lab oratory diagnosis of parasitic diseases
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LAB ORATORY LAB ORATORY DIAGNOSIS OF DIAGNOSIS OF
PARASITIC DISEASESPARASITIC DISEASES
DEPARTEMENT OFDEPARTEMENT OF PARASITOLOG PARASITOLOGYY
FAFACCULTULTY OF MEDICINEY OF MEDICINE
UNIVERSITAS PADJADJARANUNIVERSITAS PADJADJARAN
DEPARTEMENT OFDEPARTEMENT OF PARASITOLOG PARASITOLOGYY
FAFACCULTULTY OF MEDICINEY OF MEDICINE
UNIVERSITAS PADJADJARANUNIVERSITAS PADJADJARAN
INTRODUCTION
DIAGNOSIS FOR PARASITIC INFECTION
Anamnesa – ( The history should include details of the presenting complaint )
Physical examination Laboratory examination espectially
Parasitological diagnosis Imunodiagnosis
DIAGNOSIS FOR PARASITIC INFECTION
Anamnesa – ( The history should include details of the presenting complaint )
Physical examination Laboratory examination espectially
Parasitological diagnosis Imunodiagnosis
AN
PHYSICAL EXAMINATIONPHYSICAL EXAMINATION
ANAMNESIS ANAMNESIS
ASPESIFICASPESIFIC
CLINICAL FEATURES (Symptom and Sign)
CLINICAL FEATURES (Symptom and Sign)
SPESIFICSPESIFIC
LABORATORY EXAMINATIONLABORATORY EXAMINATION
1
3
2
INTRODUCTION
-Fever
-Gastrointestinal symptoms
-Fever with respiratory system
-Neurological symptoms
-Fever and meningitis / Encephalitis
-Cutaneous symptoms
-Fever
-Gastrointestinal symptoms
-Fever with respiratory system
-Neurological symptoms
-Fever and meningitis / Encephalitis
-Cutaneous symptoms
INTRODUCTION
DEPEND ON : Habitat ParasiteParasite distribution
DEPEND ON : Habitat ParasiteParasite distribution
•Very important for examination Helminth parasite and Protozoa parasite
• Repeating of Laboratory examination occasionally be needed
*Should be understood about examination technique,morphology of
parasite and life cycle of parasite
•Very important for examination Helminth parasite and Protozoa parasite
• Repeating of Laboratory examination occasionally be needed
*Should be understood about examination technique,morphology of
parasite and life cycle of parasite
TYPE OF SPECIMEN :1. Stool 2. Blood3 Urine4 Sputum5. Vaginal discharge, urethral
discharge6. Skin scrapings7. Liquor Cerebro Spinal 8. Tissue biopsy9. Nasal discharge10. Corneal scrapings
TYPE OF SPECIMEN :1. Stool 2. Blood3 Urine4 Sputum5. Vaginal discharge, urethral
discharge6. Skin scrapings7. Liquor Cerebro Spinal 8. Tissue biopsy9. Nasal discharge10. Corneal scrapings
LABORATORY EXAMINATIONLABORATORY EXAMINATION3
IMUNODIAGNOSISIMUNODIAGNOSIS4
IMPORTANTIMPORTANT
INTRODUCTION
FECAL SPECIMENSFECAL SPECIMENS
Immediatelly have to examine :
Liquid specimens should be examined within 30 minute of passage
Soft (semiformed) specimens 1 hour
Formed specimens 24 hour after passage
If this time is not possible, the specimen should be placed in one of the available fixatives :
Formalin 10%, MIF (Merthiolate Iodine Formalin), PVA (Polyvinyl Alcohol)
Generally Helminthic eggs more endure without preservation than intestinal protozoa
Immediatelly have to examine :
Liquid specimens should be examined within 30 minute of passage
Soft (semiformed) specimens 1 hour
Formed specimens 24 hour after passage
If this time is not possible, the specimen should be placed in one of the available fixatives :
Formalin 10%, MIF (Merthiolate Iodine Formalin), PVA (Polyvinyl Alcohol)
Generally Helminthic eggs more endure without preservation than intestinal protozoa
INTRODUCTION
FECAL SPECIMENSFECAL SPECIMENS
The specimens should not be contaminated with
Water – because water may contain free-
living organisms that can be mistaken for
human parasites
Urine – may destroy motile organisms
Prior to examination , fecal specimens should
never be incubated or frozen.
A chatartic with an oil base should not be used,
and a stool softener (taken either orally or as a
suppository) is usually inadequate for obtaining a
purged specimen.
The specimens should not be contaminated with
Water – because water may contain free-
living organisms that can be mistaken for
human parasites
Urine – may destroy motile organisms
Prior to examination , fecal specimens should
never be incubated or frozen.
A chatartic with an oil base should not be used,
and a stool softener (taken either orally or as a
suppository) is usually inadequate for obtaining a
purged specimen.
INTRODUCTION
FECAL SPECIMENSFECAL SPECIMENS
Repeating fecal examination after therapy :
Ascariasis, 2-3 weeks after therapy
Protozoa infection, 3-4 weeks after
therapy
Taeniasis, 5-6 weeks after therapy
Repeating fecal examination after therapy :
Ascariasis, 2-3 weeks after therapy
Protozoa infection, 3-4 weeks after
therapy
Taeniasis, 5-6 weeks after therapy
EXAMINATION OF HELMINTH PARASITE
SPECIMENS( most important )
SPECIMENS( most important )
FECAL SPECIMENS FECAL SPECIMENS
BLOOD AND TISSUE SPECIMENS BLOOD AND TISSUE SPECIMENS
&&
EXAMINATION OF HELMINTH PARASITE
FECAL SPECIMENS FECAL SPECIMENS ?
For examination of helminth egg Most important for examination intestinal
nematode including “Soil Transmitted Helminths” :
Ascaris lumbricoides Trichuris trichiura Hook worm : - Necator americanus
and Ancylostoma duodenale Strongyloides stercoralis
For examination of helminth egg Most important for examination intestinal
nematode including “Soil Transmitted Helminths” :
Ascaris lumbricoides Trichuris trichiura Hook worm : - Necator americanus
and Ancylostoma duodenale Strongyloides stercoralis
EXAMINATION OF HELMINTH PARASITE
FECAL SPECIMENS FECAL SPECIMENS
DIRECT WET SMEAR
QUALITATIVE EXAMINATIONQUALITATIVE EXAMINATION
ANOTHER QUALITATIVE EXAMINATION AND
QUANTITATIVE EXAMINATION
TO BE STUDIED IN FECAL EXAMINATION
LABORATORY TECHNIQUE FOR EXAMINATION OF HELMINTH PARASITE
LABORATORY TECHNIQUE FOR EXAMINATION OF HELMINTH PARASITE
LABORATORY TECHNIQUE FOR
EXAMINATION HELMINTH PARASITE
INTESTINAL
HELMINTH
BLOOD AND
TISSUE
HELMINTH
FECAL SPECIMENS
BLOOD AND TISSUE
HELMINTH
Click “Esc” buttonWhen finished
MACROSCOPIC
EXAMINATION
MICROSCOPIC
EXAMINATION
EXAMINATION TECHNIQUE FORHELMINTH PARASITE IN FAECES
LABORATORY TECHNIQUE FOR EXAMINATION OF INTESTINAL
HELMINTH
QUALITATIVE
QUANTITATIVE
*Consistency (hard,formed,soft,diarrhea)
*Colour
*Odor
*Foreign bodies (blood, mucus,parasite)
Click “Esc” buttonWhen finished
MICROSCOPIC
QUALITATIVE
STOLL DILLUTION
METHOD
KATO – KATZ
CELLOPHANE THICK
SMEAR METHOD
QUANTITATIVE
EXAMINATION TECHNIQUE OF INTESTINAL HELMINTH
Click “Esc” buttonWhen finished
DIRECT WET SMEAR METHOD
FLOTATION METHOD
MODIFICATION MERTHIOLATE IODINE
FORMALDEHYDE (MIF)
CELLOTAPE TAPE METHOD
FORMALDEHYDE ETHER SEDIMENTATION
(RITCHIE’S METHOD)
METODA KATO
Fast Severe infection Reagens
NaCl Physiologis (0,9%), or Eosin 2%
DIRECTDIRECT WET SMEAR METHOD WET SMEAR METHOD
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
Click “Esc” buttonWhen finished
DIRECT WET SMEAR METHODDIRECT WET SMEAR METHOD
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
1-2 dropsNaCl 0,9%
oreosin 2%
Place 2 mg faeceson microscope slide
Emulsify 2 mg faeces in NaCl 0.9% dropPlace 1-2 drops NaCl 0,9% or Eosin
2% on microscope slide
Click “Esc” buttonWhen finished
Place 2 mg faeces on microscope slide
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
SALINE SALINE WET SMEAR METHODWET SMEAR METHODPlace 2 mg faeces
on microscope slide
Emulsify 2 mg faeces in NaCl 0,9% drop
Place coverslip overthe suspension
1-2 dropsNaCl 0,9%
Emulsify 2mg faeces in the saline dropPlace coverslip over suspension
Examine using the lower power objective 10x or 40x
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
FLOATATION METHODFLOATATION METHOD
Berdasarkan BJ telur < BJ larutan
Berguna untuk infeksi ringan Larutan yang dipergunakan :
NaCl jenuh, atau Gula jenuh
FLOTATION METHODWITH CENTRIFUGATION
WITHOUT CENTRIFUGATION
Click “Esc” buttonWhen finished
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
NaCl saturatedfaeces
stirringglass
Ose
20 minutes
10 gr faeces mixed with 200 ml NaCl saturated until homogenous solution
Take down 1-2 drops of Surface layer with Ose andPlace on microscope slide
10 gr. faeces + 200 cc NaCl saturated
Place coverslip overthe solution
FLOATATION METHODFLOATATION METHODWITHOUT CENTRIFUGATION
Take down 1-2 drops the surface layer with OsePlace coverslip over microcope slide
Examine under low power objective
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
NaCl saturatedfaeces
Stirringglass
filtered
Centrifuge 100 rpmfor 5 minutes
Transfer 10 gr faeces and emulsify in 200 ml NaCl saturated until
homogenous solution10 gr. faeces + 200 cc NaCl saturated
FLOATATION METHODFLOATATION METHODWITH CENTRIFUGATION
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
NaCl saturatedfaeces
stirringglass
filtered
centrifuge 100 x/mnt5 minutes
Filtered with tea filter10 gr. faeces
+ 200 cc NaCl saturated
FLOATATION METHODFLOATATION METHODWITH CENTRIFUGATION
NaCl saturatedfaeces
stirringglass
filtered
centrifuge 100 rpmFor 5 minutes
Decant into centrifuge tube
10 gr. faeces + 200 cc NaCl saturated
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
FLOATATION METHODFLOATATION METHODWITH CENTRIFUGATION
NaCl saturatedfaeces
Stirringglass
filtered
centrifuge 100 rpmFor 5 minutes
Centrifuge 100 rpm for 5 minutes
10 gr.faeces + 200 cc NaCl saturated
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
FLOATATION METHODFLOATATION METHODWITH CENTRIFUGATION
NaCl saturatedfaeces
stirringglass
filtered
Centrifuge 100 rpmFor 5 minutes
Take down solution from surface layer with Ose
10 gr. faeces + 200 cc NaCl saturated
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
FLOATATION METHODFLOATATION METHODWITH CENTRIFUGATION
Place solution on microscope slide and Place coverslip over the microscope slide.
Examine under low power objective 10 x or 40 x.
Place the solution onmicroscope slide Place coverslip over
microscope slide
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
FLOATATION METHODFLOATATION METHODWITH CENTRIFUGATION
For identification eggs from intestinal helminth, Ameba and Giardia lamblia
Solution 1 : 250 ml aquadest 200 ml thimerosal (1:1.000) 25 ml formalin 5 ml glycerin
Solution 2 : Fresh lugol solution5%
MERTIOLAT IODIN FORMALIN MODIFICATION MERTIOLAT IODIN FORMALIN MODIFICATION (MIF MODIFICATION)(MIF MODIFICATION)
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
MERTMERTHHIOLAT IODIN FORMALIN MODIFIIOLAT IODIN FORMALIN MODIFICATIONCATION (MIF MODIFI(MIF MODIFICATIONCATION ) )
It’s good for staining and conservation of cyst of intestine protozoa and worm egg
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
(+) 7 ml. ether(40 C)
MERTMERTHHIOLAT IODIN FORMALIN MODIFIIOLAT IODIN FORMALIN MODIFICATIONCATION (MIF MODIFI(MIF MODIFICATIONCATION ) )
Gelaspengaduk
5 ml base solution 10,5 ml base solution 2
0,5 gr faeces
filtered Shake until homogenous
centrifuge1.000-3.000 x/mnt1 minutes
shake until homogenous
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
Add 7 ml ether (40 C)
Open the tube, let it 2 minutes, centrifuge for 1 minutes (1.000-3.000 rpm)
MERTMERTHHIOLAT IODIN FORMALIN MODIFIIOLAT IODIN FORMALIN MODIFICATIONCATION (MIF MODIFI(MIF MODIFICATIONCATION ) )
Throw away the supernatan,take sediment with pipette
Place the sediment onmicroscope slide
Place coverslip overmicroscope slide
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
Examine under low power objective
CELLOTAPE METHODCELLOTAPE METHOD
The egg adhere on perianal area, so rarely found in
faeces (5 %). To find this parasite we need Scotch
Adhesive tape Swab from Graham or Cellotape
Method
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
CELLOTAPE METHODCELLOTAPE METHOD
To examine the egg of Enterobius vermicularis Children 1-10 years old Doing in the morning before take a bath or wash
the anus with water after defecating Transparent plastic plaster (2 x 1,5 cm)patched
to skin around the anus Press the plaster, then lift slowly Patched to the object glass, examine under the
microscope
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
CELLOTAPE METHODCELLOTAPE METHOD
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
Concentration MethodConcentration Method
practical, simple, for ova examination in stool : 1 gr faeces, put into the reaction tube, add aquadest,
mixed until homogenous, put imyo the centrifuge tube Centrifuge with velocity 3.000 rpm for 1 mnt Throw away the supernatan, take the sediment with
pipette Place the sediment on microscope slide, place
coverslip on microscope slide
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
Put in faeces solution to the centrifuge tube
Centrifuge 3.000 x/mnt,1 minutes
Concentration MethodConcentration Method
Shake until homogenous
1 gr faeces
Stirring glass
Aquadest
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
Throw away supernatan, take sediment with pipette
Place sediment on microscope slide
Concentration MethodConcentration Method
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
place coverslip on microscope slide
Examine under the microscope
CELLOPHANE THICK SMEAR METHOD (KATO METHOD)(KATO METHOD)
Practical, simple, and cheap Can be used in mass examination Examination needs more stool, so the eggs can be found
much more Morphology of egg is clear
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
CELLOPHANE THICK SMEAR METHOD
(KATO METHOD(KATO METHOD))
Substance : Solution
100 ml aquadest/fenol 6% 100 ml glycerin (supaya kotoran tinja jadi jernih) 1 ml malachit green solution (supaya mata tidak
silau)
Cellophane tape, 2,5 x 3 cm, soak in solution for >24 hours
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
CELLOPHANE THICK SMEAR METHOD(KATO METHOD)(KATO METHOD)
Technique : Take 20-50 mg faeces ( as large as red bean ) Put on object glass, spread out Cover with cellophane, pressing the faeces until
flat and spread out under the cellophane Drain the excessive fluid with filter paper Let it 20-30 minutes Examine under the microscope
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
CELLOPHANE THICK SMEAR METHOD(KATO METHOD)(KATO METHOD)
100 part. Aquadest/fenol 6%100 part. Glycerin1 part Malachit green solution
Cut the cellophane2,5 x 3 cm
Soaked
> 24 hours
20-50 grfaeces
Soaked cellophane
faeces
MIMICCROSROSCCOPIOPICCQQUALITATIUALITATIVEVE
Solution used : NaOH 0,1 N (faeces solvent) or KOH 10%
Good for severe and moderate infection
Less good for mild infection
STOLL METHODSTOLL METHOD
MIMICCROSROSCCOPIOPICCQQUANTITATIUANTITATIVEVE
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
56 m l60 m l
56 m l60 m l
Butir G elas
Shake
Let it 1 night
or3-4 hours but shake it
longer
56 m l60 m l
faeces
NaOH solution 0,1N
STOLL METHODSTOLL METHOD
56 ml 60 ml
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
Shake and take 0,15 ml
56 m l60 m l
STOLL METHODSTOLL METHOD
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
Formula :
amount of egg in 1 gram feces = amount of seen egg (miroscopic) x 100
STOLL METHODSTOLL METHOD
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
ENUMARATION NaOH = 56 ml, feces 4 ml ~ 4gr 4 gr feces in 60 ml or 1 gr feces in 15 ml In the 0.15 ml of stool solution,
can be found y eggs So, in the 15 ml, is found y x 100
eggs(~ 1 gr stool)
STOLL METHODSTOLL METHOD
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
Infection level based on amount of egg in each gram feces and amount of helminth
MILD MODERATE SEVERE
< 7.000 7.000-35.000 > 35.000
A. lumbricoides
5 or less 6-25 > 25
MILD MODERATE SEVERE
A. duodenale
< 3.000 3.000-10.000 > 10.000
20 or less 21-100 > 100
MILD MODERATE SEVERE
N. americanus
< 2.000 2.000-7.000 > 7.000
50or less 51-200 > 200
AMOUNT OF HELMINTH
AMOUNT OF EGG PER-GRAM
FAECES
INFECTION LEVEL
Infectid by
SOURCE : PARASITIC DISEASES PROGRAME. WHO. GENEVA, 1981
STOLL METHODSTOLL METHOD
KATO-KATZ METHODKATO-KATZ METHOD
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
Tolls : Object glass cellotape, thick 40 , size 3 x 3 cm Thick carton, make hole with fixed
volume to print faeces as weight as 30 mg
Palm leaf rib, oily papper, wire netting
KATO-KATZ METHODKATO-KATZ METHOD
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
Malachite-Green solution : 100 ml aquadest 100 ml glycerin (in order to make the
feces clear) 1 ml malachite green solution
Soak the cellotape in solution for > 24 hours
KATO-KATZ METHODKATO-KATZ METHOD
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
FILTERING FAECES
Put 5 gr faeces on the oily papper, put wire netting on it then press
Put the holed carton on the object glass, print the filtered faeces on holed carton
Lift the carton Cover the faeces
with soaked cellotape
Press, spread out Let it 30 mnt Examine under the
microscope
PRINTING FAECES MAKING PREPARAT
KATO-KATZ METHODKATO-KATZ METHOD
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
Filtering faeces
Oily papper
Wire netting
press
faeces (5 gr)
TEKAN
Wire netting
faeces (5 gr)
Object glass
Holed carton
Filtered faeces
printed
Printed faeces Object glass
Soaked cellotape
Printing faeces Making THE SPECIMEN
EXAMINE UNDERTHE MICROSCOPE
KATO-KATZ METHODKATO-KATZ METHOD
MICROSCOPICMICROSCOPICQUANTITATIVEQUANTITATIVE
WHO (1981) EGG PRODUCTION PER-DAY :
A. lumbricoides : 200.000 A. doudenale : 10.000-25.000 N. americanus : 5.000-10.000
WEIGHT OF FAECES PER-DAY Children : 70 gram Adult : 2 x children
FORMOL ETHER SEDIMENTATION METHODFORMOL ETHER SEDIMENTATION METHOD(RITCHIE)
MICROSCOPICMICROSCOPICQUALITATIVEQUALITATIVE
(0,5 ml faeces) + (1-2 ml aquadest), shake, + (10-12 ml aquadest), shake
Filter Centrifuge 1.000 rpm, 1 mnt + (1 ml formalin 10%) + (formalin 10% sampai
volume 8 ml), let it 10 mnt + (3ml ether), shake 15-20 second Centrifuge 2.000 rpm, 1-2 mnt Throw away the supernatan, place the
sediment on microscope slide, examine under the microscope
MICROSCOPICMICROSCOPICQUALITATIVEQUALITATIVE
shake
0,5 ml. faeces
1-2 ml. aquadest
shake
10-12 ml. aquadest
filter
centrifuge1.000 rpm, 1 minute
FORMOL ETHER SEDIMENTATION METHODFORMOL ETHER SEDIMENTATION METHOD(RITCHIE)
centrifuge 1.000 rpm, 1 minute
MICROSCOPICMICROSCOPICQUALITATIVEQUALITATIVE
+ (1 ml formalin 10%) + (formalin 10% until volume 8 ml), let it 10 minutes
LET IT(10 minutes)
3 ml. ether
1 ml. Formalin 10%+ Formalin 10% until
volume 8 ml
SHAKE(15-20 seconds)
CENTRIFUGE2.000 rpm, 1-2 minutes
FORMOL ETHER SEDIMENTATION METHODFORMOL ETHER SEDIMENTATION METHOD(RITCHIE)
Throw away the supernatan, place the sediment on microscope slide, examine
under the microscope
USING FORMALIN SOLUTION 3,5 % OR 4%USING FORMALIN SOLUTION 3,5 % OR 4%
CONSERVATION AND STORAGE METHODCONSERVATION AND STORAGE METHODEGG AND ADULT WORM IN FAECESEGG AND ADULT WORM IN FAECES
Making formalin solution 3,5 % Or 4% : 1 part of formalin solution 35% or 40% mixed with 9
part of running water, then put into closed bottle
Put the faeces into the bottle and closed tightly For organ that attacked by parasite, wash cleanly
before put into the bottle
Laboratory tecknique for examination of Filaria sp.Laboratory tecknique for examination of Filaria sp.
BLOOD AND TISSUE NEMATODEBLOOD AND TISSUE NEMATODE((Blood specimensBlood specimens))
DIRECT METHOD Blood taken of from finger tip Thin blood smear
Qualitative Determine microfilariae in peripheral blood
vessels Not for species identification
Thick blood smear Quantitative Measured blood releasefrom finger tip with
mikropipet (160 m), make thick blood smear. CONCENTRATION METHOD
Take blood vein More sensitive than direct method
Click “esc” buttonWhen finished
SUBJECT MATERIAL SUBJECT MATERIAL � HOW TO EXAMINE AND INTERPRET INTESTINAL PROTOZOA
- Direct wet mount - Modified Merthiolate-Iodine -Formalin (MIF)
� HOW TO EXAMINE AND INTERPRET BLOOD PROTOZOA - Thin Blood smear with Giemsa staining - Thick Blood smear with Giemsa staining
� EXAMINATION FOR Trichomonas vaginalis� HOW TO PREPARE PERMANENT (FIXED) MOUNT� - Blood parasites
- Trichomonas vaginalis- Ameba, using Heidenhein method and staining
� METHODS FOR PREPARING COLOR STAININGS
� HOW TO EXAMINE AND INTERPRET INTESTINAL PROTOZOA - Direct wet mount - Modified Merthiolate-Iodine -Formalin (MIF)
� HOW TO EXAMINE AND INTERPRET BLOOD PROTOZOA - Thin Blood smear with Giemsa staining - Thick Blood smear with Giemsa staining
� EXAMINATION FOR Trichomonas vaginalis� HOW TO PREPARE PERMANENT (FIXED) MOUNT� - Blood parasites
- Trichomonas vaginalis- Ameba, using Heidenhein method and staining
� METHODS FOR PREPARING COLOR STAININGS
(1). Direct wet mount exam (1). Direct wet mount exam
Purpose :
For quick and simple examination
Purpose :
For quick and simple examination
LAB METHODS FOR INTESTINAL PROTOZOA LAB METHODS FOR INTESTINAL PROTOZOA LAB METHODS FOR INTESTINAL PROTOZOA LAB METHODS FOR INTESTINAL PROTOZOA
- For trophozoite form of ameba, use 2% eosin solution- For trophozoite form of ameba, use 2% eosin solution
- For cyst and nucleus of amoeba use lugol solution
(2% of Iodine + 3% potasssium Iodine)- For cyst and nucleus of amoeba use lugol solution
(2% of Iodine + 3% potasssium Iodine)
Using a small stick, add small amount of feces on top of an object glass
Add few drops of physiological saline solution NaCl/lugol/eosin 2% on top
PreparationPreparation : :
Distribute/spread evenly and cover with coverglass
Examine under low power microscope
LAB METHODS FOR INTESTINAL PROTOZOALAB METHODS FOR INTESTINAL PROTOZOA LAB METHODS FOR INTESTINAL PROTOZOALAB METHODS FOR INTESTINAL PROTOZOA
(1). (1). Direct wet mount examination Direct wet mount examination
(2). Modified Method Merthiolate-Iodine-Formaline (MIF)
PurposePurpose : :Good for diagnosis of Ameba and Giardia in feces
LAB METHOD FOR INTESTINAL PROTOZOA LAB METHOD FOR INTESTINAL PROTOZOA
Click “Esc”buttonWhen finished
Ingredients used Ingredients used ::Basic solution (1) :
–250 ml aquadest
–200 ml tincture of merthiolate
–25 ml formaldehyde
Basic solution (2) :
–lugol 5 % (not to be kept > 3 weeks)
Both basic solutions should be kept in brown colored bottle
Modified Method Merthiolate-Iodine-Formaline (MIF)
LAB METHOD FOR INTESTINAL PROTOZOA LAB METHOD FOR INTESTINAL PROTOZOA
Click “Esc”buttonWhen finished
Acidic in character Foul smelling Produce less mucus compared to
bacillary dysentery, less sticky With or without blood (blood may be
found in solid feces) In some cases mucosal wall may come
out
Acidic in character Foul smelling Produce less mucus compared to
bacillary dysentery, less sticky With or without blood (blood may be
found in solid feces) In some cases mucosal wall may come
out
CHARACTERISTIC OF FECES WITH AMEBA CHARACTERISTIC OF FECES WITH AMEBA
MACROSCOPIC MACROSCOPIC
CHARACTERISTIC OF FECES WITH AMEBA CHARACTERISTIC OF FECES WITH AMEBA
MACROSCOPICMACROSCOPIC
Source :A Colour Atlas of Clinical Parasitology. Tomio Yamaguchi. Alih Bahasa : Lesmana Padmasutra, dkk.
Feces of amebic dysenteric patient, plenty of trophozoite stage
CHARACTERISTICS OF FECES WITH AMEBACHARACTERISTICS OF FECES WITH AMEBA
MACROSCOPICMACROSCOPIC
Source : Color Atlas of Medicine and Parasitology. 1977. W. Peters & H.M. Gillers
Feces of amebic dysenteric patient, loose, slimy and bloody feces
CHARACTERISTICS OF FECES WITH AMEBACHARACTERISTICS OF FECES WITH AMEBA
MICROSCOPICMICROSCOPIC Plenty of bacteria Entamoeba histolytica (+) containing erythrocytes Erythrocytes in reuleaux (chain) formation Charcot-Leyden crystals (unspecific for ameba dysentery, can
be found also in other parasitic infection e.g. Strongyloides stercoralis) – from eosinophil desintegration
Pus less abundant compared to bacillary dysentery, if no secondary infection
Macrophages in bacillary dysentery mimics Entamoeba histolytica but nucleus and pseudopods differs
Cyst found in carrier patients and cases with light infection
Plenty of bacteria Entamoeba histolytica (+) containing erythrocytes Erythrocytes in reuleaux (chain) formation Charcot-Leyden crystals (unspecific for ameba dysentery, can
be found also in other parasitic infection e.g. Strongyloides stercoralis) – from eosinophil desintegration
Pus less abundant compared to bacillary dysentery, if no secondary infection
Macrophages in bacillary dysentery mimics Entamoeba histolytica but nucleus and pseudopods differs
Cyst found in carrier patients and cases with light infection
CHARACTERISTIC OF FECES WITH AMEBA CHARACTERISTIC OF FECES WITH AMEBA
microscopicmicroscopic
Charcot-Leyden crystals(From eosinophil desintegration)
Source;Color Atlas of Medical Parasitology. Prayong Radomyos, dkk.
Blood slides with Giemsa staining
PurposePurpose : :For the examination of blood protozoa, e.g. :
Plasmodium,
Trypanosoma, Babesia etc.
Prepared in two stagesPrepared in two stages : :(1). Prepare the blood smear
(2). Do the color staining
EXAMINATION METHODS FOR BLOOD PROTOZOA EXAMINATION METHODS FOR BLOOD PROTOZOA
Place blood slide in upright position, allow to dry, keep away from dust or small insects.
EXAMINATION METHOD FOR BLOOD PROTOZOA EXAMINATION METHOD FOR BLOOD PROTOZOA
Blood slides with Giemsa staining
Preparation of thin blood film
EXAMINATION METHOD FOR BLOODEXAMINATION METHOD FOR BLOOD PROTOZOA PROTOZOA
Blood slides with Giemsa staining
Staining procedure
Fix with methyl alcohol (3-5) minutes
Stain with standard Giemsa for 45 minutes
Wash slowly with tap water
ALLOW TO DRY
EXAMINATION METHOD FOR BLOODEXAMINATION METHOD FOR BLOOD PROTOZOAPROTOZOA
Thick blood smear with Giemsa stain
Purpose :Rapid examination of blood protozoa
(for mass survey and screening )
Conducted in two stages :(1). Prepare thick blood film(2). Staining the blood film
EXAMINATION METHOD FOR BLOODEXAMINATION METHOD FOR BLOOD PROTOZOAPROTOZOA
(1). Preparation of thick blood smear
Thick blood smear with Giemsa stain
Blood is prepared similar to thin blood smear Place 1-2 drops of blood on a glass slide Spread evenly, forming a circle of 1 - 1,5 cm in diameter Allow to dry, keep away from dust or insect
EXAMINATION METHOD FOR BLOODEXAMINATION METHOD FOR BLOOD PROTOZOAPROTOZOA
(2). Staining procedure
ALLOW TO DRY
Stain with standard Giemsa for 45 minutes
Rinse slowly with tap water
Thick blood smear with Giemsa stain
NO FIXATION !!!
� THIN BLOOD SMEAR– Morphology and stages of Plasmodium clearly defined– Erythrocytes are intact (due to fixation)– Slow to prepare – Used for examination of moderate and heavy infection
� THICK BLOOD SMEAR – Morphology and stages of Plasmodium not clearly defined– Erythrocytes are lysed, only stroma from erythrocytes are seen – Preparation and staining are faster (can be used for mass survey
examination)– Commonly used for light infection
COMPARISON OF THIN AND THICK BLOOD SMEAR
EXAMINATION METHOD FOR BLOODEXAMINATION METHOD FOR BLOOD PROTOZOAPROTOZOA
EXAMINATION OF Trichomonas vaginalisEXAMINATION OF Trichomonas vaginalis
Methods of examination :(1). Direct examination
(2). Culture
Methods of examination :(1). Direct examination
(2). Culture
Direct method :(1). Direct wet mount
(2). Staining
EXAMINATION OF Trichomonas vaginalisEXAMINATION OF Trichomonas vaginalis
Sample for examination :(1). Women : vaginal, or urethral discharge
(using vaginal spiculum)(2). Men : discharge from urethra or prostate,
and centrifugated urine sample
DIRECT EXAMINATION
Materials used :-Test tube containing 5% glucose (in physiological saline solution)
- Cotton bulb
Cotton bulb
Glucose 5%
Examination for Trichomonas vaginalisExamination for Trichomonas vaginalis
Terima kasih ………………….
April2005
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