SUMMARY PRACTICAL GUIDE OF SOME

BASIC LABORATORY TECHNIQUES

FOR

STUDENTS OF MEDICAL LABORATORY

TECHNICIAN

BY MR ALHASSAN EGBA (MLS)

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 THE RESPONSIBILITIES OF THE LABORATORY WORKER
The laboratory worker carries out laboratory examinations/investigations to
provide information for doctors (or their representatives) in order to benefit
patients. He therefore playts an important role in helping patients to get better. At
the same time, in the course of his work he gains a lot of information about
patients and their illness. The laboratory worker, like the doctor, must regard this
information as strictly confidential; only the doctors who requests examination
should receive the report on them. When patients enquires about test report they
should be told to ask the doctor.

In most countries of the world there are high moral and professional standards of
behaviour for doctors and qualified laboratory personnel. Every laboratory
worker handling clinical materials must maintain these standards.

PARASITOLOGY

FORMOL ETHER CONCENTRATION METHOD FOR OVA AND

CYST IN STOOL

METHOD: Formol Ether Concentration

GIVEN: Stool sample, test tube, clean grease, free glass slide, Microscope,
Coverslip, Centrifuge, Applicator, Strainer, 10% formol water.

AIM: To carryout concentrstion for ova & cyst

PROCEDURE:

 About 10ml of 10% formol water was put into a clean test tube.
 1g of stool sample was added and mixed with the use of applicator stick
 Drops of formol water was added.
 It was covered and shaked vigorously.

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  It was sieved to remove the larger particles and the suspension was
collected in a test tube.
 3ml of ether was added and centrifuge at 2,000 revolution per minutes for
5 minutes
 The fatty plug was loosen
 The supernatant was poured out quickly by i8nverting the tube without
shaking.
 The deposit was recollected by the side of the test tube by placing it on
the tube rack for 2 minutes
 An applicator stick was used to pick a droop of the deposit on a clean
slide.
 It was covered with a coverslip and examined under x 10 and x 40
objectives of the microscope respectively.

RESULT:

 Ova of ascaris lumbricoides seen

 Cyst of Entamoeba histolytical seen

NOTE: If no parasite is seen in the preparation; report as

- No parasite seen

EXAMINATION OF HEAMOPARASITE
These are the parasites tha can be found in the blood, which may be carried out
as follows:
1. Direct wet preparation for micro filarial and trypanosomes
2. Thin blood film for micro filarial and malaria parasite.
3. Thick blood film for trypanosomes and malaria parasite.

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 1. WET PREPARATION METHOD

GIVEN: Normal saline, coverslip, blood sample, clean grease-free glass slide

AIM: To Examine heamoparasite on the given blood sample.

PROCEDURE
 A drop of normal saline was placed on a clean slide
 The blood sample was mixed gently, few drop was added to the normal saline
 It was covered with a cover slip.一
 It was examined under the microscope一using x 10 and x 40 objectives
respectively.

2.THIN BLOOD FILM

GIVEN: Blood sample, Spreader,Clean slide
AIM: To examine haemoparasite on the givenblood sample.

PROCEDURE
i. A drop of blood was placed at one end of the clean slide.
ii. Cover spreader was used to touch thedrop of blood.
iii. it was allowed to spread to the edge.
iv. It was slanted at an angle of 45° and thenpushed forward in a gently
steady motionto make a thin film.
v. The slide was placed on the bench to air dry.

3.THICK BLOOD FILM

GIVEN: Blood sample,Spreader,Clean slide
AIM:To examine haemoparasite on the givenblood sample.
PROCEDURE

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 i. 2 drops of blood was placed on a cleanslide.
ii. The corner of spreader was used to makea circular motion.
iii. It was mixed and allowed to cover an areaof about 2 cm in diameter.
iv. It was placed on the bench and allowed toair dry.

NECESSARY PRECAUTION

1. Protect from flies

2. Protect from dust
3. Avoid using large quantity of blood sample.
4. Allow proper air dry.

EXAMINATION OF FAECAL SPECIMEN

The identification of Intestinal Parasite in faecal specimen.

NECESSARY PRECAUTION

Appearance: colour of the faeces

Consistency: semi, soft or hard form, watery, presence of blood mixed with
faeces

The direct or wet preparation is examined with a microscope.

GIVEN: Grease free slide, Cover slip, Applicator Stick, Microscope, Normal
saline, Iodine, stool sample

AIM: To examine the given stool sample microscopically

PROCEDURE:

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 A drop of normal saline and iodine were dropped at the end of a clean
grease free slide respectively.
 Using an applicator stick small amount of feaces were emulsified in the
saline and iodine respectively
 Each preparation were examined under the microscope using x 10 and x 40
objectives respectively
RESULT
— Ova of ascaris
— Cyst of ciliates
ADVANTAGES

1. It is fast.
2. t is cheap
3. It is easy to perform

HOW TO MAKE THIN BLOOD FILM

— A drop of blood was placed on one end of slide

— Cover spreader was used to touch the drop of blood
— The spreading was allowed to spread to the edge
— It was slanted at angle 450
— The spreader was pushed forward gently, in gentle steady motion to
make a thin film
— It was allowed to air dry

HOW TO MAKE THICK BLOOD FILM

— A drop of blood was placed on a clean slide at the centre

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 — With the corner of another slide it was used to make a circular
motion to mix the drop of blood and spread to cover an area of about
2cm in a diameter
— It was left on the bench, and allowed to air dry.

Precaution

i. Allow proper air dry

ii. Protect from flies and dust
iii. Little quantity of blood should be used.

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HAEMATOLOGY

ANTICOAGULANTS
Anticoagulants are chemical substances which remove or inactivate a number of
factors which are involved in clotting of blood thereby preventing coagulation to
take place. Anticoagulant can be described as a chemical, dried or liquid, that
prevents blood from clotting immediately blood is collected into bottle containing
a known anticoagulant. Anticoagulants used in haematology are:-
1. Ethylene Damine Tetra acetic Acid [E.D.TA]
2. Trisodium citrate
3. Ammonium and potassium oxalate mixtures (Heller and Paul mixtures) or
[double oxalate].
4. Heparin.
ETHYLENE DIAMINE TETRA ACETIC ACID
 Practical Guide for MLS
 E.D.T.A also known as sequestrene is a chelatin agent. Three salts are available
namely:-
 Disodium salt

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 Dipotassium salt
 Dilithium salt

MODE OF ACTION
 E.D.T.A removes calcium ions (Ca) which ions (Ca++) which is essential for
clotting stage (Thrombokines for thrombin).
 This is done by binding it and preventing further the ionization of Ca, this process
is called chelation i.e. binding; for blood to coagulate there is need for the factors
to be present.
 Thrombokinase
 Prothrombin
 Fibrinogen
 Calcium

PREPARATION AND COMPOSITION

E.D.T.A salt – 10g

Distilled water – 100ml

It is prepared as 10% solution i.e. 10 g per 100 ml of distilled water.

It is used at concentration of 1-2 mg per ml of blood.

ADVANTAGES

— Preventing platelets clumping.

— Red blood cells, white blood cells and packed cell volume (PCV)
estimation can be done even after 24 hrs.
— Morphology of red blood cells preserved.

DISADVANTAGES

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- It’s unsuitable for coagulation studies because of complete removal of calcium,
prevent adhesion which is a measure of their activity.

- It cannot be used for anticoagulant therapy.

STAINING PROCEDURES

LEISHMAN STAINING PROCEDURE

— A thin blood film was prepared.
— It was allowed to air - dry.
— It was flooded with leishman stain for 2 mm.
— It was double-diluted with Buffer solution (PH & minutes.
— It was differentiated with buffer solution.
— It was allowed to drain dry at room temperature.
— The back of the slide was cleaned and examine under x 100 objective.

RESULT:

 Nuclei of leucocyte - Purple

 Eosinophil granules - Orange-red or pink
 Basophil granules - Dark blue

GIEMSA STAINING PROCEDURE

 A thin blood film was pre-pared.
 It was allowed to dry.
 It was flooded with 1 in 10 diluted giemsa stain.
 The flooded film was allowed to stand for 10 minutes.
 It was rinsed and differentiated with buffer solution.
 It was allowed to drain dry at room temperature.
 The back of the slide was cleaned and examined under x 100 objective.

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RESULT:
Cytoplasm of lymphocyte - Violent - purple
Erythrocyte - yellowish red
PREPARATION OF LEISHMAN'S STAIN
CONSTITUENTS
Solution I
i. Methylene blue - 1.0g
ii. 0.5% sodium bicarbonate - 100 ml
iii. 0.1% eosin - 100 ml
iv. Absolute methyl alcohol - 100 ml

Solution II (Buffer solution of pH 6.8)

 Disodium hydrogen phosphate (Na2HPO4) - 49.6 mi

 Potassium dihydrogen phosphate (KH2PO4) - 50.4 ml

Note: Dissolve the phosphate in water and check the P.H

PREPARATION

 Dissolve the methylene blue in sodium bicarbonate solution.

 Heat at 65°c for 12 hours.
 Allow to cool and allow to stand for 10 days.
 Add an equal volume of eosin.
 Mix well and allow the mixture to stand for 6 — 12 hours
 Filter and collect the precipitate.
 Wash the precipitate with several changes of distilled water until no more
colours is extracted.
 Dry the precipitate in 37°c.
 Incubate & grind into powder form in glass mortar.
 Weigh out 0i. g of the powder and triturate in a mortar with methyl alcohol.

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  Pour off the supernatant and add more methanol until all the powder is
dissolved.
 Make the volume to 100 ml with methanol and store in a tightly stoppered
dark bottle and label.
 Allow the stain to stand for 24 hours before use.

SICKLING TEST FOR ABNORMAL HAEMOGLOBIN

PRINCIPLE
One drop of blood is mixed with one drop of Sodium metabisulphate reagent, on
a slide, if the red cells contain abnormal haemoglobin called haemoglobin S, they
will become sickle shape i.e. half moon shaped.
Sodium metabisulphate:- removes oxygen from the cells, allowing sickling to
take place.
GIVEN: Glass slide, Cover slip, Pasteur pipette 20g Sodium Metabisulphate,
Petri dis Blood sample, Filter paper
AIM: To detect abnormal haemoglobin
PROCEDURE
— A small drop of capillary blood was dropped at the centre of a slide.
— An equal drop of sodium metabisulphate was added.
— It was covered with a cover slip.

— It was placed in the petri dish that has wetted filter paper in the bottom
— It was allowed to at for 15 minutes.
— It was examined under x 40 objectives

RESULT
Negative:- The red remain round
Positive:- The red cell appears sickle shape or banana shape

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 WESTERGREEN METHOD OF ESR
This method makes use of the following; westergreen tube, westergreen rack, rubber teat, citrated blood
(1ml of sodium citrate to 4 ml of blood).
GIVEN: Westergreen rack, Rubber teat, Citrated blood
AIM: To carry out ESR using westergreen method

PROCEDURE

 The citrated blood was mixed properly.

 Rubber teat was used to fill the westergreen tube by sucking
in blood to reach 20 cm marked.
 It was allowed to stand in westergreen tube for hour.
 It was read after 1 hour the level of fall of plasma was
expressed in mm fall per 1hr westergreen.

RESULT
Men: 3 — 5 mm fall/1 hr westergreen
Women: 4 — 7 mm fall/1 hr westergreen

FACTORS AFFECTING E.S.R

1. Physical factors
2. Physiological factors
3. Pathological factors

PHYSICAL FACTORS

1. Vibrauon or disturbance of sedimentation.

2. Presence of clot in the sample.

3. Haemolysed blood.
4. Short Pasteur pipette being used in filling the tube.

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5. Presence of direct sunlight on the tube.
6. Prolong venous congestion.
7. Reading the tube before or after Inc scheduled time.
8. High room temperature.
9. Presence of air bubbles when tilling the tubes.

PRECAUTIONS

1. Avoid air bubbles.

2. Avoid vibration of the bench.

3. Read the level of fall at the appropriate time
4. Use of clean tubes.

HAEMOGLOBIN ESTIMATION

CYANMETHAEMOGLOBIN METHOD

Principle: Haemoglobin is converted to methaemoglobin by potasium ferric

cyanide which is further converted to cyanmethaemoglobin by the action of
potassium cyanide
GIVEN: Drabkin's solution, Anticoagulated blood, test tubes
AIM: To carry out haemoglobin estimation
PROCEDURE
— 4M ml of Drabkin's solution was poured into a test tube.
— 0.02 ml of blood was added.
ADVANTAGES

1. The standard can be easily prepared, stable and convenient for routine use.
2. It eliminates colour blindness.
3. All forms of haemoglobin are converted to methaemoglobin.

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 DISADVANTAGES

1. It is poisonous.

2. It is not fast
3. The reagent is expensive.

WHITE BLOOD CELL COUNT

GIVEN: 1 ml graduated pipette, Blood sample, Counting chamber and cover

slip; Turk’s solution, Microscope, Test tube

PROCEDURE

— 0.38 ml of diluting fluid was poured into a clean test tube.

— 0.02 ml of blood was added, it was mixed gently together
— Cover slip was placed properly on the counting chamber to form
rainbow colour or Newton’s ring.
— The diluted blood was mixed well using a Pasteur pipette to charge
the counting chamber
— The chamber was left on the bench for 2 — 3 minutes for the cells
to settle.
— The chamber was placed. on the microscope stage and counted with
x 10 objectives
— The four corner squares were counted
— It was calculated properly.
Normal value
Man and woman: 4,000 - 11,000
Children: 4,000 - 11,000
Infant: 4,000 - 15,000

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 NECESSARY PRECAUTION
 The blood and diluting fluid must be mixed properly
 The chamber should be left on the bench undisturbed so as to all the White
Blood settle.
 The end of the tube should be well seated.
 The tube should be handled with care, its very fragile

ADVANTAGES
1. It is fast
2. It is cheap
MICRO PIPETTE FOR WHITE BLOOD CELL COUNT
GIVEN: Diluting fluid, Blood and cotton wool, Microscope, Thoma pipette

AIM: To count white blood cell

PROCEDURE

— The pipette was filled with anticoagulated blood up to 0.5

— The tip of the pipette was cleaned with cotton wool, avoiding air bubbles.
— Diluting fluid was sucked into the rubber teat up 11 cm marked.
— The rubber teat was detached and mixed properly
— The fluid was expelled at the stem of the pipette leaving only that of bulb and
charges the counting chamber.

PACKED CELL VOLUME (PVC)

There are two methods used for the estimation PCV, which include:
1. Micro haematocrite method
2. Wintrobe method

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 Plasma

Buffy coat (Platelets and WBC)

PCV

Sealant

MICRO HAEMATOCRITE METHOD

GIVEN: Anti coagulated-Blood, Micro haematocrite reaaer, Plasticine, Cotton
wool, Plain capillary tube, Micro haematocrite centrifuge.

AIM: To carry out white blood cell count.

PROCEDURE

— The plain capillary tube was filled with anticoagulated blood.

— The outer part of the tube was cleaned with cotton wool.
— One end of the tube was sealed with plasticine
— The lid of the micro haematocrite centrifuge was opened.
— The tubes were arranged on the number plate of the centrifuge.
— The lid of the centrifuge was covered and screwed down
— It was spun for 5 mm at 10,000 revolution per minute
— The light was switched off and the centrifuge was allowed to stop on its own
— The lid of the centrifuge was opened and the capillary tubes were brought out.
— It was read on the reader.

HOW TO USE MICRO HAEMATOCRITE READER

1. Place the capillary on the shading groove on the micro haematocrite reader
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2. Position it in such a way that the bottom of the red cell along the tubes
corresponds to the zero mark, and the top of the plasma fall on the sliding lines
correspond to 100 mark
3. Move the adjustable pointer to fall at interface between the top level of the
RBC and that of bottom of the plasma.
4. Read the height of the RBC as the PCV then express in percentage.
Normal range
Men: 40 - 54%
Women: 36 - 47%
Infant: 44 - 62%

WINTROBE METHOD
GIVEN: Wintrobe tube, Long Pasteur pipette, Anti coagulated blood, Centrifuge
AIM: To carry out

PROCEDURE
 The wintrobe tube was filled with blood using Pasteur pipette
 The pipette was allowed to reach the bottom the tube
 The tube was filled with blood to 10 cm mark the top level
 Air bubble was avoided
 The tube was placed inside the centrifuge buck
 And balanced with other blood
 The light was switched on and it was spun for Minutes at 3,000 revolution
per minute
 The centrifuge was allowed to stop on its own
 The tubes were brought out of the centrifuge
 The level of fall was read and expressed Percentage (%)
Note: the method does not need reader Since wintrobe is calibrated

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