2.

GENERAL LABORATORY
DIAGNOSIS OF PARASITIC DISEASES
 OUTLINE
Morphological diagnosis

Immunological diagnosis

Molecular diagnosis

Other techniques

Specimens for parasitological diagnosis

 Learning objective
Upon completion of this unit, the student will be able to:
List the common techniques used for the detection of parasites

in fecal, blood and other specimens.

List the types of microscopic methods used in parasitological

investigations.
Discuss the importance of microscope calibration
Describe the difference between immunodiagnostic techniques
Discuss the different parasite concentration methods
Discuss the significance of molecular diagnosis in the diagnosis

parasitic diseases.
Before performing lab test give attention for
Selecting parasitology tests appropriately
Collecting, transporting and storing specimens
correctly
Ensuring the request form is completed correctly
 Checking the specimen and request form when they
reach the laboratory
correct collection and transport
● Use specimen containers that are leak-proof, clean,
dry, free from traces of antiseptics and disinfectants
● If an anticoagulated blood specimen is required, use
a suitable anticoagulant,
e.g. sodium citrate for microfilariae and EDTA
(sequestrene) for malaria parasites and trypanosomes.
Mix the blood well but gently with the anticoagulant.
● Protect specimens from direct sunlight and heat.
● Ensure specimens arrive in the laboratory as soon as
is feasible after they are collected.
Some specimens must be delivered to the laboratory
for rapid examination within 15–20 minutes
– a dysenteric faecal specimen or rectal scrape for the
detection of motile E. histolytica trophozoites.
– cerebrospinal fluid for the detection of motile
trypanosomes.
– discharge specimen for the detection of motile T.
vaginalis
● When needing to transport specimens to a specialist
parasitology laboratory, use a suitable fixative or
preservative
● Label the specimen carefully with the patient’s name
and identification number, and also the date and time
of collection.
request form

A correctly completed request form (the patient’s

name and identification number, date and time of
collection & test required…)must accompany all
specimens.
it is important to provide any drugs given or taken by
the patient before the specimen was collected.
For epidemiological purposes, the patient’s village or
locality should also be written on the request form.
Specimen rejection criteria
Properly unlabeled sample should be rejected
Delayed sample
Contaminated sample stool with soil, water, urine
Insufficient sample volume
Incorrect sample (saliva rather than sputum )
 Clotted sample ( blood)
Incomplete request form
General laboratory diagnosis of parasitic diseases
1. Morphological diagnosis (Macroscopic /Microscopic
diagnosis)

2. Immunological diagnosis (Antibody and antigen

detection

4. Molecular diagnosis

5. Culture

6. Animal inoculation

7. Xenodiagnosis
Morphological diagnosis
 Laboratory procedures detect organisms within clinical

specimens using morphological criteria,

A. Macroscopic: using the naked eye

 For the presence of some adult worms

E.g. Ascaris , E. vermicularis and gravid Taenia spp.

B. Microscopic
The majority of intestinal, blood, urinary and skin parasites

are usually detected microscopically

Stained or unstained preparation of different specimens

(such as blood, stool, urine, CSF, etc…) are examined either:

Directly or following

Concentration techniques
Parasitology staining methods

There are three categories of chemicals used to preserve

stool, prepare slides for staining and stain the
preparation:

1. Fixatives

2. Preparatory reagents

3. Stains
Parasitology staining …..

Temporary stains Permanent stains

1. Eosin
1.Auramine Phenol
2. Saline
2.Field’s stain (A/B)
3. Acridine Oragne
3.Giemsa stain
4. Lugol’s Iodine
5. Thomson’s stain 4.Iron Hematoxylene (A/B)
6. Sargeant’s stain 5.Trichrome

7. Burrow’s stain 6.Modified Trichrome

7.Modified Ziehl - Neelsen
Fixatives: Why fixation ?
1. Sodium Acetate - Acetic Acid - Formalin Fixative
(SAF)
SAF fixed material is suitable for:
Direct examination

Concentration and

Permanent staining.

A ‘pea sized’ sample (0.5g) of faeces or 1ml of loose stool

should be added to about 5ml of SAF fixative.

Mix thoroughly
 Preparation of SAF preserved smears

1. Wash specimen at least twice – three times in saline

 Centrifuge after each wash at 1000g for at least 1 minute

2. Shake the Mayer’s albumin (MA) with glycerine.

 Place a small drop on the centre of a pre-labelled slide.

3. Transfer a drop of the washed sediment onto MA & mix well

4. Spread the mixture over the slide

5. Allow the slides to dry thoroughly before staining.

6. Proceed to Staining Method.

Preparation of smears for staining from fresh
stool specimens
1. Place a small drop of well-mixed MA on a slide.

2. Prepare a saline-washed sediment (using Physiological Saline.

3. Add a small amount of wet sediment to a drop of MA and

prepare a smear.

4. Place the smears directly into Schaudinn’s fixative for 1 hr or

24 hr
 Preparation of smears…

5. Wash the smears in alcoholic iodine solution for 10 minutes.

(coagulate albumin and fix with slide)

6. Proceed to the first 70% alcohol as outlined in the Iron

Haematoxylin Staining Procedure.
 Fixatives

Preparation of smears for staining from SAF-preserved

stool specimens
The Ethyl Acetate concentration method for ova and cysts

should be used.
1. Each stool specimen should be treated on an individual basis
depending on its composition.
2. Centrifuge for 10 minutes

3. When using SAF as a preservative, it is essential to wash the

specimen two or three times
 Preparatory Reagents
Mayer’s Albumin (Egg Albumin + Glycerol-Thymol as
Preservative)
Used when preparing slides for staining.
Albumin for adhesion to the slide
Glycerin retains moisture to prevent distortion of organisms
Method
1. Mix the MA well. Place a drop in the centre of the slide.
Add a drop of washed sediment to the MA & mix well.
2. Spread the mixture on the slide ensuring gradual thickness.
3. Allow the slides to dry well at room temperature
 Stains
1. Acridine Orange (In Acetate Buffer pH4.0)

Method
1. Prepare Faecal Concentrate using Parasep®.
2. Add an equal volume of stain to Faecal Concentrate.

3. Mix and allow to stain for 30 minutes.

4. Spread small amount of deposit on slide-cover slip and

examine under UV light.
Result
 Chromidial Bars Fluoresce - bright green
 Stains
2. Auramine Phenol (Lempert)
Method

1. Make faecal smears as for ZN and fix in methanol.

2. Stain with Auramine-Phenol (Lemperts) for 10 - 15 min.

3. Rinse thoroughly in tap water.

4. Decolourise in acid alcohol (as for ZN).

5. Rinse thoroughly in tap water.

 Method…
6. Counterstain with 0.1% potassium permanganate for 30 sec.

7. Rinse thoroughly in tap water, allow to air dry. Do not blot

dry, many brands of blotting paper will fluoresce!

Results
 Oocysts appear as bright yellow discs against a dark

background.
3. Field Stain A & Field Stain B

 Enables rapid staining of fixed thin films.

 This particular method is very useful for staining films of

unformed feces, fecal exudates, duodenal aspirates etc.

Method

1. Make a thin film of feces/exudate and allow to dry.

2. Fix in methanol for 1 min.

3. Flood slide with 1 ml of Field's stain B.

 Method…
4. Immediately add an equal volume of Field's stain A mix well on
slide and allow to stain for 1 min.

5. Rinse well in tap water and drain dry.

6. Examine the film using the oil immersion objective and

immersion oil
Results
 Parasite nuclei and structures containing chromatin - red
 Cytoplasm - bluish-grey
 Leukocyte nuclei - purple
 Yeasts and bacteria - dark blue
 4. Giemsa Stain - Rapid
Giemsa stain can also be used to stain films of unformed feces,

fecal exudate, duodenal aspirates etc.

Method
1. Make a thin film of faeces/exudate. Allow to dry.
2. Fix in methanol for 1 min.
3. Tip off the methanol and flood the slide with Giemsa stain
diluted 1:10 with buffered distilled water pH 7.2 The diluted
stain must be freshly prepared each time.
4. Stain for 20-25 min.
 Method…
5. Run tap water on to the slide to float off the stain and prevent
deposition of precipitate on to the film. Allow to drain dry.
6. Examine the film using the oil immersion objective and
immersion oil.

Results
 Parasite nuclei and structures containing chromatin - red
 Cytoplasm - bluish-grey
 Leucocyte nuclei - purple
 Yeasts and bacteria - dark blue.
5. Lugol’s Iodine (Aqueous)
Temporary Stain for Protozoa.

Method
1. Make wet preparations following concentration by the
formol-ether method with Parasep®.
2. Add an equal volume of Lugol's Iodine to 25% glacial acetic
acid.
3. Place a drop of the wet preparation on a slide and add a drop
of the Iodine/acetic acid mixture prepared in 2 above.
4. Cover slip and examine.
Method…

Result
Iodine Stains:

Glycogen - brown

Nuclear Chromatin of Amoebic Cysts - brown/black.

6. Iron Haematoxylen Solution A/B

Method: preparation of Working Iron Haematoxylin Solution

1. Mix equal volumes of the two solutions and filter.
2. Allow to stand at least two hours.
Parasite stain blue if used immediately after preparation
Mature stains stain light blue with grey back ground
If a slide appears cloudy, then dehydration has been inadequate.
Agitation in the final alcohols can improve the clarity of the smear.

The stain is normally useable for a week. Shelf life can be extended
by storage in a stoppered bottle in the dark after each use.
Procedure: Iron Haematoxylin Staining

1. Prepare smear using Mayer’s Albumin

2. Treat with 95% ethanol for at least 10 minutes(to coagulate
albumin, fixing smear to slide)
3. Slides should be brought to water stepwise through 70% and 25%
alcohols.

4. Stain slides for 10 minutes in Haematoxylin stain

5. Wash for 30 seconds in distilled or de-ionised water.

6. Treat for 10 minutes using 50% saturated Picric Acid (destains

background).

7. Wash slides in running tap water for at least 10 minutes.

Procedure…

8. Transfer to alkalinised tap water for 2 minutes.

9. Treat for 10 minutes in alkalinised 70% alcohol).
10. Bring to 100% alcohol stepwise through 95% and absolute
alcohol (twice)
11. Clear in Xylene for at least 10 minutes twice.
12. Mount in DPX or any similar neutral mounting medium.
13. Examine using Immersion Oil under oil immersion lens.
7.1 Trichrome for Microsporidia

Method
1. Make smears from unconcentrated stool specimens.
2. Fix in methanol for 5 mins.
3. Stain in modified Trichrome stain for 90 mins.
4. Rinse in acid alcohol for 10 sec then in 95% alcohol.
5. Place in 95% alcohol for 5 mins.

7. Place in 100% alcohol for 10 mins.

8. Clear in Xylene for 10 mins.

9. Examine under x 100 oil immersion objective.

Method…

Interpretation
Microsporidial spores are ovoid, refractile and the spore wall

stains bright pinkish-red.

The background debris & bacteria are stained faint green.
7.2. Trichrome For Protozoa

May be used to stain fresh faeces, prefixed faeces or cultured

organisms.
The method varies slightly depending on the sample

preparation used.

Method

1. Make thin smears of fresh stool or cultured amoebae on a clean

microscope slide and immediately fix in Schaudinn’s Solution*
Method…

Leave for at least 30 mins, preferably overnight.

2. Transfer to 70% ethanol for 5 mins).

3. Transfer to alcoholic-iodine for 2 mins.

4. Transfer to 70% ethanol for 5 mins.

5. Transfer to 70% ethanol for 2-5 mins.

6. Transfer to Trichrome stain for 10 mins.

Method…

7. Differentiate in 0.5% acetic acid/alcohol for 1-2 secs.

8. Transfer to 100% ethanol for 1 sec only.

9. Transfer to 100% ethanol for 2-5 mins.

10. Transfer to 100% ethanol for 2.5 mins.

11. Transfer to xylene for 2-5 mins.

12. Transfer to xylene for 2 mins.

13. Mount with DPX or similar neutral mountant.

2.2 Immunological Diagnosis (Immunodiagnosis)

Pertaining to diagnosis by immune reactions.

Is based on the detection of :

A. Antibody (Ab) from person's serum

 Ab is produced in response to a particular parasitic
infection.
2.2 Immunological …

B. Antigen (Ag) detection

 Ag. is excreted by parasites and can be found in the
serum, urine, CSF, feces or other specimens.
 Antigen tests provide evidence of present infection
Why Immunodiagnostic techniques are required?
Parasites live in the tissue of internal organ and can not easily
obtained for examination.
Parasites can be found in specimens only in certain stages of
infection,
 E.g. in the acute stage not in the chronic stage.

Parasites are present intermittently or in too few numbers to be

easily detected in the specimens.
The techniques used to detect parasites are complex or time
consuming.
Immunodiagnosis is of particular value for:

South American trypanosomiasis •Amoebic liver abscess

, Chronic stage •Trichinosis

African trypanosomiasis, when •Toxoplasmosis

•Toxocarisis
parasitaemia is low
•Hydatid disease
Leishmaniasis
•Schistosomiasis
Filariasis
• Malaria
2.3 Culture

Growth medium is provided for a specific parasite.

A specimen is tested for the presence of an infectious agent

able to grow within that medium

Culture allows identification of infectious organisms by

examining their
Microscopic features,

Substances produced by the pathogens, and

 Directly identifying an organism by its genotype

2.3 Culture…

Relatively few of protozoa and helminths parasites, can be

cultured.
E. g. E.histolytica, T.vaginalis, T.cruzi and Leishmania

species
2.4 Xenodiagnosis (XD)
I. Procedure involving the feeding of laboratory-reared
triatomid bugs on patients suspected of having Chagas’
disease; after several weeks the faeces of the bugs are
checked for intermediate stages of Trypanosoma cruzi.
II. Diagnosis of trichinosis by means of feeding laboratory-bred
rats or mice on meat suspected of being infected with
Trichinella, and then examining the animals for the parasite.
2.5 Molecular Diagnosis
Microscopic examination is still considered the “gold

standard” for the diagnosis of parasitic diseases. 

The stool specimen can be analyzed using molecular

techniques such as polymerase chain reaction (PCR). 

PCR amplified fragments can be analyzed by using:

 Restriction fragment length polymorphisms (RFLP) or

DNA sequencing if further characterization is needed.

2.6. Specimens for parasitological diagnosis

2.6.1. Examination of stool

2.6.2. Examination of blood

2.6.3. Examination of urine

2.6.4. Examination of other specimens

2.6. 1. Examination of stool

What is stool?
Feces comes from the Latin word "faex,“
It means "dregs." Dregs means the most undesirable part.
 Stool comes from the Anglo Saxon word "stol," which means
"seat”
Feces (Stool): waste product or substance formed in the

digestive tract and excreted out through the rectum (rear

end).
 Specimen collection

1. Collect the stool in a dry, clean, leak-proof container. 

2. Not contaminated with urine, water, soil or other material
 Specimen collection…

3. Fresh stool should be examined, processed, or preserved

immediately. 
4. Preserve the specimen as soon as possible. 
 Add one volume of the stool specimen to three volumes of

the preservative.

5. Insure that the specimen is mixed well with the preservative. 

6. Insure that the specimen containers are sealed well. 

 Specimen collection…
5. Certain drugs and compounds will render the stool specimens
unsatisfactory for examination. 
 Antacids, kaolin, mineral oil and other oily materials, non-
absorbable anti-diarrheal preparations, barium or bismuth
(7-10 days needed for clearance of effects)
 Antimicrobial agents (2-3 weeks)

 Gallbladder dyes (3 weeks)

6. Specimen collection may need to be repeated if the first

examination is negative.
 
Preservation of fecal samples

Preservation of parasites in faecal samples is not only

important for maintaining parasite structure during

transportation but also as a means of preserving parasites for
future quality control and training purposes.
The following preservatives are most commonly used in

clinical laboratories and have different effects on the various

stages of parasites.
1. 10% formalin fixative in water

Formalin (10%) in distilled water:

This preservative is a good overall fixative and will fix both ova

and cysts although it only preserves the internal morphology

of the cysts for up to 6 months, after which the cytoplasm of
the organism becomes granular with poor nuclear definition.
Trophozoites do not preserve well in formalin and parasite

morphology is not maintained adequately for a permanently

stained faecal smear.
2. Formalin 10% neutral saline buffered
Formalin (10%) in 0.85% saline:
This preserves the morphology of cysts better than that of 10%

formalin solution.
As with 10% formalin, trophozoites do not preserve well in

formalin-saline and parasite morphology is not maintained

adequately for a permanently stained faecal smear.
It is recommended that 1 part of stool be mixed with 3 parts of

formalin/formalin-saline preservative for the storage of bulk

specimens.
3. Bayer’s solution
A mixture of CuCl2 in 20% v/v formaldehyde and Glacial acetic

acid.
Dilute stock solution 1 in 10 with distilled water before use. 1

part of faeces is mixed with 1 part of Bayer’s solution.

This technique is useful for the preservation of cyst

morphology.
4. Merthiolate – Iodine - Formalin (MIF)
A mixture of Glycerol, formaldehyde and thimerosal (or

Tincture of Merthiolate 1:1000) in distilled water.

For use the stock solution is mixed with Lugol’s iodine.
Add approximately 1 gram of feces to 5 ml of MIF solution and

emulsify well. Ova, cysts and larvae can be preserved in MIF for
several months.
5. Schaudinn’s fixative
Saturated mercuric chloride (HgCl2) in distilled water.
The Stock solution is mixed 2:1 in ethyl alcohol.

For use:
Add 5 ml glacial acetic acid to 95 ml of the stock solution.
Prepare faecal smears without allowing the smears to dry and

place them immediately in Schaudinn’s fixative for 1 hour.

For use…
The reagents used in the preparation of this fixative are

hazardous and should be handled with care.

The working reagents should be prepared fresh for use.
This fixative is particularly good for making permanently

stained faecal smears of protozoan trophozoites.

6. Polyvinyl alcohol (PVA)
95% ethyl alcohol in Glycerol with Schaudinn’s fixative and
Polyvinyl alcohol powder.

To use:
Emulsify 1 part of faeces in 3 parts of PVA solution.
This method will preserve ova, larvae and trophozoites well,

but cysts may show some distortion.

However some ova and cysts do not concentrate well when

preserved in PVA.
To use…
Faecal smears made from the faeces/PVA mixture and allowed

to dry can be used for the permanent staining of trophozoites.

Before staining, the slides must be placed in 70% ethyl alcohol

containing 10 drops of Lugol’s iodine to remove the mercuric

chloride.
7. Sodium acetate-acetic formalin acid
Solution (SAF)

Sodium acetate in Glacial acetic acid with 40% Formalin

solution in distilled water.

This preservative has the advantage of not containing mercuric

chloride which both Schaudinn’s and PVA fixatives contain.

This renders it less hazardous and more user friendly.
This method preserves helminth eggs and larvae, protozoan

cysts and trophozoites.

SAF…

This preservative can also be used for the formol-ethyl acetate

concentration technique and for making permanent faecal

smears.
Purpose of examining stool:

To identify intestinal parasitic infection that require

treatment
To identify chronic infection with serious complication if

untreated
To identify the parasitic causes of blood and mucus in faeces

and differentiate amoebic dysentery from bacterial

dysentery
To assist in surveillance & control of parasitic infection
 Specimen Processing

Stool specimens can be examined fresh or preserved.

A. Examination of fresh specimens
Examination of fresh specimens permits the observation of
motile trophozoites, but this must be carried out without
delay. 
Liquid (diarrheic) specimens (more likely contain

trophozoites) should be examined within 30 minutes of

passage (not within 30 minutes of arrival to lab)
Soft specimens (which may contain both trophozoites and

cysts) should be examined within one hour of passage. 

Specimen Processing…

If delays cannot be avoided, the specimen should be

preserved to avoid disintegration of the trophozoites. 

Formed specimens (less likely to contain trophozoites) can

be kept for up to one day, with overnight refrigeration if

needed, prior to examination.
The following flow chart shows how specimens preserved in

formalin and PVA are processed and tested at CDC:

Specimen Processing…
Specimen Processing…

B. Examination of specimens preserved in formalin

Specimens preserved in formalin can be tested directly (wet

mount, immunoassay, chromotrope stain, UV epifluorescence)

or can be concentrated prior to further testing.
Concentration procedures separate parasites from fecal

debris and increase the chances of detecting parasitic

organisms when these are in small numbers. 
They are divided into flotation techniques and

sedimentation techniques.
Concentration techniques may also be required:
– To detect small parasites like Strongyloides larvae, the
eggs of Taenia, cysts of G. lambli
e.g. small fluke eggs, or the oocysts of intestinal
coccidia prior to staining
– To check whether treatment has been successful
– To quantify intestinal parasites
Specimen Processing…

1. Flotation techniques
 (most frequently used: zinc sulfate ) use solutions which

have higher specific gravity than the organisms to be floated

so that the organisms rise to the top and the debris sinks to
the bottom. 
 The main advantage of this technique is to produce a cleaner

material than the sedimentation technique. 

 The disadvantages of most flotation techniques are that the

walls of eggs and cysts will often collapse, thus hindering

identification.  Also, some parasite eggs do not float.
Flotation technique using sodium chloride solution (Willis)

This method is recommended for the detection of eggs of

Ancylostoma duodenale and Necator americanus (best

method), Ascaris lumbricoides, Hymenolepis nana, Taenia spp.
and Trichuris trichiura.
It is not suitable for the detection of eggs of flukes and

Schistosoma spp., larvae of Strongyloides stercoralis, or

protozoal cysts or trophozoites.
Flotation…
Principle:
The stool sample is mixed with a saturated solution of

sodium chloride (increasing the specific gravity). The eggs are

lighter in weight and float to the surface where they can be
collected

Materials and reagents

Microscope

Microscope slides

Coverslips
Flotation…
Wide-mouth bottle, 10ml

Wooden applicators

Gauze

Petri dishes

95% Ethanol

Ether

Willis solution (reagent no. 64)

Petroleum jelly

Wax.
Flotation…
Method:

1. Place approximately 0.5g of stool in a wide-mouth bottle. Fill

the bottle to the 2.5-ml mark with Willis solution.

2. Using an applicator, crush the portion of stool and mix it well

with the solution. Then fill the bottle to the top with Willis
solution; the suspension should be completely uniform.

3. Place a coverslip carefully over the mouth of the bottle.

4. Check that the coverslip is in contact with the liquid, with no

air bubbles. Leave for 10 minutes.
Flotation…
5. Remove the coverslip with care; a drop of liquid should
remain on it. Place the coverslip on a slide and examine
under the microscope (using the x10 objective) at once
because the preparation dries very quickly. Otherwise seal
the coverslip with petroleum jelly and wax.

Use the fine adjustment of the microscope to examine every

object in the field (eggs tend to stick to the coverslip and are
not immediately distinct).
Other floatation methods include:
ZnS04 floatation used for the concentration of
parasite has a specific gravity (relative density) of 1.180 -
1.200
Its merit for examination of both cyst of protozoa and
eggs of most helminths.
Demerit Operculated eggs of trematodes and
cestodes, infertile ascaris ova and larvae of nematodes
are not concentrated
Saturated sugar floatation
ZnS04 centrifugal floatation
Specimen Processing…

2. Sedimentation techniques use solutions of lower specific

gravity than the parasitic organisms, thus concentrating the
latter in the sediment.
 Sedimentation techniques are recommended for general

diagnostic laboratories because they are easier to perform

and less prone to technical errors.
Sedimentation techniques include: 
Formalin-Ethyl Acetate Sedimentation
1. Mix the specimen well.
2. Strain 5 ml of the fecal suspension (more or less depending on
its consistency) through wetted cheesecloth-type gauze placed
over a disposable paper funnel into a 15 ml conical centrifuge
tube.  (Conical paper cups with the tips cut off are sufficient.)
3. Add 0.85% saline or 10% formalin through the debris on the
gauze to bring the volume in the centrifuge tube to 15 ml. 
Distilled water may be used; however, Blastocystis hominis
may be deformed or destroyed.
Formalin-Ethyl…
4. Centrifuge at 500 × g for 10 minutes.
5. Decant supernatant.  Add 10 ml of 10% formalin to the
sediment and mix thoroughly with wooden applicator sticks.
6. Add 4 ml of ethyl acetate, stopper the tube, and shake
vigorously in an inverted position for 30 seconds.  Carefully
remove the stopper.
7. Centrifuge at 500 × g for 10 minutes.
8. Free the plug of debris from the top of the tube by ringing the
sides with an applicator stick. Decant the top layers of
supernatant.
Formalin-Ethyl…
9. Use a cotton-tipped applicator to remove debris from sides of
the centrifuge tube.
10. Add several drops of 10% formalin to resuspend the
concentrated specimen.  Proceed with applicable testing.
Sedimentation technique for larvae of Strongyloides
stercoralis (Harada–Mori)

Principle:
A strip of filter-paper is partially submerged in a test-tube

containing water.
Any larvae of Strongyloides stercoralis present in the specimen

migrate against the current of water that rises by capillary

action and accumulate at the bottom of the tube.
Harada Mori…

Materials and reagents

Microscope

Cellophane tape

Test-tubes

Test-tube rack

Strips of filter-paper (30mm x 150mm)

Spatula

Lugol iodine, 0.5% solution

Harada Mori…
Method

1. Use the spatula to spread a small quantity of the faecal

specimen along a strip of filter-paper (previously folded
lengthwise to keep it straight), but leave the last 4 or 5cm
clean to be put into water.

2. Put the strip of filter-paper, clean end first, into a test-tube

containing filtered or boiled water 2.5–3.0cm deep; fold the
strip at the top so that the bottom does not touch the bottom
of the tube.
Harada Mori…
1. Record the serial number or name of the patient indelibly on
the tube.

2. Plug the tube with cotton wool or, preferably, seal with
cellophane tape and keep for 7–8 days at room temperature.

3. Look for the larvae at the bottom of the tube. Stain with
iodine solution for 1 minute and then examine under the
microscope, using the x 10 objective.
Harada Mori…
 The larvae usually seen in fresh stool specimens are the

rhabditiform (first-stage) larvae of S. stercoralis.

 However, if the stool was passed more than 12 hours earlier,

the larvae may have hatched into filariform (infective-stage)

larvae.
 These must be differentiated from larvae of Ancylostoma

duodenale and Necator americanus (hookworm), which may

also hatch in stools 12–24 hours after passage.
Harada Mori…
 The appearance of filariform larvae of S. stercoralis may

indicate a systemic hyperinfection.

 The genital primordium will be more visible in preparations

stained with iodine. The iodine kills the larvae and makes
the features easier to see. You will need to use the x 40
objective to see these structures.
 If you see a larva with a short mouth opening and a

prominent (clearly visible) genital primordium, it is S.

stercoralis.
Harada Mori…
If you see a larva with a long mouth opening and do not see a

genital primordium, it is A. duodenale or N. americanus.

Anal swabs for pin worm

Anal swabs are used to detect the presence of pinworms

(Enterobius vermicularis).
Pinworms are more common in children than adults. Often,

however, if one child in a family has pinworms other members of

the family will be infected.
Therefore, if a child is found positive, it is desirable to examine

swabs from all members of the family group, especially the

children
Anal swabs…

Pinworm eggs are usually found in the folds of skin around

the anus. They rarely appear in the stool.

Collection of specimen:
Materials for method A

Centrifuge

Cotton swabs

Microscope slides

Pipettes, Pasteus, with rubber bulbs

Saline solution to moisten cotton swabs

Anal swabs…
Saline solution to moisten cotton swabs

Test tubes, 100 x 13 mm

Technique – Method A

Spread buttocks apart, and rub cotton swab over the area around the

anus, but do not insert into the arus. Place the cotton swab in the tube.
Materials for Method B

Transparent adhesive tape

Tongue depressor /plastic spoon with handle 10 cm long

Microscope slide
 Anal swabs…
Technique – Method B
1. Fold a strip of transparent adhesive tape over the end of a
spoon handle or tongue depressor
2. Separate the patient’s buttocks with the other hand. Press the
end of the spoon covered with the tape against the skin
around the anus in several places
3. Place the tape with the sticky side down on a microscope
slide. Before examining the slide, lift the tape up & place a
drop of immersion oil under the middle of the tape and
replace the tape.
Anal swabs…

 This will improve the transparency of the tape

 Wash your hands after sample collection; otherwise, eggs

that may have contaminated your hands could get into your
mouth and lead to infection.
 In order to increase the chances of picking up eggs, the swab

should be taken between 22hr and midnight, or early in the

morning before the patient urinates defecates, or batches. It
may be necessary to collect several swabs before a positive
diagnosis can be made.
Anal swabs…

 Tape – swab slides may be examined directly. For cotton

swabs, proceed as follows:

1. Into the tube containing the swab, pour enough saline to

cover the cotton swab (about 5ml)

2. Let stand for 4 – 5 minutes

3. Remove the swab from the saline. Roll it against the side
of the tube to squeeze out the saline

4. Discard the swab

5. Concentrate by centrifugation for 1 minute

Anal swabs…

6. With a pipette, remove the supernatant fluid carefully so

as not to disturb the small quantity of sediment present

7. With a pipette, transfer the sediment to a slide for

examination. Reduce the microscope illumination and
focus up and down to see the Enterobius eggs
Cellophane fecal thick – smear for diagnosis of intestinal
Schistosomiasis (Kato – Katz technique
The cellophane fecal thick – smear examination technique
has proven to be an efficient means of diagnosis of
intestinal schistosomiasis and intestinal helminthes.
Cellophane thick – smear slides can be prepared in the
field, stored in microscopic slide boxes, and shipped great
distances, for examination at a central laboratory if
required.
 Cellophane…
The technique is not suitable for examining larvae, cysts,

or eggs from certain intestinal parasites

Materials and reagents:

Applicator sticks, wooden

Screen, stainless steel, nylon or plastic (60 – 105 mesh)

Template, stainless steel, plastic or cardboard

Microscope slides
Cellophane…
Cellophane, 40 – 50 m thick, strips 25 x 30 or 25 x 35

mm
Flat bottomed jar

Forceps

Toilet paper or absorbent tissue

Newspaper

Glycerol – malachite green (or methylene blue) solution

Cellophane…
Technique
Care must be taken during collection of stool

specimens. Always wear gloves to avoid contamination

of the fingers

1. Soak the cellophane strips in the 50% glycerol –

malachite green (or methylene blue) solution for at

least 24 hours before use

2. Transfer a small amount of faeces on to a piece of scrap

paper (news paper is ideal) why ?

Cellophane…
3. Press the screen on top of the faecal sample

4. Using a flat – sided applicator stick, scrape across the

upper surface of the screen to sieve the fecal sample

5. Place a template on a clean microscope slide

6. Transfer a small amount of sieved fecal material into

the hole of the template and carefully fill the hole.

Level with the applicator stick
Cellophane…
7. Remove the template carefully so that all the fecal
material is left on the slide and none is left sticking to
the template

8. Cover the fecal sample on the slide with a glycerol –

soaked cellophane strip

9. If an excess of glycerol os present on the upper

surface of the cellophane, wipe off the excess with a

small piece of toilet paper or absorbent tissue
Cellophane…
10. Invert the microscope slide and press the fecal sample

against the cellophane on a smooth surface ( a piece

of tile or flat stone is ideal) to spread the sample
evenly

11. Do not lift the slide straight up. The cellophane may

separate. Gently slide the microscope slide sideways

holding the cellophane.
Chemical test for occult blood in
stools
This test is used for screening for parasitic infection, for

example intestinal schistosomiasis, or for detection of

bleeding in the intestine caused by polyps, tumours or
inflammation.
It was originally developed using benzidine. However, the use

of benzidine is no longer recommended because it has been

shown to be carcinogenic.
Chemical test…

Note: For 1 day before the examination, the patient should not:

— eat any meat; — take any drugs containing iron compounds;

— brush his or her teeth vigorously.

Principle
Oxygen is produced when the haemoglobin in blood comes

into contact with hydrogen peroxide. The liberated oxygen

reacts with aminopyrine (aminophenazone) to yield a blue
colour.
Chemical …

Materials and reagents

Centrifuge

Conical centrifuge tube

Applicators

Measuring cylinder, 20ml

Test-tubes

Test-tube rack
Materials …

Materials and reagents

Positive control tube (containing a 1% solution of blood in water)
Negative control tube (containing distilled water)
Acetic acid, 10% solution
Hydrogen peroxide (fresh 10% solution)
95% Ethanol
Aminopyrine, crystalline.

Note: The glassware used for the test must be clean, with no traces of
blood .
Method

1. Immediately before carrying out the test, prepare a solution

of aminopyrine: — put about 0.25g of aminopyrine in the
bottom of a test-tube — add 5ml of 95% ethanol.

2. Put a portion of stool (approximately 4ml) in a centrifuge

tube. Add 7ml of distilled water and mix thoroughly.

3. Centrifuge at low speed (1000g) for about 5 minutes, or until

the solids are precipitated.

4. Decant the supernatant fluid into another test-tube and

keep it.
Method

5. Add to the test-tube containing the supernatant fluid, without

mixing: — 10 drops of 10% acetic acid solution — 5ml of the
aminopyrine solution. To prevent mixing, hold the tip of the
pipette containing the aminopyrine solution against the inside
wall of the test-tube and allow the liquid to run down the wall.

6. Add 10 drops of the 10% hydrogen peroxide solution. Do not

mix. Let it stand for 1 minute. The results must be read
within 5 minutes of adding the hydrogen peroxide
solution.
Method

Results
If the reaction is positive a red colour appears between the

two layers of liquid.

Report the results as follows:

Pale red = positive reaction (+ )

Red = strong positive reaction (++ )

Dark red = very strong positive reaction (+++)

No change in colour = negative reaction (- ).

Specimen processing…
C. Specimens preserved in PVA are mostly used for permanent
staining with trichrome.  Prior to staining, they are processed
as follows:
1. Insure that the specimen is well mixed.
2. Prepare a smear using 2 to 3 drops of the specimen depending
on density.
3. Heat fix on slide warmer set at 60oC for 5 minutes or air dry
completely at room temperature.
4. Slides may be trichrome stained or kept for several months in
a protective slide tray or box for future staining.
Methods of stool examination
I. Macroscopic examination
II. Microscopic examination
I. Wet mount preparation
II. Stained slide preparation
III. Calibration of ocular micrometer

IV. UV Fluorescence Microscopy Procedure

Methods of stool examination
Recovery and identification depends on:

Proper collection and fixation

Proper container

 Clean, dry, water-proof, wide-mouth container

Sufficient amount

 About 20-40 grams of well-formed stool or 5-6 table

spoonfuls of watery stool (10ml)
I. Macroscopic examination
A. Color
B. Consistency

C. Abnormal elements

D. Odor

E. Shape and size

A. Color of stool
Normal:
Adult: brown

New born infants: Black (meconium: babys’ first stool)

Breast feed infants:- scrambled egg

Infant feed on animal milk:- “ curd like”

Meconium: Baby’s first stool
First stool a baby will pass thick
Green, tar-like substance
Lines the intestines of the fetus
First bowel movement within a few hours after birth.

Transitional Stool - Stage One

Newborn slowly begins to pass the meconium after birth

Meconium will begin to change in consistency

Slightly lighter in color than meconium.

Transitional Stool - Stage Two
Stool is lighter in color and slightly less thick than meconium.

Transitional Stool - Stage Three

Much lighter and thinner than meconium.

Occurs just before regular stooling begins

Breastfed Stool

Yellow

Runny

Small seed like objects in the stool

Often called baby poop mustard

Color of stool …

Changes in the color, consistency, and frequency of bowel

movements is known as a "change in bowel habits.

In some cases, an unusual stool color is harmless and can be

attributed to a particular food or medication

Changes in stool color that persist can be a serious matter and

should always be investigated by a physician.

Color of stool …

Abnormal:
Clay or white:

 Absence of bile pigment (bile obstruction) or

 Diagnostic study using barium
Black or tarry:

 Drug

 Bleeding from upper gastrointestinal tract (e.g.

Stomach, small intestine)
 Diet high in red meat and
 Dark green vegetables (e.g. Spinach)
Color of stool …

Red:

 Bleeding from lower gastrointestinal tract,

 Hemorrhoids

 Some foods
 Red gelatin,
 Tomato juice or soup
 Large amounts beets
Pale:

 Malabsorption of fats,
 Diet high in milk and milk products and low in meat
B. Consistency of stool

Varies due to diet but provides information on the stage of

protozoa that is present

1. Hard- resists puncture

2. Formed- can be punctured

3. Soft-can be cut with applicator

4. Mushy- can be reshaped

5. Loose- shaped in to container

6. Diarrhea- can flows

7. Watery- can pour

Consistency…
Normal: Formed, soft, semisolid or mushy
Abnormal:
Hard, dry, constipated stool

 Dehydration, decreased intestinal motility resulting from

lack of fiber in diet, lack of exercise, emotional upset,
laxative abuse
 Diarrhea

 Increased intestinal motility (e.g., irritation of the colon by

bacteria)
Cleary watery, loose mixed with mucus and blood
Bristol stool form scale

Classification of the form,( appearance in a toilet) of feces

into seven groups.

The form of the stool depends on the time it spends in the

colon
Chart breakdown
Types 1 and 2 indicate constipation

Types 3 and 4 are usually the most comfortable to pass,

Types 5-6 tend to be associated with urgency, while

Type 7 is diarrhea
C. Shape

Normal: Cylindrical (contour of rectum) about 2.5 cm (1 inch)

in diameter in adults
Abnormal: Narrow, pencil-shaped, or string like stool

Obstructive conditional of the rectum

D. Amount and size
Normal:
Amount

 Varies with diet

 About 100 to 400 g per day
Size

A healthy piece of feces is about one foot long.

 Shorter sized feces suggest that the colon is not able to
process the food correctly and
 the feces produced does not have the correct amount of
moisture in it.
Frequency

How often does the average person poop?

Normal range

Three times a day to once every three days.

 Average person poops about once a day.

Abnormal

Four times a day or more and the stool has a liquid

consistency – diarrhea
Less than two or three days a week and the stool is hard,

dry, and difficult to pass-constipation

E. Odor
What makes feces smell so bad?
The bacteria produce various compounds and gases that

lead to the infamous smell of feces.

Gut flora produce compounds such as indole, skatole, and

thiols (sulfur containing compounds), as well as the

inorganic gas hydrogen sulfide
The bad smell of feces will usually be reduced by eating

more natural foods that do not contain any artificial flavors

or chemicals
Odor…

Normal: Aromatic, affected by ingested food and person’s

own bacterial flora

Abnormal: Pungent

Infection, blood
F. Constituents
Normal:
Water (about 75%).

Rest constitute:

 Dead bacteria that helped us digest our food, living

bacteria,
 Undigested food residue (known as fiber),
 Cellular linings, sloughed epithelial cells
 Substances released from the intestines (such as mucus)
and the liver.
Constituents…

 Fat, protein, dried constituents of digestive juices (e.G.,

Bile pigments),
 Inorganic matter (e.G., Calcium, phosphates)
Abnormal:
Pus: bacterial infection

Mucus: inflammatory condition

Parasites

Blood: gastrointestinal bleeding

Large quantities of fat: malabsorption

Foreign objects: accidental ingestion

Blood and mucus may be found in faeces from
patients with amoebic dysentery, intestinal
schistosomiasis, invasive balantidiasis (rare infection),
and in severe
T. trichiura infections.
Other non-parasitic conditions in which blood and
mucus may be found include bacillary dysentery
II. Microscopic examination
Calibration of ocular micrometer

Preparation of smear

 direct Smear
 Smear after concentration
 Permanent stained smear

UV Fluorescence Microscopy

II. Microscopic…
a. Calibration of ocular micrometer
 The size of microorganisms or substructures of organisms

can be measured by microscopy using an ocular with a

calibrated micrometer disc
 The micrometer disc has a scale that is usually divided

into 0.1mm and 0.01mm subdivisions

Calibration…

 A stage micrometer is used to calibrate the ocular

micrometer
 Materials

 Binocular microscope
 Ocular with a x10 magnification
 Ocular micrometer disc
 Stage micrometer
 Lens paper
 Immersion oil
Method
1. Unscrew the eye lens of the ocular
2. Place the micrometer with the engraved scale face – down
in the ocular. Use lens paper to handle the disc
3. Replace the lens carefully
4. Place the ocular with the micrometer in the ocular tube
of the microscope
5. Put the calibrated stage micrometer on the stage of the
microscope and focus on the scale. You should be able to
clearly distinguish the 0.1mm and 0.01mm subdivisions
Method
6. Adjust the stage micrometer so that the 0mm line
coincides with the 0mm line of the ocular micrometer
7. Look for another set of lines where the scale of the stage
micrometer coincides with that of the ocular micrometer.
This set of lines should be as far away from the 0mm line
as possible. The distance between the two coinciding sets
of lines varies, depending on the magnification of the
objective of the microscope
Method
8. Count the number of 0.1mm subdivisions of the stage
micrometer scale between the 0 – line and the second set
of coinciding lines
9. Count the number of subdivisions of the ocular
micrometer scale between the 0-line and the second set
of coinciding lines
10. Calculate the proportion of a millimeter that is measured

by a single ocular unit using the following formulla:

Method

Ocular units (m) = Stage reading (mm) x 1000m

Ocular reading x 1mm
II. Microscopic…
b. Wet mount examination
 A wet mount can be prepared directly from fecal material or from
concentrated specimens
 Basic types:
1. Saline
2. Iodine
3. Buffered Methylene Blue (BMB)
Wet mount…
Saline wet mount:
 For initial microscopic examination of stool

 Employed primarily to demonstrate worm eggs, larvae,

protozoan trophozoites, and cysts

 Can also reveal the presence of RBCs and WBCs

Iodine wet mount:

 Used mainly to stain glycogen and the nuclei of cyst
Wet mount…
Buffered Methylene Blue (BMB) wet mount:
 Prepared each time amobic trophozoites are seen in a

saline wet mount, or when their presence is suspected

 BMB stains amoebic trophozoites, but does not stain

amoebic cysts, flagellate trophozoites, or flagellate cysts

 BMB stain is appropriate only for fresh unpreserved

specimens
Wet mount…
Materials and reagents:
 Covers lip

 Dropping bottles containing:

 Saline solution, Isotonic

 Lugol’s iodine (1% solution)
 BMB
 Microscope slides

 Pens or markers for labelling

 Wire loop (or applicator sticks, matchsticks, or toothpicks)

Wet mount…
Direct saline and Iodine mounts:
1. With a wax pencil write the patient’s name or number and the date at the left –
hand end of the slide
2. Place a drop of saline in the center of the left half of the slide and place a drop of
Iodine solution in the center of the right half of the slide
NB: use warm saline (37 0) if the presence of amoebic trophozoite is suspected
3. With an applicator stick, pick up a small portion of the specimen (size of a
match head) and mix with the drop
of saline
Direct saline…
4. Similarly, pickup a small amount of the stool and mix
with the drop of iodine, to prepare an iodine mount
5. Cover the drop of saline and the drop of iodine with a
cover slip. Hold the cover-slip at an angle, touch the edge
of the drop, and lower gently on to the slide. This will
reduce the chance of including air bubbles in the mount
BMB mount
1. Proceed as in steps 1 – 5 for “Direct saline and iodine”, but place
a large drop of BMB on the slide instead of saline or iodine
2. Wait 5 – 10 minutes before examining, to allow the stain to
penetrate the trophozoites. BMB will overstain the trophozoites
in about 30 minutes. Therefore, the slide must be examined
within 30 minutes after preparation.
 Stained slide preparation

Permanent stained slides are used for identification of protozoan

trophozoites and cysts and for confirmation of species. 

It also permits consultation reference and diagnosis when needed as

well as providing a permanent record of organism(s) observed. 

Normally 3 × 1 slides are used to prepare permanent stained slides. 
 Stained…

If the specimen is unpreserved, prepare a thin, even smear of the material by

streaking the material back and forth on the slide with an applicator stick. 
If necessary dilute feces with saline. 
For PVA fixed specimens, apply two or three drops of the specimen to the slide

and with a rolling motion or an up and down dabbing motion spread the
specimen evenly to cover an area roughly the size of a 22 by 22 mm cover slip. 
For other fixatives, check manufacturers instructions.
 Stained…

After the staining process is complete, systematically examine

the smear microscopically utilizing the 100× oil objective. 

Examine at least 200 to 300 oil immersion fields. 
Report protozoa seen as either trophozoites and/or cysts as

applicable.
d. UV Fluorescence Microscopy Procedure

 The demonstration of Cyclospora oocysts in wet

preparations is greatly enhanced by using UV

fluorescence microscopy. 
 Despite the age of the specimen or sample, Cyclospora

oocysts exhibit intense blue color when observed under a

fluorescence microscope (UV excitation filter set at 330-
365 nm). 
UV fluorescence…
 Under bright-field (differential interference contrast or

DIC) microscopy, Cyclospora oocysts appear as refractile

spheres (8-10 µm) with a distinct oocyst wall.  
 The utilization of both bright-field (DIC) and fluorescence

microscopy provides an efficient and reliable approach to

diagnosis. 
 However, it does not provide a permanent stained slide

that can be archived.

Biosafety

Potential risks with stool specimens includes:

 Ingestion of eggs or cysts,

 Skin penetration by infective larvae, and

 Infection by non-parasitic agents found in stool and

biologic fluids. 
These risks can be minimized by:
 Adopting universal precautions as well as

 Standard microbiological laboratory practices (Biosafety 

Level 2).  
Biosafety…
Wear protective safety glasses, gloves and laboratory coat

when processing specimens.

Use biological safety cabinets as needed.
Do not eat, drink, smoke, apply cosmetics or manipulate

contact lenses in work area.

Decontaminate work surface at least once a day and after

any spill of potentially infectious material.

If you have cuts or abrasions on the skin of your hands,

cover them with adhesive dressing.

Biosafety…
If you use any sharp instruments, dispose of them in a

“sharps” container for decontamination.

Remove gloves and wash your hands after completing any

task involving the handling of fecal material.

 

Specimen Collection
Timing:
 Whenever possible, specimens should be collected before

treatment is initiated.
 When malaria and babesiosis are suspected, blood smears

should be obtained and examined without delay. 

 Since the parasitemia may fluctuate, multiple smears might be

needed.  These can be taken at 8 to 12 hour intervals for 2 to 3

days.
 Timing…
Microfilariae exhibit a marked periodicity depending on the

species involved; therefore the time of specimen collection is

critical. 
If a filarial infection is suspected, the optimal collection time

for demonstrating microfilariae is:

Loa loa - midday (10 AM to 2 PM)

Brugia or Wuchereria - at night, after 8 PM

Mansonella - any time

Onchocerca - any time

Type of Sample:
Venous blood samples provide sufficient material for

performing a variety of diagnostic tests, including

concentration procedures (filariasis, trypanosomiasis). 

However, in some parasitic diseases (e.g., for diagnosis of

malaria in particular), anticoagulants in the venous blood

specimen can interfere with parasite morphology and

staining characteristics 

In such cases, capillary blood samples are preferable. 

Type of Sample…
Capillary blood obtained by fingerstick:

Label pre-cleaned slides (preferably frosted-end) with the

patient’s name (or other identifier) and date and time of

collection.
Clean the site well with alcohol; allow to dry.

Prick the side of the pulp of the 3rd or 4th finger (alternate

sites include ear lobe, or in infants large toe or heel).

Wipe away the first drop of blood with clean gauze.

Prepare at least 2 thick smears and 2 thin smears.

Venous blood obtained by venipuncture:
Label collection tubes and pre-cleaned slides (preferably

frosted-end) with the patient’s name (or other identifier) and

date and time of collection.
Clean the site well with alcohol; allow to dry.

Collect the venous blood in a vacuum tube containing

anticoagulant (preferably EDTA); alternatively, collect the

blood in a syringe and transfer it to a tube with anticoagulant;
mix well.
Prepare at least 2 thick smears and 2 thin smears as soon as

possible after collection.

Specimen Processing: Preparing Blood Smears

If you are using venous blood, blood smears should be

prepared as soon as possible after collection. Why ?

Two types of blood smear can be prepared:

Thick blood film

Thin blood film

 Thick smears (TBF)  
Thick layer of dehemoglobinized (lysed) red blood cells

(RBCs). 

The blood elements (including parasites) are more

concentrated (30×) than in an equal area of a thin smear. 

More sensitivity thin film. 

Not adequate for species identification of malaria parasites

If the TBF is positive for malaria parasites, the thin smear

should be used for species identification.

 TBF…
Prepare at least 2 smears per patient!

Place a small drop of blood in the center of the pre-cleaned,

labeled slide.
Using the corner of another slide or an applicator stick, spread
the drop in a circular pattern until it is the size of a dime (1.5
cm2).
A thick smear of proper density is one which, if placed (wet)
over newsprint, allows you to barely read the words.
Lay the slides flat and allow the smears to dry thoroughly
(protect from dust and insects!). 
 TBF…

Insufficiently dried smears (and/or smears that are too

thick) can detach from the slides during staining. 

At room temperature, drying can take several hours; 30

minutes is the minimum

Protect thick smears from hot environments to prevent heat-

fixing the smear.

Do not fix thick smears with methanol or heat. 
If there will be a delay in staining smears, dip the thick smear

briefly in water to hemolyse the RBCs.

 Thin BF
Purpose:

To differentiate WBCs

RBC morphological study

WBC abnormality study

Plasmodium and Borrelia species identification

Thin smears consist of blood spread in a layer such that the

thickness decreases progressively toward the feathered edge. 

In the feathered edge, the cells should be in a monolayer, not

touching one another.

 Thin BF…
Prepare at least 2 smears per patient!

Place a small drop of blood on the pre-cleaned, labeled slide,

near its frosted end (approximately ¼ inch from the frosted

area of the glass slide).
Bring another slide at a 30-45° angle up to the drop, allowing

the drop to spread along the contact line of the 2 slides.

Quickly push the upper (spreader) slide toward the unfrosted

end of the lower slide.

Thin BF…
Make sure that the smears have a good feathered edge. 

This is achieved by using the correct amount of blood and

spreading technique.
Allow the thin smears to dry.  (They dry much faster and less

subject to detachment. Why ?

Fix the smears by dipping them in absolute methanol.
Thin BF…
The thickness of the smear is influenced by:

Angle of the spreader slide (the greater the angle the

thicker the smear).

Size of the blood dropped

Speed of spreading
 Thin BF…
Feature of well-made film:
There is no line extending across or down through the film
The film is smooth at the end (No jugged tail
The film is not too long or too short
The film is not too thick
The film does not contain holes due to grease in the slide.
The thicker area makes a gradual translation to the thin slide
The blood on the thin area does not extend to the end of the

slide.
Should cover ½ to ¾ of the area of the slide.
 Thin BF…
 
Staining the blood films
Methanol fixing thin blood film

When the smear completely dry, fix with absolute

methanol
Place the film on the staining rack and add 1-2 drops of

moisture free methyl alcohol allow it to dry on the air.

Alternatively the blood film can be immersed in a

container of absolute methanol for about 2 min, but this

is more expensive
Staining the…
-
Wright stain
Wright's stain is polychromatic stain producing

multicolored staining reaction from application.

Preparation
Pure weight stain (0.2g) is added 100ml unhydrous acetone

free methanol alcohol

The mixture is shaken to suspend and dissolve the stain

and is placed on an electric plate 60ºC until the stain has

dissolved completely
-Staining the…
The stain is then allowed to cool and is filtered through dry

coarse filter paper to remove any large partials.

The filtered stains is aged or oxidized by placing it in a 37ºC

cubature for a proximately 3o to 4o days shake the reagent

to respond and dissolve any particulate material that may
be present.
The stain is filtered before use.
 Staining the…
Procedure
 
To prevent the plasma back ground the film from staining

BF should be stained with in a few hrs of preparation or

fixed
Fixation may be accomplished by immersion of the slide

sling reagent filed jars.

Fixation is for one or two min with absolute methanol. Add

the stain on the smear.

Without removing, the stain from the horizontal slide

equal amount of buffer carefully added and mixed by

blowing gently.
Staining …
Allow these dilute stains to act for s- 10min.

Without disturbing the slide filed with distilled water

Stand the glass slide an end to dry. Films stained well with

Wright stains have a pink color when viewed with necked

eye.

Staining problem
Problems associated with Wright staining
Too acidic Wright stain - may be due to
 Insufficient staining time
Prolonged buffering or washing

Too acidic stain buffer or water which may be due to exposure

of stain or buffer to acid fumes or it may be old stain the

methyl alcohol has slowly oxidized to formic acid.

Remedies for acidic dyes

prolonged the staining time

Exact the PH of stain and buffer and/or correct with

alkaline if necessary
Shorten buffering time.
-Too alkaline Wright's stain
  Thick blood smears

Proposed staining

New stain solution which was not stood for 2-3 weeks may

be too basic
Remedies
Check PH of stain, buffer and water

Shorten a staining time

Proposed buffering time

Check the incubation time of the stain.

 Precipitation on the film may be due to:

  Unclean slide – greasy dust,

Drying during the period of staining

Inadequate washing of the slide as the end of the staining

period
Failure to hold the slide horizontally during initial

washing.
Inadequate filtration of the stain

Permiting to settle dust on the slide or smear

Giemsa stain
Preparation:
1. Powder giemsa (1.09) is dissolved in 66 ml pure an hydrous
acetone free methanol alcohol.
2. Equal volume of glycerol is added ,heat the stain on an
electric plate until the stain dissolved completely by shaking
at interval then, the stain is allowed to cool to room
temperature. The solution is mixed and allowed to stand for
nearly 30 days in direct sun light or it is in cubated at 37º0 for
30-45 days to ripe. The stain is mixed daily to redissolve any
precipitated stain
3. The stain is filtered before use
Giemsa…

Method
1. The blood smear is fixed by paling in coplin jar of anhydrous
acetone free methyl alcohol for 1-2 min, the purpose of
fixation is to preserve and prevent disruption by sub sequent
procedures
2. The concentrated giemsa stain is diluted by adding 1- volume
of stain to 9ml volumes of buffered diluted water.
3. The fixed blood smear is place either on a level staining rack
or in a second coplin jar.
4. The 1.10 diluted giemsa stain is added and allowed to stand
for 10 minutes The stain is washed off with a stream of
distilled water.

5. The water is removed under side of the slide is wiped free of

stain and allowed to air dry

The buffer is used to dilute the Romanuwisky stains

Microscopic examination of thick smears
  Since the erythrocytes (RBCs) have been lysed and the

parasites are more concentrated, the thick smear is useful

for screening for parasites and for detecting mixed
infections.
First screen the entire smear at a low magnification (10× or

20× objective lens), to detect large parasites such as

microfilaria.
Then examine the smear using the 100× oil immersion

objective lens. 
Select an area that is well stained, free of stain precipitate and

well populated with white blood cells (WBCs) (10-20

WBCs/field).
If you see parasites, make a tentative species determination

on the thick smear and then examine the thin smear to

determine the species present. 
Most often, the thin smear is the appropriate sample for

species identification.
Determination of "No Parasites Found" (NPF):

For malaria diagnosis, WHO recommends that at least 100

fields, each containing approximately 20 WBCs, be screened

before calling a thick smear negative. 
In non-immune patients, symptomatic malaria can occur at

lower parasite densities, and screening more fields (e.g., 200,

300, or even the whole smear) might be warranted,
depending on the clinical context and the availability of
laboratory personnel and time. 
NCCLS standards recommend examination of at least 300

fields using the 100× oil immersion objective.

Examining thin smears
 a rapid screen while the thick smear is still drying.

Species identification

Screen at low magnification (10× or 20× objective lens) if this

has not been done on the thick smears.

Carefully examine the smear using the 100× oil immersion

objective lens. 
NCCLS standards recommend examination of at least 300

fields using the 100× oil immersion objective.

Quantifying parasites:
In some cases (especially malaria) quantification of

parasites yields clinically useful information. 

If this information is needed by the physician, malaria

parasites can be quantified against blood elements such as

RBCs or WBCs.
 To quantify malaria parasites against RBCs, count the
  parasitized RBCs among 500 - 2,000 RBCs on the thin smear
and express the results as % parasitemia.
 % Parasitemia = (parasitized RBCs/total RBCs) × 100
If the parasitemia is high (e.g., >10%) examine 500 RBCs;

If it is low (e.g., <1%) examine 2,000 RBCs (or more); count

asexual blood stage parasites and gametocytes

separately. 
Only the former are clinically important and gametocytes of

P. falciparum can persist after elimination of asexual stages

by drug treatment.
 
 
To quantify malaria parasites against WBCs:
 
On the thick smear, tally the parasites against WBCs, until

you have counted 500 parasites or 1,000 WBCs, whichever

comes first; express the results as parasites per microliter of
blood, using the WBC count if known, or otherwise
assuming 8,000 WBCs per microliter blood.
 
Parasites/µl blood = (parasites/WBCs) × WBC count/ µl
Urine specimen examination
Usually examined for Schistosoma hematobium eggs

Trichomonas vaginalis trophozoite may also be seen

Microfilaria of Wuchereria bancrofti and Onchocerca

volvulus may be found in the uncentrifuged sediment of some

milky urine from patients
Urine…
Collection of urine for diagnosis of Schistosoma infection:
The number of ova in the urine varies throughout the day,

being highest in urine obtained between 10hr and 14hr

The specimen should be collected between these times and

consist of a single, terminal urine of at least 10ml

Alternatively a 24 hr collection of terminal urine can be made

The whole specimen must be examined as ova may be very

scanty
Collection…
If the urine must stand for an hour or longer, add 1ml of

undiluted formalin (37% formaldehyde solution) to each

100ml of urine. This will preserve
SUMMARY
Parasite diagnosis may be done by morphological,

immunological, molecular, culture and xenodiagnostic

methods
Stained or unstained preparation of different specimens (such

as blood, stool, urine, CSF, etc…) are examined either directly

or following Concentration techniques
There are three categories of chemicals used to preserve stool,

prepare slides for staining and stain the preparation: Fixatives,

Preparatory reagents and Stains
Reference
1. Guerrant R.L. Walker D.H. Weller P.F. Tropical Infectious
Diseases. Elsevier Inc. 2nd 2006.
2. Gillespie S. & Pearson R.D. Principles and Practices of Clinical
Parasitology. John Wiley & Sons Ltd. 2001.
3. Cheesbrough M. District Laboratory Practice in Tropical
Countries. 2nd edition. Part one. 2005
4. Awole M., Cheneke W. Medical Parasitology for Medical
laboratory Technology students. Upgraded lecture Notes
Series .2006.
5. Jaffeey and Leach. Atlas of Medical Helminthology and
Protozoology 2nd edition.
6. Murray P.R., Rosenthal K.S., Kobayashi G.S., Pfaller M.A.,
Medical Microbiology, 4th edition. Mosby, 2002.
7. www. cdc.gov.