lab examination parasitology unibraw
TRANSCRIPT
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
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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
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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
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CONSISTENCYCONSISTENCY
FORMEDFORMED
SOFTSOFT
LOOSELOOSE
WATERYWATERY
CYSTS
TROPHS
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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
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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))
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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.
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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
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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,
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Capillary blood administrationCapillary blood administration
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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
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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
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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
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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
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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
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ThankyouThankyouThankyouThankyou