOLOGY
Definition:- It is a branch of pathology which cells deals with the study of disease in a
tissue section. The tissue undergoes a series of steps before it reaches the examiners desk to
be thoroughly examined microscopically to arrive at a particular diagnosis.

To achieve this it is important that the tissue must be prepared in such a manner that it is
sufficiently thick or thin to be examined microscopically and all the structures in a tissue may
be differentiated. The objective of the subsequent discussions will be to acquaint the staff
with their responsibility, the basic details of tissue handling, processing and staining.

The term Histochemistry means study of chemical nature of the tissue components by
histological methods.

The cell is the single structural unit of all tissues. The study of cell is called cytology. A
Tissue is a group of cells specialized and differentiated to perform a specialized function.
Collection of different type of cells forms an organ.

Type of material obtained in laboratory:

The human tissue comes from the surgery and the autopsy room from surgery two types of
tissue is obtained:-

1). Biopsy-A small piece of lesions or tumor which in sent for diagnosis before final
removal of the lesion or the tumor (Incision biopsy).

2). If the o of the tumor or lesion is sent for examination and diagnosis by the
pathologist. It is called excision biopsy.

3). Tissues from the autopsy are sent for the study of disease and its course, for the
advancement of medicine.

Types of Histological preparation:

The Histological specimen can be prepared as

1. Whole mount

2. Sections

3. Smears

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 1. Whole mounts - These are preparation entire animal e.g. Fungus, parasite. This
preparation should be no more than 0.2-0.5 mm in thickness.

2. Sections - The majority of the preparation in histology are sections. The tissue is
cut in about 3-5 mm thick piece processed and 5 microns thick sections are cut on
a microtome. These are then stained and permanently mounted.Microtomes are
special instruments which have automatic mechanism for cutting very thin
sections. To cut the sections on the microtome, The tissue must be made hard
enough to not get crushed. There are 2 methods of hardening the tissues.One is by
freezing them and the other is by embedding them in a hard material such at
paraffin wax or gelatine.

3. Smears- Smears are made from blood, bone marrow or any fluid such as pleural
or ascetic fluid. These are immediately fixed in alcohol to presence the cellular
structures are then stained. Smears are also made by crushing soft tissue between
tow slides or an impression smear in made by pressing a clean slide in contact
with the moist surface of a tissue. By doing this, the cells are imprinted on the
slide and these may be stained for cytological examination.

 Responsibility of a technician
The technician is responsible for

1. Specimen preservation.
2. Specimen labelling, logging and identification.
3. Preparation of the specimen to facilitate their gross and microscopy.
4. Record keeping.

To obtain these aims the following point need consideration.

1. As soon as the specimen is received in the laboratory, Check if the specimen is

properly labelled with the name, age, Hospital registration No. And the nature of
tissue to be examined and there question form is also duly filled.
2. Also check if the specimen is in proper fixative. Fixative should be fifteen to twenty
times the volume of the specimen and fixative matters have been taken care off.
3. Check if the financial matters have been taken care off.
4. Make the entries in biopsy register and give the specimen a pathology number called
accession number. Note this number carefully on the requisition form as well as the
container. This number will accompany the specimen everywhere.
5. If the specimen is large inform the pathologist who will make cut in the specimen so
that proper fixation is done. Container should be appropriate to hold the specimen
without distorting it.
6. Blocks of tissues taken for processing should be left in % formaline 60°C till
processing. These would be fixed in 2 hours.

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 7. Slides should be released for recording after consultation with the pathologist.
8. Specimens should be kept in their marked container and discarded after checking with
pathologist.
9. Block must be stored at their proper number the same day. Note the blocks have to be
kept preserved for life long. Slides should be stored in their proper number after 3
days. It gives time for the slides to be properly dried.

 FIXATION:-

Definition:-It is a complex series of chemical events which brings about changes in the
various chemical constituents of cell like hardening, however the cell morphology and
structural detail is preserved. Unless a tissue is fixed soon after the removal from the body it
will undergo degenerative changes due to autolysis and putrefaction so that the morphology
of the individual cell will be lost.

Principle of fixatives
1. Coagulation and precipitation as described above.
2. Penetration fixation is done by immersing the tissue in fluid containing the fixative.
Faster a fixative can penetrate the tissue better it is penetration power depends upon
the molecular weight e.g formalin fixes faster than osimic acid.
3. Solubility of fixatives- All fixatives should be soluble in a suitable solvent, preferably
in water so that adequate concentrations can be prepared.
4. Concentration- It is important that the concentration of fixative is isotonic or
hypotonic.

Amount of fixative
The fixative should be at least 15-20 times the bulk of tissue. For museum specimens the
volume of fixative is > 50 times.

Note: If the specimen is large then see that the sections are made to make slices which have a
thickness of 1.5 cm so that fixative can penetrate the tissue easily.

Reagents employed as fixatives (simple fixatives)

I. Formaldehyde- Formaldehyde is gas but is soluble in water to the extent of 37-

40% w/v. This solution of formaldehyde in water is called formalin or full
strength formalin. Formalin is one of the commonly used fixative in all
laboratories since it is cheap penetrates rapidly hours for small biopsies- 4-6 hours
at 65°C fixative occurs in 2 hours.
II. Alcohol (Ethyl Alcohol)- Absolute alcohol alone has very little place in routine
fixation for histopathology. It acts as a reducing agents, become oxidized to
acetaldehyde and then to acetic acid. It is slow to penetrate, hardens and shrinks
the tissue. Alcohol penetrates rapidly in presence of other fixative hence in

11
 combination e.g Carnoy‟s fixative is used to increase the speed of tissue
processing.
III. Acetone- Cold acetone is sometimes used as a fixative for the histochemical
demonstration of some tissue enzymes like phosphatises and lipases. Its mode of
action as fixative is similar to that of alcohol.

Preparation of the specimen for fixation

1. For achieving good fixation it is important that the fixative penetrates the tissue well
hence the tissue section should be > 4mm thick, so that fixation fluid penetrates from
the periphery to the centre of the tissue. For fixation of large organs perfusion method
is used i.e fixative is injected through the blood vessels into the organ. For hollow
viscera fixative is injected into the cavity e.g. urinary bladder, eyeball etc.
2. Ratio of volume of fixative to the specimen should be 1:20:3. Time necessary for
fixative is important routinely 10% aqueous formalin at room temperature takes 12
hours to fix tissue. At higher temperature i.e. 60-65°C the time for fixation is reduced
to 2 hours.

Fixatives are divided into three main groups

A. Micro anatomical fixatives- Such fixatives preserves the anatomy of the tissue.
B. Cytological fixatives- Such fixation are used to preserve intracellular structures or
inclusion.
C. Histochemical fixatives- Fixative used to preserve the chemical nature of the tissue
for it to be demonstrated further. Freeze drying techniques best suited for this
purpose.
Zenker‟s fluid
a. Mercuric chloride 5 gm
b. Potassium dichromate 2.5 gm
c. Sodium sulphate 1.0 gm
d. Distilled water to 100 ml
e. Add immediately before use : Glacial acetic acid : 5 ml

 Specific features
- Good routine fixative
- Give fairly rapid and even penetration
- It is not stable after the addition of acetic acid hence acetic acid (or formalin ) should
be added just before use.
- Washing of tissue in running water is necessary to remove excess dichromate.

12
 Zenker formal (Helly’s fluid)
a. Mercuric chloride 5 gm
b. Potassium dichromate 2.5 gm
c. Sodium sulphate 1.0 gm
d. Distilled water to 100 ml
e. Add formalin immediately before use 5 ml

 Specific features
- It is excellent microanatomical fixative.
- Excellent fixative for bone marrow spleen and blood containing organs.
- As with Zenker‟s fluid it is necessary to remove excess dichromate and mercuric
pigment

Bouin‟s fluid
a. Saturated aqueous picric acid 75 ml
b. Formalin 25 ml
c. Glacial acetic acid 5 ml
Specific features
- Penetrates rapidly and evenly and caused little shrinkage
- Excellent fixatives for testicular and intestinal biopsies because it gives very good
nuclear details, in tests is used for oligospermia and infertility studies.
- Good fixatives for glycogen
- It is necessary to remove excess picric acid by alcohol treatment.

Carnoy‟s fluid
a. Absolute alcohol 60 ml
b. Chloroform 30 ml
c. Glacial acetic acid 10 ml
 Specific features
- It penetrates very rapidly and gives excellent nuclear fixtion.
- Good fixative for carbohydrates.
- Nissil substance and glycogen are preserved.
- It causes considerable shrinkage.
- It dissolves most of the cytoplasmic elements. Fixation is usually complete in 1-2 hours.
For small pieces 2-3 mm thick only 15 minutes in needed for fixation.

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  DECALCIFICATION:-

Definition- Decalcification is a process of compete removal of calcium salt from the tissues
like bone and teeth and other calcified tissues following fixation.

Decalcification is done to assure that the specimen is soft enough to allow cutting with the
microtome knife. Unless the tissues in completely decalcified the sections will be torn and
ragged and may damage the cutting edge of microtome knife.

The steps of decalcification

1. To ensure adequate fixation and complete removal of the calcium it is important that
the slices are 4-5 mm thick. Calcified tissue needs 2-3 hours only, for complete
decalcification to be achieved so it in necessary to check the decalcification after 2-3
hours.
2. Fixative of choice for bone or bone marrow is zenker formal or Bouin‟s fluid.
Unfixed tissue tends be damaged 4 times greater during decalcification than a
properly fixed tissue.

Decalcifying Agents :
Nitric acid- 5%-10% aqueous solution used. They decalcify vary rapidly but if used for
longer than 24-48 hours.

Hydrochloric acid- 5%-10% aqueous solution decalcification slower than nitric acid but still
rapid. Fairly good nuclear staining.

Formic acid

1. Brings out fairly rapid decalcification.

2. Nuclear staining in better.
3. But requires neutralization and thorough washing prior to dehydration.

Aqueous nitric acid

Nitric acid 5-10 ml

Distilled water to 100 ml

Procedure
1. Place calcified specimen in large quantities of nitric acid solution until decalcification
is complete (Change solution daily for best results).
2. Washing running water for 30 minutes.
3. Neutralize for a period of at least 5 hours in 10% formalin to which excess of calcium
or magnesium carbonate has been added.
4. Wash in running water over night.
5. Dehydrate, clear and impregnate in paraffin or process as desired

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  TISSUE PROCESSING

Specific objective-The tissue processing is the heart of any tissue section which will be
cut adequately only if the tissue is properly preserved and processed. The study of this topic
is to understand the coarse and fine details of tissue processing so that excellent sections are
obtained.

Definition-The term tissue processing refers to treatment of the tissue necessary to

impregnate it into a solid medium so that the tissue is rendered sufficiently firm yet elastic for
the tissue sections of desirable thickness to be cut on microtome.

Principle of tissue processing-The tissue is embedded in a solid medium by the help of

first removing the tissue water which is then replaced by any solid medium such as paraffin
wax so that the tissue is rendered firm enough to enable thin sections to be cut, at the same
time, the tissue is fort (not so hard) to enable microtome knife to cut the sections. Prior to
paraffin wax embedding and impregnation the tissue must be subjected to the following
steps:

1. Fixation
2. Dehydration
3. Clearing- With a substance which is totally miscible with both the dehydrating agent
which precedes it, and embedding agent which follows it.
4. Embedding all these 4 processes depend upon complete impregnation of the tissue by
the agent like paraffin wax being used. Before going into the details of these 4 stages
it is important to understand the factors which influence the rate ad efficiency of
tissue impregnation.

 Steps of Paraffin Wax Embedding :-

Fixation-Usually tissue that is received at the laboratory is already fixed but before
proceeding further check if the fixation is complete.

Dehydration-After fixation in aqueous solvent the delicate tissue needs to be dehydrated

slowly starting in 50% ethyl alcohol. The other routine tissue specimen may be put in 70%
alcohol. A higher concentration of alcohol initially is in inadvisable because this may cause
very rapid removal of water may produce cell shrinkage. An exception to this is in case of
Heidenhain‟s fixed tissue where it may be placed directly in 95% alcohol. Tissue transferred
from alcoholic based fixative like Carnoy‟s fixative may be placed in higher grades of
alcohol or even in absolute alcohol36 for routine biopsy and post-mortem tissue of 4-7,
thickness 70%, 90% and absolute alcohol (2-3 changes for 2-4 hours each) are sufficient to
give reasonably satisfactory result.

15
Clearing-It means appearance of tissue after it has been treated by the fluid chosen to remove
the dehydrating agent.

Clearing agent is required when the dehydrating agent is not miscible with the impregnating
medium. It is essential for a clearing agent to bemiscible both in dehydrating agent as well as
embedding agent. Commonly used clearing agents are as follows.

1. Xylene- It has a rapid action. Biopsy specimens of 3-4 mm thickness are cleared in 2-
4 hours.
2. Toluene and Benzene are similar in properties to xylene but are less damaging to the
tissues on prolonged exposure.

 Impregnation :-

Definition- It is the complete removal of clearing reagents by substitution of paraffin or any

such similar media.

Impregnation with wax

Impregnation with paraffin wax takes place in an oven heated to 56-60°C depending upon the
melting point of the wax in use. Frequent check of the temperature of paraffin baths is
required since temperature 5°C above the melting point of the paraffin will cause tissue
shrinkage and hardening.

Embedding- It is the orientation of tissue in melted paraffin which when solidified provides
a firm medium for keeping intact all parts of the tissue when sections are cut.

 Section Cutting:-
Section objective

1. To ensure good section cutting.

2. To overcome trouble shooters during section cutting.
3. To familiarise the staff with the equipment used for section cutting.

Microtome Knives: The knife is probably the greatest single factor in producing good
sections.

Types of microtome knives: Microtome knives are classified by the manner in which they
are ground and seen in their cross section.

1. Plane wedge
2. Plano concave
3. Biconcave
4. Tool edge

Sharpening of microtome knives

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Honing

Definition- Grinding of knife on a hone to restore straight cutting edge and correct level.

Method of honing

1. The hone is placed on a bench on a non-skid surface.

2. A small quantity of light lubricant oil is poured on the centre of the hone and lightly
smeared over the surface.
3. The knife complete with handle and backing sheath is laid on the hone with the
cutting edge facing away from the operator, and the heel in the centre of the nearest
end of hone. Correct positioning of the fingers is achieved by holding the handle of
the knife between the thumb and forefinger with the cutting edge facing away from
the operator (so that the thumb in on the back). When the knife is on the hone the tips
of finger and thumb of other hand rest on the other endfo the knife ensuring even
pressure along the whole edge of knife during honing.
4. The knife is pushed forward diagonally from heel to toe, turned over on its back and
moved across the hone until the heel is in the centre 53 with the cutting edge leading,
and then brought back diagonally. It is turned to its original position, thus completing
figure of 8 movement.
5. The process is continued until all jagged edges have been removed now the knife is
ready for stropping.

Stropping:

Definition: It is the process of polishing an already fairly sharp edge. It removes burrs
formed during honing. Fine quality leather is used leather strops may be either
flexible/hanging or rigid. In stropping usually firm surface is preferred. Action is reverse of
honing to heel direction of stropping is also opposite.

Assessment of the sharpened knife edge

Examine the edge the knife by reflected light and under microscope to assess the honing and
stropping.

Microtome:-
These are mechanical devices for cutting uniform sections of tissue of appropriate
thickness. All microtomes other than those used for producing ultra thin sections for election
microscopy depend upon the motion of as crew thread in order to advance the tissue block on
knife at a regulated.

Number of microtome ;

Types of microtome

1. Hand microtome- Limited for use in botanical sections.

2. Rocking microtome.

17
 3. Rotary microtome.
4. Freezing microtome.
5. Base sledge microtome.
6. Vibrating knife microtome.

Section cutting of paraffin embedded tissue.

Fixing of block:

1. Fix the block in the block holder on the microtome knife in such as position that it
will be clear of the knife when it is in position, block may be fixed directly or it may
be fixed to a metal carrier which in turn is fixed to the microtome.

2. Insert the appropriate knife in the knife holder and screw it tightly in position. Adjust
if required. The clearance angel should be set at 3-4 degree and angle of slope should
be set permanently at 90 degree. It is important to tighten the knife clamp screw
securely and block clamp screws most also be firm. The exposed ends of the knife
must all the times be protected by magnetic or clip on knife guards to avoid any
accidents.

3. Trimming of tissue block: Move the block forward so that the wax block is almost
touching the knife. To trim away any surplus wax and to expose a suitable area of
tissue for sectioning. The section thickness adjusters are at 15 microns.

4. On exposing a suitable are of tissue the section thickness is set to the appropriate level
for routine purposes to 4-6 microns.

5. Apply ice to the surface of the block for a few seconds and wipe the surface of block
free of water. This step is optional but make sections cut easily.

6. Note that the whole surface of the block will move parallel to the edge of the knife in
order to ensure a straight ribbon of sections.

7. The microtome is now moved in an easy rhythm with right hand operating the
microtome and left hand holding the sections away from the knife. The ribbon is
formed due to the slight heat generated during cutting, which causes the edges of the
sections to adhere. If difficulty is experienced in forming the ribbon it is sometimes
overcome by rubbing one of the edges of the block with finger.

18
 8. During cutting the paraffin wax embedded sections become slightly compressed and
creased. Before being attached to slides the creases must be removed and the section
flattened. This is achieved by floating them on warm water. Thermostatically
controlled water baths are now available with the inside coated black. These baths are
controlled at a temperature 4-6°C below the melting point of paraffin wax. It is easy
to see creases if the inside of water is black.

9. The action in floating out muse be smooth with the trailing end of ribbon making
contact with water first to obtain flat sections with correct orientation, floating out
with the shiny surface towards the water is essential. When the ribbon has come to
rest on water the remaining wrinkles and folds are removed by teasing apart by using
forceps or seeker.

10. Picking up sections- The ribbon of sections floating on water is split into individual or
groups of sections by use of forceps or seekers. Picking up a section on slide is
achieved by immersing the slide lightly smeared with adhesive vertically from the
water, the section will flatten on to the slide. The sections are then blotted lightly with
moistened blogging paper to remove excess water and to increase contact between
section and slide. For delicate tissues or when several ribbons of sections are placed
on the slide, omit the blotting instead keep the slide in upright position for several
minutes to drain.

11. Drying of section- Sections are then kept in incubator with a temperature 5-6°C above
the melting point of wax i.e. at 60°C for 20-60 minutes. It is better to overheat than
under heat. If the sections are not well dried they may come off during staining.
The sections should not be allowed to dry without a good contact with the slide, such
sections will come off during staining.

Faults in cutting

1. Fault - Tear or scratch across the section or splitting of ribbon.

2. Fault - Tear or scratch across part of section.
3. Fault - Holes in the sections.
4. Fault – Cracks across the section parallel to knife.
5. Fault – Section curl as they are cut.
 Staining:-
The sections, as they are prepared are colorless and different components cannot be
appreciated. Staining them by different colored dyes, having affinities of specific components
of tissue, makes identification and study of their morphology possible.

19
Direct staining- Application of simple dye to stain the tissue in varying shades of colors.

Indirect staining- it means use of mordant of facilitate a particular staining method or the
use of accentuater to improve either the selectivity or the intensity of stain.

Progressive staining

Stain applied to the tissue in strict sequence and for specific times. The stain is not
washed out or decolorized because there is no over staining of tissue constituents. Staining is
controlled by frequent observation under microscope.

Regressive staining

Tissue is first over stained and then the excess stain is removed from all but the
structures to be demonstrated. This process is called differentiation and should always be
controlled under microscope.

Mordents

Substance that causes certain staining reactions to take place by forming a link
between the tissue and the stain. The link is referred as lake. Without it, dye is not capable of
binding to and staining the tissue. e.g. Ammonium and Potassium alum for haematoxylin.
The most common stain applied for histological study is Haematoxylin and Eosin.

Haematoxylin and Eosin staining

Haematoxylin is extracted from the wood of the logwood tree. When oxidized it
forms haematin, a compound that forms strongly colored complexes with certain metal ion,
notably Fe(III) and A(III) salts. Metal-haematin complexes are used to stain cell nuclei prior
to examination under a microscope. Structures that stain with iron – or aluminium-haematein
are often called basophilic, even though the mechanism of the staining is different from that
of staining with basic dyes.

Ingredients:

 Haematoxylin powder - 1g

 95% Ethyl alcohol - 10ml
 Ammonium or Potassium alum - 20gm
 Distilled water -200ml
 Mercuric Oxide -0.5ml

Blueing - Alum Haematoxylin stains nuclei and red color, which is converted to bluish back
color, when the section is washed in weak alkali. Tap water is usually alkaline enough to
produce this color change.

20
Procedure

i) Deparaffinze in hot air oven.

ii) Hydrate the section.
iii) 3 dips in xylene (2 Min each)
iv) 3 dips in acetone/alcohol (2 Min. Each)
v) In running tap water for 5 minutes.
vi) Mayer‟s haematoxylin for 15 minutes.
vii) Wash in running tap water for 20 minutes.
viii) Counter stain with eosin for 2 minutes.
ix) Dehydrate the section in 95% and absolute alcohol/acetone 2 changes (2 minutes
each).
x) Clear in xylene 3 changes (2 minutes each)
xi) Mount in DPX.

Results : Nucleus –blue cytoplasm and background – pink

Causes of poor quality of staining

1. Poor or inadequate fixation of tissue.

2. Over or under – ripened Haematoxylin.
3. Over used or worked out Haematoxylin. Insufficient blueing following differentiation.
4. Failure to wash blueing agent out of section before counter staining with eosin
(especially when ammonia is used)
5. Insufficient differentiation of eosin during washing or dehydration.
6. Insufficient dehydration and clearing of sections.

MCMANUS FOR GLYCOGEN (PAS)

Aim –staining and identification of the various types of carbohydrate‟s (Polysaccharides &
mucopolysaccharides).

Principle: Tissue structures like liver & heart, striated muscles are studied by periodic acid
stiff stain. Periodic acid reacts with aldehyde group of the carbohydrates and after wards
reaction with the Schiff‟s reagent produces a red or purple red color.

Reagents

1. 0.5% w/v periodic acid solution.

2. Schiff‟s reagent –
(a) Dissolve 1.0 gm of basic fuchsine in ≈ 100 ml of boiling distilled water cool to
about 60 degrees and filter.
(b) Add 20 ml of 0.1 N hydrochloric acid, cool further and add 1.0 gm of sodium
metabisulphite and mix well.
(c) Keep in the dark for 24-48 hours. When the solution becomes straw coloured, add
300 mg of activated charcoal, shake vigorously, filter and store.

21
 3. 1 N Hydrochloric acid.
4. 0.1 gm of light green in 100 ml of 0.1% (v/v) acetic acid.
5. Harris haematoxylin stain.

Procedure
1. Deparaffinise and hydrate to distilled water.
2. Oxidize in periodic acid solution for 5 minutes.
3. Rinse in distilled water.
4. Schiff‟s reagent solution for 15 minutes.
5. Wash in running water for10 minutes for pink colour to develop.
6. Harris haematoxylin for 6 minutes or light green counter stain for a few seconds.
7. Wash in running water.
8. Differentiate in 1% acid alcohol solution 3-10 quick dips.
9. Wash in running water.
10. Dip in ammonia water to blue the sections.
11. Wash in running water for 10 minutes
12. Dehydrate in 95% alcohol, absolute alcohol, clear in xylene two changes each.
13. Mount in DPX.

Results
With haematoxylin counter stain.

1. Nuclei – blue
2. Glycogen, mucin, hyaluronic acid, reticulin, colloid droplets, amyloid infiltration,
thrombi. – purple red.
3. Fungi – red
4. Background – pale green (with light green counter staining).

b) CLINICAL PATHOLOGY

1. Urine test (Physical, Chemical & Microscopically).

2. Stool test (Physical, Chemical & Microscopically).

3. Occult blood test in urine and stool.

Working in clinical pathology laboratory are required to examine various body fluids, like
blood, urine, spinal fluid, Pleural fluid, serous fluid and synovial fluid. Laboratory
investigation in clinical pathology primarily focuses on physical examination of fluid, clinical
microscopy and simple chemical screening.

22
  EXAMINATION OF URINE
PHYSICAL EXAMINATION

Color: Normal urine color is nearly colorless to dark yellow or straw color:

Specific :The Specific Gravity at a constant temperature is the ratio of the weight of volume
of urine to the weight of same volume of distilled water. The specific gravity determination is
used to measure the concentration and diluting power of kidney. The specific gravity of urine
thus varies throughout the day and the normal range is 1.003 to 1.035.

To measure the Sp. Gravity of urine we normally use Refractometer method Gravity :

Materials:

I. Refractometer scale.
II. Urine sample.

Refractometer method:

1. Clean the glass surface of the instrument with distilled water and soft cloth.
2. Close the cover over the glass surface and insert a drop of urine sample with the help
of Pasteur pipette.
3. Hold the instrument up to a light source.
4. Read the Sp. Gravity from the scale at the point where the dividing line between the
bright and dark fields meets.

CHEMICAL EXAMINATION OF URINE

pH Test : pH normal urine varies from 6 to 6.8 which is acidic.

A rough estimated of pH is made by .indicator paper, blue litmus or wide range indicator
paper.

Urine Albumin Test:

To estimate the albumin of urine “Heat and Acetic Acid test” is require.

Materials:

1. Urine sample.
2. 3% glacial acetic acid.

23
Heat and acetic acid test
a. The urine should be acidic, a few drops of 3% acetic acid is added to an alkaline urine
to make it slightly acidic.
b. Urine should be clear, turbid.
c. At first centrifuge the turbid urine in a test tube.
d. Taken out the supernatant portion for examination.
e. Take urine in a clean test tube filling 2/3 portion.
f. Boil upper half inch of the tube.
g. A white cloud appears in a heated portion, due to presence of protein or phosphate.
h. Add 2 to 3 drops of 3% glacial acetic acid.
i. If cloudiness disappears ------- Phosphate.
If cloudiness persists ---------- Albumin.

Observation:

Negative --- No cloudiness.

Trace --- Barely visible cloudiness.

+ --- Definite cloud without granular flocculation.

++ --- Heavy and granular cloud without flocculation.

+++ --- Dense cloud without marked flocculation.

++++ --- Thick, cloudy precipitate and coagulation.

Urine glucose Test :-

To sugar test of the urine “Benedict’s Test” is essential.

Benedict’s Test is used as a simple test for reducing sugars. A reducing sugar is a
carbohydrate possessing either a free aldehyde or free ketone functional group as part of it s
molecular structures. This includes all monosaccharides (i.e, Glucose, Fructose, Galactose)
and many Disaccharides, including Lactose and Maltose. Benedict‟s Test is most commonly
used to test for the presence of glucose in urine. Glucose found to be present in urine is an
indication of Diabetes Mellitus.

 Principle of “Benedict’s Test”

Reducing sugar under alkaline condition tautomerise form enediols. Enedios are powerful
reducing agents. They can reduce cupric ions (Cu2+ ) to cuprous ions (Cu+ ), which is
responsible for the change in colour of the reaction mixture. This is the basis of “Benedict‟s
Test”. When the conditions are carefully controlled, the colouration develop and the amount
of precipitate formed (Cuprous Oxide) depends upon the amount of reducing sugars present.

24
  Composition and Preparation of Benedict’s Reagant
One litre of Benedict‟s Solution can be prepared from 100 gm of anhydrous sodium
carbonate, 173 gm of sodium citrate and 17.3 gm of copper (II) sulfate pentahydrate.

Component Amount Functions

Copper Sulphate 17.3 gm Furnishes cupric ions (Cu++ )
Sodium Carbonate 100 gm Makes medium alkaline
Complexes with the copper
(II) ions so that they do not
Sodium Citrate 173 gm
deteriorate to copper (I) ions
during storage
Distilled water Upto 1000ml Solvent
Material:

I. Benedict‟s qualitative reagent.

II. Urine sample.

Benedict’s test
a. 5ml Benedict‟s qualitative reagent taken in a test tube.
b. Boil for 1 minute.
c. Add 8 drops urine drop by drop.
d. Boil for 3 to 5 minutes.
e. Then cool the mixture.

Observation
Negative --- No change in color.

Trace --- Pale green with slide cloudiness.

+ --- Green deposit.

++ --- Yellow deposit.

+++ --- Orange to red precipitate.

++++ --- Brick red precipitate.

Occult Blood Test in Urine :-

Method –Benzidine method.

Principle –The peroxides activity of hemoglobin decompose hydrogen peroxide and the
liberate active oxygen oxidases the compound benzidine.

25
Reagents –
1. Saturated solution of benzidine in glacial acetic acid.
2. Hydrogen peroxide (3% v/v in distilled water).

Procedure –
1. Mix equal parts of two reagents.
2. Add equal volume of urine.
3. The appearance of a green or blue colour within 5 min indicates presence of blood.
Report as follows.
Trace --- Faint green
+ --- Green
++ --- Greenish blue
+++ --- Blue
++++ --- Deep blue

MICROSCOPIC EXAMINATION OF URINE:

Materials:

I. Urine sample,
II. Centrifuge tube,
III. Glass slide,
IV. Cover slip,
V. Microscope.

Method:

1. Mix the urine very well and pour into a 10 ml centrifuge tube.
2. Centrifuge with another balanced test tube for 3-5 minutes at 2500 RPM.
3. Pour off the supernatant quickly and completely into another test tube.
4. Re-suspended the deposit by shaking the tube.
5. Place 1 drop of the deposit on a glass slide.
6. Cover it with a cover slip.
7. Observe it first under low power objective in a subdued light.

Observation:
1. Red cells, pus cells, renal epithelial cell are identified under high power objective.
2. Casts are identified under low power objective, but finer structures are identified
under HP [High power] objective.
3. Crystals are examined under low power objective.
4. Bacteria, yeast cells, trichomonas are examined under HP objective.

26
 DETERMINATION OF BILE PIGMENTS:

Requirements
i) Centrifuge tubes or test tubes 10 X 75 mm and 15 X 125 mm.
ii) Pasteur pipette
iii) 10 gm / dl barium chloride
iv) Fouchet‟s reagents
v) Ehrlich reagents
vi) Whatman no. 1 filter paper (if necessary)

Procedure
i) Place 3-4 ml of urine in a centrifuge tube by using a pasture pipette
ii) Add equal amount of 10 gm / dl barium chloride, mix well
iii) Centrifuge at 1500 RPM for 10 minutes
iv) Place supernatant in another test tube for urobilinogen test
v) Add one to two drops of Fouchet‟s reagent to the sediments
vi) Add about 0.5ml of Ehrlich Reagents to the supernatant

Observation
i) Sediment (in the test tube or the filter paper)
a) No change in colour; Bile pigments absent

b) Colour change to green; Bile pigments present

ii) Supernatant
a) Development of pale pink colour; urobilinogen, present normal
b) Development of cherry red colour; urobilinogen, increased

c) HEMATOLOGY
I. Blood Sample Collection by Venipuncture:

II. Routine hematological test

 Heamoglobin estimation
 Total count of RBC
 Total count of WBC
 Differential count of WBC in peripheral blood smear
 Total platelet count
 Erythrocyte sedimentation rate (ESR)
 Packed cell volume
 Red cell indices (MCV, MCH, & MCHC)
 Reticulocyte count

27
 IV. Coagulation profile test:
 Clotting Time.
 Bleeding Time.
 Prothrombin Time.

V. Special Heamatolgical Test:

 Absolute Eosinophil Count.
 HLA Typing (Human Lymphocyte Analysis)

i). Blood Sample Collection by Venipuncture:

The Volume of blood obtained by venipuncture is sufficient to carry out multiple
tests. Venipuncture can be done either by the syringe method or vacuum tube method. The
latter is disposable and is not very popular in developing countries because of the high coast.

Procedure:-
1. All the things are assembled required during blood collection. The things are
tourniquet, cotton, alcohol, syringe, container, anticoagulant etc.
2. Patient is identified and decided the total amount of blood needed for all the tests.
3. The containers are selected and labelled them with the patient‟s identification.
4. Then the patient is asked to sit alongside the table used for blood collection, Patients
arm is laid on table and palm upwards. For indoor patients are said to lay arm in an
outstretched position.
5. The puncture is selected carefully after toeing up the tourniquet.
6. After feeling the vein in left hand the skin is disinfected with a swab dipped in
methanol or 70% alcohol.
7. The syringe is then checked it the needle is fixed tightly.
8. After holding the syringe in right hand and bevel with uppermost it is pushed firmly
and steadily into the centre of vein at 30-40° angle.
9. Then the blood is appeared in barrel and the tourniquet is released without disbursing
the needle.
10. A Swales of cotton over the hidden point of the needle is placed.
11. The needle is removed from vein and the patient is asked to press the cotton swabs for
few minutes.
12. The needle is removed from syringe and gently expelled the blood into appropriate
container.
13. The blood is mixed immediately and thoroughly but gently with anticoagulant (it
required).
14. Immediately the syringe and needle is rinsed without cold water to prevent clotting.
15. If bleeding is topped, an adhesive tape is applied on the puncture site.

28
 ii). Routine Hematological Test:
Determination of Heamoglobin Concentration ( HB%) -
Hemoglobin is a conjugated protein present in RBCs. It carries oxygen from the lungs to
the tissue cells and carbon dioxide- The gaseous waste from the cells to the lungs. Hemoglobin
consists of two components-haem (Iron+Proteoporhyrin) and globin (amino acid chains).
There are three types of hemoglobin present in adult persone-

Structure Normal %

a. HbA (adult HB) --------- α2β2 97-98%

b. HbA2 --------- α2δ2 1.5-3%
c. HbF (foetal Hb) --------- α2γ2 Absent

Clinical significance:

A decrease in haemoglobin conc in blood below normal is a sign of anaemia and this leads to
reduce oxygen carrying capacity leading to anoxemlaischemic changes and ultimately to necrosis.
Elevated haemoglobin levels can be observed in Ploycythemiasandcongenital heart diseases.
Normal Values:
Male-15-18 gm%
Female-11-16 gm%
Children (at1yr)-10-14 gm%
Infants-14-20 gm%

Cyanmethaemoglibin Method

Principle :
In the presence of potassium Ferricyanide at alkaline pH, haemoglobin and its
derivatives are oxidized to methemoglobin. Methemoglobin, so formed reacts with potassium
cyanide to form cyanmethemoglobin, a red coloured complex, which is measured
colorimetricallyat 540 nm (green filter). The color intensity is proportional to the Hb
concentration of the blood sample.

Haemoglobin + Potassium fericyanide-Methemoglobin.

Mehemoglobin + Potassium cyanide- Cyanmethemoglobin.

Reagents:
Reagent-1: Drabkin‟s Solution (contain Potassium cyanide-50mg & Potassium
ferricyanide 200mg & Distilled water-1000 ml)

Reagent-2: Cyanmethemoglobins.
Sample: Whole blood sample (0.02 ml) is used.
Procedure:-

29
 Take test tubes for three test tubes for blank rendered and test. Level them and
proceed as following steps.

Pipette into test tubes Blank Standard Test

Cyanmeth Reagent 1.0ml --- 5.0ml

(Drabkins solution)

Cyanmethemoglobins --- 1ml ---

Sample --- --- 20µl

It is mixed thoroughly and after 5minutes the test sample is measured against blank at 540nm.

Standard: The O.D. of Reagent 2 (Cyanmethemoglobin standard) is directly measured either

on the spectrophotometer or on the colorimeter against Drabkin‟s solution.

Calculations: -
Blood Hb in gm %= OD of test/OD of std ×concentration of std. Mg% × Dilution factor
= OD of test/OD of std × 60 × 0.250
= OD of test/OD of std × 15

Precaution:
Store Drabkin‟s reagent in brown bottle, as it decomposes on light exposure. Once the
haemoglobin cyanide solution has been prepared, Hbestimation must be carried out within 6
hours.
Drabkin‟s solution should be clear and pale yellow. If it is turbid, it should be discarded. It
can be stored at cool temperature, 4-6°C in the refrigerator.

Acid haematin method (SahiMehod)

It is recommended for place where colorimeter is not available. It is not recommended
because all forms of Hb are not measured by this method.

Principle:
Hb is converted to acid hematin by reaction with HCL. The acid haematin solution is
further diluted with HCL acid until its colour matches exactly that of permanent standard of
comparator box. He Hb concentration is read directly from the hemoglbinometer tube.

30
Reagent and Equipments:
N Hydro Choric Acid, Sahli Haemoglobinometer, Haemoglobinometer pipette.
Aemoglobinometer tube.

Procedure:
1. Fill the haemoglobinometer tube up to 20 marks with 0.1 N Hydro Chloric Acid.
2. Draw the blood onto haemoglobinometer pipette (20µl) from anticoagulated test
blood sample.
3. Wipe out surface of the pipette with the wet cotton.
4. Blow the blood into acid solution of haemoglobinometer tube by adding 0.1 N
HCL, until the colour match.
5. Read the Hb concentration directly from the mark reached. The reading may be in
% or in gm/dl.

Determination of Total Count Of RBC :-

Introduction: - The Human red cell is normally a circular, non-nucleated, biconcave disc.
The red blood cell contains haemoglobin. The surface area of red cell is much greater than
that of the sphere of the same size. There is exchange of oxygen & carbon dioxide is maximal
with the biconcave configuration.

Clinical significance: - The total RBC count is performed to assess the red cell mass in
the blood. The change in erythrocyte number is frequently detected in clinical practice by
odering estimation of haemoglobin rather than total RBC count, as estimation of
heamoglobinis easy and less expensive more over total RBC count is still performed in some
conditions to detect he red cell population, especially if the count is expected to be a very
high as in poly cythemia.

Principle: - The blood specimen is diluted with RBC diluting fluid which does not remove
the WBC but allows the RBC to be counted under 400X magnification in a known volume of
fluid. Finally, the number of cells in undiluted blood is calculated.

Specimen: - Well mixed anticoagulated (EDTA) venous blood is mostly used.

Requirements: -
i. RBC diluting fluid
 Trisodium citrate 3.2 gm.
 Formalin 1.0 gm.
 Distilled water 100 ml
ii. Micro-pipette
iii. Graduated pipette
iv. Improved neubauer chamber with coverslip

31
Method:- Hemocytometry method [include improved haemocytometer chamber]
Normal Range:- 10 adult

Male : 4.5-6.0×106 Cell/µl of Blood

Female : 4.0-4.8×106 Cell/µl of Blood

Procedure:-

1). Collect all the equipment which needed for practical, & clean all the equipment
properly.

2). Take adequate RBC diluting fluid in a watch glass.

3) Make a finger prick under aseptic conditions & such blood in to the pipette &
dilute the blood with RBC diluting fluid.

4). After proper mixing take some amount of mixed diluting fluid through pipette.

5). Discard first two drops of fluid from the pipette.

6). Charge the neubauer‟s chamber and allow two to five minutes for proper charge
by which the cells settle down easily.

7). Place the counting chamber.

8). Switch to low power objective, adjust light and locate the large square in the
centre.

9). Now switch to high power objective.

10). The red blood cells are counted in the four squares including centre of
neubauer‟schamber.

Calculation:-
Total RBC Count=Total NO of RBC × Diluting factor/Total no of area count × area of each
square × depth of fluid

If Total NO of RBC=N

Diluting factor=200

Total NO of area=5

32
 Total NO of each area square=1/5

Depth of fluid=1/10-0.1

So, Total RBC Count=N × 200/5 × 1/5 × 1/5 × 1/10= N × 200 × 50=N × 10,000

Determination of Total Count Of WBC ( Leukocyte)

Introduction:- Leukocyte [White Blood Cell] are nucleated cells that are involved in the
defense mechanism of the body, Unlike red cells, White cells use the blood stream primarily
for transportation to their place of function in the body tissue. Leukocytes are classified as
granulocytes, & agranulocytes. Granulocytes are neutrophils, eosinophils, basophils &
agranulocytes are lymphocytes & neonocytes.

Clinical Significance:- Total leukocyte counter is a part of the routine heamotologic

investigation to assesss the naturo& severity of an infection in some diseases, alteration in
leukocyte count alone may be diagnostic, but frequently the leucocyte count is ordered with
other investigations especially with the differential leukocyte count to aid in diagnostic.When
the total leukocyte count increase above the normal the condition is called leucocytosis and
when the count decrease below normal range then the condition is called leucocytopenia.

Specimen :- EDTA mixed venous blood or fresh capillary blood.

Principle:- Blood is diluted with acid solution, which removes the red cells by heamolysis
& accentuates the nuclei of the white cells. The counting of the white cells then becomes easy
counting is done using a microscope under low power objective and with knowledge of the
volume of fluid examined & the dilution of the blood obtained. The number of white
cells/mm2 of undiluted whole blood is calculated.

Method :- Haemocytometric method [include improved haemocytometer chamber]

Requirement:-

i). Microscope
ii). Improved neubauer chamber

iii). WBC pipette

iv). WBC diluting fluid [Turk‟s fluid]

Composition of WBC diluting fluid

i). 1% glacial acetic acid solution.

ii). Creation violet stain or aqueous methylene blue [0.3%]

iii). Distilled water.

33
Normal Range :- Adults : 4,000-11,000/mm3 of blood

Newborns : 10,000-25,000/mm3 of blood

Infants : 6,000-18,000/mm3 of blood

Children : 5,000-15,000/mm3 of blood

Procedure:-

1). Assemble all equipment needed for practical, clean it properly.

2). Take adequate WBC diluting fluid in a watch glass.

3). Make a finger prick under aseptic conditions & such blood into the pipette &

dilute the blood with WBC diluting fluid.

4). After proper mixing take some amount of mixing diluting fluid through pipette.
5). Discard the first two drops of fluid from the pipette.

6). Charge the neubauer‟s chamber allow two-five minutes for proper charge by
which the cells settle down easily.

7). Place the counting chamber.

8). Switch to low power objective, adjust light & located the large square in the
centre.

9). Now switch to high power objective.

10). The white blood cells in the four corner squares are counted.

Calculation:-
White cell total count = Total NO of WBC × diluting factor/Total number of area count ×
area of each square × depth of fluid

Where i) Dilution = 1:20,

ii) Area counted = 4

iii) Each square = 1mm

iv). Depth of fluid = 1/10 = (0.1)

v). No of white cells counted = N

Now Total white blood cells = N × 20/4 × 1 × 1 ×1/10 = N × 20 × 10/4 = N × 50

34
Determination of Differentional Count Of WBC

Introduction:- Differential count is the percent (%) distribution of various white cells in
the peripheral blood as determined from a blood smear stained with a polychromatic stain.

Clinical Significance:- Differential count is useful to identify change in the distribution

of white cells which may be related to specific type of disorders. It also gives idea regarding
the severity of the diseases and the degree of response of the body.

Principle:- The polychromic staining solution contain methylene glue and casing. The
basic and acidic dyes induced multiple colors when applied to cell. Methanol acts as fixative
and also a solvent the natural components of the cell are stained by both dyes.

Speciman:- EDTA-Anti coagulated blood or fresh capillary blood.

Normal Range:-
i). Neutrophils -40%-75%
ii). Eosinophils -1%-6%
iii). Basophils -0%-1%
iv). Lymphocytes -20%-45%
v). Monocytes -2%-10%

Requirements:-

a). Microscope slider & A glass spreader

b). Cederwood oil

Reagent:-
a). Leishman stain:
i). Leishman powder -0.5gm
ii). Acetone free methyl alcohol -100ml

iii). Eosin -7gm

35
b). Buffer (pH7)

Procedure:- Thin smear is prepared by spreading a small drop of blood evenly a slide.

Blood film preparation:

 Take a clean dry grease free slide.
 Transfer a small drop of blood near the edge of the slide.
 Place the spreader slide at an angel of 30°degree. Pull back the spreader until it
touches the drop of blood. Let the blood run among the edge of the spreader.
 Push the spreader forward to the end of the slide with a smooth movement.
 Dry the blood smear at room temperature.

Staining the film:

 Cover the smear with the staining solution by adding 10-15drops on the smear.
Wait for 2 minutes.
 Add equal number of the drops of buffer solutions. Mix the reaction mixture
adequately by blowing on it through a pipette. Wait for 10 minutes.
 Wash the smear by using tap water.
 Stand the slide in a draining rack.

Examination of film:
 First examine the stained smear under the low power (40 X) and adjust the film.
 Then examine the film under high power (60 X).
 Examine the film by moving from one field to the next field. Record the type of
Leukocytes seen in each field.
 Count at least a total of 100 Leucocytes.

Determination of Platelet Count

Introduction:- Platelets are smallest cells in blood circulation, they participate in blood
clotting process. They are membrane encapculated fragments of magacayocytes. Although
they are encecleated and have a diameter of 1-4 (µ) microne. The life span of platelets is 9-10
days. Its determination is requested in investigation of bleeding disorders.

Clinical Significance:- Decrease of platelet count is known as Thrombocytopenia is

often associated with pro longed bleeding and poor clot retraction. It also occurs in-

i). Aplastic anemia.

ii). Megeloblastic anemia.

iii). Hypersplenism.

36
 iv). Acute leukaemia.

v). Cytotoxic chemotherapy.

vi). HIV infection.

vii). Septicaemia.

Increase in platelet count is known as Thrombocytosis, Improtantly a high platelet count

may also place a risk for a bleeding. It occurs in conditions like.

 Polycythemia vera.
 Chronic myelogonous leukaemia.
 Spleenctomy.
 Renal failure.

Normal Range:- 1.5-4.5 lakhs/mm3

Speciman:-

1). EDTA anti=coagulated blood is the recommended specimen for platelet count.

2). Capillary blood can be used but venous blood generally for satisfactory results as
capillary blood gives lower values than venous blood.

Principle:- The diluents prevents coagulation as it fixes the platelets and prevents them
from clumping. No attempt is made to lyses the RBC‟s. Platelets are identified by their size,
shape and darker. The dye provides the background during cell counting. This dye does not
stain the platelet and it is essential for counting procedure.

Reagents:

Diluting fluid:-

1). Tri Sodium Citrate (.106 M) -3.8gm

2). Neutral formaldehyde (40%) -0.2ml

3). Brilliant cresol blue -0.1gm

4). De-ionised water -100ml

Equipments:-

1). Haemocytometer.
2). Sahli‟s pipette.

37
 3). Microscope.

4). Test tube.

5). Petri dish with filter paper.

Procedure:-
1). Transferred 3.98ml. of diluents into a test tube.
2). The blood specimen is gently mixed for about 2minute.

3). 20/µl of fresh non-coagulated blood is added into the test tube and the contents of
the pipette is rinsed with the diluting fluid for 3-4 minutes.

4). Immediately the diluents is mixed with the specimen for at least 5 minutes.

5). The haemocytometer chamber is then placed on the stage of the micro scope. By
using Sahli‟s pipette a drop of specimen is transferred on each side of the counting
chamber.

6). Place the mounted haemocytometer into a moist surface and let it stay undisturbed
for 15 minutes. The cover slip is placed on the counting chamber.

7). The red cell counting is focused under low magnification and then under high
magnification of microscope.

Observation:-

Platelets are bluish and must be distinguished from debris. They are oval, round or shaped
with varies size from 1-5µm.

Calculation:-

Platelet count/µm or mm3 = Number of platelets × dilution/ volume of fluid

= (N × 200)/.1

Where, volume of fluid = 1 × 0.1 = 0.1 µm

Dilution = 400/.02 = 200

38
Determination of Erythrocyte Sedimentation Rate (ESR) :-

Introduction:- The whole blood is allowed to settle, sedimentation of the erythrocyte will
occurs the rate at which the red cells fall is known as the erythrocyte sedimentation rate.

Clinical Significance:-
Erythrocyte sedimentation rate or ESR is a none specific test that reflects changes in plasma
protein which accompany most of the acute & chronic infections some of these pathologic
condition. Its means if there is high ESR, we can say that there is a disease but we cannot
diagnosis of the disease ESR does not change in functional disease

Principle:-
The erythrocyte sedimentation rate of ESR of blood may determined by the height of mm of
column of clear plasma lying about a ventral column of blood at the end of 1hour where a
sample of blood treated with an anti coagulant is left standing in an long tube place vertically.

Specimen:-Citrated ant coagulated blood determine ESR within 2 hours after blood
collection.

Method:-WESTERGREN Method, wintrobe method

Normal Range:-
1). Westergern method - Male-5-15 mm/hr

- Female-5-20 mm/hr

2). Wintrobe method - Male-0-9mm/hr-

- Female-0-20mm/hr

Reagent:-
1). Anticoagulant - 1mm of 3.8% Na citrate sol

2). Patient blood - 4ml

Westergren Method:-
1). Westergren‟s pipette (open at both ends) is about 30 cm long a bore diameter of
about 2.5mm.
2). The lower 20 cm are marked from 0 (top) to 200 (bottom).

3). Anticoagulant used is 3.8% trisodium citrate solution. One part of anticoagulant is
added of 4 parts of blood.

39
 4). The pipette accepts about 1 ml of blood. Fill the pipette by sucking till the 0 mark
and clamp it vertically in the Westergre‟s rack.

5). Read the upper level of red cells exactly after 1 hour.

This is a better method than windtrobe‟s since the reading obtained is magnified as column is
lengthier.

Calculation:

Mean ESR = 1st hour reading + 2nd hour reading/2/2

Hematocrit Packed Cell Volume ( PCV)

Introduction:- Hematocrit means “Blood separation” the hematocrit measures the

percentage of volume of the packed red cells. Therefore, hematocrit also known as packed
cell volume [PCV].

Clinical Significance:-
i). The value of hematocrit is used in determination of blood indices, especially MCV
& MCHC indices help in the diagnosis‟s classification of varies types of anaemia.

ii). Hematocrit is one of the important factors that determines viscosity of blood.

Principle:- Anticoagulated blood is taken in a wintrobe tube filled to the graduation mark
& then centrifugal for the prescribed length of time the volume of packed cells is read
directly from the graduations mark on the wintrobe.

Method:- Wintrobe method

Normal Range:-
Adult Male - 46% [42%-52%]

Adult Female - 42%[38%-48%]

Reagent:-

A sample of venous blood [fresh or EDTA blood]

Procedure:-
i). Fill the wintrobe tube with blood with the help of the Pasteur pipette to the 10cm
mark

ii). Place the wintrobe tube in one of the cup of the centrifuge.
iii). Centrifuge for 30 minutes at 3000 rpm [Rapid/minute]

40
 iv). After 30 minutes, stop the centrifuge stake out the tube & read the packed cell
volume.

Determination of Erythrocyte Indices

1) DETERMINATION OF MEAN CORPUSCULAR VOLUME (MCV)

MCV is derived from PCV and total erythrocyte count. This is the average volume of red
cells. The volume is expressed in fimtolitres (fl). MCV is calculated by following formula.

MCV =PCV × 10/RBC count in million

Normal Values: MCV : 86±10 fl

2). DETERMINATION OF MEAN CORPUSCULAR HEMOGLOBIN (MCH):

The MCH is the average haemoglobin content of a red cell. The weight is expressed in
picogram (pg). It is calculated as follows:

MCH =Hb × 10/RBC Count in millions

Normal Values: MCH : 29.5±2.5 pg

3).MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION

(MCHC):
It is expressed of the average haemoglobin concentration per unit volume (100) of
packed red cells. It is expressed in g/dl. MCHC is calculated by following two fannulas.

MCHC= (MCH/MCV) × 100

MCHC= Hb (g/dl) × 100/PCV (%)

Normal Values: MCHC : 32.5±2 g/dl or (%)

Determination of Reticulocyte count

Introduction:- Reticulocytes are Juvenile red cells that pass into the blood stream from
the bone marrow. Roticulocytes stay in circulation for about 24 hours and mature in to
erythrocytes.

Clinical Significance:- The number of Reticulocytes in the blood circulations indicates

the degree of activity of bone marrow, and when the marrow is very active their number
increase this known as Reticulocytosis, in case of aplastic anemia the reticulocyte count is
decreased.

41
Principle:- Supra vital staining method is used for reticulocyte count. Blood is mixed with
the stain and the stain enters the cells in living condition. The RNA in the cell is precipitated
by staining as dark blue network or reticulum. Blood smear is made afterwards, since a direct
count is not possible, a relative count is taken against the number of red blood cells and
expressed as a percentage of blood cells.

Specimen:- EDTA-Anti coagulated blood is commonly used or fresh capillary blood.

Normal Range:- 0.2-2% of RBC

Requirements:-

i). Grease free glass slides

ii). Test tube.

iii). Pasteur pipette with rubber teats.

iv). Capillary tube.

v). Test tube rack.

vi). Micro scope.

vii). Reagent- Staining solution: It is prepared as follow:

 Brilliant cresyl blue 1.0gm or New methylne blue powder

 Sodium citrate 0.4gm
 Sodium chloride 0.85gm
 Distilled water 100 ml

Dissolve first sodium citrate in normal saline and then the dye. Filter it and store in a plastic
container. It is stable at 2-8°C.

Procedure: -

1). Take 2-3 drops of blood and 1 drop of methylne blue powder and mix it properly.
2). After gently mixing stay it for 20 Minutes at rom tempreture.

3). Now take this mixture through Pasteur pipette

4). Now drop one drop of this mixture on slide.

5). Prepare a thin smear of the stained blood specimen with the help of a spreader
slide and air dry it.

6). Examine the smear first under the low power objective for scanning, and locate a
thin portion of the smear when red cells are evenly dristributed.

42
 7). Change to the oil immersion objective. Reticulocyte are identified by fine deep
violet filament and granules arranged in a network.

Calculation:
Reticulocyte percentage (%) = Number of reticulocyte count × 100/ Total number of
RBC Count

iii). COAGULATION PROFILE TEST:

Determination of Coagulation Time (CT)

Introduction:- This is also known as Lee & White clotting time, this method seems for
coagulation factor.

Clinical Significance:- Only severe clotting factor deficiency is recognised by this method
prolonged clotting time. This is indicting the missing coagulation factor.

Normal Range:- 4-9 minutes

Method:-
Lee & White method

 Make a clean veinepuncture with as little trauma as possible to the connective tissue
between skin and vein.
 Withdraw 4-5 ml blood by dry glass syringe. Delivered 1ml of blood in each of the
three 8 × 75 mm test tubes.
 The three tubes with blood are allowed to stand in upright position at room
temperature.
 Coagulation in the tube can be ascertained by tilting the tube or by gentle tipping. The
first tube is gently tilted every minute and the remaining tubes are examined every
half minute. The average of the clotting time in three tubes gives the result.
Since the speed of coagulation increased with temperature, the test should be done at
the particular temperature (37°C).

43
DETERMINATION BLEEDING TIME (BT)

Introduction:- Determination of bleeding time recognizes vascular defect andplatelet

disorder.

Clinical Significance:- Prolonged bleeding time is generally found with

thrombocytopenia (Platelet count < 50,000 cells/µl) and where there is a dysfunction,
bleeding time is high with a normal platelet count.

Normal Range:-

1). Duke method: 2-5 minute

2). IVY method: 5-11 minute

Method:- IVY‟s method

 A sphygmomanometer cuff is wrapped around the patient‟s arm above the elbow and
inflated to 40mm hg and the same pressure is maintained throughout the test.
 Volar surface of forearm is cleansed with rectified spirit and an area of skin devoid of
superficial veins is selected. The skin is stretched laterally by thumb between thumb
and fore finger of left hand, and two separate punctures, 5-10 cm apart, are made in
quick succession by free hand, using a disposable lancet or No.11 surgical blade. Any
microlancet with about one mm width a cutting depth of 2.5 mm is suitable.
 Timing is begun as soon as the punctures are made and bleeding starts.
 Blood extruding from the cut is blotted of gently but completely with filter paper at
15/30 seconds interval till the bleeding spots. The time is noted.
When the bleeding has ceased, a sterile adhesive strip is place on the wounds.

Determination Of Prothrombin Time ( PT)

Introduction:- Prothrombin or factor II is synthesized in the liver under the influence of

fat soluble vit-E.

Clinical Significance:- Prothrombin (factor II) is synthesized in the liver in the presence
of vitamin K. Factor VII is all so synthesized in the liver, which is related to prothrombin. In
clotting mechanism in stage 2, prothrombin is converted to thrombin, which transforms
soluble fibrinogen into insoluble fibrinclot. Abnormal prothrombin time suggests stage 2
defect. Prolonged prothrombin time is related to the deficiencies of factors II, V, VII, and X.
Since excess of coumarin group drugs may lead to hemorrhagic conditions, prothrombin time
determination is also used to monitor the drug therapy.

Normal Range:- 14± 2 seconds

44
Specimen:- Citrated plasma.

Name of the method:- Quick‟s method

Principle:- Thronbokinase preparation (Thromboplastin preparation of human or rabbit

brain) containing calcium ions is added to citrated plasma. IN the presence of factor VII,
stage 2 of coagulation mechanism is triggered and the clotting time is recorded after the
addition of thronbokinese in the presence of calcium ions. Since factors XII, Xi, VIII and
platelets are bypassed, the test depends upon the activity of factors VII, V, X, II and I.
Deficiency of any of these factors may cause prolongation of clot formation in this test.

Requirements:-

1). Water bath (37°C)

2). Stop watch.

3). Test tubes (10 × 100 mm)

4). Brain thromboplastin (or commercially available thronbokinase tablets)

5). 0.15 g/dl calcium chloride

Procedure:-

1). Place a test tube containing about 2 ml of calcium chloride at 37°C

2). Pipette 0.1 ml of plasma in a small test tube (10 × 100 mm).

3). Add 0.1 ml of brain thromboplastin and mix (or use thrombokinase tablets
according to manufacturer‟s directions).

4). Wait for 2 minutes.

5). Add 0.1 ml of pre-warned calcium chloride solution, mix and start the stop watch.

6). Hold the tube in front of a source of light and keep tilting the tube gently. At the
first appearance of fibrin clot, stop the watch immediately.

7). Record the time.

8). Repeat the steps 1 to 7 twice to check the reliability of results.

9). Repeat the procedure by using normal plasma.

10). Report prothrombin time in seconds.

45
IV) SPECIAL HAEMOTOLOGICAL TEST

Determination of Absoult Eosinophils Count

Clinical Significance:- Increased eosinophils count is often associated with allergic

reactions. Parasitic infections, brucellosis and in certain leukemia. Increase in the adrenal
function. (Hyperadrenalism or Cushing‟s syndrome) is associated with a fall in eosinophils
count.

Normal Range:- 40-440/cu mm (µl)

Specimen:- EDTA or heparinised blood

Priciple:- Blood is diluted with a special diluting fluid, which removes red cells and stains
the eosinophils red. These cells are then counted under low powder (10X) in a known volume
of fluid by using Neubauer counting chamber.

Requirements:-

1). Microscope.
2). Improved Neubauer chamber of Fuch Rosenthal counting chamber

3). Diluting fluid: (Hingleman‟s solution)

It is prepared as:

a) Yellow eosin, b) 95% phenol, c) Formalin, d) Distilled water.

Procedure:-
1). Pipette 0.36 ml of diluting fluid in a test tube.

2). Add 0.04 ml of blood (use Hb pipette, twice).

3). Mix and keep for 10 minutes.

4). Mix the diluents and charge the counting chamber.

5). Let it stand under a moist Petridis for about 2 to 3 minutes.

6). Count the cells under low power objective with reduced light.

- if improved Neubauer counting chamber is used, count cells in all nine squares.

Calculations:- Total number of eosinophils, cu mm (µl) = Number of cells counted X

10/0.9 X volume of fluid X area counted X depth.

46
 d) CYTOLOGY
Specimen collection: In the collection and preparation for smears for cytological
examination, the major objectives are:

1. Specimens should have a sufficient number of cells from the area in question.
2. Smears should contain well preserved cells uniformly distributed so that each cell can
be individually examined.
3. The staining procedure should be clearly define the details of all structures. Scraping,
obtained from the vagina, uterus, cervix, mouth or ulcerated skin area is spread
directly on a clean microscope slide. The smear is immediately fixed with a
cytological spray fixative or in an alcohol-ether dip. Fixation or preservation is one of
the most important steps in the procedure.

PAPANICOLAOU STAINING

The Papanicolaou staining procedure is used for examining exfoliated cells in secretions,
exudates, transudates or scrapings of various internal organs and tissue. Cells are fixed to a
slide and stained first with haematoxylin, which stains the nuclei followed by OG_6 and EA-
50 or EA-65 as a counter stain.

Reagents:

EA-65 Multiple Polychrome stain

Light green S.F. Yellowish fast green FCF, Bismark brown eosin y, Phosphotungstic acid,
Glacial acetic acid. Filter before use.

OG-6 Orange G stain

Orange G, Phosphotungstic acid. Filter before use.

Haematoxylin, Potassium alum, Glacial acetic acid, Sodium iodate. Filter before use.

Procedure:

Filter the Harris haematoxylin immediately before use.

1. Dip slide (s) gently 5-10 times in 95% ethanol.

2. Dip slide (s) gently 5-10 times in 70% ethanol.
3. Dip slide (s) gently 5-10 times in distilled water.
4. Stain 5 minutes in Harris Haematoxylin.
5. Place smears in distilled water. Rinse in successive changes of distilled water until the
water remains colourless.
6. Dip slide (s) gently – times in 70% ethanol.
7. Dip slide (s) in a 1% solution of HCL in 70% ethanol until the smear shows
asalmoncolour.

47
 8. Rinse slide (s) well in 2 changes of 70% ethanol.
9. Dip slide (s) gently in 3% solution of ammonium hydroxide in 70% ethanol until the
smear takes on a bule colour.
10. Rinse the slide (s) in two changes of 70% ethanol.
11. Dip slide (s) 5-10 times in 95% ethanol.
12. Stain slide (s) in OG-6 for 2 minutes.
13. Rinse slide (s) in two changes of 95% ethanol.
14. Stain slide (s) in EA-50 or EA-65 for 3-6 minutes.
15. Rinse slide (s) well in two changes of 100% methanol.
16. Rinse slide (s) in one part absolute methanol on part xylene.
17. Clean smear in xylene.
18. Mount with DPX or Canada Balsam.

Results: Nuclei are stained blue while cytoplasm displays varying shades of pink, orange,
yellow and green.

48
 4.CLINICAL MICROBIOLOGY
A. Media :

Preparation:
1. Sterilization in the Autoclave and Hot air oven.
2. Preparation of culture media.

B. Bacteriology:
1. Sample collection.
2. Staining of bacteria.
3. Methods of Inoculation.
4. Culture of Bacteria.
5. Biochemical test.
6. Anti-microbial Sensitivity test.
7. Diagnosis of leprosy.

C. Serology:
1. VDRL or RPR test.
2. ASO test.
3. CRP test.
4. RA test.
5. WIDAL test.
6. HBsAG test.

D. Mycology:
1. Diagnosis of Pathognic fungi.

E. Protozoology:
1. Examination of blood for malarial parasite.

I. INTRODUCTION TO DIAGNOSTIC MICROBIOLOGY

Microbiology involves the study of microscopic organisms. Although microorganisms are

generally beneficial and essential for life, some are however pathogenic and cause infectious
diseases. The diagnostic microbiology laboratory is engaged in the identification of infectious
agents.

The infectious agents are broadly classified as viruses, bacteria, mycosis agents, and
parasites.

Identification of the infectious agents is the principle function of the diagnostic microbiology
laboratory.

Following are the steps which are involved in microbiology laboratory:

49
 II. Collection of specimens for microbiological examinations: Collection of
specimens for microbiological examinations needs great attentions to avoid all risk of
contamination. The specimen should be collected in a properly labelled sterile
container obtained from the department of microbiology.

1. Urine- to avoid contamination of genital flora, collection of midstream urine is

suggested. Urine should be collected in a sterile wide-mouthed container having
cotton plug. Before collecting urine the area is washed with soapy water and dried.
If there is delay of processing, the urine should be stored in a refrigerator at 4oC.

2. Blood: The blood is drawn by vein puncture, through disinfection of skin. Three
bottles of broth should be incubated; one for aerobic growth, one for anaerobic
growth and other is incubated in 5-10% carbon dioxide.

3. Stool: Care should be taken to avoid contamination during collection of stool.

Cartons of waxed cardboard, which can be easily inserted, prove to be a good
container to collect faeces.

4. Sputum: For sputum collection, the patient is asked to rinse the mouth with clean
water to avoid contamination of oral micro flora. The first morning sample is best
for most of the purposes. It should be collected in a wide mouthed, sterilised and
transparent bottle with a screw-capped lid.

5. Pus swab: In case of superficial lesions, such as skin abscess or urethral pus, the
local lesion is first cleaned with spirit and then swab soaked in a warmed normal
saline is rubbed against the lesion.

6. Throat swab: Throat swab is suggested in case of throat infection. The patient‟s
mouth should be opened as widely as possible. The tongue is depressed against
spatula and the sterilised swab is rubbed against lesion.

I. Types of Culture Media generally used in Microbiological

Laboratory:
The most common media which are generally used in clinical microbiology laboratory are
described here-

Pepton Water:- This is a common liquid medium which is used as a base in sugar
fermentations and for the Indole test. It can be used for seed culture for antibiotic sensitivity
testing by the Kirby-Bauer method.

50
Formula:-
Peptone-10gm
Sodium chloride – 5 gm
Distilled water-1000ml
Ph- 7.6

Preparation:
 All the ingredients are mixed with gentle heating and the pH is adjusted.
 The solution distributed in small test tubes in 4 ml quantities.
 Then autoclaved at 121oc for 15 minutes.

Nutrient Agar :- It is a basic transport culture medium used in the preparation of blood agar
and other media. it is used to maintain cultures of control organism semisolid form and in the
solid form.

Formula:-
 Peptic digest of animal tissue- 5gm.
 NaCl– 5gm.
 Beef extract -8gm.
 Yeast extract – 1.5 gm
 Agar -15 gm
 Distilled water – 1000ml.

pH should be maintained between 7.2-7.6

Preparation:-

 At first solid ingredients except agar is kept in 1 litter conical flask.

 Then 800 ml of D/W is added in that flask.
 Then the ingredients are dissolved by using a glass rod, if necessary heat is
provided to dissolve the ingredients.
 The pH is adjusted in the range of 7.2-7.6
 At last agar is added and the solution boiled for 1-2 mins.
 The volume is made1000 ml by adding D/W.
 The total volume is distributed in conical flasks, bottles and tubes and sterilised
by autoclaving at 121oC before 15 mins.
Blood Agar :- It is a general purpose enriched and solid medium, which supports the growth
of most ordinary bacteria. Blood supplies a number of substances for the growth of fastidious
organisms.

Formula:-
 Nutrient agar – 200ml.
 Sterile defibrinated sheep blood – 25 ml.

Preparation:-
 At first sterilised nutrient agar is transferred to 50oC in water bath.
 When it is collected to 50oC, sterile defribinated blood is added to it.
 It is allowed to mixed gently and dispense aseptically in sterile petridishes.
 pH of the medium should be adjusted to 7.3

51
Macconkey Agar :- It is a differential and the low selectivity medium used to distinguish
lactose fermenting from non-lactose fermenting bacteria.

Formula:
 peptic digest of animal tissue – 20 gm.
 Lactose -5 gm.
 Neutral red – 0.07 gm.
 Agar - 20 gm.
 D/W – 1000 ml.

Preparation:-
 At first 500 ml of D/W is taken in a conical flask and weight lactose, neutral
red and peptic digest of animal tissue are added.
 It is then allowed to mix properly.
 Then sodium taurocholate and agar are added and mixed.
 Rest 500 ml of D/W is added to make the total preparation 1000 ml.
 pH is maintained in the range of 7.2-7.6.
 the suspended media is autoclaved and plating is done in sterile petriplates.
 After cooling it is kept in the refrigerator for maintaining the sterility.

MULLET- HINTON AGAR

This medium is used for diffusion method of anti-microbial susceptibility testing of bacteria
(Kirby-Bauer Method).
Formula:
 Beef infusion – 300gm.
 Casein acid hydrolysate – 17.5 gm
 Starch – 1.5 gm
 Agar – 17 gm
 D/W – 1000 ml.

Preparation:-

 The solid ingredients are allowed to dissolve in 1000ml of hot D/W in

a conical flask.
 pH is maintained in the range of 7.2-7.6.
 it is then sterilised by autoclaving at 121oC before 15 mins.
 Then it is cooled to 50 – 55oC and dispense aseptically in the
petridishes.

Sterilization in The Autoclave

An autoclave is the most commonly used equipment for sterilization. It works on the
principle that when water is heated in a closed container, saturated steam is produced under
pressure. Usually the pressure used is 15 lb/square inch for 15- 20mins. At this point, the
temperature in the autoclave will be 121oC.

52
 1. The boiler is fixed with water to a point just below the level of the bottom of the
basket.
2. The plugs of the flask containing media are covered with wrapping paper tied with
a string around the neck of the flasks. All the media to be sterilised are placed in
the basket.
3. Then the lids are closed and screws are tightened.
4. Air outlet valve should be opened and safety valves are adjusted to the required
pressure (15-20 lbs).
5. The source of the heat is turned on and all air should be expelled out through the
vent cock as steady flow of steam.
6. Then the vent-cock is closed and it is allowed to increase pressure up to the
required level.
7. The sterilization technique should be continued for about 20 minutes. The heaters
are then put off and pressure gauze is allowed to turn to zero (O).
8. The vent-cock is opened slowly and the internal pressure is allowed to adjust itself
with that of the outside atmosphere (it takes about 10-15 mins).
9. At last the autoclave is opened and all the satirized materials are taken out
carefully.

Method of Inoculation a number of techniques have been employed for the isolation of
microorganisms for natural environments. Most commonly used techniques for isolation is :

Streak plate technique:

Streak culture is the routine procedure for bacterial isolation in pure culture. The surface of a
semisolid medium in petridishes may be inoculated with the specimen by several methods.
The primary inoculation may be made by a loop, swab or other suitable devices.
A platinum or nichrome wire loop of 2-4 mm diameter with 2-3 inch long wire is
first sterilised in the Bunsen burner and cooled by touching an uninoculated part of the
medium. A loop full of specimen is gently smeared on to the surface of a well dried plate of
medium near the peripheral area. The inoculums are then thinly spread in parallel lines in
different segments of the plate. The loop is sterilised between different sets of streaks. the
plate is incubated at 37oC for overnight. Confluent growth occurs at the primary site of
inoculation and well separated colonies appear on the final series of streaks.

II. IDENTIFICATION OF BACTERIA AFTER CULTURING

The following characteristics have to be studied when a bacterium has been obtained in a
pure culture.

Motility testing:
The motility test of bacteria gives idea of their shape, approximate size and general structure.
This test is particularly important in differentiating certain groups of gram negative rod
shaped bacteria.

53
Method:
Hanging drop method.

Requirements:
 Cavity slide.
 Coverslip.
 Petroleum jelly.
 Microscope.
 Sterile saline.

Procedure:
1. Petroleum jelly is applied at the four corners of the cover slip.
2. A loopful of culture is placed at the middle of the cover slip (if the specimen is dry,
a drop of saline is put on the specimen and mixed it properly).
3. Then a cavity slide is inverted over the drop placing concave portion downwards.
4. The whole preparation is inverted so that the cover slip is on the top.
5. The suspension is examined at first under low power lens and then high power lens.
6. The results of the observations are recorded by indicting “Motile” or “Nonmotile”
organisms.
7. After finishing the observations, the cavity slide is placed in disinfectant solutions.

STAINING METHODS:

GRAM STAINING:-

This is a useful differential staining procedure used in bacteriology which in addition to

determining gross morphology, serves to differentiate bacteria in two distinctly separate
groups.
a. Gram positive.
b. Gram negative.

Principle: When a bacterium is heated with Methyl violet/Zenson violet/ Crystal violet and
later decolourised with acetone alcohol mixture, it is seen that some organisms resists the
decolourising effects of acetone or alcohol or both and remains coloured violet, they are
called gram positive. Those become colourless are called gram negative. For proper
visualisation of colourless they are counterstained with safranin and becomes coloured pink.

Reagents:
1. Crystal violet stain:-
a. Crystal violet- 10 gm.
b. Absolute methyl alcohol- 500 ml.

54
After well mixing the staining solution is kept for 24 hours and then filtered and stored in
amber coloured dropping bottle.
2. Grams iodine solution:-
a. Iodine crystal- 6 gm.
b. Potassium iodide – 12gm.
c. Distilled water – 800 ml.
It is stored in amber coloured dropping bottle after mixing.
3. Decolouriser :- 95% alcohol and acetone are mixed in equal proportion. If
alcohol is not available, rectified spirit can be used . the mixture is stored in a
white dropping bottle.
4. Safranin:-
a. Safranin- 10 gm.
b. Distilled water – 1000ml.
The mixture solution is mixed well, filtered and stored in an amber coloured dropping bottle.

Stability of the reagents:-

All the reagents are stable at room temperature.

Procedure:-

Smear preparation:-
 A clean grease free dry slide is taken and one drop of normal saline is placed in
the centre of the slide.
 The inoculating loop (nichrome) is sterilizedon the flame of a Bunsen burner.
 A suspension is made by adding specimen by the sterile nichrome loop.
 The suspension is spreaded in an oval shaped and smear is made.
 The smear is allowed to dry in the air.
 Smear is fixed by passing the slide 3-4 times through the flame quickly with the
smear side facing up.
STAINING:-
 The slide is placed on the staining glass rods.
 The smear is covered with crystal violet stain and wait for one minute.
 After that it is washed under running tap water.
 Then smear is flooded with grams iodine solution and wait for one minute.
 The iodine is drained off.
 The smear is decolourised with alcohol-acetone (or rectified spirit) for 20 to 30
seconds until purple stain just stops coming on the slide.
 Then the smear is counterstained with safranin for one minute .
 The smear is washed out in running tap water and is dried carefully by using a
blotting paper.
 The dried smear is observed microscopically at first under low power objective
afterwards under high power objective and finally under oil-immersion
objective.

Observation:-
Gram positive organism------------ dark violet.
Gram negative organism ----------- light pink.

55
 1. Gram – positive organisms are:
Cocci in clusters : Staphylococcus species.
Cocci in chains : Streptococcus species.
Cocci in pairs : Pneumococcus sp.(streptococcus pneumoniae)
Bacilli without spores: Corynebacterium sp.
Bacilli with spores : Anthraxsp(aerobic)
Clostridium sp(anaerobic)

2. Gram- negative organism are:

Cocci in pairs : neisseriasp
Bacilli : enterobacteriaceae(E. coli)
Pseudomonas sp.

Comma shaped : vibrio sp.

Coccobacilli: haemoplylus sp.
ACID FAST STAIN
The acid fast stain is another differential stain used mainly to detect Mycobacterium that
causes tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprea).

Ziehl-Neelsen method (hot stain) :

This is recommended for the preliminary diagnosis of tuberculosis infection by direct
method. It serves to differentiate bacteria into two distinctly separate groups –
a. Acid-fast
b. Non acid-fast

Principle:
When some organisms are stained with colour-dye derivative like basic fuchsine and later
decolourised with dilute mineral acid like 20% H2so4 and remains colour red, they are called
acid-fast.
Those become colourless due to decolourising effect of H2SO4 are called non-acid fast
organisms. For proper visualisation of the colourless they are counterstained with Methylene
blue and becomes colour blue.

Reagents:
1. Carbol-fuchsine solution ( saturated solution of basic fuchsine)
a. Sol- A:
Basic fuchsine powder-0.3 gm
95% ethanol -10ml.
The fuchsine is dissolved in alcohol and gentle heat may necessary to dissolve.

b. Sol- B:
Phenol crystal – 5gm.
Distilled water – 100ml.

The phenol crystal is mixed with water heated to 56oC

c. Sol- C:
1o ml of sol-a is mixed with 90 ml of solution B. It is allowed to remain
overnight at room temperature and then it is filtered.

56
 2. Acid-alcohol solution:
Conc. HCl : 3 ml.
95% ethaol : 97ml.
OR
Conc.H2SO4 : 25 ml.
D/W 75 ml.

3. Methylene blur counter stain

Methylene blue : 0.3 gm
Distilled water : 100 ml.
It is allowed to dissolve thoroughly and filtered.

Procedure:
a. A film of specimen is prepared and heat fixed.
b. The heat fixed slide is placed on the staining rack.
c. The carbol-fuchsin stain is heated in a test tube on a Bunsen burner and then
that heat fixed slide is flooded with stain and wait for 8 to 10 minutes.
d. The slide is washed with water. It is continued until the water that runs off is
colourless.
e. Then the slide is covered with 20%H2SO4 for 1 minute. Finally it is washed
well with running tap water.
f. The slide is then covered with methylene – blue and left for 30 to 1 min.
g. Then it is washed with running tap water allowed to dry or blot carefully.
h. The slide is observed microscopically first under low power objective and
then under high power objective and finally under the oil- immersion
objective.

Observation:-

Acid fast organism bright red.

Non acid fast organism dark blue.

Number of acid fact bacteria in 10 fields Report

None Absent
1 to 2 Positive,+
2 to 10 Positive,++
10 to 100 Positive,+++
Above 100 Positive,++++

 DIFFERENT BIOCHEMICAL TESTS

Following biochemical methods are useful in identification of the bacteria isolated from
wound swab.

Coagulase Test :-

57
This test is used to differentiate Staphylococcus aureus from S.Epidermis and S.aprophyticus.

Principle:-
S.aureus produces the enzyme coagulase which causes plasma to clot by converting soluble
fibrinogen to insoluble fibrin.

Requirements:-
 Oxalate or citrated plasma.
 Glass slides.
 Normal saline.

Procedure:-
1. A drop of physiological saline is placed on each end of a slide.
2. Thick suspensions of the organisms are made in each drop.
3. A drop of plasma is added to one of the suspension and it is mixed gently.

Observation:-
Look for clumping of the organisms within 10 secs.

Results:-
1. Clumping within 10 secs – S.aureus.
2. No clumping within 10 secs- No production of coagulase.

Tube test:-
1. 1.0 ml of saline suspension of the organism is prepared .
2. 1.0 ml of plasma is added.
3. It is then incubated at 37oCfor 24 hours.
4. Formation of clot indicates the presence of S.aureus organisms.

Catalase Test :-
This test is used to differentiate catalase producing bacteria such as Staphylococci from non-
catalase producing bacteria such as streptococci.

Principle:-
Catalase produced by the organisms act on hydrogen peroxide to produce water and
oxygen (indicated by bubbles).
Requirements:-
 3% hydrogen peroxide.
 Test tubes.
Procedure:-
1. 2-3 ml of hydrogen peroxide sample is poured in a testtube.
2. The growth of organisms is immersed in the test tube solution by using
sterile glass rod.
3. Look for immediate bubbling.
Observation:-
1. Appearance of bubbles: presence of catalase producing organisms.
2. No formation of bubbles: presence of non catalase producing organisms.

Oxidase test :-

58
This test is used to help in the identification of organism which produce the enzyme oxidase.
For example Pseudomonas, Neisseria, vibrio, and Pasturella sp.

Principle:-
A colony of the test organisms is smeared on a filter paper, soaked with few drops of oxidase
reagent. If the organisms are oxidase producing, the phenylenediamine in the reagent is
oxidised to a deep purple colour.

Requirements:-
 Oxidase reagent : 1.0 gm/dl tetramethyl-p-phenylnediamine dichloride in D/W (it
should be prepared freshly).
 Filter paper strips.

Procedure:-
1. A piece of filter paper is place in a clean petridish.
2. Two- three drops of freshly prepared oxidase reagent are added.
3. A colony of organisms is smeared on the filter paper by using a glass rod.
4. The reaction is observed.

Observation:-
1. Blue purple colour – positive test.
2. No blue purple colour – negative test.

INDOLE TEST
This test is important in the identification of enterobacteria such as E.coli, P. Vulgaris, etc
and differentiates from Klebsielia and Salmonella.

Principle:-
This test organism is cultured in a media containing tryptophan. The organism breakdown
tryptophan and indole is released , it is detected by Ehrlich reagent.

Requirements:-
 Motility indole urea (MIU) medium.
 Kovac‟s reagent.
Kovac’s reagent:-
Formula and preparation :-
a. P-dimethylaminobenzaldehyde – 2.0 gm.
b. Isoamyl alcohol :- 30 ml
c. Conc. HCl : 10 ml.
This ingrediants (a) and (b) are dissolved in 10 ml of conc. HCl and is stored in a brown
ottle.

Procedure:-
1. The MIU medium is inoculated with test organism colonies.
2. Kovac‟s reagent (about 0.5 ml is added and it is inoculated at 37oC).

Observation:-
Looked for forming red colour of the reaction mixture.

59
Results:-
1. Reddening of mixture : Positive test.
2. No red colour :- Negative test.

METHYL RED TEST

This test is performed to differentiate enterobacteria. This test is employed to detect
production of sufficient acid during fermentation of glucose by bnacteria and sustained
maintenance of pH below 4.5.

Principle:-
Some enterobacteria when cultured in a buffered glucose peptone water, it ferments glucose
to produce sufficient acidity, which gives red colour with methyl red indicator.

Procedure:-
1. A colony of test organisms is inoculated into 0.5 ml of sterile glucose phosphate
broth.
2. It is then incubated at 35-37oC.
3. A drop of methyl red indicator is added and the colour is observed.

Observation:-
1. Bright red colour – positive test.
2. Yellow/Orange colour – Negative test.

Citrate utilization test:-

This test is performed for the identification of Enterobacteria.

Principle:-
This test organism is cultured in a medium containing Na- citrate, an ammonium salt and
bromothymol blue indicator. The organisms use citrate (the only source of carbon) and
ammonia (the only source of nitrogen). The citrate utilisation is followed by alkaline reaction
(change of the colour from light green to blue) and growth in the medium is indicated by
appearance of turbidity.

Requirements:-
1. Simmon‟s citrate medium:-
Formula and preparation:-
a. Potassium difydrogen phosphate: 1.0 gm.
b. Sodium ammonium phosphate: 1.5 gm.
c. Magnesium sulphate: 0.2 gm.
d. Sodium citrate: 2.5 gm.
e. Bromothymol blue: 0.016 gm.
f. Agar : 15 gm.
g. D/W : 1000ml.
These ingredients are dissolved in about in about 900ml of D/W and after diluting to 1 lit, pH
of this medium is adjusted to 6.7- 6.9.

60
Test tube method:-
1. 3-4 ml of sterile Simmon‟scitrste medium is inoculated with a broth culture of test
organisms.
2. It is then incubated at 37OC upto 4 days.

Results:-
1. Turbidity and blue colour: Positive.
2. No growth (no turbidity and persistence of original colour) : Negative.

UREASE TEST
This test helps in differentiating enterobacteria. Proteus srains are strong urease producers.

Principle:-
The test organisms are cultured in MIU medium (or in Christensen‟s urea broth). If the
strain produces urease, it acts on urea ammonia carbonate is formed with the release of
Urease production is not to be considered negative till a for day old culture is tested.

Requirements:-
 Moyility Indole Urea Medium.
 Test tubes.

Procedure:-
1. MIU medium is inoculated with a colony of the test organisms.
2. It is then incubated at 35oC overnight.
3. The medium is examined by looking for a red-pink colour.

Observations:-
1. Red pink medium:-Positive test.
2. No red pink colour: Negative test.

Biochemical reactions on triple sugar iron agar(TSI):

Principle:-
1. Alkaline reactions(red colour) is shown by the organisms, who fail to ferment any one
of the sugars.
2. Fermentation of the sugars are indicated by yellow colour since pH range of phenol is
6.8- 8.4 and colour change from yellow to red. Since the glucose present on the
surface of the medium is used up and since the surface of the is exposed to
atmosephere, under anaerobic conditions, the acid reaction of the surface reverts to
alkaline(red colour) in 18-24 hrs(which is critical duration for this observation). In the
butt, since anaerobic conditions exists, the colour of the butt remains yellow.
3. Gas production (CO2) is indicated by splitting of the agar.
4. Production of H2S imparts black shade to the slant by reacting with ferrous ions. It is
an indication of H2S producing organisms.

Procedure:-
1. Streak the TSI slant with a loop and stab with a straight needle.

61
 2. Incubate at 37oC for 18- 24 hrs.

Observation:-

The various reactions on the slants are as follows:-

Colour of the Gas H2 S Sugar fermented Possible organisms

Slant/Butt
Yellow/Yellow + -  Glucose fermented E.coli
 Lactose fermented Klebsiella sp.
Acid / Acid  Or both lactose and Enterobacter sp.
sucrose fermented
Yellow / + +  Glucose and sucrose Proteus vulgaris
Yellow fermented
Red / yellow _ _  Glucose fermented Shigella sp.
 Lactose or sucrose not Proteus morganil
Alkaline / Acid fermented Providencialrettgeri.

Red / yellow - +  Glucose fermented S.typhi

 Lactose or sucrose not
Alkaline/ acid fermented.

Red / Yellow + +  Glucose fermented Other salmonella

 Lactose or sucrose not sp.
Alkaline / Acid fermented.
Proteus mirabilis

Red / Yellow + -  Glucose fermented Providencia

 Lactose or sucrose not
Alkaline / Acid fermented. Alcalifaciens

Red / Yellow - -  None of the suigar Donot belong to

enterobacteriaceae.
Alkaline / Acid

Antimicrobial Susceptibility Test

In bacterial infections, the somististy of the causative organism to the different therapeutic
antimicrobial agents may be determined. Because of the changes of the driug resistant
variants of many of the infecting microorganisms, antibiotic sensitivity tests are not

62
conducted, an important part of the bacteriological investigation. The physician is dependent
on the antibiotic susceptibility report from the pathological laboratory to find most effective
antibiotic on a particular infectious disease.

Importance of Antimicrobial Susceptibility Test :-

Antimicrobial sensitivity test is mainly necessary if usually effective agents fail to produce
the desired effects. In the treatment and control of infectious diseases which are caused by
pathogens that are drug resistant, sensitivity testing is helpful in selecting effecting
antimicrobial drugs.

Determination of Antibiotic Susceptibility by Disc Diffusion Technique (Kirby –Bauer

Method) :-
A sterile cotton swab is inoculated with the inoculums to the Muller-Hinton agar plate by
streaking the swab three times over the entire agar surface. Then 3-5 mins are allowed to dry
the surface of the agar before inoculating the antibiotic discs, using sterile forceps. O a plate
of 100 mm diameter seven discs are applied, one in the centre and six in the periphery. The
plates are then incubated at 370C for 16-18 hrs.
The zones of complete growth inhibition around each of the discs are measured. The
diameter of the discs is including this measurement. The interpretation of zone size into
sensitive, intermediate or resistant is based on interpretation chart.

Observation Report
Zone less than 4mm Resistant
Zone 4-12 mm Intermediate
Zone more than 12 mm Sensitive

Specimen of the Diagnosis of Leprosy :-

Leprosy caused by Mycobacterium leprae is a disease of great antiquity and even today.
Mycobacterium leprae causes diseases of the skin, nerves, and often the nose, it is most easily
found in smears made from skin or occasionally from the nose. Laboratory diagnosis is
primarily based on direct examination of the smear, because unlike Mycobacterium
tuberculosis, Mycobacterium leprae cannot be cultured in the laboratory on artificial media.

Procedure for skin scrapings:-

1. At first before taking the scrapings, the skin is disinfected with 80% alcohol using an
alcohol- dipped gauze which is firmly rubbed over the scraping area. A sharp clean
scalpel is sterilised in a flame.
2. The skin is held tightly between thumb and first finger to prevent bleeding at the time
of cutting.
3. A cut (5mm long and 2-3 mm deep) is made by a sterile scalpel blade into the
dermis(papillary layer).
4. Skin scrapings are scraped without bleeding by holding the scalpel sideways.
5. A small circular smear of the scraped material is made on a clean grease-free slide.
6. The smear is gently heat fixed and stained by using Ziehl-Neelsen method.

63
  LABORATORY DIAGNOSIS OF PATHOGENIC FUNGI :-

Fungi are saprophytic or parasitic eukaryotic microorganisms, which posses a complex cell
structure. Very few fungi are pathogenic. Most of the fungi are beneficial to man as they are
utilised in making of bread, fermented drinks, cheese, antibiotics etc.

Specimen collection:-
Skin scrapings, nails, and hairs:-
1. Specimens for dermatophyte infection are skin scrapings and hair. At first, the infected
area is cleansed by 70% alcohol and then with sterile water.
2. From skin lesions about 2- 3 mm scales should be scraped by using a blunt scale.
3. The nails are scrapped with a scalpel. The first superficial scrapings are discarded. The
friable material from under the nail is preferred for a laboratory test.
4. The infected hairs are plucked by using a sterile forcep and placed in a sterile petridish.

Microscopic examination:-
1. Specimens are placed on a glass slide in a drop of 10g/dl, sodium hydroxide or
potassium hydroxide. It is then covered by a cover slip.
2. After 10-30 minutes it is observed under microscope.

Observations:-
Skin, nails – Branching hyphae
Hair -Spores.

Culture:-
Specimens are inoculated on a Saboraud‟s agar slants and incubated for 2-3 weeks at room
temperature.

III. SEROLOGY
The laboratory procedure in serology consists of collection and preparation of serum
specimen and immunologic test procedures for specific serodiagnosis. The most common
routine test are :-

 Determination of Rapid Plasma Reagin ( RPR)

(Carbogen Antigen for Syphilis Testing):-

Syphilis is a sexually transmitted (venereal) disease caused by the spirochete

Treponemapallidum. After infection the-host forms treponemal antibodies to
Treponemapallidum in addition the host also forms non-treponemalantilipoidal antiobodies in
response to the lipoidal material released from the damaged host cell. These antibodies are
traditionally referred to as 'reagins' .

64
Reagents:-
1. A particulate carbon suspension coated with lipid complexes.
2. Positive control serum.
3. Negative control serum.

Principle:-
During the test procedure, the specimen, serum or plasma is mixed with the
reagent and allowed to react for four minutes. If anti lipoidal antibodies are present in the
specimen, they will react with the reagent forming visible black floccules.

Specimen: - Fresh serum or plasma is used for testing. Samples not tested immediately may
be stored at 2-8oC up to 48 hours.

Test procedure:-
All the reagents and samples are brought to room temperature before testing.

Qualitative method

i) One drop of test specimen, positive control and negative control are pipette onto
separate reaction circle of the disposable slide using a sample dispensing pipette.
ii) One drop of reagent is added next to the test specimen.
iii) Using a mixing stick the serum and reagent are mixed uniformly over the entire circle.
The dropper tips should not be touched to the liquid on the slide.
iv) The slide is rocked gently back and forth for exactly eight minutes for observing
agglutination macroscopically.

Interpretation:-
Agglutination visible within two minutes is to be interpreted as a positive test result.

 VDRL TEST

VDRL test also perform to diagnosis Syphilis. It is the old procedure and the modem
procedure is RPR. In this case used Syphilis causing Ag (inactivated) and heat inactive serum
or plasma (Ab). -

Reagents:-
1. Heat inactivated VDRL Ag.
2. Positive control serum.
3. Negative control serum.

Principle:-
During the test procedure, the specimen, serum or plasma is mixed with the reagent

65
(Ag) and allowed to react for four minutes. If antilipoidal antibodies are present in the
specimen, they will react with the reagent forming antigenic clumps which is visible under
low power microscope.
Specimen:-
Fresh serum or plasma is used for testing. Samples not tested immediately may be
stored at 2-8°C up to 48 hours.

Test procedures:-
- All the reagents and samples are brought to room temperature before testing.

 DETERMINATION ASO LATEX

Streptococcus belongs to the family of lactobacillacae and the majority are facultative
anaerobes. The facultative anaerobic streptococci are divided into two categories
a) those which produce soluble hemolysin
b) those which do not produce soluble hemolysin.
The first group of streptococci are called β-hemolytic streptococci which can be
further
subdivided into group(a),group(b),group(c) and group(d).most of the strains of streptococcus
which are pathogenic for human beingsbelong to group(a).the group(a)β hemolytic
streptococci produce various extoxins such as streptolysin O.

Clinical significance:-
The detection of ASO is very helpful for the diagnosis of streptococcal infections.
Increased ASOtitres be associated with rheumatic fever and glomerulonephritis. An elevated
ASOtiter of more than 200 IU/ml indicates past or present acute streptococcal infection. The
titer of ASO should be observed repeatedly over a time of 4-6 weeks to follow the course of
an infection.

Principle:-
ASOslide test for detection of antibodies to streptolysin-O is based on the principle of
agglutination. The test specimen (serum) is mixed with polystyrene latex particles and
allowed to react. If antibodies to streptolysin-O are present in concentration more than 200
IU/ml but less than 4000 IU/ml, then a visible agglutination is observed. If antibodies to
streptolysin-O are not present or are in concentrations less than 200 IU/ml then no
agglutination will be observed.
Sample:-It is recommended that the test be performed only on serum. It is not necessary to
heat inactive the test or control serum. If delay is anticipated before testing test sera should be
stored in a refrigerator (2-8°C).

Reagents:-
Reagent 1: ASO latex antigen

Reagent 2: Positive control serum

Reagent 3: Negative control serum

Accessories:-
i. Glass slide with reaction circles

66
 ii. Disposable applicator sticks
iii.Disposable plastic droppers
iv.Rubber teats.

Storage and stability:-

The reagents and control sera are stable at 2-8°C till the expiry date mentioned on the
individual level.

Procedure:-
All the reagents and samples (Ire-brought to room temperature.

A. Qualitative slide test:-

i. With the help of disposable -plastic dropper one drop of undiluted test serum is
placed within the circled area on me special slide provided in the kit.
ii. One drop of well mixed ASO latex reagent is added to the drop of test sample on
the slide.
iii. Using a mixing stick the serum and reagent are mixed uniformly over the entire
circle. The dropper tips should not be touched to the liquid on the slide.
iv. The slide is rocked gently back and forth for exactly two minutes for observing
agglutination macroscopically.

Interpretation:-
Agglutination visible within two minutes is to be interpreted as a positive test result
corresponding to ASO titer of more than 200 IU /ml. A test is considered as negative when
there is absence of agglutination indicating ASO titer of less than 200 IU/ml.

B. Semi quantitative slide test:

i. Using isotonic saline serial dilutions of the serum sample positive in the qualitative
method are prepared as for example 1:2, 1:4, 1:8, and 1:16 and so on.
ii. With the help of disposable plastic dropper, one drop of diluted serum is dispensed
into one of the circled area.
iii. One drop of well mixed ASO latex reagent is then added to it.
iv. The reagent and the drop of diluted serum are mixed together using applicator
stick.
v. It is observed for macroscopic agglutination for two minutes.

Interpretation:-
Agglutination in the highest serum dilutions correspond to the amount of ASO in IU/ml
Present in the test specimen.

Serum dilution Corresponding IU/ml

1:2 400
1:4 800
1:8 1600
1:16 3200

67
  DETERMINATION OF CRP
C-reactive protein (CRP) is an acute phase protein found in concentrations upto 5µg/rnl in the
serum of healthy persons.

Clinical significance:
Elevated CRP level are usually observed in a variety of infections and inflammatory
conditions where there is tissue destruction. This increase in CRP level may be detected as
early as 5-10 hours after tissue damage.The changes in concentration of CRP occur more
rapidly than in the ESR. Elevated level of CRP can usually be demonstrated in cases of acute
myocardial infarction, rheumatoid arthritis, bacterial and viral infections, acute rheumatic
fever with or without carditis and in several types of malignancies.

Principle:
Uniform latex particles are coated with anti-human CRP. The specimen containing
CRP, on mixing with latex reagent agglutinates, showing the positive test result. If CRP is
absent there will be no agglutination, indicating a negative test result.

Sample:
Fresh serum is preferable, in case of delay in testing, it should be stored at 2-8°C.
Plasma, haemolysed or Iipaemic serum should not be used.

Reagents:
Reagent 1: CRP latex reagent

Reagent 2: Positive control serum

Reagent 3: Negative control serum

Accessories:
i) Glass slide with reaction circles
ii) Disposable applicator sticks
iii) Disposable plastic droppers
iv) Rubber teats.

Storage and stability:

The reagents and control sera are stable at 2-80C till the expiry date mentioned on the
individual level.

Procedure:
All the reagents and samples are brought to room temperature.

A. Qualitative slide test:

i. With the help of disposable plastic dropper one drop of undiluted test serum is
placed within the circled area on the special slide provided in the kit.
ii. One drop of well mixed CRP latex reagent is added to the drop of test sample on
the slide.
iii. Using a mixing stick the serum and reagent are mixed uniformly over the entire
circle. The dropper tips should not be touched to the liquid on the slide.
iv. The slide is rocked gently back and forth for exactly two minutes for observing

68
agglutination macroscopically.

Interpretation:

Observation Conclusion
Coarse agglutination Strongly positive
Finer agglutination Weakly positive
Smooth suspension without any noticeable change Negative

B. Semiguantitative slide test:

i) Using isotonic saline serial dilutions of the serum sample positive in the
qualitative method are prepared as for example 1:2, 1:4, 1:8 , 1: 16 and so on.
ii) With the help of disposable plastic dropper, one drop of diluted serum is
dispensed into one of the circled area.
iii) One drop of well mixed CRP latex reagent is then added to it.
iv) The reagent and the drop of diluted serum are mixed together using applicator
stick.
v) It is observed for macroscopic agglutination for two minutes.

Interpretation:

CRP level can be calculated in terms of µg/ml by multiplying the highest dilution
giving clear cut agglutination with a factor of 6 (sensitivity of antigen 6 µg/ml).
Serum dilution Corresponding µg/ml
1:2 12
1:4 24
1:8 48
1:16 96

 DETERMINATION OF RA

Rheumatoid arthritis is an auto immune disorder, where auto-antibodies are produced against
self-antigen (lgG). These antibodies are termed as "Rheumatoid Factor"(lgM).

Clinical significance:
The RA test is done in order to detect the presence of rheumatoid factor in the serum of
patients with rheumatoid arthritis. The factor is non specific and is found in other disease as
well as both related and none related to rheumatoid arthritis.

Principle:
RA test antigen consists of polystyrene latex particle coated with specially modified

69
preparation of human gammaglobulin (lgG) in order to avoid non-specific agglutination. The
suspension of coated latex particles agglutinate visibly when mixed with serum containing
rheumatoid factor.

Sample:
Fresh serum is preferable, in case of delay in testing; it should be stored at 2-80C. Plasma,
haemolysed or lipaemic serum should not be used.

Reagents:
Reagent 1: latex gammaglobulin reagent

Reagent 2: Positive control serum

Reagent 3: Negative control serum

Accessories:
i). Glass slide with reaction circles
ii). Disposable applicator sticks
iii). Disposable plastic droppers
iv). Rubber teats.

Storage and-stability:
The reagents and control sera are stable at 2-8°C till the expiry date mentioned on the
individual level.

Procedure:
All the reagents and samples are brought to room temperature.

A. Qualitative slide test:

i) With the help of disposable plastic dropper one drop of undiluted test serum is
placed within the circled area on the special slide provided in the kit.
ii) One drop of well mixed RA latex reagent is added to the drop of test sample on the
slide.
iii) Using a mixing stick the serum and reagent are mixed uniformly over the entire
circle. The dropper tips should not be touched to the liquid on the slide.
iv) The slide is rocked gently back and forth for exactly two minutes for observing
agglutination macroscopically.

Interpretation:
Observation Conclusion
Clearly visible agglutination Positive result
Granularity of doubtful clumping Negative result

B. Semi quantitative slide test:

i) Using isotonic saline serial dilutions of the serum sample positive in the qualitative
method are prepared as for example 1:2, 1:4, 1:8, and 1:16 and so on.

70
 ii) With the help of disposable plastic dropper, one drop of diluted serum is dispensed
into one of the circled area.
iii) One drop of well mixed RA latex reagent is then added to it.
iv) The reagent and the drop of diluted serum are mixed together using applicator
stick.
vi) It is observed for macroscopic agglutination for two minutes.

Interpretation:

RA level can be calculated in terms of IU/ml by multiplying the highest dilution giving
clear cut agglutination with a factor of 10(sensitivity of antigen 10 IU/ml).

Serum dilution Corresponding IU/ml

1:2 20
1:4 40
1:8 80
1:16 160

 DETERMINATION OF WIDAL

Typhoid is an enteric fever caused by various species of Salmonella such as S.typhi,

S.paratyphi A and S.paratyphi_B. The disease is characterized by sustained high fever, severe
headache, nausea, anorexia- and constipation initially and later diarrhea. Humans are only
reservoir of the bacteria.

Clinical significance:
This test is applied for the diagnosis of the enteric fever that include typhoid
(Salmonella typhi) and paratyphoid (Salmonella paratyphi) which are clinically similar and
cannot be differentiated by laboratory cultures. The pathogen can be recovered from the
blood during the first 7 to 10 days of illness but after two weeks the laboratory culture may
not yield any useful information. Specific antibodies (agglutinins) are usually detectable in
the patient's blood after six days of enteric fever.

Principle:
This test is based on the principle of direct agglutinations reaction. The smooth
suspension of killed Salmonella bacilli carries homologous "0" and "H" antigens. When
patient's serum (containing antibodies to S. typhi and S. Paratyphi) is incubated with
respective antigens, visible agglutination occurs. A rising titer of antibodies is indicative of
enteric fever.

Reagents:
1) S. typhi "H"
2) S. typhi "0"
3) S. Paratyphi "AH"
4) S. Paratyphi "BH"
5) Positive control

Materials:
1. Applicator sticks

71
2. Disposable plastic droppers
3. Rubber teats
4. Test tubes
5. Pipettes
6. Normal saline

Sample:
Fresh serum is preferable, in case of delay in testing, it should be stored at 2-8°C.
Plasma, haemolysed or lipaemic serum should not be used.

Procedure:

A. Rapid Slide (Screening) Test

i) Cleaned the glass-slide supplied in the kit properly and wiped it free of moisture.
ii) Placed one drop ot' undiluted test serum in each of the 1st four circles (1 to 4) and
one drop of positive control serum in each of the last two circles (5 &6).
iii) Placed one drop of antigens 0, H, A(H),B(H) in circles 1,2,3 and 4 respectively
and O antigen in circle 5 and anyone of the 'H' antigens {H, A(H) or B(H)} in circle 6.
iv) Mixed the contents of each circle with separate applicator stick and spread it to the
whole area of the individual circle.
v) Rocked the slide for 1minute and observed for agglutination. If agglutination is-
visible within 1minute the test is positive, proceed for quantitative slide test or tube test for
the quantitative estimation of the titer of the appropriate antibody. If no agglutination is
observed, the test is negative.

B. Semi-quantitative slide test

i) Cleaned the glass slide supplied in the kit well and wiped it free of moisture.
ii) Placed 5µl, 10 µl, 20µl, and 40µl, 80µl of undiluted serum in 1 st, 2nd, 3rd, 4th and 5th
circles respectively on the slide.
iii) Added 1 drop of the appropriate antigen suspension which showed agglutination
in screening slide test, to each of the above circles.
iv) Mixed the contents of each circle with separate applicator sticks.
v) Rotated the slide slowly for 1 minute and observe for agglutination.
vi) The titer of the antibody is the highest dilution of serum up to which there is clear
agglutination.
vii) Repeated steps through with all the antigens, which showed agglutination in
screening slide test.

The serum volumes in the quantitative slide test correspond approximately to the tube test
titers as follows:
Serum Volume Approximate Tube Test Titer
80µl 1:20
40µl 1:40
20µl 1:80
10µl 1:160
5µl 1:320

72
  DETERMINATION OF HEPATITIS B SURFACE ANTIGEN (HBsAg)

The HBsAg test is a test to diagnose viral hepatitis/Australia antigen.

Principle –
The test is an immunoassay for the detection of hepatitis B surface antigen in serum. The
membrane is precoated with anti-HBs antibody on the test line and anti mouse antibody on
the control line. During testing, the serum or plasma reacts with the dye, which has been
precoated in the test strip. The mixture migrates upwards on the membrane
chromatographically by capillary action to react with anti-HBs antibodies on the membrane
and generate red lines. Presence of this red line indicates positive test, while its absence
indicates negative test.

Requirements-
Blood sample
HBsAg test strip

Procedure:
i) The strip is removed from the pouch.
ii) The test strip is immersed in serum sample within arrows pointing towards sample.
iii) It has to be wait for red lines appearing.

Observation-
The test is negative if only on-e line appears on control window. The test is positive if
lines appear on test window as well as on-control window.

IV. Mycology
 Study of candida albicans
Specimens:
- Swabs and scrapings from surface lesions
- Sputum
- Exudates
- Material removed from intravenous catheters
- Vaginal discharge

Requirements:
- 10 g/dl, sodium hydroxide or potassium hydroxide
- Glass slides and cover slips
- Microscope

Direct examination
1) Mount skin and nail scrapping on a slide in a drop of 10%KOH
(or NaOH). Place a coverslip and observe under the microscope after
15-30 minutes.
2) Make a thin film of the specimen. Place under a coverslip and observe
immediately,first under low power objective and afterwards under high
power objective.
3) Make stain for Gram staining.

Requirements:

73
 - 10 g/dl, sodium hydroxide or potassium hydroxide
- Glass slides and cover slips
- Microscope

Observation:
1) 2 to 4 um size, thin walled oval, yeast like calls are observation. Occasionally,
mycelia threads are seen with bidding cells.
2) Gram staining: budding Gram positive cells.

V. Proto Zoology

Microscopic Identification of Malarial Parasite:

1. Sample collection.
2. Standard procedure for staining.
1. Sample collection:
Blood sample are collected from with symptoms of fever for routine examination of
malaria parasite. A thick and a thin film are made on same slide.

• Items needed for making blood films

 Cleaned, wrapped slides.
 Sterilized lancets.
 70% methanol.
 Absorbent cotton wool.
 Labels.
 Register or record form.

Procedure of sample collection:

a. With patient's left hand palm upwards, select the ring finger.
With the pellet of cotton wool lightly socked in alcohol clean
the finger. With the clean cotton wool dry the finger using firm
strokes to stimulate blood circulation.

b. With a sterile lancet puncture the ball of finger.

c. By applying gently pressure to express the first drop of blood

and wipe it away with cotton wool.

d. Apply gently pressure to the finger and collect a single small

drop of blood on to middle of the slide. This is for the thin
smear.

e. Apply further pressure to express more blood and collect two to

three large drops of blood onto the slide, about 1 cm. from the
drop intended for the thin smear.

74
 f. Using another clean slide as "spreader" and with the slide with
the I-st blood drop resting on flat firm surface touch the small
drop of blood with the spreader and allow the blood to run
along its edge. Firmly push the spreader along the slide keeping
the spreader at an angle of the 45°. Make sure the spreader is in
even contact with the surface of the slide all the time the blood
is being spread.

g. For thick smear using the corner of the spreader, quickly join
the drops of blood and spread them to make an even thick
smear. Blood should not be excessively stirred but can be
spread in a circular or rectangular form with 3-6 movements.

h. Label the dry thin smear with the soft lead pencil by writing
across the thicker portion of the smear the patient's name and
date.

i. Allow the thick smear to dry in flat, level position protected

from flies, dust and extreme heat.

J. The slide used for spreading the blood films may now be used for
the next patient and another clean slide from the pack will be used as a spreader.

2. Standard 'procedure for staining:

TECHNIQUE USED: GIEMSA STAINING

i. Preparation of stock solution of giemsa

Staining formula
Giemsa powder : 0.75 gm
Methanol 65 ml
Glycerol: 35 ml

• Preparation:
a. A dark bottle or a chemically clean and dry, clear hard glass or polythene bottle is
taken.
b. Put about 50 solid glass beads of methanol.
c. Pour in measured amount of methanol.
d. Add the stain powder.
e. Tightly stopper the bottle.
f. Allow the stain powder to sink slowly through the methanol until it settles down to
the bottom. Shake the bottle with circular motion for 2-3 minutes.
g. Add measured amount of glycerol and repeat the shaking process. Continue to
shake for 2-3 minutes at half hourly intervals for at least 6 times.
h. Leave the bottle unused for 2-3 days; shaking it 3-4 times each day until the stain is
thoroughly mixed.

75
 i. Labe the bottle with date of preparation. Keep the bottle tightly Stoppard in a cool,
place, away from direct sunlight. This can be used for on month.

ii. Preparation of buffer water

Disodium hydrogen phosphate : 1.0 gm
Potassium Di- hydrogen phosphate : 0.7 gm
Distilled water : 10000 cc
pH : 7.2

STAINING PROCEDURE:

a. Dehaernoglobinize the thick smear by tap water with the help of a pipette; care
should be taken not to touch the thin smear.
b. Allow to dry.
c. Fix both thick and thin smear by methanol for one minute.
d. Allow to dry & place the slid on staining tray.
e. Add the freshly prepared working solution of Giemsa stain (1 :3) (1 part Giemsa
stock solution & 3 part buffered water, pH 7.2) gently onto the slides, untile the slide is
completely covered.
f Allow the stain to remain for 10minutes.
g. Wash the slides with clean water.
h. Dry the slides and observe under oil immersion object.

76
 5. CLINICAL BIOCHEMISTRY
1. CARDIAC PROFILE TEST
a. Total cholesterol
b. Triglyceride
c. HDL
d. LDL
e. VLDL
f. Serum LDH
g. Amylase
2. LIVER FUNCTION TEST (LFT)
a. Total bilirubin
b. Direct bilirubin
c. Indirect bilirubin
d. SGOT
e. SGPT
f. ALP
g. Total protein
h. Serum albumin
i. Serum globulin
3. KIDNEY FUNCTION TEST (KFT)
a. Urea
b. Creatinine
c. Creatinine clearance test
d. ACR
4. THYROID PROFILE TEST
a. TSH
b. FT3
c. FT4
5. DIABETIC PROFILE TEST
a. Fasting plasma glucose
b. Post Prandial plasma glucose
c. Random glucose

Clinical biochemistry deals with the biochemistry laboratory to find out cause of a disease.
The clinical constituents of various body fluids such as blood (serum/plasma), urine, CSF,
and other body fluids are analyzed in clinical biochemistry laboratory. These determinations
are useful in diagnosing various clinical conditions such as diabetes mellitus, jaundice, gout,
hyperlipidemia, pancreatitis, rickets, etc. The biochemistry tests are very useful in
determining the severity of disease of many organs such as liver, stomach, heart, kidneys,
brain as well as endocrine disorders and related status of acid-base balance of the body.

Instruments, used in this department: ( all steps must correlate with my project & literature)

77
 CARDIAC PROFILE TEST
Clinical Significance:
Elevation of the total cholesterol values in plasma is considered to be a prime risk factor for
coronary heart disease.

Increased triglycerides and VLDL values are taken as primary risk factors. A low serum
triglyceride level is suggestive of intravascular lipolysis and enhanced formation of HDL.
Hypertriglyceridemia, on the other hand indicates less effective intravascular lipolysis and a
reduced formation of HDL, which associated with a higher atherogenic risk.

Elevated LDL is suggestive of atherogenic risk. Low level of HDL-Cholesterol indicate high
risk of coronary heart disease.

 DETERMINATION OF SERUM TOTAL CHOLESTEROL

Clinical significance( ref.no.17):

Elevated levels of serum cholesterol are associated with atherosclerosis, nephrosis, diabetes
mellitus, obstructive jaundice & myxedema. Decrease levels are observed in
hyperthyroidism, malabsorption & anemia.

Method:

Enzymatic method ( application on auto-analyzers). For photometric determination of

cholesterol according to the “CHOD-PAP” method.

Normal range: 150-250 mg/dl

Sample material:

Fasting serum or heparinized plasma

Test principle:

Cholesterol esters are hydrolyzed by cholesterol ester hydrolase to free cholesterol & fatty
acids. The free cholesterol produced & pre-existing one are oxidized by cholesterol oxidase
to cholesterol-4-en-3-one & hydrogen peroxide. Peroxidase acts on H₂O₂& liberated o2 reacts
with the chromogen (4-aminophenazone/phenol) to form a red colored compound which is
read at 510nm (505-530nm).

Requirements:

1. Test tube
2. Micropipettes
3. Stop watch
4. Semi auto analyser
5. Auto analyser

78
 Reagents:

1. Cholesterol working reagent

2. Chlosterol standard: 200mg/dl

Stability of the reagents:

All the reagents are stable at 2-8⁰C

Procedure:

Dispense in the tubes labeled as follows-

Test Std. Blank

Working reagent, ml 1.0 1.0 1.0
Serum, ml 0.01 - -
Cholesterol Std, ml - 0.01 -
Distilled water, ml - - 0.01

Mixed well & kept at 37⁰C for 10 minutes. Read the absorbance of test & standard against
blank.

Calculation: Serum total cholesterol, mg/dl= X 200

DETERMINATION OF SERUM HDL-CHOLESTEROL

Principle (ref. no. 17):

When the serum is reacted with the polyethylene glycol contained in the precipitating
reagent, all the VLDL and LDL are precipitated. The HDL remains in the supernatant and is
then assayed as a sample for cholesterol using the cholesterol reagent.

Clinical significance of HDL-Cholesterol:

HDL-Cholesterol values below 40 mg% indicate towards the risk factor. The clinical risk
factor is better represented by the ratio of total cholesterol to HDL Cholesterol in case of
heart ailments.

Risk Factor Total Cholesterol/HDL Cholesterol

Female Male
Average x 0.5 3.3 3.4
Average x 1.0 4.5 5.0
Average x 2.0 7.0 9.6
Average x 3.0 11.0 24.0

79
Normal reference values:

Prognostically Standard Risk Favourable Risk level indicator

HDL Cholesterol Male>55 35-55 <35
Female>65 45-65 <45
LDL Cholesterol Male<150 150-190 >190

Total Cholesterol Male>3.8 3.8-5.9 <5.9

Female>3.1 3.1-4.6 <4.6

Contents:

L1: Precipitating Reagent

S: HDL Cholesterol Standard

Specimen collection & preservation:

Blood should be collected in a clean dry container. Fasting blood is preferred for HDL-
Cholesterol assay.

Plasma should be separated immediately from the cells. For plasma separation any of the
following anticoagulants may be used.

EDTA 10 mg/ ml blood

HEPARIN 200 IU/ ml blood

HDL- Cholesterol value is stable in serum for 24 hours at 2-8⁰C & 30 days when stored at
20⁰C.

Procedure:

HDL separation:- Pre warm at room temperature (25⁰-30⁰C) the required amount of
Precipitating Reagent & Cholesterol working solution before use.

Perform the assay as given below:

Pipetted into clean dry test tube:

Precipitating Reagent(L1): 0.1 ml

Sample : 0.1 ml

Mix well (cyclo- mixture machine may be used) & centrifuge at 2500-3000 r.p.m. for 10
minutes in a common laboratory centrifuge (1800 x g) to obtain a clear supernatant.
Procedure for the Cholesterol assay ( give from own performed literature).

Wavelength/filter: 505 nm (Hg 546 nm)/ Green

80
Temparature: 37⁰C/r.t

Light path: 1 cm

Pipetted into clean dry test tubes labeled as Blank, Standard, & Test:

Addition Sequence Blank(ml) Standard(ml) Test(ml)

Working reagent 1.0 1.0 1.0
Distilled water 0.05 - -
HDL Standard(s) - 0.05 -
Supernatant - - 0.05

Incubation:

Incubate the assay mixture for 10 minutes at 37⁰C or 30 minutes at room temperature(25⁰-
30⁰C). After completion of the incubation , measure the absorbance of assay mixture against
blank at 510 nm. Final color is stable for two hours , if not exposed to direct light.

Calculation:

HDL-Cholesterol in mg/dl = (Abs. test/ Abs. standard) x 50 x 2

(Where 2 is the dilution factor due to the deprotinization step)

Calculation of VLDL Cholesterol (mg/dl) = Triglycerides/5

Calculation of LDL Cholesterol (mg/dl) = Total cholesterol- ( VLDL cholesterol+ HDL

cholesterol)

 DETERMINATION OF SERUM TRIGLYCERIDES

Principle (ref. no. 18):

Determination of triglycerides involves enzymatic splitting with lipoprotein lipase. Indicator

in quinoneimine, which is generated from 4-aminoantipyrine & 4-chlorophenol by hydrogen
peroxide under the catalytic action of peroxidase.

Lipase

Triglyceride (Glycerol+ Fatty acids)

GK

Glycerol + ATP Glycerol 3-phosphate +ADP

81
Glycerol 3- phosphate + o₂ →GPO Dihydroxyacetone phosphate + H₂O₂

2H₂O₂ + Aminoantipyrine + 4-Chlorophenol POD Quinonemine + HCL + 4H₂O

Reagent:

1. Triglycerides working reagent

2. Triglycerides standard

Sample material:

Serum, heparin plasma or EDTA plasma.

The stability in serum or plasma is 2 days between 20-25⁰C. 7 days between 4-8⁰C & min 1
year at -20⁰C.

Assay procedure:

Wavelength 550 nm , Hg 546 nm

Light path 1 cm

Temperature 20-25⁰C/37⁰C

Measurement against blank

Pipette in the tubes as follows:

Test Standard Blank

1. Working reagent(ml) 1.0 1.0 1.0
2. Serum (ml) 0.01 - -
3. Standard(ml) - 0.01 -
4. Distilled water(ml) - - 0.01

Mixed & incubated at 10 min at 20-25⁰C or 5 min at 37⁰C. Read the absorbance against the
blank with in 60 mins.

Calculation:

Serum triglycerides = OD Test/OD Standard x 100mg/dl

Clinical interpretation:

Epidemiological studies have observed that a combination of plasma triglycerides > 180
mg/dl (>2.0 mmol/l) & HDL cholesterol <40 mg/dl (1.0 mmol/l) predict a high risk of CHD.

Borderline levels (>200 mg/dl) should always be regarded in association with other risk
factors for CHD.

82
  DETERMINATION OF LDH

Method: UV-Kinetic( ref. no. 19)

Principle: LDH

L- lactate + NAD⁺ ↔ Pyruvate + NADH + H

Increase in OD is measured after 45 sec by the interval of 1 min

Normal range: 70-240 IU

Sample: serum or heparinized plasma. Hemolysed serum should not used for the test.

Reagent:

1. Buffer substrate
2. NAD⁺ solution

Stability of the reagents: the reagents are stable at 2-8⁰C

Procedure:

Wavelength 340 nm

Cuvette 1 cm light path

Temperature 37⁰C

Buffer substrate, ml 1.0

NAD⁺ solution, ml 0.2
Serum or plasma, ml 0.02
Mix, take after 45 sec & then by the interval of 1, 2, & 3 minutes. Determine the mean

absorbencies change/ min ( ∆ A/ min)

Calculation: LDH, IU = 9807 x ∆ A/ min. If the absorbance change ∆ A/ min exceeds 0.100
at 340 nm , dilute serum 1:10 by using normal saline ( result x 10). (i.e. 0.1 ml serum
+ 0.9 ml normal saline).

DETERMINATION OF SERUM AMYLASE

Introduction( ref. no. 20):

Amylase was originally referred to as diastase amylases are a group of hydrolyses which split
polysaccharides such as starch & glycogen. The enzymes splits straight chains of polyglucans
(amylase) at alternate α-1-4 links, forming maltose & some residual glucose. It also acts on
branched starch amylopectine& glycogen forming, dextrins& residual glucose. Human serum
amylase has an optimum pH at 6.9-7.0. fullactivity is displayed in the presence of various
icon such as mono hydrogen phosphate, chloride, bromide ctc.

83
Clinical significance:

Determination of serum & urine amylase largely used in the diagnosis of disease of the
pancreas & the investigation of the pancreatic function an acute pancreatitis, a transitant rise
in serum amylase activity occur within 2-12 hours of the onset. Serum amylase levels return
to normal by the 3rd or 4th day. Usually high levels of amylase (4-6 food elevations above the
reference limit) are observed during 12-72 hours.

Parallel increase in serum lipase confirms acute pancreatitis . elevation of serum amylase
activities are reflected in the rise of urinary amylase activity. The urinary clearance of
amylase is markedly increase in acute pancreatitis. Serum amylase also increases in traumatic
lesions of pancreas (including surgical trauma & radiological investigations) & carcinoma of
pancreas. Tumors of lungs & serous tumors of the ovary can produce high levels of serum
amylase with elevation as high as 50 times the upper reference limit. Both kinds of tumor can
produce pleural effusion.

Salivary gland lesions caused by infection , obstruction, surgery, irradiation, & tumor have all
been reported to elevate serum amylase (S. Type hyperamylasemia). Mumps & maxillofacial
surgery can cause a twofold.

Biliary tract disease ( such as cholecystitis) can cause up to four fold elevation of the serum
amylase activity ( due to primary or secondary pancreatic involvement). Macroamylase are
complexes between ordinary amylase ( mainly S-type) & IgA, IgG or other normal or
abnormal high moleculer weight proteins. There presence may increase serum amylase
activity( 6-8 fold over the observed in health), since macroamylasecan not be filtered through
the glomeruli of the kidney due to their large size. In macroamylasemia, amylase activity in
the urine is lower then normal because the kidney clears less amylase.

The amylase/creatinine clearance ratio (ACCR) has been defined & expressed as percentage
as follows:

Urine amylase ( U/L) X Urine Creatinine (mg/l)

ACCR % = X 100
serum amylase (U/L) X Urine creatinine (mg/l)

Normal ACCR = 1-5%

In macroamylasemia: <1%

In acute pancreatitis: >5%

Elevations in ACCR are also observed renal insufficiency, burns, ketoacidosis & in light
chain proteinuria.

Laboratory methods: Amylase was previously measured by iodometric&saccharogenic

methods which required long incubation periods. Turbidimetric methods were relatively first
but they required special instrumentation & it was difficult to prepare stable & reproducible
starch solutions. The method based on modification of wallenfels et al is a short & simple

84
rate of reaction ( visible kinetic) method. A substrate used in this method is silyl-blocked-p-
nitrophenyl-α-D-maltoheptoside, which undergoes spontaneous degradation by α-glucosidase
&glucoamylase. Resultant nitrophenol is yellow colored complex which can be measured at
405 nm.

Method: Colorimetric ( amyloclastic, iodometric)

Principle:

Amylase in specimen acts on the substrate, such starch the products formed are dextrins&
maltose. After the incubation, when the end product are treated with the color reagent ( iodine
reagent), decreased in the blue color is observed, comparing to that is product with the blank .
The disappearance of blue color is directly to the amylase concentration in the specimen &
gives the measure of amylase concentration in the specimen.

Enzyme units: Caraways unite: Caraway defined the enzyme unit as the amount of
enzyme that will hydrolyse 10 mg of starch in 30 mins to colorless stage.( in the procedure,
the substrate should be completely hydrolysed by 800 units of amylase in 100 ml serum)

Sample material: Serum. The enzyme is quite stable. Activity loss is negligible even at
room temperature it is stable for 3 month refrigerated.

Requirements:

1. Test tubes
2. Stop watch
3. Water bath
4. Photometer

Procedure:

Test Blank
Buffer substrate, ml 2.5 2.5
Keep at 37⁰C for 5 min.
Serum, ml 0.1 -
Mix , 37⁰C for 7 min.
Working color reagent, ml 2.5 2.5
Serum, ml - 0.1
Distilled water, ml 20 20

Mix & read test & blank against distilled water, at 660 nm.

85
Calculation:

OD of blank – OD of test
Serum amylase = X 400

OD of blank

 DETERMINATION OF SERUM URIC ACID

Summary ( ref. no. 22):

Uric acid is the end product of purine metabolism. Uric acid is excreted to a large degree by
the kidneys & to a smaller degree in the intestinal tract by microbial degradation. Increased
levels are found in gout, arthritis, impaired renal function, & starvation. Decreased levels are
found in Wilson‟s disease, Fanconis syndrome & yeitow atrophy of the liver.

Method:Uricase/ PAP method

Principle:

Uricase converts uric add to allantoin & hydrogen peroxide. The hydrogen peroxide formed
further reacts with a phenolic compound & 4-aminoantipyrine by the catalytic action of
peroxidase to form a red colored quinoneimine dye complex. Intensity of the color formed is
directly proportional to the amount of uric acid present in the sample.

Uricase→Uric acid alantoin + H₂O₂→Peroxidase →H₂O₂ + 4- aminoantipyrine→ Red

quinoneimine + H₂O₂ + Phenolic Compound.

Normal reference values:

Serum/Plasma (male): 3.4 – 7.0 mg/dl

( Female): 2.5 – 6.0 mg/dl

It is recommended that each laboratory establish its own normal range patient population.

Sample material:

Serum, plasma, uric acid is stable in the sample for 3 – 5 days when stored at 2 – 8⁰C.

Procedure:

Wavelength/filter : 520 nm (Hg 546 nm)/ Yellow green

Temperature : 37⁰C/ r.t

Light path : 1 cm

Pipette into clean dry test tubes labeled as blank, standard, & test

86
Addition sequence Blank Standard Test
Working reagent, ml 1.0 1.0 1.0
Distilled water, ml 0.02 - -
Uric acid standard, ml - 0.02 -
Sample, ml
Mix well & incubate at 37⁰C for 5 min. or at r.t. (25⁰C) for 15 min. Measure the absorbance
of the standard , & test sample against the blank, within 30 min.

Calculation:

Uric acid in mg/dl = ( OD of test/ OD of standard) X 8

Linearity:

This procedure is linear up to 20 mg/dl. If values exceed this limit , dilute the serum with
normal saline (NaCl-0.9%) & repeat the assay. Calculate the value using the proper dilution
factor.

 LIVER FUNCTION TEST (LFT):

Clinical significance of LFT ( ref. no. 23):

 In the differential diagnosis of the different types of jaundice.

 To asses the severity of liver damage in known liver disease.
 To follow the trend of the disease.
 To screen the suspected case during outbreak of infective hepatitis.

 DETERMINATION OF SERUM BILIRUBIN

Principle:

Bilirubin is estimated by reacting it with diazotized sulfanilic acid obtained from sodium
nitrate &sulfanilic acid. It forms a pink colored azo compound. Direct bilirubin ( conjugated
or soluble fraction) reacts very quickly & is read by measuring color developed in 15 seconds
of reaction. The unconjugated or free bilirubin tekes longer time to react & requires an
accelerator. Hence total bilirubin is measured using caffeine as an accelerator allowing the
reaction to proceed for 5 minutes.

87
Clinical significance:

Total & direct bilirubin estimation in serum or plasma is used for the diagnosis,
differentiation & follows up of jaundice.

The following table gives the findings:

HEMOLYTIC HEPATIC OBSTRUCTIVE

DIRECT/SOLUBLE BILIRUBIN NORMAL INCREASED INCREASED
INDIRECT/FREE BILIRUBIN INCREASED INCREASED NORMAL

Specimen collection:

Fasting, fresh, clear, non hemolysed serum is preferred. Plasma collected using heparin may
be used.

Reagent:

1. Reagent 1 ( sodium nitrate)

2. Reagent 2 ( sulfanilic acid)
3. Reagent 3 ( caffeine)
4. Bilirubin standard (5mg/dl) (ready to use)

Reagent storage & stability:

All reagents included in the kit are stable at room temperature until the expiry stated on the
table. Reagent 1 is stable at 2 – 8⁰C.

System parameters:

TOTAL BILIRUBIN DIRECT BILIRUBIN

Type of reaction End point End point
Wavelength 546 nm 546 nm
Flow cell temperature 3⁰C 30⁰C
Incubation time 5 min. at r.t 15 sec. at r.t
Factor 16.2 16.2
(refer insert) (refer insert)
Sample volume 100µl (6.1 ml) 100µl (0.1 ml)
Reagent volume 1.10 ml 1.10 ml
Zero setting Distilled water Distilled water
Light path 1.0 cm 1.0 cm

88
Procedure:

TOTAL DIRECT
BLANK TEST BLANK TEST
REAGENT 1 - 0.05 - 0.05
REAGENT 2 0.1 0.05 0.1 0.05
REAGENT 3 1.0 1.0 - -
NORMAL SALINE - - 1.0 1.0
SAMPLE 0.1 0.1 0.1 0.1
Incubate for 5 min. at r.t. (25⁰- Incubate for 15 min. at r.t (25⁰-
30⁰ C) 30⁰C)

Read absorbance against distilled water at 546 nm ( 530 – 570 nm) or green filter

Calculation:

Serum bilirubin (mg/dl) = (OD of test – OD of sample blank) X F

F = factor 16.2

The use of a ready to use dye standard instead of pure bilirubin is a practical way & a
compromise for convenience.

Procedure for factor determination:

Read the absorbance of standard (5 mg/dl) at 546 nm or at green filter(530- 570 nm) against
distilled water for several times till you get consistence reading. Record it, take the average
absorbance of the standard & calculate the factor as follows.

Factor determination:

F = 5/ average absorbance of standard

Under standard given parameters factor range from 14 – 17. Once a factor is obtain this can
be stored in the instrument, under normal conditions recalibration is not required for the same
batch of reagent.

Normal values:

Total bilirubin : 0 – 1.0 mg/dl

Direct bilirubin: 0 – 0.3 mg/dl

Linearity:

20 mg/dl for total & direct bilirubin.

89
  DETERMINATION OF SERUM SGPT (ALT)

Summary & clinical significance:

Serum Glutamate Pyruvate Transaminase (SGPT), also called Alanine Aminotransferase

(ALT), belongs to the transferase class of enzymes. It is found to be distributed mainly in the
liver & to a lesser extent in the kidney & muscles. In hepatitis of different etiologies, SGPT is
an important indicator not only in the diagnosis of the ailment but also in assessing the
prognosis & process of the disease. An elevated SGPT level is characteristic in alcoholics.

Principle:

L-Alanine + 2- Oxoglutarate --------------→ Pyruvate + Glutamate

Pyruvate + 2, 4 DNPH --------------------- →Brownish red colored complex

SGPT (ALT) catalyses the transfer of amino group from L- Alanine to 2- Oxoglutarate to
form pyruvate & L-Glutamate. The pyruvate thus formed reacts with 2, 4 Dinitrophenyl
Hydrazine (2,4 DNPH) to form a corresponding Hydrazone, a brownish red colored complex
in an alkaline medium. The color intensity is directly proportional to the SGPT concentration
in the serum & is measured photometrically at 505 nm (490- 546 nm) or with green filter.

Reagent composition:

1. Reagent 1---------Substrate reagent --------L- Alanine 200 mmol/l

2- oxoglutarate 10 mmol/l
2. Reagent 2---------Color reagent ------------ 2, 4- DNPH 5 mmol/l
3. Reagent 3---------Sodium Pyruvate --------- 170 U/L
4. Reagent 4---------Alkaline reagent --------- Sodium Hydroxide 4N

Reagent preparation & storage:

Dilute reagent 4 (Alkaline reagent) 1:10 with deionised water ( 1 part Alkaline reagent + 9
parts Deionised water). All the other reagents are ready to use. All the reagents are stable at 2
– 8⁰C up to the expiry date mentioned on the lebel when properly stored.

Specimen:

Clear, unhemolysed serum is preferred ( serum should be removed from the clot as soon as
possible after collection because of the presence of approximately 10 folds greater
concentration of SGPT in erythrocytes than in serum). For similar reason hemolysis of the
specimen must be avoided.

Normal range:

Male: Upto 42 U/ L at 37⁰C

Female: Upto 38 U/L at 37⁰C

90
Procedure:

Pipette into test tubes labeled as blank, calibrator, control, test & proceed as per given below.

Reagent Blank Calibrator Control Test

Substrate reagent, ml 0.5 0.5 0.5 0.5
Deionised water, ml 0.1 - - -
Serum sample, ml - - - 0.1
Calibrator - 0.1 - -
Mix & incubate at 37⁰C for 30 minutes

Color reagent, ml 0.5 0.5 0.5 0.5

Serum sample, ml - - 0.1 -
Mix & incubate at 37⁰C for 30 minutes

Alkaline reagent, 3.0 3.0 3.0 3.0

ml(diluted)
Read absorbancies of all the tubes against distilled water at 505 nm( 490- 546 nm) or with
green filter.

Calculation:

SGPT (ALT) activity in U/L = OD of sample X Concentration of calibrator.

 DETERMINATION OF SERUM SGOT (AST)

Summary & clinical importance:

Serum Glutamate Oxaloacetate Transaminase ( SGOT), also called as Aspartate

aminotransferase (AST), belongs to the transferase class of enzymes. This enzyme shows
high levels of activity in the heart, liver, skeletal muscles & kidneys. Since its level seems to
be increasing enormously following Myocardial Infection (MI). Elevated levels are also seen
in viral/ toxic hepatitis, hepatic & cardiac necrosis, muscular dystrophy & pulmonary
embolism.

91
Principle:

L- Aspartate + 2-Oxoglutarate ---------------→Oxaloacetate + L- Glutamate

Oxaloacetate + 2,4 DNPH --------------------- →Brownish red colored complex

SGOT (AST) catalyses the transfer of amino group from aspartic acid to 2-Oxoglutarate to
form Oxaloacetate & L- Glutamate. The Oxaloacetate thus formed reacts with 2,4-
Dinitrophenyl Hydrazine (2,4-DNPH) to form a corresponding Hydrazone , a brownish red
colored complex in an alkaline medium. The color intensity is directly proportional to the
SGOT concentration in the serum & is measured photometrically at 505 nm (490- 546 nm) or
with green filter.

Specimen:

Clear, unhemolysed serum is preferred ( serum should be removed from the clot as soon as
possible after collection because of the approximately 10 fold greater concentrations of
SGOT in erythrocytes than in serum). For similar reason hemolysis of the specimen must be
avoided.

Normal range:

Male: Upto 40 U/L at 37⁰ C

Female: Upto 37 U/L at 37⁰ C

(The expected value should be used as a reference only. It is recommended that each
laboratory should establish its own normal range)

Procedure:

Pipette into test tubes labeled as blank, calibrator, control, test & proceed asper given below

REAGENT BLANK CALIBRATOR CONTROL TEST

Substrate reagent, ml 0.5 0.5 0.5 0.5
Deionised water, ml 0.1 - - -
Serum sample, ml - - - 0.1
Calibrator, ml(conc. - 0.1 - -
160U/L)
Mix & incubate at 37⁰C for 20 minutes

Color reagent, ml 0.5 0.5 0.5 0.5

Serum sample ,ml - - 0.1 0.1
Same serum sample which is used above

Mix & incubate at 37⁰C for 20 minutes

Alkaline reagent, ml 3.0 3.0 3.0 3.0

(diluted)

92
Read the absorbance‟s of all the tubes against distilled water at 505 nm ( 490- 546) or with
green filter.

Calculation:

SGOT (AST) activity in U/L = ∆A/ min. X 1746

Linearity:

This method is linear upto 300 U/L . For samples above the linearity limit , dilute suitably
with saline & re assay. Multiply by the dilution factor to calculate the end result

 DETERMINATION OF SERUM ALKALINE PHOSPHATASE

Summary:

Alkaline phosphatase (ALP) is an enzyme of the hydrolase class of enzymes & acts in an
alkaline medium. It is found in high concentrations in the liver, billiary tract epithelium & in
the bones. Normal levels are age dependent & increase during bone development. Increased
levels are associated mainly with liver & bone disease. Moderate increase are seen in
Hodgkins disease & congestive heart failure.

Principle:

ALP at an alkaline pH hydrolyses p-Nitrophenylphosphate to form p-Nitrophenol&

Phosphate. The rate of formation of p-Nitrophenol is measured as an increase in absorbance
which is proportional to the ALP activity in the sample.

ALP
p- Nitrophenylphosphate --------------- →p-Nitrophenol + phosphate

Normal range:

Serum ( Adult) --------- 80-290 U/L at 37⁰ C

(children) ------ 245- 770 U/L at 37⁰ C

(it is recommended that each laboratory establish its own normal range representing its
patient population)

Reagent:

1. Buffer reagent (L1) ---------- 2X150 ml

2. Substrate reagent (T1) ----- 2X 10 Nos.

Storage/ stability:

Content are stable at 2 – 8 ⁰C till the expiry mentioned on the lebels.

93
Reagent preparation:

Working reagent: Dissolve 1 substrate tablet in 15 ml of buffer reagent. The working reagent
is stable for at least 15 days when stored at 2 – 8 ⁰C.

The substrate is light & temperature sensitive. Take adequate care, especially after
reconstitution.

Sample material:

Serum ( free from hemolysis. ALP is reported to be stable in serum for 3 days at 2 -8 ⁰C.

Procedure:

Wavelength / filter : 405 nm

Temperature: 37⁰C

Light path: 1 cm

Pipette into a clean dry test tube labeled as test (T)

Addition sequence Test

Working reagent, ml 1.0
Incubate at the assay temperature for 1 minute & add
Sample, ml 0.02

Mix well & read the initial absorbance Aₒ after 1 minute & repeat the absorbance reading
after every 1, 2, & 3 minutes. Calculate the mean absorbance change per minute ( ∆A/ min.)

Calculation:

ALP activity in U/L = ∆A/min. X 2754

Linearity:

The procedure is linear upto 700 U/L at 37⁰C . If the absorbance change (∆A/min.) exceeds
0.250, use only the value of the first two minutes to calculate the result, or dilute the sample
1+9 with normal saline ( NaCl 0.9%) & repeat the assay ( Result X 10)

TOTAL SERUM PROTEIN DETERMINATION

Clinical significance:

Total serum protein values decrease below normal range in different clinical conditions
associated with nephritic syndrome, malnutrition, cirrhosis, of liver & other liver disease in
which liver cells are severely damaged. Increased total protein values may be found in
multiple myeloma & conditions associated with high globulin concentration.

94
Normal range:

Serum protein: 6 – 8 gm/dl

Method:

Biuret method

Principle:

Proteins react with cupric ions in alkaline medium to form a violet colored complex. The
intensity of the color produced is directly proportional to proteins present in the specimen can
be measured on a photometer at 530 nm( or by using a green filter).

Requirements:

1. Test tubes
2. Micro pipette
3. Test tube stand
4. Semi auto analyser

Preparation of the reagent:

1. Protein reagent
2. Protein standard

Stability of the reagent: Depends on kits expiry date

Procedure:

Mono step method

Pipette in three tubes lebeled as follows

Addition sequence TEST STANDARD BLANK

Protein reagent, ml 5.0 5.0 5.0
Serum, ml 0.05 - -
Protein standard (6 gm/dl), ml - 0.05 -
Distilled water, ml - - 0.05

Mix thoroughly & keep at room temperature ( 25⁰- 30⁰C) for exactly 10 minutes . Measure
the intensities of the test & standard by setting blank at 100% T, by using 530 nm(green
filter)

Calculation:

Serum protein = (OD of test/ OD of standard) X 6 gm/dl

95
  DETERMINATION OF SERUM ALBUMIN

Method:

Bromocresol green method

Principle:

Albumin present in serum binds specially with bromocresol green at pH 4.1 to form green
colored complex, intensity of which can be measured colorimetrically by using 640 nm or a
red filter.

Normal range:

3.3 – 4.8 gm/dl

Requirements:

1. Test tubes ------15X125 mm

2. Serological & graduated pipettes, 10 ml, 5 ml
3. Test tube standard
4. Push button pipette of 0.05 ml or serological pipette of 0.1 ml
5. Semi auto analyser
6. Serum

Preparation of reagent:

1. Albumin reagent
2. Albumin dtandard
3. Distilled water as sample blank

Stability of the reagent:

Depends on kits expiry

Procedure:

Mono step method

Pipette into three test tubes lebeled as follows ----- like protein estimation

Calculation:

Serum albumin = (OD of test/ OD of standard) X 4

96
  DETERMINATION OF SERUM GLOBULIN

Normal range:

1.8 – 3.6 gm/dl

Calculation: Serum globulin gm/dl= (Total protein – Serum albumin)gm/dl

A/G ratio = (Serum albumin, gm/dl/ Serum globulin gm/dl)

KIDNEY FUNCTION TEST

Kidney function test contains with:

1. Determination of blood urea

2. Determination of blood creatinine
3. Creatinine clearance test

DETERMINATION OF BLOOD UREA

Clinical significance:

Elevated levels of urea are observed in pre- renal, renal, & post renal conditions. Pre-renal
conditions such as diabetes mellitus, dehydration, cardiac failure, severe burn, high fever etc,
& renal conditions such as disease of kidneys, post-renal conditions such as enlargements of
prostate, stone in the urinary tract, tumor of the bladder. Decreased values have been reported
in severe liver disease, protein malnutrition & pregnancy.

Principle:

Urea reacts wiyh hot acidic Diacetylmonoxime in presence of thiosemicarbazide& produces a

rose-purple colored complex, which is measured colorimetrically.

Method:

DAM (Diacetylmonoxime method)

Sample:

Serum/plasma( 0.01 ml is required)

Reagent:

1. Reagent 1: Urea reagent

2. Reagent 2: DAM
3. Reagent 3: Working urea standard, 30 mg%

97
Normal range:

20 – 40 mg/dl

Procedure:

BLANK TEST STANDARD

Reagent 1, ml 2.5 2.5 2.5
Sample, ml - 0.01 -
Reagent 3, ml - - 0.01
Mix well
Reagent 2, ml 0.25 0.25 0.25
Mixed well & kept the tubes in the boiling water exactly for 10 minutes & cooled
immediately under running tap water for 5 minutes. Mixed by inversion & measured the
color intensity within 10 minutes using a green filter against blank.

Calculation:

Serum/Plasma Urea(mg/dl) = (OD of test/ OD of standard) X 30

DETERMINATION OF BLOOD CREATININE

Clinical significance:

Creatinine is waste product formed in muscle from the high energy storage compound,
creatine phosphate. The amount of creatinine produced is fairly constant & is primarily a
function of muscle mass. Creatinine is removed from plasma by glomerular filtration & then
excreted in urine without any appreciable resumption by the tubules.

Creatinine is used to asses renal function; however, serum creatinine levels do not start to rise
until renal function has decreased by at least 50%. Congestive heart failure, shocks &
mechanical obstructions of urinary tract may also contributed to an elevated level of serum
creatinine. An elevated serum creatinine level due to obstructive may rapidly fall when the
obstruction is removed by surgery.

Principle:

Acid reagent differentiates color developed using Jaffe‟s reagent by (1) Creatine + reactive
non creatinine substances & (2) reactive non creatinine substances.

Colored developed before & after addition of acid reagent is measured photometrically at 510
nm (500 – 540 nm/ green filter) & the difference is proportional to creatinine concentration.

Creatinine + Alkaline Picrate-------- →Creatinine Picrate complex

Method:

Alkaline Picrate method

98
Normal range:

Men: 0.9 – 1.4 mg/dl

Women: 0.8 – 1.2 mg/dl

Sample:

Fresh, clear serum with no hemolysis is the specimen of choice. Plasma prepared using
heparin as an anticoagulant may also be used.

Reagent:

1. Creatinine (Picrate reagent)

2. Creatinine (Alkali reagent)
3. Creatinine (Acid reagent)
4. Creatinine standard, 3 mg/dl

Storage:

All the reagents to be stored at room temperature.

Procedure:

Deproteinization of sample is not required.

PIPETTE INTO TEST TUBES STANDARD TEST

Working reagent, ml 2.5 2.5
Sample, ml - 0.2
Standard, ml 0.2 -
Mixed well & incubated for 15 minutes at room temperature .

Read absorbance of standard(1) & test(1) at 510 nm against distilled water.

3 creatinine, ml 0.1 0.1

Mixed well & kept for 5 minutes at room tempareture.

Read the absorbance of standard(2) & test(2) at 510 nm against distilled water.

Calculation:

Creatinine concentration mg/dl = TS/STD X 3

Where , TS = corrected absorbance of sample = test(1) – test(2)

STD = corrected absorbance of standard = standard(1) – standard(2)

99
  DETERMINATION OF CREATININE CLEARANCE

Introduction:

This test gives relatively accurate & useful measure of the glomerular filtration rate & also
the excretory capacity of the kidney. The reasons for the degree of accuracy of creatinine
clearance are (1) creatinine is not absorbed by the tubules (2) the effect of fluid intake &
excretion on creatinine clearance is much less than that of urea & (3) the blood creatinine
values relatively stable. The creatinine clearance values may be greater than the actual
glomerular filtration rate, when plasma creatinine levels increase considerably above the
normal range.

The clearance of any substance is defined as the number of ml of plasma which contains the
amount of that substance, excreted in urine in one minute.

UV 1.73
Clearance = X
S (P) A

In the case of creatinine clearance

U = mg/ml of urine creatinine

S (P) = mg/ml of serum (or Plasma) creatinine

V = ml of urine excreted per minute

1.73 = standard average surface area of the normal individual

A = surface area of the patient

Normal range:

a) For male: 105 ± 20 ml/min

b) For female: 95 ± 20 ml/min

Preparation of the patient:

 Requirement
A polythene container to collect 24 hours urine & it should contain few thymol
crystals (preservative)
 Urine collection
a) The patients should be instructed to empty the bladder at the beginning of the
period (8 am) & discard the urine.
b) Collect all urine passed until 8 o clock the next morning, emptying the bladder
at the time & adding this urine to the 24 hours specimen.
c) The urine should be kept in a cool place.
d) Note: The diuretic drinks & drugs should not be given to the patients during
the 24 hours of urine collection

100
 e) A blood sample is collected in a test tube for serum
f) Patients height & age are noted

Laboratory requirements:

1. Patients serum
2. 24 hours urine specimen
3. All other requirements are the same as those used for the determination of
serum & urinary creatinine

Procedure:

a) Measure volume of the collected urine specimen.

b) Determination serum & urine creatinine
c) Calculate creatinine clearance from the following formula:

Urine creatinine mg/dl V 17.3

Creatinine clearance, ml/min = X X

Serum creatinine mg/dl min A

 DETERMINATION OF BLOOD GLUCOSE

Clinical significance:

Accurate measurement of glucose in body fluid is important in diagnosis & management of

diabetes, hypoglycemia, adrenal dysfunction & various other conditions.

High levels of serum glucose may be seen in case of diabetes mellitus, in patients receiving
glucose containing fluids intravenously, during severe stress & in cerebrovascular accidents.

Decreased levels of glucose can be due to insulin administration , as a result of insulinoma,

inborn errors of carbohydrate metabolism or fasting.

Method:Trinder‟s method ( GOD – POD)

101
Principle:

α- D- glucose in the sample is rapidly converted to the β-isomer by the action of mutarotase
which then is oxidized to yield gluconic acid & hydrogen peroxide in the presence of Glucose
oxidase. The enzyme peroxidase catalyses the oxidative coupling of 4- aminoantipyrine with
phenol to yield a colored quinonemine complex, with absorbance proportional to the
concentration of glucose in sample.

Mutarotase
α- D- glucose ---------------- →β- D- glucose

Glucose oxidase
β- D- glucose + H₂O + O₂ ------------- →Gluconic acid + H₂O₂

Peroxidase
H₂O₂ + phenol + 4AAP ---------------- →Red dye + 2H₂O

Reagent composition :

1. Tris-Phosphate buffer ------------ 50 mmol/l

2. Glucose oxidase ------------------ ≥20000 U/L
3. Peroxidase ------------------------- ≥800 U/L
4. Mutarotase ----------------------- ≥1000 U/L
5. Phenol 10 mmol/l

Reagent preparation:

Reagents supplied are ready for use. Protect from bright light.

Stability & storage:

The unopened reagents are stable till the expiry date stated on the bottle & kit lebel when
stored at 2 – 8⁰C. The reagent develops a pink coloration on storage, this however does not
affect the performance of the test.

Specimen collection & handling:

Use fresh unhemolysed serum. The stability of glucose in specimen is reduced by bacterial
contamination & by glycolysis. Serum or plasma should be separated from the cells, as soon
as possible, to prevent glycolysis. The addition of sodium fluoride is recommended to inhibit
glycolysis. Serum/ plasma is stable for 3 days at 2 – 8⁰C. It is recommended to perform the
assay with freshly collected samples.

102
Requirements:

1. Semi-auto analyzer
2. cuvettes
3. pipettes
4. reagent
5. sample
6. Glucose standard (100 mg/dl)

Procedure:

Pipette in the tubes labeled as follows

TEST STANDARD BLANK

Glucose reagent, ml 1.0 1.0 1.0
Serum/Plasma, ml 0.01 - -
Glucose standard; 100 mg/dl , ml - 0.01 -
Distilled water, ml - - 0.01
Mix & keep at 37⁰C for 15 minutes or at room temperature for 30 minutes. Measure the
intensity of the color at 505 nm.

Calculation:

Serum/Plasma glucose, mg/dl = (OD of test/ OD of standard) X 100

Linearity:

Linearity of the method is up to 500 mg/dl.

103
 6.BLOOD BANK & BLOOD TRANSFUSION

 Blood bank & blood transfusion unit:

a. In-house blood collection

b. Urgent laboratory
1. Blood grouping & Rh factor
2. Compatibility tedting or cross matching
3. Direct & indirect Coombs test
4. Du test
c. Component separation
1. R.B.C. or Packed cell separation
2. Platelet separation
3. F.P.P. or plasma separation
4. Wash R.B.C preparation
5. Cryoprecipitate

Clinical significance of blood transfusion:

Blood transfusion today is a major medical service that is rendered to patients needing
replacement of whole blood or blood components. Since the beginning to the Second World
War, transfusion of blood & blood products has become accepted as a routine & relatively
safe procedure in the management of patients. The blood bank tries to select a donor‟s blood
that will be compatible with recipient‟s blood. The recipient‟s plasma should not have any
reactive antibody towards the red cell or donor which may lead to haemagglutination or
hemolysis of donor‟s red cells.

ABO blood groups:

During blood transfusion, an identical ABO blood group of the donor is ideal. Persons
belonging to blood group “O” are considered to be “universal donors”, i.e. their blood can be
given to individuals of any other blood groups. The red cells do not carry either A or B
antigen & hence they do not react with their corresponding antibodies. On the other hand,
those with blood group AB are “universal recipients”. They will accept blood from any of the
blood groups-A, B, AB or O. this is because they neither have anti-A or anti-B.

Transfusion of specific component blood, as needed by the patient, is preferred over

transfusion of whole blood. Whole blood is needed in case of large scale blood less. The
whole blood arrangements the recipient‟s total blood volume which may lead to congestive
heart failure. Anemic patients with adequate blood volume but who are deficient in red blood
cell population require packed red cells. Transfusion of plasma, plasma components &
platelet concentrates are the therapeutic measures recommended for bleeding patients.

104
IN-HOUSE BLOOD COLLECTION

Blood collection procedure:

Preparation:

Before starting to draw blood, sauce that the following materials are available & within
reachable distance properly labeled blood collection bottle with donors identification
member; bleeding set & air way attached; pilot tubes; tourniquet/pacer cuff; forceps; stripper;
adhesive tape; rubber bond; savlon; alcohol; iodine swab; local anesthetic & syringe & needle
for injection.

Procedure:

1. The donor is advised to lie on the bleeding table & made sure he/she is relaxed &
comfortable. The bottle is placed approximately 30 – 40 on below the level of table.
2. The tourniquet/pacer cuff is applied to the upper arm & select a prominent antecubital
vein.
3. The tourniquet is released & disinfected venipuncture site using savlon, iodine &
alcoholswab in that order. The skin is allowed to dry before inserting the needle.
4. The tourniquet is re-applied & injected local aesthetic subcutaneously at the site of
venipuncture using aseptic technique.
5. The venipuncture is performed with a 15 or 16 gauge needle. The bottle or bag is
gently agitated to mix the blood with anticoagulant. For this purpose agitating
machine may be used.
6. When the required amount of blood has been collected, tube is clamped; the
tourniquet is released & removed the object from the donor‟s hand.
7. When the required amount of blood is collected before the needle has been removed
from vein, the needle is drawn from the bottle & putted 5 – 10 ml. Sample into 2
sterile, dry pilot test tubes, each labelled with the same number as that or the main
blood collection bottle.
8. An alcohol swab is placed over the needle & then removed it. Pressure is applied over
the puncture site with alcohol swab. The donor is asked to press swales firmly over
the area for 3 – 5 minutes & preferably with arm hold straight up in an extended
position.
9. The air way needle from the blood collecting bottles is promptly removed & replaced
the cays after applying 70% alcohol swab an open areas.
10. Finally, tally the members on the bottle with the donor slip, record the book.
11. A rubber band is putted around the pilot tubes & collecting bottles.
12. Did not let the donor‟s stand up immediately after blood donation. Make sure that any
bleeding from the venipuncture had stopped with a Band-Aid wound is covered.
13. The bottle is stored at 2 – 4⁰C.

105
  BLOOD BANK LABORATORY

DETERMINATION OF BLOOD GROUP BY CROSS MATCH

Principle:

The house of blood transfusion has become steadily more frequent now a days. To detect or
to decide whether the donor‟s blood matches the recipient‟s blood, the blood group test is
done.

Requirements:

1. Glass slide
2. Pasteur pipette
3. Applicator sticks

Reagents:

1. Anti-A sera
2. Anti-B sera
3. Anti-D sera
4. Normal saline

Procedure:

1. A suspension of RBC‟s in normal saline is prepared.

2. On 1 half of glass slide is placed 1 drop of Anti-A blood grouping sera.
3. On the other half of the glass slide placed 1 drop Anti-B blood grouping sera & Anti-
D sera.
4. Using a Pasteur pipette added 1 drop of cell suspension to each half slide.
5. With separator applicator sticks mixed each cells sera mixture well.
6. Tilt the slide back & forth & observed for agglutination.

ANTI-A ANTI-B ANTI-D PROBABLE BLOOD GROUP

+ - + A(+)
- + + B(+)
- - + O(+)
+ + + AB(+)
- - - O(+)
Agglutination: +

Not agglutination: -

Significance: The use of blood groups are:-

1. To ensure compatible blood transfusion,

2. To eliminate haemolytic disease of the newborns due to Rh incompatibility,
3. To detect susceptibility to various diseases.

106
  DIRECT & INDIRECT METHODS MUST GIVE COMPATIBILITY
TESTING OR CROSS MATCHING

Before the recipient receives blood transfusion, a compatibility test must be run within the
laboratory with the donor‟s red cells & the recipient‟s serum. This is called major cross
matching. The primary purpose of major cross match is to find out any incompatibility of
donor‟s cells with patient‟s serum is order to avoid transfusion reactions. The minor
crossmatch is rarely requested when the compatibility of the recipient‟s red cells is tested
against donor‟s serum.

Compatibility test or cross matching is performed subsequent to the ABO grouping & Rh
typing of the recipient‟s & donor‟s blood. It is the final criterion as to the suitability of a
particular donor blood for a particular recipient.

The recipient‟s blood is obtained fresh while the donor‟s blood is obtained from the pilot
tube. ACD anti coagulated donor‟s blood should not be more than 21 days & constantly
stored at 4⁰C.

Principle:

Serum of the recipient is tested against the red cells of the donor under different conditions in
order to establish their compatibility or non- agglutination. Agglutination in any of the
conditions indicates the presence of incompatible antibody in patient, natural or immune.

The three phases of compatibility testing are listed below & illustrated in saline phases,
where the immunologic reaction between red cells suspended in saline medium & the
antibody occurs at room temperature.

1. Thermophase with protein: Where the red cells are suspended in the antibody (serum)
with 22% albumin (protein) & incubated for 30 minutes at 37⁰C.
2. Antihuman globulin(AHG) phase: Where the incubated cells are washed ( to remove
free globulin) & reacted with antihuman globulin serum ( Coombs reagent or
antihuman globulin).

 CROSS MATCHING BY SLIDE METHOD

The above slide method is commonly done in blood bank for quick cross matching.

Requirements:

1. Glass slide, marker

2. Disposable plastic sticks
3. Microscope
4. 4% red cell suspension of donor‟s & recipient‟s
5. Donor‟s & recipient‟s serum

107
Procedure:

1. Take a slide & draw a line centrally to divide into 2 parts.

2. Marked 1 part „P‟ for major cross matching & part „D‟ for minor cross matching.
3. On the „P‟ slide add 1 drop of patient‟s serum & 1 drop of donor‟s 4% cell
suspension.
4. On the part of „D‟ add 1 drop of donor‟s serum & 1 drop of patient‟s cell suspension.
5. Mix the content of each slide by gently rotating the slide.
6. Incubate at room temperature (21 -25⁰C) for 10 minutes.
7. Examine both macroscopically & microscopically for agglutination.

Result:

There is no agglutination in both sides of the side that indicates donor‟s blood compatible
with recipient‟s blood.

 COOMBS TEST

Antihuman globulin technique is very useful in recognizing weak immunologic reactions. It

is widely used in identification of Du compatibility testing, antibody screening &
identification of sensitized red cells.

DIRECT COOMBS TEST

This recognizes sensitized red cells when the sensitizing occurs within the body, i.e, in
haemolytic disease of new born (HDN) & autoimmune hemolytic anemia. This test is
performed to detect anti-D antibody or other antibodies attached to the red cell surface within
the blood stream.

Principle:

Antihuman globulin or Coombs test detect sensitized re cells where the red cell get coated
with IgG antibody or globulin but do not agglutinate when sensitize red cells come in contact
with anti human globulin reagent they agglutinate.

Reagents:

1. Anti human globulin or Coombs reagent

2. Pre sensitized red cells or Coombs sensitive cells
3. Saline (0.85%)

Specimen:

Collected blood is preferred over whole blood (citrate) is required for direct Coombs test. In
case of indirect Coombs test for antibody screening serum specimen will be needed.

108
Procedure:
1. Prepare 4% cell suspension in isotonic saline of the red blood cell to be tested.
2. With a clean pasture pipette add 1 drop of the prepared red cell suspension to a small
test tube.
3. Wash 3 times with normal saline to remove all trace of serum or free globulin.
4. Decant completely after last washing.
5. Add 2 drops of AHG serum to the sedimented cells.
6. Mix well & centrifuge for 1 minute at 1500 r.p.m.
7. Re- suspend the cells by gentle agitation & examine macroscopically for
agglutination.
8. Examine for agglutination by holding the tube against a lighted background & tapping
the bottom of the tube.

Observation:

If agglutination occurs ---------------------- Positive Coombs test

If no agglutination --------------------------- Negative Coombs test

 INDIRECT COOMBS TEST

Principle:
Here the sensitization of red cells is done in the laboratory by incubating the red cells
with the corresponding antibody at 37⁰C for 30 minutes. The test is applied in detecting
the presence of unexpected antibody in the serum which will react with corresponding
antigen on the red cells.

Specimen:
In case of indirect Coombs test for antibody screening, is needed it need not to be a
fasting sample.

Procedure:
1. Label there test tubes as „T‟ (test serum), „PC‟ (positive control), „NC‟ (negative
control).
2. In the „T‟ marked tube add 1 drop of test serum.
3. In „PC‟ marked tube add 1 drop of Anti-D serum.
4. In „NC‟ marked tube add 1 drop of saline or bovine albumin (22%).
5. In each test tube add 1 drop of 4% saline suspension of the pooled Rh +ve („O‟
group) cells or Coombs sensitive cells.
6. Incubate all the three tubes at 37⁰C for 30 minutes.
7. Wash the cells 3 – 4 times with normal saline to remove excess serum or free
globulin.
8. Add 2 drops Coombs serum (AHG serum) to each tube & mix well.
9. Keep for 5 minutes & centrifuge for 1 minute, at 1500 r.p.m.
10. Re-suspend the cells & examine macroscopically or microscopically for
haemagglutination.

109
 Observation:

If agglutination occurs ------------ Positive Coombs test

If no agglutination ---------------- Negative Coombs test

Du TEST

Requirements:

1. Test tube
2. Micro-pipette
3. Pasteur pipette
4. Centrifuge machine
5. Water bath
6. Microscope

Reagents:

1. Anti-human serum (Coombs serum/ Anti-IgG)

2. Bovine albumin

Procedure:

1. Prepare 5% red blood cell suspension in isotonic saline.

2. Level one tube as „T‟ & add 1 drop of anti-D serum in it.
3. Level second tube as „C‟ & add 22% Bovine albumin to it.
4. By a Pasteur pipette add 1 drop of cell suspension to each & mix well.
5. Incubate at 37⁰C for 15 minutes.
6. After incubation wash the cell with normal saline for three times.
7. Decant the supernatant after washing.
8. Add 2 drops of anti-human serum to each tube.
9. Mix & centrifuge at 15,000 RPM for 1 minute.
10. Re-suspend the cells by gentle agitation & examine macroscopically & confirm the
result microscopically.

Interpretation:

OBSERVATION NO. ANTI-D (ANTI-Rho BOVINE ALBUMIN INTERPRETATION

SERUM) CONTROL
1 0 0 D(Rho) – NEGATIVE
2 + - D(Rho) – POSITIVE
3 + + TEST INVALID

110
 7. PATIENT’S REPORT
RESULTS OF CLINICAL PATHOLOGY
RESULTS OF PHYSICAL, BIOCHEMICAL & MICROSCOPICAL
TEST OF URINE

SL.N NAM AG SE DATE CHEMICAL MICROSCOPICAL

O E E X PHYSIC
yrs M/ AL
F CO SP. Pᴴ SU AL RB PU E CAS BAC
LO GR G B C S P T
R
01 P. 33 M 14/11/21 Yel 102 Ac Trac ---- 1-2 2-3 Fe Hyal Trac
Bauri low 5 idi e - w ine e
c
02 K. 32 F 17/11/21 ,, 102 ,, ---- ---- 2-3 1-3 ,, ,, ,,
Pal 3 -

03 S. 45 M 17/11/21 ,, 101 ,, Trac ---- ---- 1-2 6- ,, Oce

Pan 6 e 9

04 N. 32 M 17/11/21 ,, 101 ,, --- ---- ---- 1-2 5- ,, ----

Khatun 5 - 8

05 J. 42 F 18/11/21 ,, 102 ,, ---- Tra ---- 2-3 Fe ,, 2+

Sen 5 ce w
06 T. Pal 75 M 18/11/21 ,, 102 ,, --- ---- 1-2 1-4 ,, ,, ----
2 --
07 B. 25 F 19/11/21 ,, 101 ,, --- ---- --- 1-3 ,, ,, Oce
Nandi 9
08 R. 68 M 19/11/21 ,, 102 ,, 2+ 1+ 0-1 8- ,, ---- ---
Murm 5 10
u
09 B. 53 F 20/11/21 ,, 102 ,, ---- Tra ---- 1-2 1- ----- ---
Dey 0 ce - 2
10 V. 75 F 20/11/21 ,, 102 ,, Trac 2+ 2-4 ple 1- ---- 1+
Mandi 4 e nty 4

11 M. 36 F 21/11/21 ,, 102 ,, Trac --- 0-1 1-3 6- Hyal Trac

Das 0 e 9 ine e
12 S. Dan 20 M 21/11/21 ,, 102 ,, ----- ---- ---- 1-2 2- ,, ---
0 -- - 7
13 R. 57 F 22/11/21 ,, 101 ,, ------ ---- 1-2 1-4 1- ---- ----
khan 7 5
14 S. 55 M 23/11/21 ,, 101 ,, ------ ---- ---- 1-2 1- Hyal Oce
Panja 9 - 3 ine
15 R. 36 F 24/11/21 ,, 102 ,, Trac 2+ ---- 1-2 2- ,, ----
Panda 2 e 4

112
16 R. Dey 29 M 25/11/21 ,, 102 ,, ---- ,, ---- 1-2 2- ,, 1+
0 - 6
17 S. 60 M 26/11/21 ,, 102 ,, --- ,, 1-2 1-3 1- ,, ---
Kundu 2 2

18 A.Dey 75 M 26/11/21 ,, 102 ,, ---- ,, ---- 2-4 1- ,, ----

3 - 3
19 D. 26 F 27/11/21 ,, 102 ,, ---- ---- ---- 2-3 Fe ,, -----
Ghosh
3 - w
20 C. 65 M 28/11/21 ,, 102 ,, 2+ 2+ 1-2 Ple ,, ,, 2+
Barik 1 nty
21 R. 24 M 30/11/21 ,, 102 ,, Trac ---- 0-1 1-3 ,, ----- -----
Dutta 5 e

22 P. 29 F 01/12/21 ,, 101 ,, ---- ---- --- 1-2 ,, --- 1+

Gupta 6

23 B. 52 M 06/12/21 ,, 101 ,, ------ ---- ---- 1-4 3- ---- ----

Monda 8 4
l
24 S. 12 F 07/12/21 ,, 102 ,, Trac ---- ---- 1-3 5- ---- Oce
Saha 6 e 8
25 B. 36 F 07/12/21 ,, 102 ,, ,, --- ---- 1-2 4- --- ---
Mandi 3 - 9

26 P. 64 M 09/12/21 ,, 102 ,, ,, Tra ---- 1-4 Fe ----- ----

Sen 2 ce - w

27 A. 69 M 09/04/21 ,, 102 ,, ------ ---- 1-2 1-2 ,, Hyal Oce

Khan 3 ine
28 H. 10 F 09/04/21 ,, 102 ,, ------ ---- 0-1 1-3 ,, ,, ----
Dey 0
29 K. 39 F 09/04/21 ,, 101 ,, Trac ---- ---- 1-4 ,, ,, ----
Mandi 5 e - -
112
 DAILY SAMPLE REPORT OF HEMATOLOGY

RESULT OF RBC, TC, DC, PLATELET COUNT

SL NAME A SE DATE RBC TC DC % PLATE

. G X MI NEU EOS LY MO B LET
N E M/ LLI T I MP NO E (Lakh)
O F ON S
O
1 K.MANDI 54 F 13/12/21 2.2 7500 76 3 33 0 1 2.3

2 S.NANDI 49 M 13/12/21 3.4 6800 90 4 32 0 0 1.3

3 B. DUTTA 29 F 13/12/21 2.6 15000 87 8 28 0 0 3.8

4 S.ROY 57 M 13/12/21 2.3 6700 87 9 34 0 0 3.6

5 S.DEY 67 F 13/12/21 2.5 13400 86 6 22 1 1 1.3

6 P. PAL 48 M 13/12/21 4.6 7600 89 8 54 2 1 3.4

7 K.MANDI 36 F 14/12/21 3.0 13300 76 6 24 0 0 1.5

8 K.DULEY M 15/12/21 3.7 13000 80 7 25 3 0 2.4

49
9 P.MONDA 65 M 15/12/21 5.3 12500 88 0 22 1 0 1
L
10 B.DULEY 42 F 15/12/21 3.2 15000 90 0 20 5 0 1.3

11 M.KALIND 55 F 15/12/21 3.4 9000 70 4 23 0 0 2.2

I
12 G.PAL 44 M 15/12/21 4.2 8200 75 2 24 0 0 2.4

13 P.MAN 24 F 16/12/21 1.3 12500 84 5 22 3 1 3.6

14 D. SINGH 55 M 16/12/21 3.3 13000 75 4 33 2 1 2.5

15 C.DEY 22 F 17/12/21 3.5 14500 70 5 23 0 0 2.2

16 M.GHOS 55 M 17/12/21 5.5 5700 67 4 19 0 0 1.1

H

112
17 M. BIBI 34 F 18/12/21 3.5 7300 51 5 28 1 0 1.5

18 S. DAS 52 M 18/12/21 4.8 6500 86 2 34 0 1 3.4

20 B.MITRA 42 F 19/12/21 5.5 14000 80 1 25 0 1 2.4

21 K. PAL 76 M 19/12/21 2.3 13400 86 6 22 1 1 1.3

22 S. 78 M 19/12/21 2.6 6500 89 8 54 2 1 3.4

CHOWDHU
RY
23 P.SAREN 26 F 19/12/21 3.4 12100 76 6 24 0 0 1.5

24 D.MAL F 19/12/21 4.5 6210 80 7 25 3 0 2.4

54
25 M.BEGAM 65 M 19/12/21 3.3 7500 88 0 22 1 0 1

26 B.LAHA 42 F 19/12/21 2.2 10100 90 0 20 5 0 1.3

27 L.SEN 55 F 19/12/21 4.4 17000 70 4 23 0 0 2.2

28 A.SEN 44 F 19/12/21 2.3 11100 75 2 24 0 0 2.4

29 MD.SAJAH 24 M 19/12/21 5.3 8000 84 5 22 3 1 3.6

AN
30 T.MAHANT 55 F 19/12/21 3.2 12000 75 4 33 2 1 2.5
Y
31 22 M 20/12/21 2.8 9000 70 5 23 0 0 2.2
M.NAND
I

112
 RESULT FOR HB% , ESR,BT,CT,PT
SL.NO NAME AGE SEX DATE HB% ESR BT CT PT

1 T.DEY 28 F 20/11/21 12.0 19 02 04 14.2

2 S.PATI 34 M 20/11/21 14.5 16 03 03 13.4

3 R.MAJHI 38 M 20/11/21 13.4 21 02 05 12.5

4 B.DULEY 45 F 21/11/21 10.1 65 03 04 13.0

5 R.CHALAK 39 M 21/11/21 14.6 11 02 03 14.2

6 B.PAL 54 F 21/11/21 12.1 16 01 05 11.5

7 SK.ROY 50 M 21/11/21 14.2 26 02 06 12.2

8 M.DAS 63 F 22/11/21 10.5 42 03 08 11

9 S.BERA 27 M 22/11/21 12.8 23 05 07 25.2

10 S.MUKHERJEE 37 M 22/11/21 13.9 24 05 09 16.2

11 M.SAHA 57 F 22/11/21 8.2 85 02 02 19.3

12 K.MONDAL 29 M 23/11/21 15.0 16 02 02 20.3

13 N.BAURI 38 M 23/11/21 14.2 17 2.4 05 14.2

14 G.NANDI 61 F 24/11/21 11.9 38 03 06 14.5

15 S.DEY 76 M 26/11/21 13.6 19 01 02 13.0

16 S.MAHANTY 57 M 26/11/21 12.8 25 1.5 03 14.2

17 G.DUTTA 19 F 27/11/21 9.0 45 2.22 04 14.1

18 H.SEN 16 M 28/11/21 13.4 13 02 06 15.7

19 D.TUDU 29 F 29/11/21 9.2 45 01 02 17.2

20 S.PAN 48 M 30/11/21 14.2 13 02 01 13.2

112
 BACTERIOLOGICAL LABORATORY
Sl. Date Name Age/S specime Identifie Sensitivity Pattern
No ex n d
Organis Sensiti Intermedi Resistant
m ve ate
1 14/01/22 Giridhari 57/M Eye S.aureus AZK,B IZ, FD,CP,DX,ER,AK,
Patra swab A,CD, VA,AG,GF,ZN,CB,
P,TE,Q FG,NA.
B
2 21/01/22 Kojol Das 58/M Stool Vibrio GM,T CP. PC,RC,AG,BA,GF,
cholera B,NT, FD,TE,CB,NA,NX,
CB,ZN PC,PR,CZ.
,AK,
FG,
QB.
3 21/01`/2 ParikshitBa 24/M Fluid Preoteus GM,T PC,RC,A PR,CZ,CP,BA,GF,
2 la spp. B,NT, G. FD,TE,
CB,ZN CB,NA,NX,PC.
,AK,
FG,QB
.
4 22/01/22 Ashok 53/M Urine Acineto GN,TB CB,NA. PC,RC,AG,PR,CZ,
Mondal bacter ,NT,C CP,BA,
spp. B,ZN, GF,FD,TE,CB,NA,
AK,FG NX,PC.
,QB.
5 23/01/22 R.G.Ghosh 62/M Burn pPseudo GM,T FG,QB. PC,RC,AG,BA,GF,
swab monas B,NT, FD,TE,CB,NA,NX,
sup CB,ZN PC.
,AK,P
R,CZ,
CP.
6 23/01/22 Atis Malik 48/M Wound Preteus CB,FG CB,GF,B AG,RC,CZ,PC,NX,
swab vulgaris ,TE,A A. NT,PR,TB,GM,DX
K,FD, .
CP,ZN.
7 23/01/22 S.P.Chatter 35/M Blood Preteus CB,FG CB,DX. GF,BA,AG,RC,CZ,
jee. vulgaris ,TE,A PC,NX,NT,PR,TB,
K,FD, GM.
CP,ZN.
8 23/01/22 Sarajani 85/F Urine E.Coli PR,NT, GM,TB, NX,PC,CB,CZ,RC,
Nandi ZN, TE. AG,BA,GF,FD,CB,
CP,DX QB,NA
,AK,
CZ.
9 03/05/21 Subtra 56/M Wound Streptoc VA,CB TE,QB AG,GF,FD,BA,AK,
Mondal swab occus ,FG,N IZ,AZK,ER,CD,P
spp. A,ZN,
CPDX.
10 23/01/22 Alok Nath 25/M Ear Streptoc TE,QB ZN,CP AG,GF,FD,CB,FG,
discharg occus ,DX,B NA,IZ,AZK,ER,C
e spp. A,AK, D,P

112
 VA.

11 23/01/22 Sourav 22/M Urine E.Coli CB,DX GF. TB,FG,TE,AG,RC,

Roy ,BA,A CZ,PC,NX,NT,PR,
K,FD, QB,GM
CP,ZN.
12 24/01/22 Jayashri 21/F Urine E.colai TB,FG, GF CB,GX,BA,AG,RC
Das TE,AK ,CZ,PC,NX,NT,PR,
,FD,CP QB,GM.
,ZN.
13 24/01/22 Banalata 22/F Urine E.colai NX,NT GF CB,GX,BA,AG,RC
Sen ,TB,FG ,CZ,PC,AK,FD,PR,
,TE,CP QB,GM.
,ZN.
14 24/01/22 Haripada 57/M Wound Streptoc ZN,CP, TE,QB. AG,GF,FD,CB,FG,
Ghosh swab occus DX,BA NA,LZ,AZK,ER,C
spp. ,AK,V D,P
A.
15 24/01/22 AsmaulMo 20/M Blood Salmone CB,FG CB,DX, GF,BA,AG,RC,CZ,
lla llaatyphi ,TE,A PC,NX,NT,PB,TB,
K,FD, GM.
CP,ZN.
16 24/01/22 NabinPatta 65/M Burn Pseudo QB,FG CB,DX. GF,BA,AG,RC,CZ,
nayak swab monas ,TE,A PC,NX,NT,PB,TB,
sup K,FD, GM.
CP,ZN
17 25/01/22 Jamirul 62/M Urine E.coli PR,NT, GM,TB, NX,PC,CB,CZ,RC,
Islam ZN,CP, TE. AG,BA,GF,FD,CB,
DX,A QB,NA.
K,CZ.
18 25/01/22 ShrihariPat 71/M Pleural Streptoc ER,CD TE,QB. AG,GF,FD,CB,FG,
ra fluid occus ,P,BA, NA,IZ,AZK,ZN,CP
spp. AK,V ,DX.
A.
19 26/01/22 ShikhaMon 26/F Urine Acineto CB,QB DX,FD. GF,BA,AG,RC,CZ,
dal bacter ,FG,TE PC,NX,NT,PB,TB,
spp. ,AK,C GM.
P,ZN.
20 26/01/22 FalguniMu 37/F Urine Proteus QB,FG CB,DX. GF,BA,AG,RC,CZ,
rmu ,TE. PC,NX.

112
 REPORT OF SEROLOGICAL TEST

SL. NAME A S DATE ASO R CRP WIDAL H VDR

NO G E A Bs L
E X Ag
1 BimalK 2 M 07/02/22 Upto Negat O-Recative upto 1.80 - -
alindi 8 400 ive dilution H-Reactive upto
IU/ 1:60 dilution AH-
ml Negative in all dilution
BH- Negative in all
dilutions BH- Negative in
all dilutions
2 AvijitP 0 M 07/02/22 - - 24ug/ - - -
atra 6 m
3 Sonia 3 F 07/02/22 <20 - - - - -
Murmu 2 0IU/
ml
4 Rena 1 F 07/02/22 - - - - Po Nega
Mondal 8 siti tive
ve
5 Sk. Ali 1 M 07/02/22 Upto - - - - -
2 400
IU/
ml
6 Reena 2 F 07/02/22 - - 24ug/ - Po Nega
Roy 2 ml siti tive
ve
7 Lotika 1 F 07/02/22 - - - O-Recative upto 1:60 - -
Bibi 8 dilution H-Recative upto
1:80 dilution AH-
Negative in all dilution
BH- Negative in all
dilutions
8 RanuTu 3 F 08/02/22 - - - O-Reactive upto1:320 - -
du 2 dilution H-Recative upto
1: 60 dilution AH-
Negative in all dilutions
BH-Negative in all
dilutions
9 Hamida 3 F 08/02/22 <40 - 12ug/ - - -
Bibi 5 0IU/ ml
ml
10 Akram 9 M 08/02/22 <20 - 24ug/ - -- -
ul Ali 0IU/ ml
ml
11 Sujit 3 M 09/02/22 <20 - - O-Reactive upto1:320 - -
Sarkar 0 0IU/ dilution H-Recative upto

112
 ml 1: 60 dilution AH-
Negative in all dilutions
BH-Negative in all
dilutions
12 Mangal 4 F 10/02/22 - - 48ug/ O-Recativeupto 1:60 - -
aMaju 5 ml dilution H-Recative upto
mder 1:80 dilution AH-
Negative in all dilution
BH- Negative in all
dilutions
13 Sarasw 2 F 11/02/22 - - 20IU/ O-Recative upto 1:60 - -
atiBera 5 Ml dilution H-Recative upto
1:80 dilution AH-
Negative in all dilution
BH- Negative in all
dilutions
14 Sarbani 3 F 11/02/22 - - 24ug/ O-Recative upto 1:60 - -
Mollick 4 <20 ml dilution H-Recative upto
0IU/ 1:80 dilution AH-
ml Negative in all dilution
BH- Negative in all
dilutions
15 PutulH 1 F 12/02/22 20IU/ - - -
ansda 8 Ml
16 Anjali 4 F 12/02/22 <20 - 24ug/ - - -
Parama 2 0IU/ ml
nik ml
17 Salim 5 M 12/02/22 Upto - - - - -
Ali 4 800I
U/m
l
18 Poli 2 F 12/02/22 - - 24ug/ - - -
Sing 3 ml
19 Tajo 3 F 12/02/22 - -- 24ug/ - - -
Bibi 4 ml
20 Munia 2 F 12/02/22 - - - - - -
Bibi 5 Upto
400
IU/
ml

112
 REPORT OF BIOCHEMISTRY

REPORT OF BIOCHEMISTRY (SUGAR, UREA,

CREATININE)
S L N A M E AGE SEX DAT E GLUCOSE GLUCOSE UREA CREATININE
NO. (Y) FASTING P.P. (mg/dl) (mg/dl)
(mg/dl) (mg/dl)

1 . P. 3 0 M 17.11.21 9 5 1 3 5 2 4 1 . 1
Chatterjee
2 . R . S i ng h 3 2 M 17.11.21 8 6 1 2 7 3 1 1 . 2
3 . D. De 3 5 M 17.11.21 1 3 5 2 0 5 5 2 1 . 4
4 . M. Raj 3 6 M 17.11.21 8 3 1 3 4 1 5 0 . 8
5 . P. Banerjee 4 2 F 18.11.21 1 2 0 1 8 0 4 5 1 . 3
6 . S. Barman 4 5 M 18 .11.21 7 9 1 3 6 1 7 0 . 9

7 . D.. 5 2 F 18.11.21 7 6 1 3 0 1 9 0 . 9
Banerjee
8 . M. 5 5 M 18.11.21 9 8 1 3 7 2 3 1 . 0
Banerjee
9 . T. Thakur 4 7 M 19.11.21 2 4 1 2 3 2 5 6 1 . 5
10 . F. Ahmed 4 3 F 19.11.21 8 7 1 3 5 3 2 1 . 2
11 . D. Das 4 1 M 20.11.21 9 2 1 3 8 3 7 1 . 3
12 . M. Sen 3 9 F 20.11.21 8 3 1 3 4 1 9 1 . 0
13 . S . Gu p t a 2 5 F 21.11.21 9 3 1 4 0 3 9 1 . 4
14 . D. Mal 2 8 F 21.11.21 1 3 7 2 1 6 4 9 1 . 4
15 . J. Gogoi 4 8 M 21.11.21 8 4 1 3 7 2 8 1 . 2
16 . B. Borah 2 8 M 21.11.21 8 8 1 4 1 2 4 1 . 1
17 . R. Gupta 4 6 M 21.11.21 1 0 5 1 4 4 4 1 1 . 3
18 . R. Sen 6 2 M 21.11.21 9 6 1 3 7 1 6 1 . 0
19 . S. Pandit 5 5 F 22.11.21 8 1 1 3 3 2 3 1 . 1
20 . H. Paja 5 6 M 23.11.21 1 1 9 1 8 9 3 7 1 . 3
21 . R. D olu i 4 2 M 23.11.21 1 0 3 1 4 6 3 9 1 . 3
22 . G. Ghosh 4 5 M 26.11.21 9 5 1 3 9 3 1 1 . 2
23 . P. Dey 4 6 M 26.11.21 8 4 1 3 2 2 6 1 . 1
24 . S. Dey 4 9 M 27.11.21 9 6 1 3 8 1 9 1 . 0
25 . M.Roy 5 2 M 01.12.21 1 1 0 1 6 1 3 4 1 . 3
26 . F. Khatun 2 7 F 01.12.21 7 6 1 2 8 2 2 1 . 1
27 . D. Kar 2 5 F 02.12.21 9 9 1 3 4 1 4 0 . 8
28 . P . P a l 4 0 F 03.12.21 7 8 1 2 3 1 6 0 . 8

112
SL NAME AGE SEX DATE Cholesterol TG HDL LDL VLDL Uric
N. (Y) (M/F) (mg/dl) (mg/ (mg/dl) (mg/dl) (mg/dl) Acid
dl) (mg/dl)

1. P.DEY 68 M 04.12.21 178 136 54 59 27 4.3

2. S.PATRA 41 F 05.12.21 205 166 61 67 41 5.6
3. D.GHOSH 45 F 06.12.21 317 276 81 89 63 6.9
4. L.SINGH 66 M 07.12.21 165 129 41 48 33 4.8
5. D.SAHA 33 M 08.12.21 312 276 79 84 62 5.6
6. A.ROY 36 M 09.12.21 227 182 76 81 45 5.9
7. S.SARKAR 54 F 10.12.21 367 324 94 119 64 6.8
8. G.DAS 45 M 11.12.21 205 168 61 68 41 7.5
9. A.GHOSH 32 F 12.12.21 186 148 46 49 37 5.0
10. R.DEY 22 M 13.12.21 194 157 45 51 39 4.7
11. P.MAL 25 M 15.12.21 273 234 71 79 54 6.8
12. F.SARKAR 28 M 17.12.21 214 175 64 72 43 7.2
13. N.GHOSH 36 M 18.12.21 194 159 56 64 39 8.5
14. M.MAITY 65 M 19.12.21 157 114 42 49 31 4.9
16. S.PATTRA 54 M 10.12.21 219 175 64 71 44 6.3
17. V.SANTRA 47 M 21.12.21 182 148 60 70 36 5.7
18. C.JANA 34 M 22.12.21 275 235 68 75 55 5.9
19. A.LAHA 58 M 23.12.21 182 144 45 52 36 6.4
20. S.PAL 37 F 24.12.21 246 208 72 80 49 7.9
21. R. DEY 21 M 26.12.21 297 254 76 89 59 8.0
22. A.SINGHA 64 M 27.12.21 216 175 68 75 43 6.4
23. P.BARMAN 44 M 28.12.21 334 294 82 102 58 4.6
24. U.MULA 35 M 28.12.21 291 256 79 79 51 8.3
25. J.GOGOI 39 M 29.12.21 193 153 68 74 38 4.3
26. S.BAURI 41 F 30.12.21 284 244 58 76 48 7.1
27. D.JANA 61 M 30.12.21 168 146 64 52 34 5.4
28. S.GUPTA 57 M 31.12.21 267 248 64 74 53 8.7
29. P.DAS 24 M 31.12.21 195 153 57 66 39 6.4

112
 REPORT OF BIOCHEMISTRY

8. OUTPUT OF INTERNSHIP TRAINING

Through Internship training I can able to gain some knowledge about the application of
ELISA Reader , Semi-automated Analyze , Auto analyzer, colorimeter, component
separation machine , HPLC machine, Flame Photometer, Rotary Microtome, Autoclave , Hot
Air oven etc. in the biological science and they also familiar with different operative parts of
the concern instruments. So, practical knowledge as well as theoretical knowledge about
some of the instruments has been clear by this Internship training.

Different research programs of human health are going in the concern laboratory have been
focused and we can able to motivated to continue our higher education in the field and this
visit is also a driving force to motivate as in research work.

The techniques of different hormone assay protocol, biochemical estimations, and protein
separation technique and issue section preparation have been learned by us, which is also
helpful for us to clear about our knowledge and confidence.

Role of discipline, importance of devotion and accountability for scientific work has been
learned by us form this training.

112
 9. CONCLUSION

The duties of medical laboratory technologist as undergone repaid change science the last
decay. The advancement of knowledge , the medical science and technology have to keep
place with the newest development in the fields of physiology , Biochemistry , Immunology ,
Endocrinology, Microbiology , etc.

The thrust of knowledge has increased the so much graduate coerces as even Doctorate
course are now being may available to us. So that the knowledge accumulating.

In the words of poet, regarding advancement of knowledge we the graduate of Medical

Laboratory Technology has miles to go, many more miles to go……………...

112
 10. ABBERVIATION

VA Vancomycin

LZ Linezolid

ER Erythromycine

CD Clindamycine

P Penicillin

GM Gentamycin

TB Tobramycin

NT Netilmicin

NX Norfloxacin

PC Piperacilin

CB Carbenicillin

CZ Cefazolin

RC Cipeofloxacin

CP Cefoparazone

DX Doxycyclin

GF Gatifloxacin

AK Amikacin

TE Tetracyclin

CF Ceftazidime

112
 11. REFERANCE

1) Manual of practical microbiology & parasitology, first edition, January 2008, p29-31.
2) Manual of practical microbiology & parasitology, first edition, January 2008,p 30-32.
3) Manual of practical microbiology & parasitology, c.p. baveja, first edition, p46-48.
4) Manual of practical microbiology & parasitology, c.p. baveja, first edition, p 50-51.
5) Manual of practical microbiology & parasitology, c.p. baveja, first edition, p 67
6) Manual of practical microbiology & parasitology, c.p. baveja, first edition, p 72-73.
7) Manual of practical microbiology & parasitology, first edition, January 2008, p 87-89.
8) Herbert K.,Lipids, In Clinical Chemistry ;Theory, Analysis and Co-relation, Kaplan
L.A. and Pesce A.J., Eds. C.V. Mosby, Toronto, 1984 . p 1182-1230.
9) Nader.R.,Poul B. John A., Lipids ,Lipoproteins and Apo lipoproteins, In Tietz Text
book of Clinical Chemistry , 3rdedition ., Burtis C.A. and Ashwood E.R. , Edition .W.
B. Saunders, Philadelphia, 1994.p 809-852.
10) Siedel J., et.al.,Clin.Chem., 29/6, p1073 (1983).
11) Crescenziol . , et.al. , Clin. Chem. , 27, p 371.(1981)
12) Manual of practical microbiology & parasitology, first edition, January 2008, p 65-69.
13) Younge D., In Effect of Preanalytical Variables on Clinical Laboratory Tests, 2nd
edition. , AACC press, Washington, 1997. P 4493-4497.
14) Warnick. , et.al.,Clin. Chem ., 41, p 1427-1433 (1995)
15) P.B. Godkar, D.P. Godkar ; Text book of Medical Laboratory Technology .
16) Kaplan A., Lavernel L. S., Lipid Metabolism , In Clinical Chemistry ; Interpretations
and Techniques , 2nd edition.,Lea and Fediger, Philadelphia, 1983.p 333-336.
17) Executive summary of the 3rd report of the National Cholesterol Education
Programme (NCEP) Expert Panel on Detection, Evolution and Treatment of High
Blood Cholesterol in Adults (Adult Treatment panel- III)
18) Mc Gowan MW.et.al., Clin.Chem.,29,p538(1983)
19) Stein A., Mayer G.L., Clin. Chem., 41,p1421-1426(1995)
20) Trinder, P., (1969) Ann. Clin. Biochem. 6:24
21) Alllain, C.C., et.al. , (1974) Clin. Chem. 20:470
22) Flegg, H.M., (1972) Amm. Clin. Bio Chem. 10:7916
23) Grillo, F., et.al. , (1981) Clin. Chem. 27:375
24) Demacker, P.N.M., et.al. , (1980) Clin. Chem 26:1775
25) Reitman, S.,Frankel, S.,(1957) Amer. J. Clin. Path. 28:56
26) Tietz, N.W., (1970) Fund. Of Clin. Chem. P. 447

112