CLINICAL PATHLOGY

Introduction
Clinical pathology is one of the two major divisions of pathology, the other being anatomical
pathology. Clinical pathology is itself divided in to subspecialties, the main ones being
clinical haematology/blood banking, clinical chemistry, haematopathology and clinical
microbiology and emerging subspecialties such as molecular diagnostics. Haematology
studies the blood and blood-forming tissues to evaluate presence of disease and assist in
therapeutic interventions as clinically indicated. Clinical chemistry (also known as chemical
pathology and clinical biochemistry) is the area of clinical pathology that is generally
concerned with analysis of bodily fluids.
Blood parameters play a critical role in diagnosis, assessing progression, and in the
characterization of disease and phenotypes in clinical and research situations. The accuracy
and reliability of the whole blood parameter analysis depends on identification and control or
elimination of variables that may affect these results. Different blood collection and handling
strategies represent source of variability that can be controlled in many instances.
Basic haematological procedures such as the complete blood count are frequently conducted
to help physicians and veterinarians arrive at a diagnosis and prognosis, also to evaluate
treatment. Haematology testing includes, but is not limited to: Haematology Automated
Testing, Lamellar Body Counts, Microscopic Cellular Analysis, Body Fluid Analysis and
Specialized Stains.
The outline of this laboratory manual deals with the basic haematological procedures and
clinical chemistry analysis which is meant for students use. The study of haematology begins
with proper sample collection and handling. Part one deals with haematology containing
different sections. Part two covers clinical chemistry including functional tests. All of the
procedures described in this manual are classical methods that are routinely performed in a
standard clinical pathology laboratory. Detail procedures including preparation of reagents,
solutions, stains and buffers are given. Calculations of blood indices and chemical analysis
are also discussed in part one and part two respectively.

Objectives of Clinical Pathology Practical Course

At the end of Clinical Pathology practical course the students will be able to:
a. Identifying the most important haematological and functional pathological tests of vet
importance.
b. Diagnose different animal diseases by confirming the pathological causes that constraint
live

stock production in general.

c. Have knowledge more about clinical pathology.
d. Write lab report pertaining the tests carried out .

QUALITY ASSURANCE IN HAEMATOLOGY

The importance and implementation of quality assurance (QA), quality management, and
standardization to achieve reliable test results and a quality service to patients, QA and good
laboratory practice are also required to ensure a safe working environment and cost-effective
use of resources. Implementing QA in haematology requires the preparation and use of
Standard Operating Procedures (SOPs) for all haematological tests and associated activities.

Appropriate use of haematological investigations

Clear guidelines must be provided on the use of haematological tests to avoid unnecessary
tests being undertaken, e.g. there should be clear clinical indications for requesting a white
cell count and differential cell count.
In some situations the result of a particular test will indicate further tests to perform which the
laboratory should undertake routinely, e.g. tests to investigate anaemia when the result of a
haemoglobin test indicates moderate or severe anaemia , tests to detect haemoglobin S when
examination of a blood film suggests sickle cell disease, or measurement of haemoglobin
when examination of a thick blood film shows many malaria parasites and the patient is a
young child.

Basic equipment use in pathology lab:

Microscope
Equipment for purifying water
Balance
Equipment for pipetting and dispensing
Centrifuge(s)
Autoclave
Incubator (for culturing micro-organisms)

MICROSCOPE
A microscope is the most expensive and important piece of equipment used in district
laboratories. Microscopy forms 70-90% of the work.

WORKING PRINCIPLE OF A MICROSCOPE

A microscope is a magnifying instrument. The magnified image of the object ( specimen) is

first produced by a lens close to the object called the objective. This collects light from the
specimen and forms the primary image. A second lens near the eye called the eyepiece
enlarges the primary image, converting it into one that can enter the pupil of the eye. The
magnification of the objective multiplied by that of the eyepiece, gives that total
magnification of the image seen in microscopes having a mechanical tube length (MTL) of
160 mm. The MTL is the distance between the shoulder of an objective and the rim of the
eyepiece.

Resolving and defining power of an objective:

An objective accepts light leaving the specimen over a wide angle and recombines the
diverging rays to form a point-for-point image of the specimen. Objectives of varying
magnifications allow a specimen to be examined in broad detail over a wide area, and in
increasing detail over a smaller area.
This increase in magnifying power is always linked to an increase in resolving power. The
higher the resolving power of an objective, the closer can be the fine lines or small dots in the
specimen which the objective can separate in the image.
The resolving power of an objective is therefore of great importance. It is dependent on what
is known as the numerical aperture (NA) of the objective. The NA of an objective is an exact
figure that has been worked out mathematically from its equivalent focal length and lens

diameter. It is not necessary to know the details of this calculation. Both the NA and
magnification of an objective are usually engraved on it. The following are the usual NAs of
commonly used objectives:
10_objective NA 0.25
20_objective 0.45
40_objective 0.65equently
100_(oil) objective 1.25

Working of an oil immersion objective:

When a beam of light passes from air into glass it is bent and when it passes back from glass
to air it is bent back again to its original direction. This has little effect on low power
objectives but with high power lenses this bending limits not only the amount of light which
can enter the lens but also affects the NA of the objective and consequently its resolving
power. The bending effect and its limitations on the objective can be avoided by replacing the
air between the specimen and the lens with an oil which has the same optical properties as
glass, i.e. immersion oil . When the correct oil used , the light passes in a straight line from
glass through the oil and back to glass as though it were passing through glass all the way. By
collecting extra oblique light, the oil provides better resolution and a brighter image. Some
50x objectives and all 100x objectives are used immersed in oil.

COLLECTION OF BLOOD
Blood must be collected with care and adequate safety precautions to ensure test results are
reliable, contamination of the sample is avoided and infection from blood transmissible
pathogens is prevented. Protective gloves should be worn when colleting and handling blood
samples Lancets, needles, and syringes must be sterile, and dry, and blood collecting
materials must be discarded safely to avoid injury from needles and lancets.

Capillary Blood:
Capillary blood is mainly used when the patient is an infant or young child and the volume of
blood required is small, e.g. to measure haemoglobin perform a WBC count, and to make
thick and thin bood films. In district laborartories, capillary blood is also used to monitor
anaemia during pregnancy and post-operatively. Haemoglobin and PCV values are slightly
higher in capillary blood than in venous blood. Thick blood films for malaria parasite are best
made from capillary blood.

Capillary blood can be collected from:

-The ring finger of a child or adult.
Do not stick the thumb or index finger as these are the most sensitive.
-The heel of an infant up to 1 year old care must be taken not to damage the heel by sticking
it too near the edge or by holding it too forcibly.

Venous Blood:
Anticoagulated venous blood is used when more than 100 ul of whole blood is required or
when serum from a clotted blood sample is needed, e.g. for compatibility tests or antibody
tests. Venous blood is preferable to capillary blood for the reasons previously described,
particularly when the patient is an adult and several tests are required.

Anticoagulants:
For haematological tests, the anticoagulants used are EDTA (ethylenediamine tetra-acetic
acid), also called sequestrene, and tri-sodium citrate. These chemicals prevent blood from
clotting by removing calcium. EDTA anticoagulated blood can be used for most tests, e.g.
haemoglobin, PCV, WBC count, ,Platelet count, reticulocyte count, and reporting blood cell
morphology. It is not suitable for coagulation Tests.

Technique for collecting venous blood:

Laboratory staff must not collect venous blood unless they have been adequately trained in
the procedure Newly qualified staff must be supervised until they have gained sufficient
experience .When venous blood is required from infants, this should be collected by a
medical officer. Do not collect blood for haematological tests from intravenous lines.
1. Select a sterile, dry, preferably plastic syringe of the capacity required, e.g. 2.5 ml , 5 ml,
or 10 ml. Attach to it a 19 or 20 SWG needle (preferably a disposable one). If the patient is
child or adult with small veins, use a 23 SWG needle.
2. Apply a soft tubing tourniquet or Velcro fastening arm band to the upper arm of the patient
to enable the veins to be seen and felt. Do not apply the tourniquet too tightly or for longer
than 2 minutes. Ask the patient to make tight fist which will make the veins more prominent.
3. Using the index finger , feel for a suitable vein, selecting a sufficiently large straight vein
that does not roll and with a direction that can be felt.
4. Cleanse the puncture side with 70% ethanol and allow to dry. Do not re-touch the cleansed
area.
5. With the thumb of the left hand holding down the skin below the puncture side, make the
venepuncture with the level of the needle directed upwards in the line of the veins. Steadily

withdraw the plunger of the syringe at the speed it is taking the vein to feel. Avoid moving
the needle in the vein.
6. When sufficient blood has been collected, release the tourniquet and instruct the patient to
open his or her fist. Remove the needle and immediately press on the puncture site with a
piece of dry cotton wool. Remove the tourniquet completely.
Instruct the patient to continue pressing on the puncture site until the bleeding has stopped.
7. Remove the needle from the syringe and carefully fill the container(s) with the required
volume of blood. Discard the needle safely. Do not attempt to re-sheath it because this can
result in needle-stick injury.
Important: Do not fill a container with the needle attached to the syringe. Forcing blood
through the needle can cause haemolysis.
8. Mix immediately the blood in an EDTA or citrate anticoagulated container. When required,
make thick blood film from the blood containing in the syringe. Immediately label carefully
all the blood samples.
9. Check that bleeding from the venepuncture site g=has stopped. Cover the area with a small
dressing.

MAKING, FIXING AND OF BLOOD

FILM
This blood films can be made from free-flowing capillary blood or well mixed EDTA
anticoagulated blood, collected. To prevent EDTA associated blood changes (explained in
subunit 8.3). It is important to make blood films from EDTA anticoagulated blood with as
little delay as possible, i.e. within 1 hour of collecting the blood.
Caution: Wear protective gloves when handling blood and follow safe working practices.

Technique of making a thin blood film:

1. Make a blood spreader from a slide which has ground glass polished sides.
2. Place a drop of blood on the end of a clean dry Slide.
3. Using a clean smooth edged spreader, draw the spreader back to touch the drop of blood
and allow the blood to extend along the edge of the spreader. Holding the spreader at an angle
of about 30_, spread the drop of blood to make a film about 40 - 50 mm in length (two thirds
of the slide).

Note: When the blood is from an anaemic patient, increase the angle of spreading and spread
the blood more quickly. When the blood is thick and viscous, reduce the angle of spreading
and spread the blood more slowly.
4. Wipe clean the end of the spreader.
5. Immediately air dry the film by waving the slide back and forth. Protect the dried film
from dust and insects.
Note: When not using a frosted ended slide, write the patient name and number on the dried
blood at the top or along the side of the film using a lead pencil.
6. When completely dry and within a few minutes of making the blood film, fix it in absolute
methanol.

Methanol fixing thin blood films:

When completely dry, fix a blood film with absolute methanol (methyl alcohol). Place the
film on a staining rack and add 1 2 drops of moisture-free methyl alcohol and allow it to
dry on the film.
Alternatively, the blood film can be immersed in a container of absolute methanol for about 2
minutes, but this is a more expensive method of fixing and also in tropical humid climates
there is a greater risk of the alcohol absorbing water from the atmosphere which will result in
poor fixing of blood cells.

Staining thin blood films:

In district laboratories, thin blood films are usually stained manually using Leishman or
Wrights stain. These stains are examples of alcohol containing
Romanowsky stains which stain blood cells differentially.

Leishman staining technique:

Many of the difficulties in reporting blood films, particularly red cell morphology, are
due to variable staining. It is important therefore for laboratories to use a reproducible
standardized staining technique.

Reagents
Leishman stain
For daily use , store the stain in an amber container, e.g. TK dropper bottle, which can be
closed in between use to prevent moisture entering the stain. Make sure the stain is kept in a
cool place(not refrigerated) and never left in direct sunlight. Bright light and heat will cause

the stain to deteriorate rapidly. Keep the stock stain in a tightly stoppered light opaque (e.g.
amber) container in a cool dark place. Renew every 3 months or more often if indicated.
Allow 3 -5 days before using freshly made stain (to obtain optimum colour reaction).

pH 6.8 buffered water

Method
1. Cover the blood film (preferably methanol prefixed) with undiluted stain but do not flood
the slide. If using a dropper bottle count the number of drops required to cover the film.
Note: The undiluted stain not only acts as a fixative but also partially stains the smear. This
stage is required to obtain the best possible staining results.
2. Add twice the volume of pH 6.8 buffered water. The diluted stain should not overflow.
Ensure the water is well mixed with the stain by blowing on the diluted stain or mixing the
stain and water using a plastic bulb pipette. Allow to stain for 10 minutes.
3. Wash off the stain with tap water. Do not tip off the stain, because this will leave a fine
deposit covering the film. Wipe the back of the slide clean and stand it in a draining rack for
the smear to dry. The blood film should appear neither too pink nor too blue (check results
microscopically).

Staining results
Red cells...........................................................................Pink-red
Nucleus of cells...........................................................Purple-violet
Cytoplasm
Neutrophils, eosinophils..................................................Pale pink
Large lymphocytes...........................................................Clear blue
Small lymphocytes............................................................Darker clear blue
Monocytes...............................................................................Grey-blue
Granules
Eosinophils................................................................................Orange-red
Neutrophils................................................................................Mauve-purple

Toxic granules............................................................................Dark violet

Basophils....................................................................................Dark blue-violet
Platelets......................................................................................Purple-blue

CELL COUNT
WHITE BLOOD CELL COUNT
Value of test:
A white blood cell (WBC) count is used to investigate HIV/AIDS, infections and
unexplained fever, and to monitor treatments which can cause leukopenia. In most situations
when a total WBC count is requested it is usual to perform also a differential WBC count.

Principle of test:
Whole blood is diluted 1 in 20 in an acid reagent which haemolyzes the red cells (not the
nucleus of nucleated red cells), leaving the white cells to be counted. White cells are counted
microscopically using an Improved Neubauer ruled counting chamber (haemocytometer) and
the number of WBCs per litre of blood calculated.

Blood sample:

EDTA anticoagulated blood or scapicallary blood can be used for

counting white cells. Heparin or sodium citrate anticoagulated blood must not be used. The
count should be performed within 6 hours (blood should not be refrigerated)
Note: The correct and safe collection of capillary and venous blood.

Equipment:

Counting chamber (haemocytometer) the counting chamber recommended for cell

counts is a metallized surface (Bright-line) double cell Improved Neubauer ruled
chamber. The rulings of an Improved Neubauer ruled chamber. The chamber grid has
an area of 9 mm2 and the depth of the chamber (space between cover glass and grid)
is 0.1 mm.
Counting chamber cover glasses Special optically plane cover glasses of defined
thickness (designed for use with hsemocytometers) are required. Other cover glasses
must not be used. Manufacturers of counting chambers provide two cover glasses
with each chamber. The laboratory should always keep in stock spare cover glasses.
Pipettes/ calibrated capillaries and safe filling device A 20_1 (0.02 ml, 20 cmm)
micropipette e.g. white shellback type, or calibrated capillary is required to measure

blood samples. A safe pipette/capillary filler should be used to aspirate and dispense
the blood. This can be a simple bulb filler or device.

Reagent:
WBC diluting fluid

Test method:
1. Measure 0.38 ml of diluting fluid and dispense it into a small container or tube.
2. Add 20_1 (0.02 ml, 20 cmm) of well-mixed EDTA anticoagulated venous blood or
free flowing capillary blood and mix.
Important: The volume of blood used in the test must be correct. This can be achieved
by using the technique illustrated on page 302.
3. Assemble the counting chamber:
_ Make sure the central grid areas of the chamber and the special haemocytometer
cover glass are completely clean and dry.
_ Slide the cover glass into position over the grid areas and press down on each side
until rainbow colours (Newtons rings) are seen. Prior moistening of the chamber
surface on each side of the grid areas will help the cover glass to adhere to the
chamber.
4. Re-mix the diluted blood sample. Using a capillary, Pasteur pipette, or plastic bulb
pastette held at an angle of about 45 degree fill one of the grids of the chamber with
the sample, taking care not to overfill the area.
Important: The chamber must be refilled if the sample overfills into the channel
beyond the grid or an air bubble forms in the grid area.
5. Leave the chamber undisturbed for 2 minutes to allow time for the white cells to
settle.
Note: To prevent drying of the fluid, place the chamber in a petri dish or plastic
container on dampened tissue or blotting paper and cover with a lid.
6. Dry the underside of the chamber and place it on the microscope stage.
Using the 10degree objective with the condenser iris closed sufficiently to give good
contrast, focus the rulings of the chamber and white cells. Focus the cells until they
appear as small black dots.
7. Count the cells in the four large corner squares of the chamber marked W1, W2,
W3, W4.
Include in the count the cells lying on the lines of two sides of each large square.

Interpretation of WBC counts:

Reference ranges for white cell counts vary with age with higher counts being found
in children. There are also gender differences with higher total WBC and neutrophil
counts being found in women of child-bearing age and during pregnancy. Counts also
vary in different populations with lower total WBC and neutrophil counts being found
in Africans and people of African descent.

WBC RANGE
Children at 1 y...............................................................6.0-18.0_109/1
Children 4-7 y...............................................................5.0-15.0_109/1

Adults............................................................................4.0-10.0_109/1
Adults of African origin.................................................2.6-8.3_109/1
Pregnant women.........................................................Up to15_109/1

AUTOMETIC CELL COUNTER

FLOW CYTOMETRY

A flow cytometer
Flow cytometry is by far the most sophisticated and expensive method for cell counting. In a
flow cytometer the cells flow in a narrow stream in front of a laser beam . The beam hits
them one by one, and a light detector picks up the light that is reflected from the cells.

Image Analysis:
Recent approaches consider the use of high-quality microscopy images over which a
statistical classification algorithm is used to perform automated cell detection and counting as
an image analysis task. Generally performs with a constant error rate as an offline (batch)
type process. A range of image classification techniques can be employed for this purpose.

FNAC (FINE
CYTOLOGY)

NEEDLE

ASPIRATION

Fine-needle aspiration biopsy (FNAB, FNA or NAB), or fine-needle aspiration

cytology (FNAC), is a diagnostic procedure used to investigate lumps or masses. In this
technique, a thin, hollow needle is inserted into the mass for sampling of cells that, after
being stained, will be examined under a microscope. There could be cytology exam of
aspirate (cell specimen evaluation, FNAC) orhistological (biopsy - tissue specimen
evaluation, FNAB). Fine-needle aspiration biopsies are very safe, minor surgical procedures.
Often, a major surgical (excisional or open) biopsy can be avoided by performing a needle
aspiration biopsy instead. In 1981, the first fine-needle aspiration biopsy in the United States
was done at Maimonides Medical Center, eliminating the need for surgery and
hospitalization. Today, this procedure is widely used in the diagnosis of cancer and
inflammatory conditions.
A needle aspiration biopsy is safer and less traumatic than an open surgical biopsy, and
significant complications are usually rare, depending on the body site. Common
complications include bruising and soreness. There is a risk, because the biopsy is very small
(only a few cells), that the problematic cells will be missed, resulting in a false negative
result. There is also a risk that the cells taken will not enable a definitive diagnosis.

Application:
This type of sampling is performed for one of two reasons:
1. A biopsy is performed on a lump or a tissue mass when its nature is in question.
2. For known tumors, this biopsy is performed to assess the effect of treatment or to
obtain tissue for special studies.
When the lump can be felt, the biopsy is usually performed by a cytopathologist or a surgeon.
In this case, the procedure is usually short and simple. Otherwise, it may be performed by an
interventional radiologist, a doctor with training in performing such biopsies under xray or ultrasound guidance. In this case, the procedure may require more extensive
preparation and take more time to perform.
Also, fine-needle aspiration is the main method used for chorionic villus sampling, as well as
for many types of body fluid sampling.
It is also used for ultrasound-guided aspiration of breast abscess, of breast cysts, and
of seromas.

Preparation:
Several preparations may be necessary before this procedure.

No use of aspirin or non-steroidal anti-inflammatory medications

(e.g. ibuprofen, naproxen) for one week before the procedure;
No food intake a few hours before the procedure;
Routine blood tests (including clotting profile) must be completed two weeks before
the biopsy;

Suspension of blood anticoagulant medications;

Antibiotic prophylaxis may be instituted.

Before the procedure is started, vital signs (pulse, blood pressure, temperature, etc.) may be
taken. Then, depending on the nature of the biopsy, an intravenous line may be placed. Very
anxious patients may want to be given sedation through this line. For patients with less
anxiety, oral medication (Valium) can be prescribed to be taken before the procedure.

Procedure:

A physicians hands are seen performing a needle biopsy to determine nature of lump either
fluid-filled cyst or solid tumor.
The skin above the area to be biopsied is swabbed with an antiseptic solution and draped with
sterile surgical towels. The skin, underlyingfat, and muscle may be numbed with a local
anesthetic, although this is often not necessary with superficial masses. After locating the
mass for biopsy, using x-rays or palpation, a special needle of very fine diameter is passed
into the mass. The needle may be inserted and withdrawn several times. There are many
reasons for this:

One needle may be used as a guide, with the other needles placed along it to achieve a
more precise position.

Sometimes, several passes may be needed to obtain enough cells for the intricate tests
which the cytopathologists perform.

After the needles are placed into the mass, cells are withdrawn by aspiration with
a syringe and spread on a glass slide. The patient's vital signs are taken again, and the patient
is removed to an observation area for about 3 to 5 hours.

For biopsies in the breast, ultrasound-guided fine needle biopsy is the most common.

STAINING TECHNIQUES IN
HISTOPATHOLOGY
TAKING PARAFFIN SECTIONS TO WATER
SAFETY NOTE: Turn on the exhaust system before commencing.
Wear protective clothing, gloves and safety glasses during the procedure.
xylene 2 minutes
xylene 2 minutes
absolute alcohol 1 minute
absolute alcohol 2 minutes
70% alcohol 30 seconds
water
Take the slides through the various solutions as follows:
1. Place slides in slide holder.
2. Lift lid off staining dish, and immerse the slides in the first solution with agitation.
3. Replace lid and allow slides to remain in solution for the specified time with periodic
agitation.
4. Remove slides from the solution (with agitation) and tilt slide holder to allow excess
solution to drain
before transferring it to the next solution.
5. Continue in this manner through the remaining solutions for the specified times
Times can be reduced if slides are agitated constantly.

DEHYDRATION AND CLEARING OF SECTIONS IN

XYLENE BEFORE MOUNTING
Dehydration 70% alcohol 15 seconds
absolute alcohol 1 minute
absolute alcohol 2 minutes
Clearing xylene 15 seconds
xylene 15 seconds
Take the slides through the various solutions as follows:
1. Place slides in slide holder.
2. Lift lid off staining dish, and immerse the slides in the first solution with agitation.

3. Replace lid and allow slides to remain in solution for the specified time with periodic
agitation.
4. Remove slides from the solution (with agitation) and tilt slide holder to allow excess
solution to drain
before transferring it to the next solution.
5. Continue in this manner through the remaining solutions for the specified times.
Times can be reduced if slides are agitated constantly.

BLOTTING DRY AND CLEARING OF SECTIONS

Blotting Dry
1. Place slide face down carefully on blotting paper.
2. Fold blotting paper over slide over and apply gentle pressure to dry slide.
3. Lift slide and move to dry section of blotting paper and repeat until section is completely
dry.
4. Allow section to air dry if necessary before clearing.
Clearing xylene 15 seconds
xylene 15 seconds
Clear dried section by dipping the slide in xylene for specified times and mount in DPX.

MOUNTING OF SECTIONS
1. Remove slide from slide holder.
2. Carefully dry the BACK of the slide with a tissue.
3. Lay the slide on blotting paper or bench-coat on the fume bench.
4. Place a drop of DPX mounting medium on the section.
5. Carefully place a cover-slip over the DPX ensuring that it covers the section.
6. Remove any bubbles by jiggling the coverslip gently.
7. Place mounted slide on small cardboard slide holder and place in 50oC oven for a few
hours.
8. Allow mounted slide to dry lying flat for at least 1 week.

ZIEHL-NEELSEN TECHNIQUE FOR ACID-FAST BACILLI

(ZN)

The histological method is similar to the classical bacteriological technique that depends
upon the resistance of certain bacilli to decolourisation by acid alcohol after being stained
with hot carbol-fuchsin.

Fixation
Most fixatives can be used. Avoid Carnoy which removes lipid from the bacilli which makes
them less acid-fast. Formalin, especially when prolonged, is said to reduce acid-fastness, but
specimens usually stain perfectly satisfactorily. The treatment of formalin fixed sections with
0.5% ammonium hydroxide before staining may improve the brightness of color of acid-fast
bacilli, but this is seldom necessary and may detach the sections from the slide.

ZN Staining Procedure
1. Place a rectangle of filter paper over the section (to prevent precipitation of the stain) and
flood the slide with carbol-fuchsin.
2. Warm the slide until the stain begins to steam; this can be conveniently done by the flame
from a throat swab soaked in alcohol.
3. Leave for 5 minutes.
4. Wash in tap water for 2 minutes.
5. Differentiate in acid alcohol (3% HCl in 95% ethanol) until no more color runs from the
slide.
6. Rinse in water to remove acid alcohol.
7. Counterstain in acidified methylene blue for 30 seconds.
8. Wash in water, dehydrate, clear and mount in synthetic resin eg DPX.

Results
Acid-fast bacilli: red
Other bacteria: blue

Cells and their nuclei: blue

Red blood cells should retain a slight red color.

PAP STAIN
Principle
The most widely used staining procedure for cytological specimens is Papanicolaous
technique. In the first staining step the nuclei are stained by a haematoxylin solution. Nuclei
are stained blue, dark violet to black. The second staining step is cytoplasmic staining by
orange staining solution, especially for demonstration of mature and keratinised cells. The
target structures are stained orange in different intensities. In the third staining step the socalled polychromatic solution is used, a mixture of eosin, light green SF and Bismarck
brown. The polychromatic solution is used for demonstration of differentiation of squamous
cells e.g. cervical cancer and cycle diagnosis for examination under microsope Samples
derived from the human body.

Application:
Papanicolaous stain gives a pale , yellow-orange staining result with mature and keratinised
squamous cells. Papanicolaous solution, Orange II solution gives a more intense reddish
staining result with mature and keratinised squamous cells. Sample material and preparation
for professional use only Gynaecological and non-gyneacological specimen as sputum, urine,
FNAB, body effusions, lavages samples derived from the human body. The collected cells are
smeared on a microscope slide and immediately wet fixed with a thin film to maximize cell
preservation In order to avoid errors, the staining process must be carried out by an expert.
National guidelines for work safety and quality assurance must be followed. Microscopes
equipped according to the standard must be used. If necessary use a centrifuge suitable for
medical diagnostic laboratory.

Fixation
Wet fixation immediately with Cytology spray fixative or wet fixation immediately in 96%
ethanol for minimum 30 min. All samples must be clearly labelled. Suitable instruments must
be used for taking samples and their preparation; manufacturer instructions for application
/use must be followed.

Reagent
Papanicolaous stain
Papanicolaous solution

Haematoxylin Harris mercury free

Procedure
Wet fixed samples on microscope slides Conventional staining, manual:
1. Wash with 96 % alcohol*.
2. Wash with 80 % alcohol*. 3. Wash with 70 % alcohol*.
4. Wash with 50 % alcohol*.
5. Wash with distilled water
6. Stain in Harris haematoxylin solution 3 min
7. Rinse under weak stream of tap water 3-5 min
8. Wash with 50 % alcohol
9. Wash with 70 % alcohol
10. Wash with 80 % alcohol
11. Wash with 96 % alcohol
12. Stain in Papanicolaous stain OG6 solution 3 min
13. Wash with 96 % alcohol
14. Wash with 96 % alcohol
15. Stain in Papanicolaous solution EA50 3 min
16. Dehydrate with 96 % alcohol
17. Dehydrate with 96 % alcohol
l 18. Dehydrate with absolute alcohol 5 min
19. Dehydrate with equal parts of absolute alcohol and xylene or xylene substitute.
20. Clear with xylene or xylene substitute.
21. Clear with xylene or xylene substitute 2 min
22. Mount with DePeX or DPX mountant

Result
The microscope used should meet the requirements of a medical diagnostic laboratory
Cytoplasm Eosinophilic (acidophilic)

Pink

Cytoplasm Keratinised

Pink-orange

Erythrocytes

Red

Nuclei

Blue

CLINICAL CHEMISTRY TESTS

Clinical chemistry in district laboratories
The clinical chemistry tests performed in district laboratories will depend on:

The health needs of the community and the requirement for clinical chemistry tests
to assist in disease diagnosis and prognosis, monitoring of treatment, and screening to prevent
ill-health.

The training and experience of laboratory staff and the support that can be provided
by the district laboratory coordinator to ensure clinical chemistry tests are performed
correctly with adequate control and safety.

Whether the appropriate equipment is available and affordable (purchasing and

running costs) and can be maintained by the user.

Whether the required chemicals and products to make reagents, standards, and
controls are available and affordable, and whether the more complex reagents can be
provided to district laboratories in ready-made form or in easy to prepare packs that have
sufficient stability.

Whether the laboratory has access to or can make its own supply of chemically pure
water .

Whether the numbers of tests performed are sufficient to avoid reagents, standards,
and controls from being wasted due to their expiry. In some situations it may be possible and
more appropriate to send patients samples to a larger laboratory for clinical chemistry tests.

Tests
The following tests are described in this chapter:
Whole blood, serum, or plasma tests
albumin, to investigate disorders of water balance, liver diseases, protein energy
malnutrition.
urea or preferably creatinine, to investigate disorders of renal function and monitor
uraemia
glucose, to diagnose and monitor diabetes
bilirubin, to diagnose and monitor jaundice, e.g. in newborn.
amylase, to assist in the diagnosis of acute pancreatitis.
alanine aminotransferase, to investigate liver disease.
potassium and sodium, to investigate and monitor disorders causing electrolyte
disturbances.

CLINICAL CHEMISTRY METHODS

DISTRICT LABORATORIES

USED

The following clinical chemistry methods are used in district laboratories:

Manual techniques.
Test kits using ready-made reagents.
Rapid solid phase (dry reagent) technologies, e.g. reagent strip tests.
Direct read-out analyzers for use with readymade liquid reagents..

Manual techniques

IN

Manual colorimetric techniques are possible and recommended in district laboratories when:

The chemicals needed can be obtained and the reagents and standards
correctly and economically made or preferably supplied to district laboratories
from the regional or central laboratory.
The required control material can be obtained and the quality of test methods
monitored.
The manual technique for a particular test is an approved test method.

For some tests where the reagents are stable and not complex and the manual
technique is easy to perform, it will be more economical for district laboratories to prepare
their own reagents and standards. When, however, reagents are complex to make or contain
several chemicals which are not easily obtainable or available only in large quantities (often
the situation), it will be difficult and expensive for district laboratories to use manual
techniques.
Whenever possible the regional or central laboratory should procure the chemicals,
prepare the reagents and standards, and distribute them with the necessary controls and
approved testing procedure to district laboratories.

Use of test kits

Commercially produced clinical chemistry test kits are used by district laboratories,
particularly when there is no local reliable production and distribution of reagents. In these
situations test kits are used because all the reagents are ready-made or easily reconstituted,
standards are often also provided, and the test method is usually rapid and simple to perform.
Compared with manual techniques, some test kits are expensive, particularly when the
standards or working reagents have poor stability and have to be discarded before they are
finished. When manual tests require complex reagents each containing several chemicals, the
use of test kits for measuring analytes can be less expensive and preferable. Many clinical
chemistry test kits are designed either for use with automatic analyzers, which are not
suitable for manual techniques, or as manual kits that require the use of a spectrophotometer
or colorimeter fitted with interference filters to provide a specific wavelength. The use of a
calculation factor in such a test kit is dependent on reading the test at the wavelength stated in
the method.

Dry reagent systems and liquid reagent analyzers

Direct readout clinical chemistry analyzers that use ready-made dry or liquid reagent
technologies are available for the rapid measurement of a wide range of analytes in whole
blood or serum. In response to the requirements of point of care (near patient testing),
many of the analyzers are small battery operated instruments designed for rapid single
specimen testing and easy to use, calibrate, and maintain.

Collection of blood specimens

Factors regarding the collection of blood specimens that can affect the correctness of
clinical chemistry test results include:

incorrect venepuncture technique,

haemolysis of red cells,
collection into the wrong container
instability of some chemical substances in whole blood, serum, and plasma.

Venepuncture technique
The following precautions need to be followed when collecting venous blood:
Do not apply the tourniquet too tightly or for too long a period because this will
cause venous stasis leading to a concentration of substances in the blood such as
haemoglobin, plasma proteins, potassium, and calcium.
Do not collect the blood from an arm into which an intravenous (IV) infusion is
being given.
If an anticoagulated specimen is required, add the correct amount of blood to the
tube or bottle and mix the blood with the anticoagulant by gently inverting the container
several time
s. Follow a safe technique and wear protective gloves (see subunit 8.3 in Part 2).
Note: A vein which can be felt is usually easier to enter than one which can only be seen.
Avoiding haemolysis
The haemolysis (rupture) of red cells can be a serious source of unreliable test results.
If red cells are haemolyzed, substances from the cells are released into the serum or plasma
leading to a false increase in the concentration of analytes, e.g. potassium. Haemolysis also
interferes with many chemical reactions.
Haemolysis can be avoided by:
Checking that the syringe and needle are dry and that the barrel and plunger of the
syringe fit well.
Not using a needle with too fine a bore.
Not withdrawing the blood too rapidly or moving the needle once it is in the vein.
Frothing of the blood must be avoided.
Removing the needle from the syringe before dispensing the blood into the
specimen
container. Allow the blood to run gently down the inside wall of the container.

 Adding the correct amount of blood to anticoagulant. Do not shake the blood but
gently mix it with the anticoagulant.
Using clean dry tubes or bottles for the collection of blood from which serum is
required and by allowing sufficient time for the blood to clot and clot retraction to take place.
Red cells are very easily haemolyzed by the rough use of an applicator stick to dislodge a
clot.
Centrifuging blood samples for a minimum period of time. Centrifuging for 5
minutes at about 1000 g is adequate to obtain serum or plasma.
Not storing whole blood samples in, or next to, the freezing compartment of a
refrigerator.

Specimen containers and anticoagulants

Specimen containers for clinical chemistry tests must be leakproof and chemically
clean. They should be well washed with detergent, rinsed in several changes of clean water,
and finally rinsed in distilled or deionized water before being allowed to dry. To avoid the
confusion which often arises from the use of several different types of container, the
following system is recommended:
Non-urgent blood tests (excluding glucose): Dispense about 5 ml of blood (see Chart
6.1) into a dry tube or bottle and allow to clot. When the clot has retracted, centrifuge the
specimen and transfer the serum to a labelled container. Tightly cap the container to avoid
water evaporating from the specimen in dry warm conditions or moisture entering in humid
environments. Avoid collecting blood into a plastic container if serum is required. Blood
takes much longer to clot and clot retraction is poorer in a plastic tube or bottle than in a glass
one.
Urgent blood tests (excluding glucose) and paediatric blood tests: Dispense the
correct amount of blood into a tube containing lithium heparin anticoagulant. Gently mix the
blood with the anticoagulant. Centrifuge the specimen and transfer the plasma to a labelled
container.
Blood glucose (non-urgent or urgent): Dispense the correct amount of blood into a
tube containing fluoride-oxalate. Gently mix the blood with the anticoagulant. Centrifuge the
specimen to obtain plasma.

Quality Control

Objective of the session : To teach the external and internal quality control
programme

Introduction :Variation in results

Biochemical measurements vary for two reasons. There is analytical variation and
also biological variation.

Laboratory analytical performance

A number of terms describe biochemical results. these include:
precision and accuracy
Sensitivity and specificity
Concentration is always dependent on two factors: the amount of solute and the
amount of solvent. The concentration of the sugar solution in the beaker can be increased
from 1 spoon/beaker (a) to 2 spoons beaker by either decreasing the volume of solvent (b) or
increasing the amount of solute (c).
quality assurance
reference ranges.

Precision and Accuracy

Precision is the reproducibility of an analytical method. Accuracy defines how close
the measured value is to the actual value. A good analogy is that of the shooting target figure
3 shows the scatter of results which might be 13 obtained by someone with little skill,
compared with that of someone with good precision where the results are closely grouped
together. Even when the results are all close, they may not hit the centre of the target.
Accuracy is therefore poor, as if the 'sights' are off. It is the objective in very biochemical
method to have good precision and accuracy.

Sensitivity and specificity

Sensitivity of an assay in a measure of how little of the analyte the method can detect,
As new methods are developed they may offer improved detection limits which may help in
the discrimination between normal results and those in patients with the suspected disease.
Specificity of a assay related to how good the assay is at discriminating between the
requested analyte and potentially interfering substances.

Quality assurance

Every laboratory takes great pains to ensure that the methods in use continue to
produce reliable results. laboratory staff monitor performance or assay using quality control
samples to give reassurance that the method is Imprecise Precise but inaccurate Precise and
accurate 14 performing satisfactorily with the patients' specimens. These are internal quality
controls which are analysed every day or every time an assay is run. The expected values are
known and the actual results obtained are compared with previous values to monitor
performance. In external quality assurance schemes, identical samples are distributed to
laboratories; results are then compared. in this way, the laboratory's own internal standards
are themselves assessed.

Biological factors affecting the interpretation of results

The discrimination between normal and abnormal results is affected by various
physiological factors which must be considered when interpreting any given result. These
include:
Sex of the patient. Reference ranges for some analytes such as serum creatinine are
different for men and women.
Age of the patient. There may be different reference range for neonates, children,
adults and the elderly.
Effect of diet. The sample may be inappropriate if taken when the patient is fasting
or after a meal.
Time when sample was taken. There may be variations during the day and night.
Stress and anxiety. They may affect the analyte of interest.
Posture of the patient. Redistribution of fluid may affect the result.
Effects of exercise. Strenuous exercise can release enzymes from tissues.
Medical history. Infection and/or tissue injury can affect biochemical values
independently of the disease process being investigated.
Pregnancy. The alters some reference ranges.
Menstrual cycle. Hormone measurements will vary through the menstrual cycle.
Drug history. Drugs may have specific effects on the plasma concentration of some
analytes.

AUTOMATION

Objective :- Advances in diagnostic methodologies and instrumentation have been

impressive but most of these are deleted in the teaching programme this chapter tries to cover
the automation required for the analysis of samples.

Introduction
During the past few year, in clinical biochemistry there has been a considerable
increase in clinical demand for laboratory investigations. When the volume of work
increased, there arose a need for work simplification. Monostep methods are introduced to
replace multistep cumbersome and inaccurate methods like Folin-Wu's blood sugar
determination. The efficiency of monostep methods was further increased by the introduction
of automatic dispensers and diluters. For the common test like blood glucose, blood urea etc.,
however, most large laboratories found this approach still inadequate to deal with work load
and instruments designed to handle the whole analytical process in mechanized fashion have
become common place in last decade. This procedure is called automation. It is a self
regulating process, where the specimen is accurately pipetted by a mechanical probe and
mixed with a particular volume of the reagent and results are displayed in digital forms and
also printed by a printer. There is an element of feed back which detects any tendency to
malfunction. The function of autoanalyser is to replace with automated devices the steps of
pipetting, preparation of protein free filtrated, heating the colour forming reagents in a
waterbath and increase the accuracy and precision of the methods.
The automated instruments not only save the labour and time but allow reliable
quality control, reduce subjective crrors and work economically by using smaller quantities
of samples and reagents. Following are the different types of autoanalysers used in clinical
chemistry laboratories

Semi automated discrete analysers

In the case of these analysers the initial part of the procedure i.e. pipetting of reagent
and specimen, mixing and incubation is carried out by the technician. Rest of the procedure
i.e. setting of incubation temperature (for kinetic determinations), zero setting, photometric
readings, result display, automatic printing and data management and processing is carried
out by the analyser.

Advantage of semiautoanalysers
1) The semiautoanalysers are cheap and compact, compared to other fully automated
analyses.
2) Specimen analysis is cheap, since volume of reagent used is 0.5 to 1.0ml.
3) Enzyme determinations by kinetic methods are performed accurately in 1 to 3
minutes.
4) The enzymatic reagents are not corrosive and involve monostep testing .

Fully automated batch analysers

These analysers carry out all the function of a semiautomated analyzer, in addition to
the pipetting of specimen and reagents and also the mixing of the reaction mixtures. The
basic working stages of these analysers, after selecting general system parameters are as
follows
1) the specimen cups are placed on the sampler.
2) The required quantity of reagent is dispensed by a reagent probe, in the reaction
cups. 26
3) The respective specimens from the sampler are pipetted into the appropriate
reaction cups by another sample probe
. 4) The reaction cups are shaken mechanically to mix the contents.
5) After observing the required incubation time (for delay time in the case of kinetic
determinations) the reaction mixture is aspirated by a probe for photometric readings.
6) The resulted values are printed and displayed in appropriate units by digital display.

Estimation of Blood Glucose

Diabetes Mellitus: It is a chronic disease due to disorder of carbohydrate metabolism,
cause of which is either deficiency or diminished level of insulin resulting in hyperglyeemia
(increased blood glucose level) & glucose (presence of glucose in urine). Secondary
metabolic defect is also seen. Such as metabolism of proteins & fats

1. Primary or Idiopathic or Essential Diabetes

(a) Juvenile

Diabetes or. Type I Diabetes or Insuline dependent Diabetes

Mewlitus (IDDM) Less Frequent Occures before the age of 15 years. Due to less
production of insulin from b cells of langerhans (Pancrea)

Maturity onset diabetes or. Type II diabetes or Non-insulin

dependent Diabetes melitus (NIDDM) More frequent in population. Occures at
(b)

middle age. Ketoacidosis is rare. B cell is degenerated to some extent but response to
glucose load is seen.

2. Secondary It is secondary to some other main disease

(a) Pancreatic Diabetes. Pancreatitis Hemochromatosis Malignancy of Pancrease.
(b)Inereased level of antagonistic hormone Hyperthyrodism Hypercorticism
Cushings disease Hyperpitnatarism Clinical Biochemical fnding in diabetes
1) Presence of large amount of glucose in urine.
2) Large volume of urine & increased frequency of micturition (Polyuria
) 3) Polyphagia i.e. eats more frequently.
4) Increased catabolisim of fat so there is increase in free fatty acid level in blood &
liver.
5) Increased acetyl coA is seen which further lead to increase formation of
cholesterol & hence at formation of atherosclerosis.
6) Increased ketone bodies in blood & its apperance in urine leads to acidosis.
7) Increased catabolism of tissue protein for energy requirement lead to loss of
weight & increased level of amino acid in blood & more formation of urea by deamination of
amino acid.

Objective :- To estimate blood glucose

Introduction :- Estimation of glucose in blood was one of the first biochemical tests to
be applied clinically and now it has become a routine in clinical biochemistry lab. In blood
quantitative estimation of glucose is done with either whole blood, plasma or serum and
several methods have been in use. Whole blood 30 values are 10-15% lower than plasma.
Arterial blood values are higher than venous values.
The term Blood Sugar is used synonymously with blood glucose but certain other
substance like glutation, glucuronic acid, threonine, uric acid, ascorbic acid, fructose etc. give
erroneously high values (5-20%) when any reduction method is adopted.

Procedure:
a) Fasting blood Sugar (FBS) : The blood sample is collected after the patient
fasts for 12 hours or overnight.

b) Post-Prandial Blood Sugar (P P B S) : After the patient fasts for 12 hours,

a meal is given which contains starch and sugar (approx. 100 gms). Blood is collected 2 hours
after the ingestion of the meal
.

c) Random Sample : Blood is collected any time without prior prepration of the

patient.

Collection of Blood Sample :

Blood is usually collected from a vein and kept in a bottle containing sodium fluoride
(Na F) and potassium oxalate mixed at proportion of 1 : 3 Usually 4 mg. of the mixture is
required. Both the substances act as anticoagulant and Na F prevents glycolysis in RBC's by
inhibiting the enzyme 'enolase'.

Methods of Estimation :
1. Enzymatic : Measure only glucose in blood.
a) Glucose Oxidase Method :
Glucose oxidase catalyses the oxidation of glucose to gluconic acid and hydrogen
peroxide. This H2 O2 is broken down to water and oxygen by a peroxidase in the presence of
an oxygen acceptor which itself is converted to a coloured compound, the amount of which
can be measured colorimetrically.

Estimation Of Blood Urea

Kidney function tests :
Since the kidneys perform a multitude of functions, a single test cannot give information
about the entire range of renal functions. A group of tests is required to evaluate the different
renal functions. Abnormal results may sometimes be obtained due to a temporary renal
dysfunction. Hence the test should be performed repeatedly and interpreted on the basis of a
series of results. Moreover, the results of renal function tests may some-times be affected by
extra-renal factors. Therefore, the results must always be interpreted in conjunction with the
clinical picture.

A large number of tests have been introduced during the past decades to assess the
renal functions. Only a few of them have stood the test of time.
The more important and commonly employed tests will be discussed below.
1) Blood urea
2) Serum creatinine

Object : To estimate blood urea.

Introduction

: Urea is main end product of protein catabolism. It is formed in the liver

and is excreted through urine. Urea represents about 45-50% of the non-protein nitrogen of
blood and 80-90% of the total urinary nitrogen exeretion. The urea concentration in the
glomerular filtrate is same as that in plasma. Tublar reabsorption of urea varies inversely with
the rate of urine flow ad hence is not a useful measure of GFR. Blood urea nitrogen varies
directly with protein intake and inversely with the rate of excertion of urea.

Estimation : Various methods are use :

(i) Enzymatic using urease :
a) 'eseler's Method : Urea is converted to ammonia by the enzyme urease. Ammonia
is made to react with Neseler's reagent (Potassium mercurie iodide) giving rise to a brown
coloured compound which is read at 450nm the enzyme acts optimally at 55oC pH 7.0 to 8.0
and is inhibited by ammonia and fluoride. Disadvantages are turbidity, colour instability and
non linear, calibration beside susceptibility to contamination with NH3 from the laboratory
and endogenous ammonia in the specimen.

b) Berthelot Reaction : In this NH3 reacts with phenol in presence of hypochlorite to

form an indophenol which gives a blue coloured compound in alkali. Nitroprusside acts as a
catalyst increasing the rate of reaction, intensity of colour obtained and its reproducibility.

Estimation Of Serum Creatinine

Object :- To estimate serum creatinine
Introduction:
Creatinine is a waste product formed in muscle by creatine metabolism. Creatine is
synthesized in the liver which then passes into circulation where it is taken up by skeletal
muscle for conversion to creatine phosphate which serves as storage form of energy in
skeletal muscles. Creatine and creatine phosphate are spontaneously converted to creatinine

at a rate of about 2% the total per day. This is related to muscle mass and body weight.
Creatinine formed is excreted in the urine. On a normal diet almost all creatinine in urine is
endogenous. Its excretion is fairly constant from day to day and has been used to check the
accuracy of 24 hours urine collection. It is independent of urine flow rate and its level in
plasma is quite constant.

Estimation of serum creatinine by Jaffe's Alkaline Picrate Method

Principle : Creatinine in alkaline medium reacts with picric acid to form a red tautomer
of creatinine picrate the intensity of which is measured at 520nm. The two chief
disadvantages with Jaffe's reaction are:
- Lack of specificity :- Only 80% of the colour developed is due to creatinine in
serum. other non specific chromogens that react with picric acid are proteins, ketone bodies,
pyruvate, glucose, ascorbate etc.
- Sensitivity to certain variables like PH temperature etc.

Reagents:
1) Picric acid 0.04M (9.16g/L)
2) Sodium hydroxide 0.75N
3) Sodium tungstate 10%
4) 2/3 N H2 SO4
5) Creatinine standard stock 100mg%
6) Working standard 3mg%

Procedure :-

In a centrigure tube, take 2ml of serum with 2 ml of distilled water.

Precipitate proteins by adding 2ml sodium tungstate and 2ml of 2/3 N sulphuric acid drop
with constant shaking. Stand for 10 minutes and filter. Use protein free filterate for test.
Make the test tubes as follows and add
B

S1

S2

S3

S4

Filtrate

3ml

Standard 3mg%

0.5

1.0

2.0

3.0

D. Water

3ml

2.5

2.0

1.0

NaOH

1ml

1.0

1.0

1.0

1.0

1.0

Picric acid

1ml

1.0

1.0

1.0

1.0

1.0

Mix well Allow to stand for 15 min. and read absorbance at 520nm.

Calculation : Serum creatinine (mg%) =

O.D. Test /O.D.Std.x Amount of Std. /Vol.of Serum x 100

Interpretation :
Normal serum creatinine levels are
males : 0.7-1 4mg/100ml
Females: 0.4-1.2mg/100ml

Estimation Total Protein

Object : To estimate total protein.
Introduction:
Serum contains a large variety of proteins. More than hundred different proteins have
been identified so far, and perhaps many more are yet to be identified. Albumin and the
various globulins constitute the bulk of the total amount of proteins present in serum.
The most widely used method is still the biuret method. (the name derives from the
reaction between biuret and cupric ions in an alkaline medium.

Biuret Method
Principle :

Cupric ions form chelates with the peptide bonds of proteins in an alkaline
medium. sodium potassium tartrate keeps the cupric ions in solution. The intensity of the
violet colour that is formed is proportional to the number of peptide bonds which, in turn,
depends upon the amount f proteins in the specimen.

Reagents
(i) Biuret Reagent 3 mg of copper sulphate is dissolved in 500 ml of water. 9 gm
of sodium potassium tartrate and 5 gm of potassium iodide are added and dissolved. 24 gm of
sodium hydroxide, dissolved separately in 100 ml of water is added. The volume is made up
to 1 litre with water. The reagent is stored in a well-stoppered polythene bottle.
(ii) Biuret blank this is prepared in the same way as the buiret reagent with the
difference that copper sulphate is not added.
(iii) Standard protein solution the best way is to determine the total protein
concentration in pooled human serum by Kjeldahl method, dilute it to bring the protein
concentration to the desired level, say 6 gm/100 ml and use it as standard. Alternatively, a 6
gm/100 ml solution of bovine albumin in water may be prepared and used as standard.

Procedure :
label 3 test tubes 'Unknown', 'Standard' and 'Blank', Measure 5 ml of biuret reagent
into each. Wash 0.1 ml of serum into 'Unknown', 0.1` ml of standard protein solution into
';Standard' and 0.1 ml of water into 'Blank'. Mix and allow to stand for 30 minutes. Read
'Unknown' and 'Standard' against 'Blank' at 540 nm or using a green filter.

Calculations
Serum total proteins (gm/100 ml) =AU/AS x 6

Interpretation
The normal range of serum total proteins is 6-8 gm/100 ml in adults. The values. Are
lower in neonates, rise gradually in infancy and reach the adult levels in early childhood. The
level decreases in pregnancy. A slight change occurs with posture also. The level is a little
higher in upright posture than in recumbent posture due to shift of water from the vascular
compartment into the interstices.

Estimation
(Transaminases)
Object :-

Of

Aminotransferases

To estimate aminotransferases. Asparate Aminotransferase (AST) SGOT

Alanine Aminotransferase (ALT) SGPT

Introduction :

The aminotransferases are a group of enzymes that catalyse the inter

conversions of the aminoacids and -Keto acids by transfer of amino groups. Distinct
isoenzymes of AST are present in the cytoplasm and mitochondria of cells.

Method of teaching: Demonastration and its measurement of serum SGOT and SGPT
by kit and taking readings. Using a colorimeter or semi auto analyzer.

Evaluation: Giving exercise of serum SGOT and SGPT.

Estimation
Phosphatase

of

Serum

Alkaline

Object : To estimate serum alkaline phosphatase.

Introduction:
Alkaline phosphatase is present in practically all tissues of the body. It occurs at high
levels in the intestinal epithelium, kidney tubules, bone (osteoblasts), liver and placenta. The
relative contributions of bone and liver isoenzymes to the total activity are makedly agedependent. There is also a significant difference in levels of serum alkaline phosphatase
between different sexes of same ages.

Principle :
The serum is incubated with the buffer substrate under optimum, conditions of
temperature and pH to release phenol. This reacts with 4- animoantipyrine in alkaline
medium to give a red coloured compound which is estimated at 520nm against a reagent
blank. Colour development is rapid and is stable for at least an hour sodium hydroxide is
added immediately after incubation to raise the pH and stop the reaction. Potassium
ferricyanide is the oxidising agent. Sodium Bicarbonate provides the alkaline medium.

Interpretations :
The normal range of serum alkaline phosphatase activity is 3-13 KA units in adults
and is up to about two and a half times greater in children particularly during periods of
active growth.
1. Increase in levels of serum alkaline phosphatase

Physiological :i. Children during periods of bone growth.

ii. Puberty
iii. Pregnancy third trimester-due to the isoenzyme of placental origin

Estimation of Serum Bilirubin

Jaundice Jaundice is yellow discoloration of the skin or sclera (fig. 1) this is due to
the presence of bilirubin in the plasma and is not usually detectable until the concentration is
greater than about 1.1 mg/dl. Normally the bilirubin concentration in serum ranges from 0.21.1 mg/dl. Biliburin is derived from the terapyrrole prosthetic group found in haemoglobin
and the cytochromes. It is normally conjugated with glucuronic acid to make it more soluble,
and excreted in the bile (fig. 2) there are three main reasons why bilirubin levels in the blood
may rise
Haemolysis. The increased haemoglobin breakdown produces bilirubin which
overloads the conjugating mechanism.
Failure of the conjugating mechanism within the hepatocyte.
Obstruction in the biliary system.

Biochemical tests:
Bilirubin metabolities are responsible for the brown coloration of faeces. If bilirubin
does not reach the gut, stools become pale in colour. Bilirubin in 66 the gut is metabolized by
bacteria to produce stercobilinogen. And may be detected by simple biochemical tests. When
high levels of conjugated bilirubin are being excreted, urine may be a deep orange colour,
particularly if allowed to stand.
Biochemical test (LFTs) include measurement of serum AST and ALT as markers of
hepatocellular damage, as well as bilirubin and alkaline phosphatase which are indicators of
cholestasis.

Object :- To estimate serum bilirubin.

Principle:
When reacted with diazotized sulfanilic acid (Ehrlich's Reagent), bilirubin is
converted to azobilirubin molecules which give a red purple colour in acid the intensity of
which is read colorimetrically .

Both conjugated and unconjugated bilirubins give purple azobilirubins with

diazotized acid. Conjugated bilirubin can react in aquous solution 67 (Direct Reaction),
whereas unconjugated requires an accelerator or solubiliser , such as methanol (Indirect
Reaction-which gives total bilirubin i.e. conjugated + unconjugated bilirubin).

Interpretations :
Normal serum bilirubin is less than 1 mg/dL. Hyperbilirubinaemia of more than 3
mg/dL results into clinical jaundice. Hyperbilirubinaemia may be due to :-

I. Unconjugated Hyperbilirubinaemia (Retention Jaundice):

Due to retention in circulation of increased amount of unconjugated bilirubin.

II.
Jaundice) :

Conjugated

Hyperbilirubinaemia

(Regurgitation

Due to regurgitation into circulation of conjugated Bilirubin which would normally

pass along the bilirubin system

Estimation of serum triglycerides

Lipid profile:
For lipid profile estimation following tests are performed
1) Total lipids : Principle lipids react with sulphuric acid, phosphoric acid and vanillin
to form pink colour complex. Normal values 400-1000 mg/dl
2) Phospholipids : It is a fully enzymatic method which uses three different
enzuymes- phospholipase D, Choline oxidase and per oxidase to developed colour which is
measured at 500 nn. Normal range 160-270 mg/dl
3) Triglycerides :
4) Cholesterol :
5) HDL :
6) LDL : Cholesterol HDL + VLDL
7) VLDL : TG/5

Object: To estimate serum triglycerides.

Introduction :
Elevated levels of triglycerides in plasma have been considered as risk factors related
to atherosclerotic diseases. The hyperlipidemias can be inherited trait or they can be
secondary to a variety of disorders of diseases including nephrosis, diabetes mellitus, biliary
obstruction and metabolic disorders associated with endocrine disorders.

Method:
Acetyl-acetone

Normal range
10-190 mg/dl 72

Principle:
The serum lipids are extracted by isopropanol, which also precipitates serum
proteins. The interfering phospholipids, containing glycerol as integral part, are removed by
adsorption on alumina. Filtrate is used for saponification and glycerol is separated from
triglycerides. Action of metaperiodate converts glycerol into glyceraldehydes, which forms a
yellow coloured complex with acetyl acetone. The intensity of the coloured complex is
measured at 410 nm. (violet filter).

Estimation of Serum Cholesterol (Total)

Object: To estimate serum cholesterol.
Introduction:
Cholesterol is a fat-like substance that is found in all body cells. The liver makes all
of the cholesterol the body needs to form cell membranes and to make certain hormones.
The determination of serum cholesterol is one of the important tools in the diagnosis
an classification of lipemia. High blood cholesterol is one of the major risk factors for heart
disease.

Determination of cholesterol by enzymatic method

Principle :

Cholesterol esters are hydrolysed by cholesterol ester hydrolase to free cholesterol &
fatty acids. the free cholesterol produced and pre-existingh one are oxidised by cholesterol
oxidase to Cholestenone-4-en-3-one and hydrogen peroxide. Peroxidase acts on hydrogen
peroxide and liberated oxygen reacts with the chromogen (4-amino phenazone/phenol) to
form a red coloured compound which is read at 510 nm (505-530 nm).

Technical contents : Using kit of serum cholesterol.

Method of teaching : Demonstration and its measurement of serum cholesterol by kit
and taking readings. Using a colorimeter or semi auto analyser.

Evaluation : Giving exercise of serum cholesterol.

Estimation of Serum HDL cholesterol

Object : To estimation serum HDL cholesterol
Introduction :
In the presence of phosphotungstic acid and magnesium chloride, LDL, VLDL &
chylomicrons are precipitated. Centrifugation leaves only the HDL in the supernatent.
Cholesterol in the HDL fraction can be tested by the usual methods.

Normal range
Men : 30-60 mg/dl.
Women : 40-70 mg/dl.

Method: Colorimetric (Watson)

Precipitating reagents:
1) Phosphotungstie acid reagent (P.T.A.): It is prepared by dissolving 2.25 g. of
phosphotungstie acid in 8.0 ml of IN sodium hydroxide and 42.0 ml of distilled water.
2) Magnesium chloride reagent : 20.34 g of magnesium chloride in distilled water. It
is diluted to 50 ml.

Procedure :
Pipette in the centrifuge tubes labeled as follows:
Reagent

Test

1)Serum

0.5ml

2)P.T.A Reagent

0.05ml

3)MgCl2

0.02ml

Mix well, centrifuge at 3000 R.P.M. for 20 minutes Separate the supernatent by using
a pipette. The clear supernatent is treated in the similar way as for method for cholesterol and
the absorbance is measured at 500 nm.

Calculations :
Serum HDL Cholesterol, mg/dl = O.D. Test /O.D. Std. x 114

Additional Information:
Heparin and manganese chloride are also used as precaution.

MEDICAL
MICROBIOLOGY
Medical microbiology is a branch within the field of microbiology of
medical interested. Medical microbiologists study organisms which can
cause diseases in people, looking at the life cycle of such organisms, how
they cause infections in infections in humans, how they spread, what they
do to the human body, and how they can be eradicated. People in this
field may work in research labs studying microorganisms, or they may

work in diagnostic labs, performing tests to identify disease-causing

organisms in patients and making treatment recommendations.
Bacteria, fungi, protozoans, and viruses can all cause disease in humans.
Humans have been colonized by microorganisms from the beginning of
time, and microorganisms are constantly evolving and changing to thwart
human attempts at controlling them. The field of medical microbiology is
engaged with identifying new microorganisms, monitoring changes in
rapidly mutating species, and dealing with ongoing challenges in
microbiology, ranging sfrom the development of resistance to antibiotics
in bacteria to contact nation of water supplies with protozoans.
Like other researchers in microbiology, medical microbiologists are
interested in identifying and categorizing the organisms they see. This
information can help people see what they are dealing with, and it can
assist with the development of treatments. Understanding the relationship
between organisms can also be important when researchers explore
methods of transmission. The identification process can also include
inquiries into the origin and history of an organism; learning where a flu
virus developed, for example, can be important to understanding how an
epidemic occurred.

The role of the microbiology laboratory in

healthcare
Introduction:
Healthcare associated infections (HAI) are a big problem worldwide,
increasing morbidity and mortality of hospitalized patients, as well as
increasing cost of healthcare. Many hospitals throughout the world have
good systems for HAI prevention and control, but others have not and are
forced to develop such systems nowadays. Having microbiology
laboratory as a part of the hospital diagnostic laboratories, is a huge
advantage for HAI prevention and control. Ideally, a microbiology
laboratory should be set up inside the hospital, working everyday on a 24
hour basis. If it is not possible, then hospital should contract microbiology
services with the geographically nearest laboratory.

Diagnosis of infections
For the accurate etiological diagnosis of infection several conditions
should be fulfilled. first, clinician should put right indication for this
diagnostics; furthermore, the right sample should be taken; then, all data
needed for the laboratory should be put on the request (name of the
patient and the physician, location of the patient, date and time of
specimen collections, antimicrobial chemotherapy if already instituted).
Specimens sent to the microbiology laboratory have to be taken on the
right time (depending on the pathogenesis of the infection), from
appropriate sites using proper techniques, in a quantity that will ensure
good workout in the laboratory. Transport conditions from the patient to
the laboratory are to be carefully maintained. Microbiology laboratory
staff can assist in ensuring good Specimens by educating clinical staff.
Rapid and accurate diagnosis and sensitivity test are of outmost
importance for the direct patient care. In the laboratory methodology it is
necessary to provide rapid tests (like pneumococcal urine antigen in the
case of community acquired pneumococcal, or rotavirus latex
agglutination for detection of rotavirus in faeces etc). For diagnosis to be
accurate, laboratory has to develop procedures for quality assurance and
has external quality control system in function.
Microbiology laboratory should be able to examine blood, cerebrospinal
fluid, urine, stool, wound exudates or swab, respiratory secretions, and
perform basic serological tests (HIV, HBV, HCV, influenza).
Methods used in the diagnosis of infections are:

1. Classical Methods, like

Direct smear
Culture
Antigen detection
Serological tests

2. Molecular methods (not very often used in

routine work)

Hybridization
Amplification

Real - time amplification

Microbial Culture
Microbiological culture is the primary method used for isolating infectious
disease for study in the laboratory. Tissue or fluid samples are tested for
the presence of a specific pathogen, which is determined by growth in a
selective or differential media.
The 3 main types of media used for testing are:

Solid culture:

A solid surface is created using a mixture of

nutrients, salts and agar. A single microbe on an agar plate can then
grow into colonies (clones where cells are identical to each other)
containing thousands of cells. These are primarily used to culture bacteria
and fungi.

Liqued culture:

Cells are grown inside a liquid media. Microbial

growth is determined by the time taken for the liquid to form a colloidal
suspension. This technique is used for diagnosing parasites and detecting
mycobacteria.

Cell Culture:

Human or animal cell cultures are infected with the

microbe of interest. These cultures are then observed to determine the
effect this new microbe has on the cell. This technique is used for
identifying viruses.

PREPARING CULTURE MEDIA

Alkaline and peptone water
To make about 50 bottles:
Peptone................................................... 5 g
Sodium chloride...................................... 5 g
Distilled water.................................... 500 ml

Dissolve the peptone and sodium chloride in the water. Adjust the reaction
of the medium to pH8.6-9.0 using 1 mol/1sodium hydroxide Dispense the
medium in 10 my amounts in screw - cap bottles. Sterilize by autoclaving
(with caps loosened) at 121C for 15 minutes. Tighten the bottles caps
after the medium has cooled. Date the medium and give it a batch
number. Label the bottles and record on each the expiry date of the
medium (2 years from preparation). Store in a cool dark place with the
bottle caps screwed tightly to prevent a change in pH. Shelf-life:Up to 2
years providing there is no change in the volume or appearance of the
medium to suggest contamination. pH of medium: This should be within
the range pH 8.69.0 at room temperature.

Nutrient agar
Nutrient agar is a general purpose medium supporting growth of a wide
range of non-fastidious organisms, and typically contains
1. 0.5% Peptone
2. 0.3% beef extract /yeast extract
3. 1.5% agar
4. 0.5% NaCl
5. distilled water
6. pH adjusted to neutral (6.8) at 25 degree centigrade.

Directions
1. Suspend 23 g of the medium in one liter of purified water.
2. Heat with frequent agitation and boil for one minute to completely
dissolve the medium.
3. Autoclave at 121 degree centigrade for 15 minutes.

Test Procedure
1. Inoculate medium with isolated colonies or a loopful of pure culture
from broth.
Streak for isolation.

2. Incubate aerobically at 35 degree centigrade for 1824 hours or

longer if necessary.

Results
Good growth of no fastidious organisms on Nutrient Agar will appear as
translucent colonies.

Storage
Store sealed bottle containing the dehydrated medium at 230 degree
centigrade. Once opened and recapped, place container in a low humidity
environment at the same Storage temperature. Protect from moisture and
light by keeping container tightly closed.

Expiration
Refer to expiration date stamped on the container. The dehydrated
medium should be discarded if not free flowing, or if appearance has
changed from the original colour. Expiry applies to medium in its intact
container when stored as directed.

BLOOD AGAR
Blood agar plate (BAP) Contains mammalian blood (usually sheep or
horse), typically at a concentration of 510%. BAP are enriched,
differential media used to
isolate fastidious organisms and detect haemolytic activity. Betahaemolytic activity will show lysis and complete digestion of red blood cell
contents surrounding colony. Examples include Streptococcus
haemolyticus. Alpha-haemolysis will only partially lyse (the cells are either
lysed or not it is the digestion that may be incomplete) the haemoglobin
and will appear green or brown (due to the conversion haemoglobin to
methaemoglobin). An example of this would be Streptococcus viridans.
Gama-haemolysis (or non-haemolytic) is the term referring to a lack of
haemolytic activity. Contains meat extract, tryptone, sodium chloride,
and agar.

Principles of the Procedure

Nitrogen, vitamin, and carbon sources are provided by Enzymatic Digest

of Casein and Enzymatic Digest of Animal Tissue in Blood Agar Base,
Improved. Yeast Extract is a vitamin source. Corn Starch is added to
ensure any toxic metabolites produced are absorbed, and enhance
organism growth. 5 Sodium Chloride maintains the osmotic balance of the
medium. Agar is the solidifying agent.

Formula / Liter
Enzymatic Digest of Casein......................................... 15 g
Enzymatic Digest of Animal Tissue............................... 4 g
Yeast Extract................................................................... 2 g
Corn Starch......................................................................1 g
Sodium Chloride..............................................................5 g
Agar...............................................................................14 g
Final pH: 7.0+or - 0.2 at 25 degree centigrade

Directions
1. Suspend 42 g of the medium in one liter of purified water.
2. Heat with frequent agitation and boil for one minute to completely
dissolve the medium.
3. Autoclave at 12 degree centigrade for 15 minutes.
4. Prepare 5 - 10% blood agar by aseptically adding the appropriate
volume of sterile defibrinated blood to melted sterile agar medium,
cooled to 45 - 50 degree centigrade.

Test Procedure
1. Process each specimen as appropriate, inoculate directly onto the
surface of the medium. Streak for isolation with inoculating loop, stab
agar several times to deposit Beta-haemolytic streptococci beneath agar
surface. Subsurface growth will display the most reliable haemolytic
reactions owing to activity of both oxygen-stable and oxygen-labile
streptolysins.6
2. Incubate plates aerobically, anaerobically, or under conditions of
increased CO2 (5-10%) in accordance with established laboratory
procedures.

Results
Examine medium for growth and haemolytic reactions after 18 - 24 and 48
hours incubation. There are four types of haemolysis on blood agar media
described as:7
1. Alpha haemolysis (square) is the reduction of haemoglobin to
methaemoglobin in the medium surrounding the colony. This produces a
green discoloration of the medium.
2. Beta haemolysis (square ) is the lysis of red blood cells, producing a
clear zone surrounding the colony.
3. Gamma haemolysis (gamma) indicates no haemolysis. No destruction
of red blood cells occurs and there is no change in the medium.
4. Alpha-prime haemolysis (alpha ) is a small zone of complete haemolysis
surrounded by an area of partial lysis.

Storage
Store sealed bottle containing the dehydrated medium at 2 - 30 degree
centigrade. Once opened and recapped, place container in a low humidity
environment at the same storage temperature. Protect from moisture and
light by keeping container tightly closed.

Chocolate agar
It is non-selective, enriched growth medium. It is a variant of the blood
agar plate, containing red blood cells that have been lysed by slowly
heating to 80 degree centigrade. Chocolate agar is used for growing
fastidious respiratory bacteria, such as Haemophilus influenza and
Neisseria meningitidis. In addition, some of these bacteria, most notably
H. influenza, need growth factors such NAD (factor V) and hemin (factor
X), which are inside red blood cells; thus, a prerequisite to growth for
these bacteria is lysis of the red blood cells. The heat also inactivates
enzymes which could otherwise degrade NAD. The agar is named for the
color and contains no actual chocolate.

MACCONKEY AGAR
MacConkey agar

is a culture medium designed to grow Gramnegative bacteria and differentiate them for lactose fermentation.
Contents
It contains bile_salts (to inhibit most Gram-positive bacteria), crystal violet
dye (which also inhibits certain Gram-positive bacteria), neutral red dye
(which stains microbes fermenting lactose), lactose and peptone.

Composition:
Peptone - 17 g
Proteose peptone - 3 g
Lactose - 10 g
Bile salts - 1.5 g
Sodium chloride - 5 g
Neutral red - 0.03 g
Crystal Violet - 0.001 g
Agar - 13.5 g
Water - add to make 1 litre; adjust pH to 7.1+/- 0.2

Uses
Acting as a visual pH indicator, the agar distinguishes those Gramnegative bacteria that can ferment the sugar lactose (Lac+) from those
that cannot (Lac-).
This medium is also known as an "indicator medium" and a "low selective
medium".
Presence of bile salts inhibits swarming by Proteus species.

SEROLOGY
VENEREAL DISEASE RESEARCH LABORATORY (VDRL)
SLIDE TESTS
TEST PRINCIPLES
The Venereal Disease Research Laboratory (VDRL) tests1-3 are slide microflocculation tests
for syphilis that use an antigen containing cardiolipin, lecithin, and cholesterol. The antigen,
suspended in a buffered saline solution, forms flocculates when combined with lipoidal
antibodies in serum or cerebrospinal fluid from syphilis patients. The VDRL tests are fast,
easy to perform, and excellent for screening of samples. The VDRL tests measure IgM and
IgG antibodies to lipoidal material released from damaged host cells as well as to lipoproteinlike material, and possibly cardiolipin released from the treponemes.4,5 The antilipoidal
antibodies are antibodies that are not only produced as a consequence of syphilis and other
treponemal diseases, but also may be produced in response to nontreponemal diseases of an
acute and chronic nature in which tissue damage occurs.6 Without some other evidence for
the diagnosis of syphilis, a reactive nontreponemal test does not confirm T. pallidum
infection.

Collection
The procedures for the collection and processing of venous blood and spinal fluid are given
in detail in Chapter 3
. 1. Collect whole blood or spinal fluid into a clean, dry tube without an anticoagulant.
2. Label each specimen with patient identifier, and date.

Handling
1. Allow sufficient time (approximately 20 minutes) at room temperature for the specimen to
clot. 2. Centrifuge the specimen at room temperature at 1000 to 1200 x g for at least 5
minutes to sediment cellular elements (see chapter 3).
3. Transfer the serum into a clean, dry, labeled test tube.
4. If serum samples are to be shipped to a testing site, specimen containers must be leakproof
and placed within a leakproof plastic bag. Paper work should be submitted in a separate
plastic bag, if included with the specimen.8

5. On the day of testing, heat the serum in a 56o C water bath for 30 minutes.
6. Remove the serum from the water bath and examine for debris. Recentrifuge all serum
specimens containing particulate debris.
7. Reheat serum at 56o C for 10 minutes if testing is delayed more than 4 hours.
8. Specimens must be at room temperature, 23o - 29o C (73o - 85o F) when tested.

PROCEDURE
1.Slide flocculation tests for syphilis are affected by room temperature. For reliable and
reproducible test results, the VDRL antigen suspension, controls, and test specimens must be
at room temperature, 23 - 29C (73 - 85F), when tests are performed.
2. Place 50 l of serum into one ring of a paraffin or ceramic-ringed slide using a safety
pipetting device. Do not use glass slides with concavities, wells, or glass rings.
3. Gently resuspend the VDRL antigen suspension.
4. Holding the VDRL antigen suspension dispensing needle and syringe in a vertical
position, dispense several drops to clear the needle of air. Then add exactly 1 free-falling drop
(17 l) of antigen suspension to each circle containing serum.
5. Place the slide on the mechanical rotator. Rotate the slide for 4 minutes at 180 2 rpm.
When testing in a dry climate, cover the slides with a moist humidifying cover during rotation
to prevent excessive evaporation.
6. Immediately after rotating the slide, remove it from the rotator and read the test results.
7. Test quantitatively, to an endpoint, all serum specimens that produce reactive, weakly
reactive, or rough nonreactive results in the qualitative VDRL slide test.

Reading and Reporting Results

1. Read slide microscopically, using 10X oculars and a 10X objective.
2. Report the results as follows:

Reading
Medium or large clumps

Report
Reactive (R)

Small clumps

Weakly reactive (W)

No clumping or very slight roughness

Nonreactive (N)

INTERPRETATION OF RESULTS
1. The VDRL test is an aid in the diagnosis of syphilis. To diagnose syphilis, clinicians
combine VDRL test results with the results of other serologic tests, darkfield examinations,
clinical signs and symptoms, and risk factors. Without other supporting evidence for a
diagnosis of syphilis, a reactive VDRL may be unrelated to T. pallidum infection. The
predictive value of a reactive VDRL in the serologic diagnosis of syphilis is increased when
combined with a reactive treponemal test, such as the fluorescent treponemal antibody
absorption (FTAABS) test or the microhemagglutination assay for antibodies to T. pallidum
(MHA-TP).
2. A reactive VDRL test may indicate past or present infection with a pathogenic treponeme;
however, the result may also be a false-positive reaction. Falsepositive reactions can result
from laboratory error and also from serum antibodies that are unrelated to syphilis infection.
Laboratory errors are detected by a nonreactive VDRL with a second serum specimen. Falsepositive VDRL tests that result from infection with nontreponemal diseases or other disease
conditions are identified by an accompanying nonreactive treponemal test.
3. A nonreactive VDRL test without clinical evidence of syphilis may indicate no current or
past infection, an effectively treated infection, or occasionally, long standing infection. A
nonreactive VDRL test with clinical evidence of syphilis can be seen in early primary
syphilis, in secondary syphilis as a result of the prozone reaction, and in some cases of late
syphilis. If a prozone reaction is suspected, the test should be repeated with the patients
serum diluted 1:16 before determining that the serum sample is nonreactive. A nonreactive
VDRL result does not rule out an incubating syphilis infection.
4. When the quantitative VDRL test is performed on patients with syphilis, a fourfold rise in
titer, e.g. 1:4 to 1:16, on a repeat specimen may indicate infection, a reinfection, or a
treatment failure. If the increase in titer occurs over a short period of time, for example two
weeks, the immunologic response is most likely due to treponemal response rather than a
biologic false-positive lipoprotein response. A fourfold decrease in titer, e.g. 1:8 to 1:2, 6 to
12 months following treatment for early syphilis usually suggests that therapy was
adequate.11
5. All reactive qualitative VDRL tests should be quantitated to an endpoint, and the endpoint
titer should be reported. Unusually high VDRL titers may be seen with concurrent HIV-1
infection. Unusually high false-positive titers may be seen in serum from some patients with
lymphomas.

ASO TITER
INTRODUCTION:
ASO TEST method is a rapid latex agglutination test for the qualitative and semiquantitative
determination of antistreptolysin-O in serum WITHOUT SAMPLE DILUTION.
Antistreptolysin-O test systems detect serum antibodies to streptolysin, an oxygen labile
hemolysin derived from gorup A streptococci. The detection of antistreptolysin-O may be the
single best test for documenting antecedent streptococcal infections.
Over 80% of patients with acute rheumatic fever and 95% of patients with acute
glomerulonephritis have elevated titers of ASO.
The group A beta-hemolytic streptococci produces various toxins that can act as antigens.
One of these exotoxins is streptoysin-O.
In the presence of ASO in the serum, the latex suspension agglutinates due to the antigenantibody reaction.

STORAGE:
Store as packaged at 20 - 80 C. DO NOT FREEZE or use beyond the expiration date printed
on the label.

SAMPLE COLLECTION & HANDLING:

Only serum should be used with this test. DO NOT use plasma. Collect venous blood by
venipuncture or convenient fingertip mehtod. Separate serum as soon as possible after
collection. Store sera refrigerated, if testing is delayed more than of 48 hours, specimen
should be frozen.

REAGENTS AND MATERIALS PROVIDED:

01. ASO latex reagent
02. ASO Positive control
03. ASO Negative control
04. Stirrers & Glass slide

PROCEDURE:
1. Bring all reagents and samples to Room Temperature prior to testing.
2. Label circles on the slide provided with appropriate sample identification.
3. Using a separate Pipet/Stirrer for each sample, presqueeze and draw up SAMPLE.
Dispense ONE drop (20ul) of sample to the appropriately identified circle on the test slide.
4. Mix the ASO Liquid Reagent by inverting bottle several times, then, holding bottle in
vertical position, gently squeeze and dispense ONE free falling drop into each circle, being
used on the test slide.
5. Using the paddle end of the Pipet/Stirrer from Step 3 mix the contents of each circle
completely together over the entire surface area of the circle.
6. Rock the card by a to and fro motion for up to 2 minutes. Alternately, an automated rocker
or rotator may be used to mix the card for the required time.
7. Observe the for any sign of agglutination.

INTERPRETATION OF RESULTS:
Agglutination indicates an ASO content of more than 200 lU/ml in the specimen. Sera with
positive results in the qualitative test should be retested in the semiquantitative test.

B. SEMIQUANTITATIVE TEST:
Prepare further dilution of the specimen (1:2, 1:4, 1:8, 1:16, 1:32 etc.) with physiological
saline 0.9%. Then proceed as in qualitative test.

INTERPRETATION OF RESULTS:
The Presence of agglutination indicates a content of ASO in the sample equal or greater than
200 lU/ml. The lack of agglutination indicates an ASO level lower than 200 lU/ml in the
sample.
NOTES: As with all serologic procedures for ASO, it is advisable to compare the results of 2
separate samples taken in 2 week intervals. The early use of pencillin, as well as other
antibiotics, will prevent the ASO titre from rising. Compare the agglutination with that of
positive and negative control for a correct interpretation.

EXPECTED VALUES: Normal sera has an ASO titer of less than 200 lU/ml, i.e.,
reportedly, 166-200 lU/mL, depending on the population. Higher titers are representative of a
streptococcal infection.

MANTOUX TEST
INTRODUCTION:
Tuberculosis (TB) remains a leading cause of morbidity and mortality in the world, especially
in developing countries. A combination of factors including high costs, limited resources and
the poor performance of various diagnostic tests make the diagnosis of TB difficult in
developing countries. Short of demonstrating viable organisms in body tissues and fluids the
tuberculin skin test (TST) is the only method of detecting M. tuberculosis infection in an
individual and is used in the diagnosis of TB in individual patients, as well as in
epidemiological settings, to measure the prevalence of tuberculous infection in populations.

IMMUNOLOGIC BASIS FOR THE TUBERCULIN REACTION:

The reaction to intracutaneously injected tuberculin is the classic example of a delayed
(cellular) hypersensitivity reaction. T-cells sensitized by prior infection are recruited to the
skin site where they release lymphokines. These lymphokines induce induration through local
vasodilatation, edema, fibrin deposition, and recruitment of other inflammatory cells to the
area. Features of the reaction include (1) its delayed course, reaching a peak more than 24 h
after injection of the antigen; (2) its indurated character; and (3) its occasional vesiculation
and necrosis.

INTERPRETATION OF TUBERCULIN REACTION

The Mantoux test does not measure immunity to TB but the degree of hypersensitivity to
tuberculin. There is no correlation between the size of induration and likelihood of current
active TB disease but the reaction size is correlated with the future risk of developing TB
disease. The test has a poor positive predictive value for current active disease. There is no
correlation between the size of post-vaccination Mantoux reactions and protection against TB
disease and routine post-BCG Mantoux testing serves no purpose.
The results of this test must be interpreted carefully. The persons medical risk factors
determine the size of induration the result is positive (5 mm, 10 mm, or 15 mm).
Five mm or more is positive in

HIV-positive person

Recent contacts of active tuberculosis cases

Persons with nodular or fibrotic changes on Chest X-ray consistent with old healed
TB

Organ transplant recipients and other immunosuppressed patients who are on

cytotoxic immune-suppressive agents such as cyclophosphamide or methotrexate.

Patients on long term systemic corticosteroid therapy (> than six weeks) and those on
a dose of prednisone 15 mg/day or equivalent.

End stage renal disease

Ten mm or more is positive in

Recent arrivals (less than five years) from high-prevalence countries

Injectable drug users

Residents and employees of high-risk congregate settings (e.g., prisons, nursing

homes, hospitals, homeless shelters, etc.)

Mycobacteriology lab personnel

Persons with clinical conditions that place them at high risk (e.g., diabetes, prolonged
corticosteroid therapy, leukemia, end-stage renal disease, chronic malabsorption
syndromes, low body weight, etc.)

Children less than four years of age, or children and adolescents exposed to adults in
high-risk categories

Infants, children, and adolescents exposed to adults in high-risk categories.

HEPATITIS C
Introduction
Laboratory tests for hepatitis C are generally divided into two categories including:

serologic assays that detect antibodies to HCV

assays that detect, quantify, and/or differentiate HCV RNA genomes within an
infected individual. These are commonly called "hepatitis C viral load" tests and
"hepatitis C genotype"

Serologic Assays
Enzyme immunoassays (EIAs)
Enzyme immunoassays (EIAs) detect the presence of antibodies in serum directed against
HCV. These tests are commonly used for initial detection of hepatitis C. However, EIAs do
not differentiate between acute, chronic or resolved infection.
There are three generations of the EIA, with the latest EIA being a highly sensitive test. An
important issue with the EIA is that it can yield both false positive and false negative results.
False negative results may occur if the test is sent during the window period before
seroconversion ("serologic window" averages 8 weeks). False negative results may also occur
in immunocompromised populations such as those with HIV. False positive results may occur
due to cross-reactivity with other viral antigens and in immunologic disorders. Recombinant
immunoblot assay (RIBA)

STERILIZATION
Sterilization (or sterilisation) is a term referring to any process that eliminates (removes) or
kills (deactivates) all forms of life and other biological agents (such as prions, as well
as viruses which some do not consider to be alive but are biological pathogens nonetheless),
including transmissible agents (such as fungi, bacteria, viruses, prions, spore
forms, unicellular eukaryotic organisms such as Plasmodium, etc.) present in a specified
region, such as a surface, a volume of fluid, medication, or in a compound such as
biological culture media.[1][2] Sterilization can be achieved with one or more of the
following: heat, chemicals, irradiation, high pressure, and filtration. Sterilization is distinct
fromdisinfection, sanitization, and pasteurization in that sterilization kills, deactivates, or
eliminates all forms of life and other biological agents.

Dry heat

Dry heat sterilizer

Dry heat was the first method of sterilization, and is a longer process than moist heat
sterilization. The destruction of microorganisms through the use of dry heat is a gradual
phenomenon. With longer exposure to lethal temperatures, the number of killed

microorganisms increases. Forced ventilation of hot air can be used to increase the rate at
which heat is transferred to an organism and reduce the temperature and amount of time
needed to achieve sterility. At higher temperatures, shorter exposure times are required to kill
organisms. This can reduce heat-induced damage to food products.
The standard setting for a hot air oven is at least two hours at 160 C. A rapid method heats
air to 190 C for 6 minutes for unwrapped objects and 12 minutes for wrapped objects. Dry
heat has the advantage that it can be used on powders and other heat-stable items that are
adversely affected by steam (e.g. it does not cause rusting of steel objects).

Heat
Steam Sterilization

Front-loading autoclave
A widely used method for heat sterilization is the autoclave, sometimes called a converter or
steam sterilizer. Autoclaves use steam heated to 121-134 C under pressure. To achieve
sterility, the article is heated in a chamber by injected steam until the article reaches a time
and temperature setpoint. The article is then held at that setpoint for a period of time which
varies depending on the bioburden present on the article being sterilized and its resistance (Dvalue) to steam sterilization. A general cycle would be anywhere between 3 and 15 minutes,
(depending on the generated heat) at 121 C at 100 kPa, which is sufficient to provide a

sterility assurance level of 104 for a product with a bioburden of 106 and a D-value of 2.0
minutes. Following sterilization, liquids in a pressurized autoclave must be cooled slowly to
avoid boiling over when the pressure is released. This may be achieved by gradually
depressurizing the sterilization chamber and allowing liquids to evaporate under a negative
pressure, while cooling the contents.

CHEMICAL STERILIZATION
Bleach
Chlorine bleach

is another accepted liquid sterilizing agent. Household

bleach consists of 5.25% sodium hypochlorite. It is usually diluted to 1/10
immediately before use; however to kill Mycobacterium tuberculosis it should be
diluted only 1/5, and 1/2.5(1 part bleach and 1.5 parts water ) to inactivate
prions. The dilution factor must take into account the volume of any liquid waste
that it is being used to sterilize. Bleach will kill many organisms immediately,
but for full sterilization it should be allowed to react for 20 minutes. Bleach will
kill many, but not all spores. It is also highly corrosive.
Bleach decomposes over time when exposed to air, so fresh solutions should be made daily.

Glutaraldehyde and formaldehyde

Glutaraldehyde and formaldehyde solutions (also used as fixatives) are accepted liquid
sterilizing agents, provided that the immersion time is sufficiently long. To kill all spores in a
clear liquid can take up to 22 hours with glutaraldehyde and even longer with formaldehyde.
The presence of solid particles may lengthen the required period or render the treatment
ineffective. Sterilization of blocks of tissue can take much longer, due to the time required for
the fixative to penetrate. Glutaraldehyde and formaldehyde are volatile, and toxic by both
skin contact and inhalation. Glutaraldehyde has a short shelf life (<2 weeks), and is
expensive. Formaldehyde is less expensive and has a much longer shelf life if some methanol
is added to inhibit polymerization to paraformaldehyde, but is much more volatile.
Formaldehyde is also used as a gaseous sterilizing agent; in this case, it is prepared on-site by
depolymerization of solid paraformaldehyde. Many vaccines, such as the original Salk polio
vaccine, are sterilized with formaldehyde.

AUTOCLAVE
An autoclave is a device used to sterilize equipment and supplies by subjecting them to high
pressure saturated steam at 121 C (249 F) for around 1520 minutes depending on the size
of the load and the contents. The autoclave was invented by Charles Chamberland in 1879,
although a precursor known as the steam digester was created by Denis Papin in 1679. The
name comes from Greek auto-, ultimately meaning self, and Latin clavis meaning key, thus a
self-locking device.

Uses
Sterilization autoclaves are widely used in microbiology, medicine, podiatry, tattooing, body
piercing, veterinary medicine, mycology,funeral homes, dentistry, and prosthetics fabrication.
They vary in size and function depending on the media to be sterilized.
Typical loads include laboratory glassware, other equipment and waste, surgical instruments,
and medical waste.[5]
A notable recent and increasingly popular application of autoclaves is the pre-disposal
treatment and sterilization of waste material, such as pathogenic hospital waste. Machines in
this category largely operate under the same principles as conventional autoclaves in that they
are able to neutralize potentially infectious agents by using pressurized steam and
superheated water. A new generation of waste converters is capable of achieving the same
effect without a pressure vessel to sterilize culture media, rubber material, gowns, dressings,
gloves, etc. It is particularly useful for materials which cannot withstand the higher
temperature of a hot air oven.
Autoclaves are also widely used to cure composites and in the vulcanization of rubber.[7] The
high heat and pressure that autoclaves allow help to ensure that the best possible physical
properties are repeatable. The aerospace industry and sparmakers (for sailboats in particular)
have autoclaves well over 50 feet (15 m) long, some over 10 feet (3.0 m) wide.

INCUBATOR
In biology, an incubator is a device used to grow and maintain microbiological
cultures or cell cultures. The incubator maintains optimal temperature,humidity and other
conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside.
Incubators are essential for a lot of experimental work in cell
biology, microbiology and molecular biology and are used to culture both bacterial as well
as eukaryotic cells.

Louis Pasteur used the small opening underneath his staircase as an incubator.Incubators are
also used in the poultry industry to act as a substitute for hens. This often results in higher
hatch rates due to the ability to control both temperature and humidity. Various brands of
incubators are commercially available to breeders.
The simplest incubators are insulated boxes with an adjustable heater, typically going up to
60 to 65 C (140 to 150 F), though some can go slightly higher (generally to no more than
100 C). The most commonly used temperature both for bacteria such as the frequently
used E. coli as well as for mammalian cells is approximately 37 C, as these organisms grow
well under such conditions. For other organisms used in biological experiments, such as the
budding yeast Saccharomyces cerevisiae, a growth temperature of 30 C is optimal.
More elaborate incubators can also include the ability to lower the temperature (via
refrigeration), or the ability to control humidity or CO2 levels. This is important in the
cultivation of mammalian cells, where the relative humidity is typically >80% to prevent
evaporation and a slightly acidic pH is achieved by maintaining a CO2 level of 5%.
Louis Pasteur used the small opening underneath his staircase as an incubator.

BACTIRIOLOGICAL STAINING
GRAMS STAIN
Gram staining is a quick procedure used to look for the presence of bacteria in tissue samples
and to characterise bacteria as Gram-positive or Gram-negative, based on the chemical and
physical properties of their cell walls. The Gram stain should almost always be done as the
first step in diagnosis of a bacteria infection.
The Gram stain in named after the Danish scientist Hans Christian Gram (1853 - 1938), who
developed the technique in 1882 and published it in 1884 as a technique to discriminate
between two types of bacteria with similar clinical symptoms: Streptococcus pneumonia (also
known as the pneumococcus) and Klebsiella pneumonia bacteria.

PROCEDURE
1. Get a tissue sample for the Gram stain. For example, if a pneumonia is suspected, get a
sputum sample. If an urinary tract infection is suspected, get a urine sample. If an intestinal
infection is suspected, get a stool sample. If a brain infection is suspected, get a cerebrospinal
fluid (CSF) sample (centrifuge the fluid and obtain the sediment for the Gram stain). If a skin
infection is suspected, get a skin biopsy sample. If an ear infection is suspected, get a middle

ear fluid sample (to increase yield, or the likelihood of finding the bacteria, centrifuge the
middle ear fluid sample to allow it to settle into three layers: supernatant (top), buffy coat
(middle), and red cells (bottom); get the buffy coat layer for Gram stain). Essentially any
human tissue can be obtained for the Gram stain.
2. Add 1-2 drops of the tissue sample onto a glass slide. Spread it evenly on the slide to
form a thin smear, which can be done by sliding the edge of another glass slide across the
glass slide containing the tissue sample. Allow it to air dry.
3. Heat fix the smear, by quickly passing it two to three times through a flame, or heat it
on top of an electric slide warmer. Do not overheat, to avoid distortion. Alternatively, the
smear may be fixed by methanol instead, by adding 1-2 drops of methanol onto the dried
smear, draining off excess methanol, and allowing it to air dry. Methanol has the advantage
that it does not lyse red cells and it minimises damage to host cells, giving a cleaner
background.
4. Flood the smear with crystal violet. Wait thirty seconds. Crystal violet (CV) dissociates
in aqueous solutions into CV+ and chloride (Cl-) ions. These ionspenetrate through the cell
wall and cell membrane of both gram-positive and gram-negative cells. The CV+ ion
interacts with negatively charged components of bacterial cells to stain the cells purple.
5. Gently rinse off the crystal violet with tap water. Do not rinse excessively, which might
remove the stain from Gram positive bacteria.
6. Flood the smear with iodine. Leave for at least three seconds. Iodine, in the form of
negatively charged ions, interacts with CV+ to form large complexes of crystal violet and
iodine (CV I complexes) within the inner and outer layers of the cell. Iodine acts as a
trapping agent to retain the purple crystal violet colour in the cell.
7. Gently rinse off the iodine with tap water.
8. Decolorize by adding alcohol or acetone to the smear while holding the slide at an
angle to allow the decoloriser to drain. Stop when runoff becomes clear, within seconds.
The decolorisation step is critical and must be timed correctly. If left on too long, the
decolorising agent will remove the crystal violet stain from both gram-positive and gramnegative cells.
8. Gently rinse off excess decoloriser with tap water.
9. Flood the smear with safranin counterstain. Wait thirty seconds. Counterstain is pplied
last to stain the decolorised gram-negative bacteria a pink or red shade. Basic fuchsin, which
stains anaerobic bacteria more intensely, may be substituted for safranin, but it is less
commonly used.

10. Gently rinse off excess safranin with tap water.

11. Drain slide and allow it to air dry. The Gram stain is done.
12. Examine the slide under the light microscope. Gram-positive bacteria appear purple as
stained by crystal violet, which is trapped within their thick cell walls. Gram-negative
bacteria appear pink as stained by the safranin counter-stain, as their thin cell walls allow the
crystals violet to wash out during decolorisation. Bacteria are further classified by their
shape under the microscope, most commonly as cocci (spherical) or rods (cylindrical). The
most common bacterial species in each of the four groups thus classified are as follows:

Gram positive cocci are generally either Staphylococci (meaning cocci in clusters) or
Streptococci (meaning cocci in chains).
Gram positive rods include Bacillus, Clostridium, Corynebacterium, and Listeria.
Gram negative cocci are most commonly Neisseria spp.
Gram negative rods are subclassified as follows:
Gram negative coccoid rods (or coccobacilli) include Bordetella, Brucella,
Haemophilus, Pasteurella.
Other Gram negative rods (not coccobacilli) include E. coli, Enterobacter, Klebsiella,
Citrobacter, Serratia, Proteus, Salmonella, Shigella, Pseud omonas, and many others.

ZIEHL NEELSEN STAIN FOR AFB

PRINCIPLE:
As the causative agents for Leprosy (Mycobacterium leprae) and Nocardiasis (Nocardia
asteroides) aremuch less acid and alcohol fast than Mycobacterium tuberculosis bacilli, a
more gentle dewaxing andminimal exposure to organic solvents is required for adequate
staining.

CAUTION: HOT SOLUTIONS, POTENTIALLY TOXIC

METHOD:
(1) Heat slides in slide dryer to facilitate dewaxing.
(2) Take sections to water.
(3) Drain slide and place in a slide mailer with 15 ml of Soft Carbol Fuchsin.
(4) Fill Slide Mailer ( ~ 15 ml volume), so that fluid comes up to slide frosting. Place Mailer
into microwave in a beaker, in case of spillage, leave cap of mailer open.
Microwave on FULL POWER,
First time 10 seconds FULL POWER

Second Time 7 seconds FULL OOWER

(Check slides each time. CAUTION halt if solution boils.)
(5) Allow to stand for 2 5 minutes.
(6) Wash excess stain from slide.
(7)Wash wel l in running water, wipe away excess stain 5 min.
(8) Differentiate individually with 0.5 % Acid Alcohol, control microscopically 1 3 min
(Use 5 % Sulphuric acid for Atypical AFB)
(9) Wash in running tap water. 5 min
(10) Filter on Working Methylene Blue 20 secs
Avoid overstaining as some weak staining bacterial staining can be replaced by methylene
blue.
(11) Wash with water.
(12) Drain and air dry thoroughly.
(13) Dip slides in xylene and mount in DPX.
RESULTS:
Acid Fast Bacilli RED (Leprae bacilli are short rods)
Nocardia RED (Nocardia organisms are long, thin, and filamentous)
Erythrocytes PALE PINK
Background BLUE
REAGENTS:

(1) CARBOL SOFT FUCHSIN

New Fuchsin (CI42520) 1.5 gm
Absolute alcohol 10 ml
Distiller water 80 ml
Glycerol 10 ml
Triton X 100 0.75 ml
Add the New fuchsin to the alcohol in a 100 ml flask and mix, on a magnetic stirrer for 30
minutes.

Add the glycerol and the Triton X 100 to the water and mix till dissolved. Add to the New
Fuchsin
Mixture and continue mixing on the stirrer overnight. Filter and store in a brown glass bottle.

(2) STOCK METHYLENE BLUE

Methylene Blue (CI52015) 2 gm
Using a magnetic stirrer dissolve in 100 ml distilled water
Then add absolute alcohol 100 ml

(3) WORKING METHYLENE BLUE

Stock Methylene Blue 40 ml
Distilled water 60 ml
Glacial acetic acid 0.5 ml

(4) 0.5 % ACID ALCOHOL

Distiller water 700 ml
Absolute alcohol 300 ml
Hydrochloric acid 5 ml

(6) 5% SULPHURIC ACID

Distilled water 475 ml
Sulphuric acid 25 ml

RESULTS:
Acid-fast bacilli
Background

bright red
blue

Inoculation of Culture Media

For the effective detection of the bacterial content of specimens, it is important to achieve
growth of individual colonies by using a good technique to inoculate the specimen on culture
media. There are many variations and personal preferences for plating out, some of which
are described in this Guidance Note.

All culture media should be checked before use for contamination and expiry date. Culture
media should have an identifiable batch or quality control number and have passed QC tests
before use. Plates that are beyond their expiry date, contaminated plates, and broth media
appearing unusually turbid should be discarded.
The initial area inoculated should cover between a quarter and a third of the total area of agar
used. Whole plates, half plates, or quarter plates can be used depending on the circumstances.
Specimens may be plated out for individual colonies, or seeded directly over an entire
segment of a plate and incubated without further spreading.
Wire loops should be flamed by holding them loop end down in a Bunsen flame until the loop
and entire wire reach red heat. Place on a rack to cool before use. This should be done before
and after use and between agar plates. It is usual to use two loops, to allow adequate cooling
of one after flaming whilst the other is in use. Different disposable loops should be used for
each plate.
For a potentially heavily contaminated sample, the loop should either be flamed between
each series of streaks, or the loop may be rotated to make the next series of streaks with the
unused side of the loop. For semi-quantitative analysis of urine, the loop should not be
flamed in this way.
All media should be incubated as soon as possible after inoculation. Plates for anaerobic
incubation should be incubated as soon as possible to prevent loss of viability (<15
minutes)4. After inoculation, the specimen, or a portion of it, should beretained for at least 48
hours after the laboratory has issued the final report.5. Most positive culture plates can be
discarded within 24-48 hours of issuing a final authorised report. Cultures of particular
epidemiological value may be retained for longer as organisms may need further work or
referral to a reference laboratory5 .
Stained routine microscope slides should be kept for seven days after issue of the final report.
Slides for examination for Mycobacterium species should be kept locked under level 3
conditions until the final report on the specimen is issued. Positive cultures of
Mycobacterium species should be retained in a locked cupboard in a Category 3 laboratory
until the final report from the Reference Laboratory has been received.

Aseptic Technique
When handling specimens or cultures, aseptic technique is important to avoid their
contamination and to protect the worker from infection from the sample.
In-house training to demonstrate the skills of aseptic technique should be given to staff who
will process specimens or cultures.
Aerosol production should be minimised by
: o Opening caps slowly as the contents of containers are sometimes under pressure
o Avoiding vigorous swirling or shaking of the sample prior to opening

o Cooling loops that have been heated before use

o Avoiding expelling the last drop from a pipette

Swabs - Plate Culture

Initial inoculum should cover between a quarter and a third of the plate to be used . The swab
should be rolled over the inoculation area to maximise transfer of organisms, taking care to
avoid the edges of the plate.
Inoculation of samples to selective media such as Sabourauds agar (when usually only a
quarter plate will be used) may not require spreading with a loop.

Swabs - Liquid Culture

Using aseptic technique, remove the broth container cap, place the swab in the broth, break
off (or cut) the swab-stick and replace the cap. The swab may be placed in the broth directly,
or after inoculating solid culture media (consideration should be given to the possibility that
contaminants may be transferred into the broth from contaminated culture plates).

Fluid Specimens and Pus

The centrifuged deposit of any fluid is re suspended in approximately 0.5mL supernatant,
and then transferred to the appropriate culture media with a sterile pipette. Thick pus may
require inoculation with the aid of a swab/swabstick. If a semiquantitative method is required,
inoculate the media with a standard loop (1L, 10L etc) or a piston-operated pipette as
appropriate.

Urine - Calibrated Loop, Surface Streak Method

The urine is mixed gently to avoid foaming. The end of a sterile calibrated loop (eg 1L, 2L
or 10L) is dipped to just below the surface of the urine and removed vertically, taking care
not to carry over any urine on the shank.

Subculture Methods
Subculture of Liquid Media to a Solid or Liquid Medium
Obtain samples for subculture with a sterile loop (1L, 10L etc) or a plastic pipette.
Immerse the loop in the fluid to be subcultured, and remove carefully without allowing
excess fluid to remain on the shank of the loop. Care should be taken not to contaminate the

loop holder with liquid culture as this will be difficult to sterilize and may cause subsequent
problems with cross contamination.
Either inoculate the loopful of fluid on an appropriate agar plate, streaking out for individual
colonies (Figure 2), or inoculate 2-3 drops from the pipette on appropriateagar plates or to
further fluid culture media. The use of a pipette is particularly recommended when subculturing organisms to multiple culture media, including those used for biochemical tests.
Subculture blood culture bottles according to manufacturer's instructions. Most continuous
monitoring systems require the use of sub-vent units or sheathed needles.

BIOCHEMICAL TESTS
While several commercial systems for identifying bacteria are available, these are often
difficult to obtain or too expensive to use in developing countries. This subunit includes a
range of conventional biochemical tests and tablet identification tests which most district
laboratories will be able to perform.
The following tests are described in this subunit:

Test Purpose

Beta-glucuronidase To identify E. coli

Bile solubility To differentiate S. pneumoniae from other alpha-haemolytic
streptococci
Catalase To differentiate staphylococci from streptococci
Citrate utilization To differentiate enterobacteria
Coagulase To identify S. Aureus
Indole To differentiate Gram negative rods, particularly E. coli
Oxidase To help identify Neisseria, Pasteurella, Vibrio, Pseudomonas
Urease To help identify Proteus, Morganella, Y. enterocolitica, H. Pylori

CATALASE TEST
This test is used to differentiate those bacteria that produce the enzyme catalase, such as
staphylococci, from non-catalase producing bacteria such as streptococci.

Principle
Catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water. An
organism is tested for catalase production by bringing it into contact with hydrogen peroxide.
Bubbles of oxygen are released if the organism is a catalase producer. The culture should not
be more than 24 hours old.

Required

Hydrogen peroxide, 3% H2O2 (10 volume solution)

Method
1 Pour 23 ml of the hydrogen peroxide solution into a test tube
. 2 Using a sterile wooden stick or a glass rod (not a nichrome wire loop), remove several
colonies of the test organism and immerse in the hydrogen peroxide solution.
Important: Care must be taken when testing an organism cultured on a medium containing
blood because catalase is present in red cells. If any of the blood agar is removed with the
organism, a false positive reaction may occur.
3 Look for immediate bubbling as shown in Plate 7.7.

Results
Active bubbling . . . . . . . . . . . . Positive catalase test
No bubbles . . . . . . . . . . . . . . Negative catalase test

Citrate utilization
This test is one of several techniques used occasionally to assist in the identification of
enterobacteria. The test is based on the ability of an organism to use citrate as its only source
of carbon.
Ways of performing a citrate test
Using a Rosco citrate identification tablet. This is the most economical method when only
a few tests are performed. The tablets have a long shelf-life and good stability in tropical
climates.
Using Simmons citrate agar but the dehydrated medium is only available in 500 g pack
size from manufacturers. After being opened the medium does not have good stability in
tropical climates.

Citrate utilization using a Rosco citrate tablet

Citrate identification tablets (code 56511) are available from Rosco Diagnostica (see
Appendix 11) in a vial of 50 tablets.
1 Prepare a dense bacterial suspension of the test organism in 0.25 ml sterile physiological
saline in small tube.
2 Add a citrate tablet and stopper the tube.

3 Incubate overnight at 3537 C.

Results
Red colour . . . . . . . . . . . . . . . . . Positive citrate test
Yellow-orange colour . . . . . . . . Negative citrate test

Coagulase test
This test is used to identify S. aureus which produces the enzyme coagulase. A tube test must
always be performed when the result of a slide test is not clear, or when the slide test is
negative and Staphylococcus has been isolated from a serious infection. A tube test may be
required to detect some MRSA (methicillin resistant S. aureus) strains although some
commercially available latex test kits to differentiate coagulase positive and coagulase
negative staphylococci, overcome this. Before performing a coagulase test, examine a Gram
stained smear to confirm that the organism is a Gram positive coccus.

Results
Clumping within 10 secs . . . . . . . . . . . . . S. aureus
No clumping within 10 secs . . . . . . . . . . No bound coagulase

Indole test
Testing for indole production is important in the identification of enterobacteria. Most strains
of E. coli, P. vulgaris, P. rettgeri, M. morganii, and Providencia species break down the amino
acid tryptophan with the release of indole.

Results
Red surface layer . . . . . . . . . . . . Positive indole test
No red surface layer . . . . . . . . Negative indole test

Oxidase test
The oxidase test is used to assist in the identification of Pseudomonas, Neisseria, Vibrio,
Brucella, and Pasteurella species, all of which produce the enzyme cytochrome oxidase.

Method (fresh reagent)

1 Place a piece of filter paper in a clean petri dish and add 2 or 3 drops of freshly prepared
oxidase reagent.
2 Using a piece of stick or glass rod (not an oxidized wire loop), remove a colony of the test
organism and smear it on the filter paper.
3 Look for the development of a blue-purple colour within a few seconds as shown in colour
Plate 3.

Results
Blue-purple colour . . . . . . . . . . Positive oxidase test (within 10 seconds)
No blue-purple colour . . . . . Negative oxidase test (within 10 seconds)

Urease test
Testing for urease enzyme activity is important in differentiating enterobacteria. Proteus
strains are strong urease producers. Y. enterocolitica also shows urease activity (weakly at
3537 C). Salmonellae and shigellae do not produce urease.

Principle
The test organism is cultured in a medium which contains urea and the indicator phenol red.
When the strain is ureaseproducing, the enzyme will break down the urea (by hydrolysis) to
give ammonia and carbon dioxide. With the release of ammonia, the medium becomes
alkaline as shown by a change in colour of the indicator to pink-red.
Ways of performing a urease test
Using a Rosco urease identification tablet.
Using modified Christensens urea broth.
Urease test using a Rosco urease tablet Urease identification tablets are available from Rosco
Diagnostica (code 57511) in a vial of 50 tablets. The tablets have a long shelf-life (34 y).
1 Prepare a dense milky suspension of the test organism in 0.25 ml physiological saline in a
small tube.
2 Add a urease tablet, close the tube and incubate at 3537 C (preferably in a water bath for a
quicker result) for up to 4 hours or overnight. Proteus and M. morganii organism give a
positive reaction within 4 hours.

Results

Red/purple colour . . . . . . . . Positive urease test

Yellow/orange . . . . . . . . . . Negative urease test

Method using an oxidase reagent strip

1 Moisten the strip with a drop of sterile water.
2 Using a piece of stick or glass rod (not an oxidized wire loop) remove a colony of the test
organism and rub it on the strip.
3 Look for a red-purple colour within 20 seconds. Red-purple colour . . . . . . . . positive
oxidase test.

Note: When using a Merck reagent strip, follow exactly the manufacturers instructions on
how to perform the test.