PARASITOLOGICAL

LABORATORY EXAMINATION

DEPARTMENT OF PARASITOLOGY
FACULTY OF MEDICINE
BRAWIJAYA UNIVERSITY

TEGUH WS ‘08
 Introduction
 Parasitic diseases:
 Are considered to be “non life threatening” 
neglected
 Symptoms are mostly non specific or asymptomatic
 Only some have specific symptoms
 Clinical Diagnosis  difficult
  to find confirmed diagnosis needs supportive
examination  laboratory examination
 To get the definitive diagnosis  basic knowledge is
needed:
 Habitat of the parasite  sample/materials
 Taking and sending sample (fresh or preserved)
 Examination technique

TEGUH WS ‘08
 MATERIALS/SAMPLES
1. STOOLS
2. BLOOD
3. URINE
4. OTHER BODY LIQUIDS:
• Sputum,
• Liquor Cerebrospinalis (LCS)
• Pleural fluid,
• Ascites etc.
5. OTHER MATERIALS:
• Skin scratch
• Tissue biopsy etc

TEGUH WS ‘08
 ROUTINE STOOL EXAMINATION
 WHY STOOLS??
 ≈ GI tract/intestine is the most habitat of parasites

 WHAT SHOULD BE EXAMINED?

 qualitative examination
 Macroscopic
 consistency: formed / soft / loose / watery
 Color: yellow / green / white
 Odor : specific
 Mucous
 Blood
 Fat
 Food materials
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 CONSISTENCY

CYSTS
FORMED

SOFT

LOOSE

TROPHS
WATERY

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Microscopic
Qualitative :
 Intestinal parasites or their derivatives :
 Worm eggs : Nematodes, Trematodes, Cestodes
 Protozoa : Cysts / Trophozoites
 Artifacts /pseudoparasites
 RBC ≈ intra luminal bleeding/ ulcer
 White cells (PMN) ≈ inflammatory processes
 Charchot-Leyden crystals (breakdown products of eosinophils) ≈
allergic phenomena associated ŵ amebiasis & worm infections
 Macrophages ≈ bacterial & parasitic infection, similar ŵ Amoeba
 Epithelial cells ≈ normal desquamation of intestinal cells
 Eggs of Arthropods, plant nematodes and spurious parasites,
yeasts etc
TEGUH WS ‘08
Microscopic
 Specific examination/procedure :
 Concentration:

 Sedimentation

 Floatation

 Culture

 Quantitative :  parasite burden in

stools
 number of worm eggs in stools
(worm burden)
TEGUH WS ‘08
 Qualitative
 Wet Mount
 Direct smear : Saline,Lugol /KI, Eosin
 Thick smear :
 Cellophane Covered Thick Smear
 Kato’s thick smear
 Preserved & Stained Film
 Concentration techniques:
 Sedimentation :
 simple
 centrifugation
 Floatation
 Simple floatation
 Centrifugal floatation
 direct
 ZnSo4
TEGUH WS ‘08
 DIRECT WET MOUNTS/ UNPRESERVED
EXAMINATION
 NORMAL SALINE
SOLUTION 0,85%
 EOSIN 2%
 LUGOL/IODINE 1%

1. Place a drop of solution

(Saline, Eosin or Iodine) on
a clean slide
2. Take about 2 grams of
stools and mix it to be
uniform suspension
3. Cover with 22 x 22 mm
coverglass
4. Examine under microscope
TEGUH WS ‘08
 THICK SMEAR
Cellophane Covered Thick Smear
(Kato & Miura, 1954)
 Cut wettable cellophane into 22 x 30 mm strips and soak them for 24
hours or longer in a mixture of
100 parts of glycerol,
100 parts of destilled water
1 part of 3% aqueous Malachite green
 Place ± 50-60 mg (≈ bean) fresh feces on a clean standard slide and
cover with one of the cellophane strips
 Invert the preparation, place it on paper towels, and press to spread the
fecal material uniformly to the egdes of the strips
 Reverse the slide and allow it to stand at 40º C for 30 minutes or at
room temperature for 1 hour
 Examine the slide under a low power objective; higher magnification
can be used to confirm identification
TEGUH WS ‘08
 PRESERVATION OF STOOL
SPECIMENS
 Polyvinyl Alkohol (PVA)
 Schaudinn’s Preservation
 Conventional
 Cupric sulfate modification
 Formalin Preservation
 Merthiolate-Iodine-Formalin (MIF) Preservation

TEGUH WS ‘08
 CONCENTRATION METHODS
 SEDIMENTATION
 For cysts, worm eggs, trophozoite
 Principle : difference of Specific gravity (SG)
 SG of solution < SG of the Parasites  Parasite will be placed on
lower level

 FLOATATION
 For Nematodes eggs
 Principle : SG > SG Parasite  Eggs will be floated/ placed on
upper level

 COMBINATION
 Centrifugal floatation
 Centrifugal sedimentation

TEGUH WS ‘08
 Simple Sedimentation

 For operculated worm eggs, larvae, cysts

 Procedure :
 Thoroughly comminute ≥ 10 g feces in 50 – 100 cc tap water in a
250 ml or larger beaker glass using a stick or tongue depressor
blade
 Strain the suspension through two layers of wet gauze into a
sedimentation flask  allow the material to sediment ± 1 hour
 Decant the supernatant carefully (so as not to lose any sediment)

 Resuspend the sediment by adding more tap water  allow ± 1

hour. This wash procedure can be repeated ≈ clarity of supernatan
 Using long capillary pipette, remove small portion of sediment,
place on a glass slide examine under microscope. If too thick
add a drop of saline, or dilute with iodine

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 CENTRIFUGATION
 For Schistosomes eggs & Protozoon cysts
 2-3 grams of stools + 20 mL tap water (± 10 x volume of
stools)
 Strain the suspension through two layers of wet gauze
into centrifugation tube
 Centrifuge with speed 1500-2300 rpm for 1-2 minutes
 Decant the supernatant. This procedure can be repeated
until supernatant is clear (usually 2 times)
 remove small portion of sediment, place on a glass slide
examine under microscope

TEGUH WS ‘08
 FLOATATION
 Principle : SG of solution > SG of Parasites  parasite will float
 Solution of NaCl /Brine solution: SG 1.200
 Ascaris, Trichuris, Hokworms eggs  Good results
 Schistosomes eggs, Ss + Hw larvae  shrinking
 Opercululated egs  sediment
 Sugar solution, Glycerin
 ZnSO4 solution (SG 1.180)  centrifugal floatation

TEGUH WS ‘08
 ZnSO4 CENTRIFUGAL FLOATATION

 Non operculated worm eggs, Protozoon cysts,larvae

 331 grams ZnSO4 crytals + 670 mL water = SG 1,18
 1 part of stools + 10 parts of water  mix
 Strain  pour into 15 mL conical centrifuge tube
 Centrifuge at 400-500 or 2300 rpm for 1-2 menit  decant the
supernatant
 Add 2 – 3 mL destilled water  recentrifuge (repeat 2-3 x)
 Add 3-4 mL ZnSO4 sol with SG 1.180
 Centrifuge at full speed for 1- 2 minutes
 Use freshly flamed wire loop (diameter 5-7 mm) to touch the center
of the surface film  object glass  examined with or without cover
glass

TEGUH WS ‘08
 Method for using wire loop to remove
surface film from ZnSO4 floatation technique

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 QUANTITATIVE
For Estimating:
 Number of worm infecting host/worm burden

  degree or severity of infection

 Especially for intestinal nematode (Ascaris, Trichuris,

Hookworms)
 Most frequent method used

 Kato-Katz’s Thick Smear

 Stoll Egg Count Technique

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Kato Katz’s Thick Smear
Procedure : As same as Kato Thick Smear,
but:
 Use wire strain for straining the stools

 Amount of stool can be calculated 

use thick paper

- 1 mm thick  a hole Φ 9 mm ≈ 50-60 mg of stools,
or
- ½ mm thick a hole Φ 6,5 mm ≈ 20-25 mg of stools
- cover with cellophane soaked with Malachyte green  allow it at
40º C for 30 minutes or at room temperature for 1 hour
- examine under the microscope and count the number of eggs

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 Stoll Egg Count Technique
 Equipments:
 Long necked “Stoll flask” 60 mL with marker on 56 and 60 mL

 Stick/applicator for taking stools

 Stoll Pipette (0,075 ml and 0,15 ml)
 Glass beads (diameter 6 mm)

 Materials :
 NaOH Solution 0,1 N

 Dissolve 4 g of NaOH in small amount of distilled water

 Add distilled water to bring total volume to 1000 ml
 Store until needed

TEGUH WS ‘08
 Stoll Egg Count Technique
Procedure:
 Add 0,1 N NaOH solution to the 56 mL mark of the Stoll flask
 Using applicator stick carefully add feces to the flask  the level of
fluid raised to the 60 cc mark
 Add 5-10 small glass beads to the flask, stopper the flask  shake
vigorously with up-and-down motion for a minute or more to obtain a
homogenous suspension. If the stool is hard,  allow the mixture to
stand for several hours or overnight
 Use Stoll pipette to remove sample 0,15 mL from the center of the
suspension
 Expel the pipette content on to a slide  cover with 22x44 mm
cover glass
 Count all of the eggs on the slide (p). The number of egg per gram
of feces, is the number of eggs obtained x 100. (NEPG = p x 100)
 Correction factors according to consistency of stools:
 Soft  x 1,5 ; Loose  x 2; Watery  x 3

TEGUH WS ‘08
 Worm burden

Daily production of eggs

 Trichuris trichiura = 5.000/ day
 Necator americanus = 9.000/ day
 Ascaris lumbricoides = 200.000/day

 If the total amount of feces be weight and the NEPG had been
calculated  the number of worms infecting host can be estimated
 Single adult Ascaris may be dangerous, due to migrating tendency
 Hookworms infection with NEPG > 2500  clinical symptoms
 Trichuris infection with NEPG > 20.000  clinical symptoms

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 Culture of feces
Aims :
 To differentiate the worms which had similar morphology of eggs,

as:
 Strongyloides stercoralis

 Necator americanus
 Ancylostoma duodenale
 Trychostrogylus spp
 The larvae of those worm can be differentiated morphologically

TEGUH WS ‘08
 HARADA MORI METHOD
 Use conical/screw cap test tube vol. 15 mL
 Use a 13x120 mm paper strip that tapers at
the end inserted into the tube. Write the
specimen identification number on it
 Spread approximately 0.5 – 1.0 of feces on to
the middle one-third of the strip,  forms a
layer several millimeters thick
 Insert the strip into conical screw cap test tube
 several millimeters of the tube bottom
 Carefully add distilled water until the water is
slightly below the fecal mass  do not wash!!
 Keep test tube upright in the test-tube rack at
room temperature (24º-28ºC) for 7-10 days
 Larvae in fluid can be heat-killed and removed
from the bottom of the tube using a glass
pipette  drop on a slide  iodineexamine

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 Polyethylene tube
 Modification of Harada Mori culture by Sasa
 Using polyethylene sac instead of screw cap tube
 Filter paper strip or newspaper strip
 Easier and cheaper
 5-10 days (about a week)
 The aim and procedure are the same as Harada Mori

TEGUH WS ‘08
 Sand culture technique
 For Soil Transmitted Helminths
 Procedure:
 Sterile Petri-dish filled by sterile sand
 Add feces on to it and covered
 Rhabditiform larva (+) after 1 day
 Filariform larva (+) after D 3-7

Filter paper/slant Culture

 write sample identity
 Smear feces on to middle 1/3 part
 Place the strip on to a slide  petri
dish so that one end rests on a short The culture can be examined under
piece of glass tubing dissecting microscope for the presence of
free living adult or larvae of S. stercoralis.
 Add a shallow layer of distilled water
Hookworm and trichostrogylid larva can
 Cover dish and allow it to stand at be found in water
24º-28ºC
TEGUH WS ‘08
 Cellulose Tape Preparation
Procedure
 Grahams-Scotch’s Adhesive Tape Swab
 For diagnosing intestinal parasites which migrate to perianal region:
 Enterobius vermicularis (adult and eggs)
 Taenia solium & T. saginata eggs (less frequency)
 In the morning, prior to using the toilet or bathing
 Cellulose tape preparations should be taken at least 3-4
consecutive days before a patient is considered to be free from
infection
 Adult male of Enterobius vermicularis
 can demonstrated by cellulose tape preparation also
 Can be seen with naked eye, crawling around the anus, but the
male

TEGUH WS ‘08
 Materials and methods
Procedures
a. Place a strip of cellulose tape on a
microscopic slide, starting 1.2 cm from
one end, and running toward the same
end. Wrap the tape over the edge of the
slide.
b. Peel back the tape, and, with adhesive
side outward over a wooden tongue
depressor.
c. Press the tape firmly against the right and
left perianal fold
d. Spread the tape back over the slide,
adhesive side down
e. Press the tape down on the slide, examine
directly under low power of microscope.

TEGUH WS ‘08
 ANAL SWAB METHOD
 For diagnosing of Enterobius vermicularis eggs
 Is an alternative procedure for diagnosing pinworm infection
 This procedure is more complex than the cellulose tape method
 Offers no advantages over it

Procedure:
 Immerse cotton swab in paraffin and petroleum jelly mixture, place
swabs into tubes, store in refrigerator until needed
 Spread the buttock and rub the swab over the perianal surface very
gently. Insert the swab a short distance into the anal opening.
Return the swab to the tube and label
 Fill the tube ± half full ŵ xylene to cover the swab  allow for 5 min.
 Remove the swab, centrifuge the suspension 500 g/ for 1 min.,
remove supernatant  examine under microscope

TEGUH WS ‘08
 Examination of Blood
 MALARIA
 FILARIASIS
 TOXOPLASMOSIS
 TRYPANOSOMIASIS
 LEISHMANIASIS
 Examination of Blood
Taking of peripheral blood samples
 Capillary blood : finger tip, heel, auricle/ear lobe

 Circulatory blood : v. mediana cubiti

Preparation blood film

 Thin blood film/smear : malaria, trypanosomiasis, babesiosis
 Because of the small amount of blood used in preparation of thin film,
they are not as sensitive for detecting parasites as one would like
 Thick blood film/smear : malaria, filaria
 The amount of blood used in preparation of thick film is more  more
sensitive, but less specific than thin film,

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Capillary blood administration

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TEGUH WS ‘08
TEGUH WS ‘08
 Examination of Microfilaria
 Direct examination (wet blood)
 Directly examining the motility of microfilaria

 Punction the finger tip with lancet  suck the blood with capillary
tube drop blood on to a clean and dry slide  spread with
other slide edge  directly examine under the microscope
 Knott Concentration Technique
 Venous blood + anticoagulant  into a tube containing formaline
solution 2% (10 cc)  centrifuge for 2 minutes
 Decant supernatant, directly examine the sediment, or stained
with Giemsa staining
 Straining using nucleophore filter
 Venous blood + anticoagulant were strained using nucleophore
filter  microfilaria will strained  stain with Giemsa examine

TEGUH WS ‘08
 Capillary
tube Wet preparation

20 l

Air dried

Dehaemoglobin
Fixed with
with water
Stained with methanol
Giemsa 1 : 15 Air dried
for 15 min.

washed with
running water

Examined under compound microscope

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 Examination of urine
(Urinalysis)
 Urinalysis  centrifugation urine sediment
 Urogenital tract parasites
 Trichomonas vaginalis
 Schistosoma haematobium
 Others : Polychaete annelids, Ren worm
 Chyluria  microfilaria of Wuchereria bancrofti, Onchocerca
volvulus
 Trophozoite of Entamoeba histolytica  fistel?
 Strongyloides stercoralis Larvae

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 Examination of sputum
 DIRECT SMEAR METHOD
 For Pulmonary parasites
 Paragonimus

 Amoeba

 Hydatid cyst

 Strongyloides stercoralis Larvae

TEGUH WS ‘08
 OTHER SPECIMENS

 Other body fluids

 LCS
 Ascites, pleural fluid
 Scratching /rub down of Skin or mucosal
 Muscle biopsy
 Duodenal fluid aspiration
 Lymph-node biopsy
 Soil
 Finger nails

TEGUH WS ‘08
Thankyou

TEGUH WS ‘08