TEGUH WS ‘08

PARASITOLOGICAL PARASITOLOGICAL LABORATORY LABORATORY

EXAMINATION EXAMINATION

PARASITOLOGICAL PARASITOLOGICAL LABORATORY LABORATORY

EXAMINATION EXAMINATION

DEPARTMENT OF PARASITOLOGYFACULTY OF MEDICINEBRAWIJAYA UNIVERSITY

DEPARTMENT OF PARASITOLOGYFACULTY OF MEDICINEBRAWIJAYA UNIVERSITY

TEGUH WS ‘08

IntroductionIntroductionIntroductionIntroduction Parasitic diseases:Parasitic diseases:

Are considered to be “non life threatening” Are considered to be “non life threatening” neglectedneglected

Symptoms are mostly non specific or asymptomaticSymptoms are mostly non specific or asymptomatic Only some have specific symptomsOnly some have specific symptoms Clinical Diagnosis Clinical Diagnosis difficult difficult

to find confirmed diagnosis needs supportive to find confirmed diagnosis needs supportive examination examination laboratory examination laboratory examination

To get the definitive diagnosis To get the definitive diagnosis basic knowledge is basic knowledge is needed: needed: Habitat of the parasite Habitat of the parasite sample/materials sample/materials Taking and sending sample (fresh or preserved)Taking and sending sample (fresh or preserved) Examination techniqueExamination technique

TEGUH WS ‘08

MATERIALS/SAMPLESMATERIALS/SAMPLESMATERIALS/SAMPLESMATERIALS/SAMPLES

1.1. STOOLSSTOOLS

2.2. BLOODBLOOD

3.3. URINEURINE

4.4. OTHER BODY LIQUIDS: OTHER BODY LIQUIDS: • Sputum, Sputum, • Liquor Cerebrospinalis (LCS)Liquor Cerebrospinalis (LCS)• Pleural fluid, Pleural fluid, • Ascites etc.Ascites etc.

5.5. OTHER MATERIALS: OTHER MATERIALS: • Skin scratchSkin scratch• Tissue biopsy etcTissue biopsy etc

1.1. STOOLSSTOOLS

2.2. BLOODBLOOD

3.3. URINEURINE

4.4. OTHER BODY LIQUIDS: OTHER BODY LIQUIDS: • Sputum, Sputum, • Liquor Cerebrospinalis (LCS)Liquor Cerebrospinalis (LCS)• Pleural fluid, Pleural fluid, • Ascites etc.Ascites etc.

5.5. OTHER MATERIALS: OTHER MATERIALS: • Skin scratchSkin scratch• Tissue biopsy etcTissue biopsy etc

TEGUH WS ‘08

ROUTINE STOOL EXAMINATIONROUTINE STOOL EXAMINATIONROUTINE STOOL EXAMINATIONROUTINE STOOL EXAMINATION

WHY STOOLS??WHY STOOLS?? GI tract/intestine is the most habitat of parasites GI tract/intestine is the most habitat of parasites

WHAT SHOULD BE EXAMINED?WHAT SHOULD BE EXAMINED? qualitative examinationqualitative examination

MacroscopicMacroscopic consistency: formed / soft / loose / wateryconsistency: formed / soft / loose / watery Color: yellow / green / white Color: yellow / green / white Odor : specific Odor : specific MucousMucous Blood Blood Fat Fat Food materials Food materials

TEGUH WS ‘08

CONSISTENCYCONSISTENCY

FORMEDFORMED

SOFTSOFT

LOOSELOOSE

WATERYWATERY

CYSTS

TROPHS

TEGUH WS ‘08

Microscopic Microscopic Microscopic 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

MicroscopicMicroscopicMicroscopicMicroscopic

Specific examination/procedure :Specific examination/procedure : Concentration:Concentration:

SedimentationSedimentation FloatationFloatation

CultureCulture Quantitative : Quantitative : parasite burden in parasite burden in

stoolsstools number of worm eggs in stools number of worm eggs in stools

((worm burdenworm burden))

TEGUH WS ‘08

QualitativeQualitative Wet MountWet Mount

Direct smear : Saline,Lugol /KI, EosinDirect smear : Saline,Lugol /KI, Eosin Thick smear : Thick smear :

Cellophane Covered Thick SmearCellophane Covered Thick Smear Kato’s thick smearKato’s thick smear

Preserved & Stained FilmPreserved & Stained Film Concentration techniques:Concentration techniques:

Sedimentation :Sedimentation : simple simple centrifugationcentrifugation

FloatationFloatation Simple floatationSimple floatation Centrifugal floatation Centrifugal floatation

direct direct ZnSo4 ZnSo4

Wet MountWet Mount Direct smear : Saline,Lugol /KI, EosinDirect smear : Saline,Lugol /KI, Eosin Thick smear : Thick smear :

Cellophane Covered Thick SmearCellophane Covered Thick Smear Kato’s thick smearKato’s thick smear

Preserved & Stained FilmPreserved & Stained Film Concentration techniques:Concentration techniques:

Sedimentation :Sedimentation : simple simple centrifugationcentrifugation

FloatationFloatation Simple floatationSimple floatation Centrifugal floatation Centrifugal floatation

direct direct ZnSo4 ZnSo4

TEGUH WS ‘08

DIRECT WET MOUNTS/ UNPRESERVED DIRECT WET MOUNTS/ UNPRESERVED EXAMINATIONEXAMINATION

DIRECT WET MOUNTS/ UNPRESERVED DIRECT WET MOUNTS/ UNPRESERVED EXAMINATIONEXAMINATION

NORMAL SALINE NORMAL SALINE SOLUTION 0,85%SOLUTION 0,85%

EOSIN 2%EOSIN 2% LUGOL/IODINE 1%LUGOL/IODINE 1%

NORMAL SALINE NORMAL SALINE SOLUTION 0,85%SOLUTION 0,85%

EOSIN 2%EOSIN 2% LUGOL/IODINE 1%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 THICK SMEAR Cellophane Covered Thick Smear Cellophane Covered Thick Smear

(Kato & Miura, 1954)(Kato & Miura, 1954)

THICK SMEAR THICK SMEAR Cellophane Covered Thick Smear Cellophane Covered Thick Smear

(Kato & Miura, 1954)(Kato & Miura, 1954)

Cut wettable cellophane into 22 x 30 mm strips and soak them for 24 Cut wettable cellophane into 22 x 30 mm strips and soak them for 24 hours or longer in a mixture of hours or longer in a mixture of

100 parts of glycerol, 100 parts of glycerol,

100 parts of destilled water 100 parts of destilled water

1 part of 3% aqueous Malachite green1 part of 3% aqueous Malachite green Place Place 50-60 mg ( 50-60 mg ( bean) fresh feces on a clean standard slide and bean) fresh feces on a clean standard slide and

cover with one of the cellophane stripscover with one of the cellophane strips Invert the preparation, place it on paper towels, and press to spread the Invert the preparation, place it on paper towels, and press to spread the

fecal material uniformly to the egdes of the stripsfecal material uniformly to the egdes of the strips Reverse the slide and allow it to stand at 40Reverse the slide and allow it to stand at 40º C for 30 minutes or at º C for 30 minutes or at

room temperature for 1 hourroom temperature for 1 hour Examine the slide under a low power objective; higher magnification Examine the slide under a low power objective; higher magnification

can be used to confirm identificationcan be used to confirm identification

Cut wettable cellophane into 22 x 30 mm strips and soak them for 24 Cut wettable cellophane into 22 x 30 mm strips and soak them for 24 hours or longer in a mixture of hours or longer in a mixture of

100 parts of glycerol, 100 parts of glycerol,

100 parts of destilled water 100 parts of destilled water

1 part of 3% aqueous Malachite green1 part of 3% aqueous Malachite green Place Place 50-60 mg ( 50-60 mg ( bean) fresh feces on a clean standard slide and bean) fresh feces on a clean standard slide and

cover with one of the cellophane stripscover with one of the cellophane strips Invert the preparation, place it on paper towels, and press to spread the Invert the preparation, place it on paper towels, and press to spread the

fecal material uniformly to the egdes of the stripsfecal material uniformly to the egdes of the strips Reverse the slide and allow it to stand at 40Reverse the slide and allow it to stand at 40º C for 30 minutes or at º C for 30 minutes or at

room temperature for 1 hourroom temperature for 1 hour Examine the slide under a low power objective; higher magnification Examine the slide under a low power objective; higher magnification

can be used to confirm identificationcan be used to confirm identification

TEGUH WS ‘08

PRESERVATION OF STOOL SPECIMENS

PRESERVATION OF STOOL SPECIMENS

Polyvinyl Alkohol (PVA)Polyvinyl Alkohol (PVA)

Schaudinn’s PreservationSchaudinn’s Preservation

ConventionalConventional

Cupric sulfate modificationCupric sulfate modification

Formalin PreservationFormalin Preservation

Merthiolate-Iodine-Formalin (MIF) PreservationMerthiolate-Iodine-Formalin (MIF) Preservation

TEGUH WS ‘08

CONCENTRATION METHODSCONCENTRATION METHODSCONCENTRATION METHODSCONCENTRATION METHODS

SEDIMENTATIONSEDIMENTATION For cysts, worm eggs, trophozoiteFor cysts, worm eggs, trophozoite Principle : difference of Specific gravity (SG)Principle : difference of Specific gravity (SG)

SG of solution SG of solution < SG of the Parasites < SG of the Parasites Parasite will be placed on Parasite will be placed on lower levellower level

FLOATATIONFLOATATION For Nematodes eggsFor Nematodes eggs Principle : SG > SGPrinciple : SG > SG Parasite Parasite Eggs will be floated/ placed on Eggs will be floated/ placed on

upper levelupper level

COMBINATION COMBINATION Centrifugal floatationCentrifugal floatation Centrifugal sedimentation Centrifugal sedimentation

SEDIMENTATIONSEDIMENTATION For cysts, worm eggs, trophozoiteFor cysts, worm eggs, trophozoite Principle : difference of Specific gravity (SG)Principle : difference of Specific gravity (SG)

SG of solution SG of solution < SG of the Parasites < SG of the Parasites Parasite will be placed on Parasite will be placed on lower levellower level

FLOATATIONFLOATATION For Nematodes eggsFor Nematodes eggs Principle : SG > SGPrinciple : SG > SG Parasite Parasite Eggs will be floated/ placed on Eggs will be floated/ placed on

upper levelupper level

COMBINATION COMBINATION Centrifugal floatationCentrifugal floatation Centrifugal sedimentation Centrifugal sedimentation

TEGUH WS ‘08

Simple SedimentationSimple SedimentationSimple SedimentationSimple Sedimentation

For operculated worm eggs, larvae, cysts For operculated worm eggs, larvae, cysts Procedure :Procedure :

Thoroughly comminute Thoroughly comminute ≥ ≥ 10 g feces in 50 – 100 cc tap water in a 10 g feces in 50 – 100 cc tap water in a 250 ml or larger beaker glass using a stick or tongue depressor 250 ml or larger beaker glass using a stick or tongue depressor blade blade

Strain the suspension through two layers of wet gauze into a Strain the suspension through two layers of wet gauze into a sedimentation flask sedimentation flask allow the material to sediment allow the material to sediment ±± 1 hour 1 hour

Decant the supernatant carefully (so as not to lose any sediment)Decant the supernatant carefully (so as not to lose any sediment) Resuspend the sediment by adding more tap water Resuspend the sediment by adding more tap water allow allow ±± 1 1

hour. This wash procedure can be repeated hour. This wash procedure can be repeated ≈ clarity of ≈ clarity of supernatansupernatan

Using long capillary pipette, remove small portion of sediment, Using long capillary pipette, remove small portion of sediment, place on a glass slide place on a glass slide examine under microscope. If too thick examine under microscope. If too thick add a drop of saline, or dilute with iodineadd a drop of saline, or dilute with iodine

For operculated worm eggs, larvae, cysts For operculated worm eggs, larvae, cysts Procedure :Procedure :

Thoroughly comminute Thoroughly comminute ≥ ≥ 10 g feces in 50 – 100 cc tap water in a 10 g feces in 50 – 100 cc tap water in a 250 ml or larger beaker glass using a stick or tongue depressor 250 ml or larger beaker glass using a stick or tongue depressor blade blade

Strain the suspension through two layers of wet gauze into a Strain the suspension through two layers of wet gauze into a sedimentation flask sedimentation flask allow the material to sediment allow the material to sediment ±± 1 hour 1 hour

Decant the supernatant carefully (so as not to lose any sediment)Decant the supernatant carefully (so as not to lose any sediment) Resuspend the sediment by adding more tap water Resuspend the sediment by adding more tap water allow allow ±± 1 1

hour. This wash procedure can be repeated hour. This wash procedure can be repeated ≈ clarity of ≈ clarity of supernatansupernatan

Using long capillary pipette, remove small portion of sediment, Using long capillary pipette, remove small portion of sediment, place on a glass slide place on a glass slide examine under microscope. If too thick examine under microscope. If too thick add a drop of saline, or dilute with iodineadd a drop of saline, or dilute with iodine

TEGUH WS ‘08

CENTRIFUGATIONCENTRIFUGATIONCENTRIFUGATIONCENTRIFUGATION

For Schistosomes eggs & Protozoon cystsFor Schistosomes eggs & Protozoon cysts 22-3-3 g gramsrams of stools of stools + 20 + 20 mL tap water (mL tap water ( 10 x volume of 10 x volume of

stools)stools) Strain the suspension through two layers of wet gauze Strain the suspension through two layers of wet gauze

into centrifugation tubeinto centrifugation tube Centrifuge with speed 1500-2300 rpm for 1-2 minutesCentrifuge with speed 1500-2300 rpm for 1-2 minutes Decant the supernatant. This procedure can be repeated Decant the supernatant. This procedure can be repeated

until supernatant is clear (usually 2 times)until supernatant is clear (usually 2 times) remove small portion of sediment, place on a glass slide remove small portion of sediment, place on a glass slide

examine under microscopeexamine under microscope

For Schistosomes eggs & Protozoon cystsFor Schistosomes eggs & Protozoon cysts 22-3-3 g gramsrams of stools of stools + 20 + 20 mL tap water (mL tap water ( 10 x volume of 10 x volume of

stools)stools) Strain the suspension through two layers of wet gauze Strain the suspension through two layers of wet gauze

into centrifugation tubeinto centrifugation tube Centrifuge with speed 1500-2300 rpm for 1-2 minutesCentrifuge with speed 1500-2300 rpm for 1-2 minutes Decant the supernatant. This procedure can be repeated Decant the supernatant. This procedure can be repeated

until supernatant is clear (usually 2 times)until supernatant is clear (usually 2 times) remove small portion of sediment, place on a glass slide remove small portion of sediment, place on a glass slide

examine under microscopeexamine under microscope

TEGUH WS ‘08

FLOATATIONFLOATATIONFLOATATIONFLOATATION

Principle : SG of solution Principle : SG of solution > SG of Parasites > SG of Parasites parasite will float parasite will float Solution of NaCl /Brine solution: SG 1.200Solution of NaCl /Brine solution: SG 1.200

Ascaris, Trichuris, Hokworms eggs Ascaris, Trichuris, Hokworms eggs Good results Good results Schistosomes eggs, Ss + Hw larvae Schistosomes eggs, Ss + Hw larvae shrinking shrinking Opercululated egs Opercululated egs sediment sediment

Sugar solution, GlycerinSugar solution, Glycerin ZnSO4 solution (SG 1.180) ZnSO4 solution (SG 1.180) centrifugal floatation centrifugal floatation

Principle : SG of solution Principle : SG of solution > SG of Parasites > SG of Parasites parasite will float parasite will float Solution of NaCl /Brine solution: SG 1.200Solution of NaCl /Brine solution: SG 1.200

Ascaris, Trichuris, Hokworms eggs Ascaris, Trichuris, Hokworms eggs Good results Good results Schistosomes eggs, Ss + Hw larvae Schistosomes eggs, Ss + Hw larvae shrinking shrinking Opercululated egs Opercululated egs sediment sediment

Sugar solution, GlycerinSugar solution, Glycerin ZnSO4 solution (SG 1.180) ZnSO4 solution (SG 1.180) centrifugal floatation centrifugal floatation

TEGUH WS ‘08

ZnSOZnSO44 CENTRIFUGAL FLOATATION CENTRIFUGAL FLOATATIONZnSOZnSO44 CENTRIFUGAL FLOATATION CENTRIFUGAL FLOATATION

Non operculated worm eggsNon operculated worm eggs,, Protozoon cysts,larvae Protozoon cysts,larvae 331 gr331 gramsams ZnSOZnSO44 crytals crytals + + 670 mL water 670 mL water = = SG SG 1,181,18 1 part of stools + 10 parts of water1 part of stools + 10 parts of water mix mix Strain Strain pour into 15 mL conical centrifuge tube pour into 15 mL conical centrifuge tube Centrifuge at 400-500 or Centrifuge at 400-500 or 2300 2300 rpmrpm for for 11-2-2 menit menit decant the decant the

supernatantsupernatant Add 2 – 3 mL destilled water Add 2 – 3 mL destilled water recentrifuge (repeat 2-3 x) recentrifuge (repeat 2-3 x) Add 3-4 mL Add 3-4 mL ZnSOZnSO44 sol with SG 1.180 sol with SG 1.180 Centrifuge at full speed for 1- 2 minutesCentrifuge at full speed for 1- 2 minutes Use freshly flamed wire loop (diameter 5-7 mm) to touch the center Use freshly flamed wire loop (diameter 5-7 mm) to touch the center

of the surface film of the surface film object glass object glass examined with or without cover examined with or without cover glassglass

Non operculated worm eggsNon operculated worm eggs,, Protozoon cysts,larvae Protozoon cysts,larvae 331 gr331 gramsams ZnSOZnSO44 crytals crytals + + 670 mL water 670 mL water = = SG SG 1,181,18 1 part of stools + 10 parts of water1 part of stools + 10 parts of water mix mix Strain Strain pour into 15 mL conical centrifuge tube pour into 15 mL conical centrifuge tube Centrifuge at 400-500 or Centrifuge at 400-500 or 2300 2300 rpmrpm for for 11-2-2 menit menit decant the decant the

supernatantsupernatant Add 2 – 3 mL destilled water Add 2 – 3 mL destilled water recentrifuge (repeat 2-3 x) recentrifuge (repeat 2-3 x) Add 3-4 mL Add 3-4 mL ZnSOZnSO44 sol with SG 1.180 sol with SG 1.180 Centrifuge at full speed for 1- 2 minutesCentrifuge at full speed for 1- 2 minutes Use freshly flamed wire loop (diameter 5-7 mm) to touch the center Use freshly flamed wire loop (diameter 5-7 mm) to touch the center

of the surface film of the surface film object glass object glass examined with or without cover examined with or without cover glassglass

TEGUH WS ‘08

Method for using wire loop to remove Method for using wire loop to remove surface film from ZnSOsurface film from ZnSO44 floatation technique floatation technique

Method for using wire loop to remove Method for using wire loop to remove surface film from ZnSOsurface film from ZnSO44 floatation technique floatation technique

TEGUH WS ‘08

QUANTITATIVEQUANTITATIVEQUANTITATIVEQUANTITATIVE

For Estimating:For Estimating: Number of worm infecting host/worm burden Number of worm infecting host/worm burden degree or severity of infectiondegree or severity of infection Especially for intestinal nematode (Ascaris, Trichuris, Especially for intestinal nematode (Ascaris, Trichuris,

Hookworms)Hookworms) Most frequent method usedMost frequent method used

Kato-Katz’s Thick Smear Kato-Katz’s Thick Smear Stoll Egg Count TechniqueStoll Egg Count Technique

TEGUH WS ‘08

Kato Katz’s Thick SmearKato Katz’s Thick SmearKato Katz’s Thick SmearKato Katz’s Thick Smear

Procedure : As same as Kato Thick Smear,Procedure : As same as Kato Thick Smear,

but:but: Use wire strain for straining the stools Use wire strain for straining the stools Amount of stool can be calculated Amount of stool can be calculated

use thick paperuse thick paper

- 1 mm thick - 1 mm thick a hole a hole 9 mm ≈ 50-60 mg of stools, 9 mm ≈ 50-60 mg of stools,

oror

- - ½½ mm thick mm thick a hole a hole 6,5 mm ≈ 20-25 mg of stools 6,5 mm ≈ 20-25 mg of stools

- cover with cellophane soaked with Malachyte green - cover with cellophane soaked with Malachyte green allow it at allow it at

4040º C for 30 minutes or at room temperature for 1 hourº C for 30 minutes or at room temperature for 1 hour

- examine under the microscope and count the number of eggs- examine under the microscope and count the number of eggs

TEGUH WS ‘08

Stoll Egg Count TechniqueStoll Egg Count TechniqueStoll Egg Count TechniqueStoll Egg Count Technique

Equipments: Equipments: Long necked “Stoll flask” 60 mL with marker on 56 and 60 mLLong necked “Stoll flask” 60 mL with marker on 56 and 60 mL Stick/applicator for taking stoolsStick/applicator for taking stools Stoll Pipette (0,075 ml and 0,15 ml)Stoll Pipette (0,075 ml and 0,15 ml) Glass beads (diameter 6 mm)Glass beads (diameter 6 mm)

Materials :Materials : NaOH Solution 0,1 NNaOH Solution 0,1 N

Dissolve 4 g of NaOH in small amount of distilled waterDissolve 4 g of NaOH in small amount of distilled water Add distilled water to bring total volume to 1000 ml Add distilled water to bring total volume to 1000 ml Store until neededStore until needed

Equipments: Equipments: Long necked “Stoll flask” 60 mL with marker on 56 and 60 mLLong necked “Stoll flask” 60 mL with marker on 56 and 60 mL Stick/applicator for taking stoolsStick/applicator for taking stools Stoll Pipette (0,075 ml and 0,15 ml)Stoll Pipette (0,075 ml and 0,15 ml) Glass beads (diameter 6 mm)Glass beads (diameter 6 mm)

Materials :Materials : NaOH Solution 0,1 NNaOH Solution 0,1 N

Dissolve 4 g of NaOH in small amount of distilled waterDissolve 4 g of NaOH in small amount of distilled water Add distilled water to bring total volume to 1000 ml Add distilled water to bring total volume to 1000 ml Store until neededStore until needed

TEGUH WS ‘08

Stoll Egg Count TechniqueStoll Egg Count TechniqueStoll Egg Count TechniqueStoll Egg Count TechniqueProcedure:Procedure: Add 0,1 N NaOH solution to the 56 mL mark of the Stoll flaskAdd 0,1 N NaOH solution to the 56 mL mark of the Stoll flask Using applicator stick carefully add feces to the flask Using applicator stick carefully add feces to the flask the level of the level of

fluid raised to the 60 cc markfluid raised to the 60 cc mark Add 5-10 small glass beads to the flask, stopper the flask Add 5-10 small glass beads to the flask, stopper the flask shake shake

vigorously with up-and-down motion for a minute or more to obtain a vigorously with up-and-down motion for a minute or more to obtain a homogenous suspension. If the stool is hard, homogenous suspension. If the stool is hard, allow the mixture to allow the mixture to stand for several hours or overnightstand for several hours or overnight

Use Stoll pipette to remove sample 0,15 mL from the center of the Use Stoll pipette to remove sample 0,15 mL from the center of the suspensionsuspension

Expel the pipette content on to a slide Expel the pipette content on to a slide cover with 22x44 mm cover with 22x44 mm cover glass cover glass

Count all of the eggs on the slide (p). The number of egg per gram 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) of feces, is the number of eggs obtained x 100. (NEPG = p x 100)

Correction factors according to consistency of stools:Correction factors according to consistency of stools: Soft Soft x 1,5 ; Loose x 1,5 ; Loose x 2; Watery x 2; Watery x 3 x 3

TEGUH WS ‘08

Worm burdenWorm burden

Daily production of eggs Daily production of eggs Trichuris trichiuraTrichuris trichiura = 5.000/ day = 5.000/ day Necator americanusNecator americanus = 9.000/ day = 9.000/ day Ascaris lumbricoidesAscaris lumbricoides = 200.000/day = 200.000/day

If the total amount of feces be weight and the NEPG had been If the total amount of feces be weight and the NEPG had been calculated calculated the number of worms infecting host can be estimated the number of worms infecting host can be estimated

Single adult Ascaris may be dangerous, due to migrating tendencySingle adult Ascaris may be dangerous, due to migrating tendency Hookworms infection with NEPG > 2500 Hookworms infection with NEPG > 2500 clinical symptoms clinical symptoms Trichuris infection with NEPG > 20.000 Trichuris infection with NEPG > 20.000 clinical symptoms clinical symptoms

TEGUH WS ‘08

Culture of fecesCulture of fecesCulture of fecesCulture of feces

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

as:as: Strongyloides stercoralisStrongyloides stercoralis Necator americanusNecator americanus Ancylostoma duodenaleAncylostoma duodenale Trychostrogylus sppTrychostrogylus spp

The larvae of those worm can be differentiated morphologicallyThe larvae of those worm can be differentiated morphologically

TEGUH WS ‘08

HARADA MORI METHODHARADA MORI METHODHARADA MORI METHODHARADA MORI METHOD Use conical/screw cap test tube vol. 15 mL Use conical/screw cap test tube vol. 15 mL Use a 13x120 mm paper strip that tapers at Use a 13x120 mm paper strip that tapers at

the end inserted into the tube. Write the the end inserted into the tube. Write the specimen identification number on itspecimen identification number on it

Spread approximately 0.5 – 1.0 of feces on to Spread approximately 0.5 – 1.0 of feces on to the middle one-third of the strip, the middle one-third of the strip, forms a forms a layer several millimeters thicklayer several millimeters thick

Insert the strip into conical screw cap test tube Insert the strip into conical screw cap test tube several millimeters of the tube bottom several millimeters of the tube bottom

Carefully add distilled water until the water is Carefully add distilled water until the water is slightly below the fecal mass slightly below the fecal mass do not wash!! do not wash!!

Keep test tube upright in the test-tube rack at Keep test tube upright in the test-tube rack at room temperature (24room temperature (24ºº-28-28ººC) for 7-10 daysC) for 7-10 days

Larvae in fluid can be heat-killed and removed Larvae in fluid can be heat-killed and removed from the bottom of the tube using a glass from the bottom of the tube using a glass pipette pipette drop on a slide drop on a slide iodine iodineexamineexamine

Use conical/screw cap test tube vol. 15 mL Use conical/screw cap test tube vol. 15 mL Use a 13x120 mm paper strip that tapers at Use a 13x120 mm paper strip that tapers at

the end inserted into the tube. Write the the end inserted into the tube. Write the specimen identification number on itspecimen identification number on it

Spread approximately 0.5 – 1.0 of feces on to Spread approximately 0.5 – 1.0 of feces on to the middle one-third of the strip, the middle one-third of the strip, forms a forms a layer several millimeters thicklayer several millimeters thick

Insert the strip into conical screw cap test tube Insert the strip into conical screw cap test tube several millimeters of the tube bottom several millimeters of the tube bottom

Carefully add distilled water until the water is Carefully add distilled water until the water is slightly below the fecal mass slightly below the fecal mass do not wash!! do not wash!!

Keep test tube upright in the test-tube rack at Keep test tube upright in the test-tube rack at room temperature (24room temperature (24ºº-28-28ººC) for 7-10 daysC) for 7-10 days

Larvae in fluid can be heat-killed and removed Larvae in fluid can be heat-killed and removed from the bottom of the tube using a glass from the bottom of the tube using a glass pipette pipette drop on a slide drop on a slide iodine iodineexamineexamine

TEGUH WS ‘08

Polyethylene tubePolyethylene tubePolyethylene tubePolyethylene tube

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

TEGUH WS ‘08

Sand culture techniqueSand culture techniqueSand culture techniqueSand culture technique

Filter paper/slant CultureFilter paper/slant Culture write sample identitywrite sample identity Smear feces on to middle 1/3 partSmear feces on to middle 1/3 part Place the strip on to a slide Place the strip on to a slide petri dish petri dish

so that one end rests on a short piece of so that one end rests on a short piece of glass tubingglass tubing

Add a shallow layer of distilled waterAdd a shallow layer of distilled water Cover dish and allow it to stand at 24º-Cover dish and allow it to stand at 24º-

28ºC28ºC

Filter paper/slant CultureFilter paper/slant Culture write sample identitywrite sample identity Smear feces on to middle 1/3 partSmear feces on to middle 1/3 part Place the strip on to a slide Place the strip on to a slide petri dish petri dish

so that one end rests on a short piece of so that one end rests on a short piece of glass tubingglass tubing

Add a shallow layer of distilled waterAdd a shallow layer of distilled water Cover dish and allow it to stand at 24º-Cover dish and allow it to stand at 24º-

28ºC28ºC

For Soil Transmitted HelminthsFor Soil Transmitted Helminths Procedure:Procedure:

Sterile Petri-dish filled by sterile sandSterile Petri-dish filled by sterile sand Add feces on to it and coveredAdd feces on to it and covered Rhabditiform larva (+) after 1 dayRhabditiform larva (+) after 1 day Filariform larva (+) after D 3-7Filariform larva (+) after D 3-7

The culture can be examined under dissecting microscope for the presence of free living adult or larvae of S. stercoralis.

Hookworm and trichostrogylid larva can be found in water

TEGUH WS ‘08

Cellulose Tape Preparation Cellulose Tape Preparation ProcedureProcedure

Grahams-Scotch’s Adhesive Tape Swab Grahams-Scotch’s Adhesive Tape Swab For diagnosing intestinal parasites which migrate to perianal region:For diagnosing intestinal parasites which migrate to perianal region:

Enterobius vermicularisEnterobius vermicularis (adult and eggs) (adult and eggs) Taenia solium & T. saginata Taenia solium & T. saginata eggs (less frequency)eggs (less frequency) In the morning, prior to using the toilet or bathingIn the morning, prior to using the toilet or bathing Cellulose tape preparations should be taken at least 3-4 Cellulose tape preparations should be taken at least 3-4

consecutive days before a patient is considered to be free from consecutive days before a patient is considered to be free from infectioninfection

Adult male of Adult male of Enterobius vermicularisEnterobius vermicularis can demonstrated by cellulose tape preparation alsocan demonstrated by cellulose tape preparation also Can be seen with naked eye, crawling around the anus, but the Can be seen with naked eye, crawling around the anus, but the

male male

TEGUH WS ‘08

Materials and methodsMaterials and methodsMaterials and methodsMaterials and methodsProceduresProceduresa. Place a strip of cellulose tape on a a. Place a strip of cellulose tape on a

microscopic slide, starting 1.2 cm from microscopic slide, starting 1.2 cm from one end, and running toward the same one end, and running toward the same end. Wrap the tape over the edge of the end. Wrap the tape over the edge of the slide.slide.

b. Peel back the tape, and, with adhesive b. Peel back the tape, and, with adhesive side outward over a wooden tongue side outward over a wooden tongue depressor. depressor.

c. Press the tape firmly against the right and c. Press the tape firmly against the right and left perianal foldleft perianal fold

d. Spread the tape back over the slide, d. Spread the tape back over the slide, adhesive side downadhesive side down

e. Press the tape down on the slide, examine e. Press the tape down on the slide, examine directly under low power of microscope. directly under low power of microscope.

TEGUH WS ‘08

ANAL SWAB METHODANAL SWAB METHOD For diagnosing of For diagnosing of Enterobius vermicularisEnterobius vermicularis eggs eggs Is an alternative procedure for diagnosing pinworm infectionIs an alternative procedure for diagnosing pinworm infection This procedure is more complex than the cellulose tape methodThis procedure is more complex than the cellulose tape method Offers no advantages over itOffers no advantages over it

Procedure:Procedure: Immerse cotton swab in paraffin and petroleum jelly mixture, place Immerse cotton swab in paraffin and petroleum jelly mixture, place

swabs into tubes, store in refrigerator until neededswabs into tubes, store in refrigerator until needed Spread the buttock and rub the swab over the perianal surface very Spread the buttock and rub the swab over the perianal surface very

gently. Insert the swab a short distance into the anal opening. gently. Insert the swab a short distance into the anal opening. Return the swab to the tube and labelReturn the swab to the tube and label

Fill the tube Fill the tube ± half full ŵ xylene to cover the swab ± half full ŵ xylene to cover the swab allow for 5 min. allow for 5 min. Remove the swab, centrifuge the suspension 500 g/ for 1 min., Remove the swab, centrifuge the suspension 500 g/ for 1 min.,

remove supernatant remove supernatant examine under microscope examine under microscope

Examination of BloodExamination of BloodExamination of BloodExamination of Blood

MALARIAMALARIA FILARIASISFILARIASIS TOXOPLASMOSISTOXOPLASMOSIS TRYPANOSOMIASISTRYPANOSOMIASIS LEISHMANIASISLEISHMANIASIS

MALARIAMALARIA FILARIASISFILARIASIS TOXOPLASMOSISTOXOPLASMOSIS TRYPANOSOMIASISTRYPANOSOMIASIS LEISHMANIASISLEISHMANIASIS

TEGUH WS ‘08

Examination of BloodExamination of BloodExamination of BloodExamination of Blood

Taking of peripheral blood samplesTaking of peripheral blood samples Capillary blood : finger tip, heel, auricle/ear lobeCapillary blood : finger tip, heel, auricle/ear lobe Circulatory blood : v. mediana cubitiCirculatory blood : v. mediana cubiti

Preparation blood filmPreparation blood film Thin blood film/smear : malaria, trypanosomiasis, babesiosisThin blood film/smear : malaria, trypanosomiasis, babesiosis

Because of the small amount of blood used in preparation of thin film, Because of the small amount of blood used in preparation of thin film, they are not as sensitive for detecting parasites as one would likethey are not as sensitive for detecting parasites as one would like

Thick blood film/smear : malaria, filariaThick blood film/smear : malaria, filaria The amount of blood used in preparation of thick film is more The amount of blood used in preparation of thick film is more more more

sensitive, but less specific than thin film, sensitive, but less specific than thin film,

TEGUH WS ‘08

Capillary blood administrationCapillary blood administration

TEGUH WS ‘08

TEGUH WS ‘08

TEGUH WS ‘08

Examination of MicrofilariaExamination of MicrofilariaExamination of MicrofilariaExamination of Microfilaria Direct examination (wet blood)Direct examination (wet blood)

Directly examining the motility of microfilariaDirectly examining the motility of microfilaria Punction the finger tip with lancet Punction the finger tip with lancet suck the blood with capillary suck the blood with capillary

tube tube drop blood on to a clean and dry slide drop blood on to a clean and dry slide spread with spread with other slide edge other slide edge directly examine under the microscope directly examine under the microscope

Knott Concentration TechniqueKnott Concentration Technique Venous blood + anticoagulant Venous blood + anticoagulant into a tube containing formaline into a tube containing formaline

solution 2% (10 cc) solution 2% (10 cc) centrifuge for 2 minutes centrifuge for 2 minutes Decant supernatant, directly examine the sediment, or stained Decant supernatant, directly examine the sediment, or stained

with Giemsa stainingwith Giemsa staining Straining using nucleophore filterStraining using nucleophore filter

Venous blood + anticoagulant were strained using nucleophore Venous blood + anticoagulant were strained using nucleophore filter filter microfilaria will strained microfilaria will strained stain with Giemsa stain with Giemsa examineexamine

Direct examination (wet blood)Direct examination (wet blood) Directly examining the motility of microfilariaDirectly examining the motility of microfilaria Punction the finger tip with lancet Punction the finger tip with lancet suck the blood with capillary suck the blood with capillary

tube tube drop blood on to a clean and dry slide drop blood on to a clean and dry slide spread with spread with other slide edge other slide edge directly examine under the microscope directly examine under the microscope

Knott Concentration TechniqueKnott Concentration Technique Venous blood + anticoagulant Venous blood + anticoagulant into a tube containing formaline into a tube containing formaline

solution 2% (10 cc) solution 2% (10 cc) centrifuge for 2 minutes centrifuge for 2 minutes Decant supernatant, directly examine the sediment, or stained Decant supernatant, directly examine the sediment, or stained

with Giemsa stainingwith Giemsa staining Straining using nucleophore filterStraining using nucleophore filter

Venous blood + anticoagulant were strained using nucleophore Venous blood + anticoagulant were strained using nucleophore filter filter microfilaria will strained microfilaria will strained stain with Giemsa stain with Giemsa examineexamine

TEGUH WS ‘08

Air dried

Dehaemoglobin with water

Air dried

Fixed with methanolStained with

Giemsa 1 : 15 for 15 min.

washed with running water

Examined under compound microscope

Capillary tube

20 l

Wet preparation

TEGUH WS ‘08

Examination of urineExamination of urine(Urinalysis)(Urinalysis)

Examination of urineExamination of urine(Urinalysis)(Urinalysis)

Urinalysis Urinalysis centrifugation centrifugation urine sediment urine sediment Urogenital tract parasitesUrogenital tract parasites

Trichomonas vaginalisTrichomonas vaginalis Schistosoma haematobiumSchistosoma haematobium Others : Polychaete annelids, Ren wormOthers : Polychaete annelids, Ren worm

Chyluria Chyluria microfilaria of microfilaria of Wuchereria bancrofti, Onchocerca Wuchereria bancrofti, Onchocerca volvulusvolvulus

Trophozoite of Trophozoite of Entamoeba histolyticaEntamoeba histolytica fistel? fistel? Strongyloides stercoralis LarvaeStrongyloides stercoralis Larvae

Urinalysis Urinalysis centrifugation centrifugation urine sediment urine sediment Urogenital tract parasitesUrogenital tract parasites

Trichomonas vaginalisTrichomonas vaginalis Schistosoma haematobiumSchistosoma haematobium Others : Polychaete annelids, Ren wormOthers : Polychaete annelids, Ren worm

Chyluria Chyluria microfilaria of microfilaria of Wuchereria bancrofti, Onchocerca Wuchereria bancrofti, Onchocerca volvulusvolvulus

Trophozoite of Trophozoite of Entamoeba histolyticaEntamoeba histolytica fistel? fistel? Strongyloides stercoralis LarvaeStrongyloides stercoralis Larvae

TEGUH WS ‘08

Examination of sputumExamination of sputumExamination of sputumExamination of sputum

DIRECT SMEAR METHODDIRECT SMEAR METHOD For Pulmonary parasitesFor Pulmonary parasites

Paragonimus Paragonimus Amoeba Amoeba Hydatid cystHydatid cyst Strongyloides stercoralis LarvaeStrongyloides stercoralis Larvae

DIRECT SMEAR METHODDIRECT SMEAR METHOD For Pulmonary parasitesFor Pulmonary parasites

Paragonimus Paragonimus Amoeba Amoeba Hydatid cystHydatid cyst Strongyloides stercoralis LarvaeStrongyloides stercoralis Larvae

TEGUH WS ‘08

OTHER SPECIMENSOTHER SPECIMENS OTHER SPECIMENSOTHER SPECIMENS

Other body fluidsOther body fluids LCSLCS Ascites, pleural fluidAscites, pleural fluid

Scratching /rub down of Skin or mucosalScratching /rub down of Skin or mucosal Muscle biopsy Muscle biopsy Duodenal fluid aspirationDuodenal fluid aspiration Lymph-node biopsy Lymph-node biopsy Soil Soil Finger nails Finger nails

Other body fluidsOther body fluids LCSLCS Ascites, pleural fluidAscites, pleural fluid

Scratching /rub down of Skin or mucosalScratching /rub down of Skin or mucosal Muscle biopsy Muscle biopsy Duodenal fluid aspirationDuodenal fluid aspiration Lymph-node biopsy Lymph-node biopsy Soil Soil Finger nails Finger nails

TEGUH WS ‘08

ThankyouThankyouThankyouThankyou