MOST OF THE PARASITE ARE SEEN IN THE GIT: i.

NORMAL IS SOFT TO
SPECIFICALLY, THE SMALL AND LARGE WELL FORMED
INTESTINE ii. OTHER MAY BE
WATERY, SOFT, HARD
SPECIMEN: STOOL
ETC.
NOTE: PREPARED FIRST FECAL SMEAR
 ENTAMOEBA HISTOLYTICA NSS AND STOOL ARE USED
 ASCARIS LUMBRICOIDES  GET A SLIDE AND PUT LITTLE
 NECATOR AMERICANUS / PORTION OF STOOL IN THE
ANCYLOSTOMA DUODENAL SLIDE AND PUT NSS =
 SCHISTOSOMA JAPONICUM UNSTAINED
 PAREAGONIMUS WESTERMANI NOTE: IODINE IS ONLY GOOD FOR CYST NOT
PURPOSE OF FECALYSIS FOR TROPHOZOITES

1. DETECT PARASITE 2. MICROSCOPIC EXAM

2. DETECT GIT BLEEDING a. MUST BE DONE IN LPO FIRST
AND SCAN ALL FIELDS AFTER
OTHER SPECIMENS: THAT SHIFT IT TO HPO
BLOOD MICROSCOPIC STRUCTURE
A. PLASMODIUM SPP A. NORMAL STRUCTURES
B. ALL FILARIAL WORMS EXCEPT O. a. AIR BUBBLES
VOVIVULUS b. VEGETABLE HAIR
C. BABESIA MICROTI c. VEGETABLE COILS
URINE d. VEGETABLE SPIRALS
e. FOOD REMINANTS
A. TRICHOMONAS VAGINALIS f. FAT GLOBULES
B. SCHISTOSOMA HAEMATOBIUM g. STARCH GRANULES
B. ABNORMAL STRUCTURES
SPUTUM
a. PARASITE EGGS/ OVA =
A. PARAGONIMUS WESTERMANI LPO
B. ASCARIS LUMBRICOIDES b. PROTOZOAN CYST/
C. ENTAMOEBA HISTOLYTICA TROPHOZOITES = HPO
c. BODY CELL (WBC’S &
PERIANAL SWAB
RBC’S) = HPO
A. ENTEROBIUS VERMICULARIS d. EPITHELIAL CELLS =
HPO
FECALYSIS
NOTE: NORMAL CAN BE MISTAKEN AS PARASITE
1. MACROSCOPIC EXAM
a. NOTE THE COLOR AND STOOL DIRECT FECAL SMEAR ( USES NSS OR IODINE)
CONSISTENCY
STAINED FECAL SMEAR – USE OF PERMANENT
i. NORMAL COLOR IS
STAIN SUCH AS IRON HEMATOXYLIN AND
BROWN
GOMORI’S TRICHROME
b. STOOL CONSISTENCY
IODINE IS ONLY TEMPORARY STAIN

PREPARING DIRECT FECAL SMEAR:

 GET A SLIDE
 PUT NSS
 COVER THE PREPARATION
USING COVER SLIP

IN FECAL SMEAR INSTEAD OF NSS IT USES

PERMANENT STAIN

KATO THICH SMEAR/ CELLOPHANE COVERED

THICK SMEAR/ GLYCERINE MALACHITE GREEN
METHOD

THIS DOESN’T USED COVER SLIP BUT USES

CELLPHONE

PROCEDURE: GET A TRANSPARENT

CELLOPHANE CUT IT LIKE THE SIZE OF COVER
SLIP THEN SOAK/IMMERSE IT IN A MIXTURE OF
GLYCERINE AND MALACHITE GREEN

STOOL:

1. SMEAR (DIRECT FECAL SMEAR,

STAINED, KATO THICK)
2. EGG COUNTING TECHNIQUE
3. CONCENTRATION TECHNIQUES
a. SEDIMENTATION
b. FLOTATION
4. CULTURE
a. HARADA MORI
b. COPRO CULTURE

READ PAGES 16-25 FOR LABORATORY

SPP COLLECTION (MUST READ)

EX. PRESERVATION IS DONE BY REFRIGERATION

(OR FORMALIN)
STOOL FOR OVA AND PARASITE EXAMINATION ( O&P) – most  Should not retrieved from toiled bowl
common procedure in performed in parasitology becasuse free living protozoa and
nematodes may be fused with human
Ova- egg stage
parasites; water may destroy parasites
GENERAL PROCEDURE (schistomsome eggs and amebic
trophozoites)
1. Macroscopic  Toilet paper in stool specimen may mask
parasites or make examination of the
 Collection
sample difficult
 3 SPECIMEN COLLECTED
 Specimen should labeled patient’s
1 specimen- collected every other day = 3
name,identification number,physcian name,
collected in 10 days
date and time of sample collection
EXCEPTION: Amebiasis 6 specimen in 14
days
 Transport : do’s and dont
 Medication and substances interfere with detection
 The specimen should be placed into a ziplock plastic
or parasites( stool sample patients theraphy)
bag
 Barium
 Paper works accompanying the specimen should be
 Bismuth
separated from the specimen container
 Mineral oil
 Gloves and protective coat should be worn
Collected prior to theraphy or not until 5-7
 Biohazard hoods should also be used
days after theraphy
 Important consideration in testing: receipt and
 Taken antibiotics or antimalarial medication –
examination
should be delayed for weeks following theraphy
 To detect motility of protozoan and trophozoites:
 Speciemen should be collected: do’s and dont
fresh specimen is required
 Clean, watertight container with tight-
 Tropozoite- sensitive; found liquid stool;
fitting lid.
should be examined 30 mins; evaluated
 2 to 5 g acceptable amount- walnut size
1hour
 Urine should not contaminate the stool can
 Protozoan, helminth eggs, larvae- not
be destroy some parasites.
sensitive; survive in longer periods
 Fixatives  Polyvinyl Alcohol comprised of plastic powder
 Substances that preserve the morphology of that acts as an adhesive for the stool specimen
protozoa when preparing slides from staining.
 prevent further development of certain helmints  Most often combined: Schaudinn solution-
eggs and larvae contains:
 ratio: 3 parts fixative to one part stool 1. zinc sulfate
 the specimen mixed well to achieve thorought 2. copper sulfate
fixation 3. mercuric chloride as base
 specimen fixed in the preservatives at least 30 mins  ADVANTAGES & DIS.A
before processing begins 1. It can be used for preparation of a
 some fixative contain: mercury & dispocsal permanent stained smear.
regulations 2. Long shelf life when stored room temp.
 Formalin recovery protozoa and helminths 3. Concentration tech. Can also perfomed
 Concentation commonly used: but recovery of the parasite not
 5% protozoan cysts effective as when formalin is used
 10% helminth eggs and larvae 4. DIS.A schaudinn solution contains
 ADVANTAGES & DISAVANTAGES merucric chloride – potential health
FORMALIN AS FIXATIVES problems cause mercury
1. Easy to prepare  Sodium acetate formalin alternative to use
2. It preserves specimens for up to PVA and schaudinn fixative is sodium acetate
several years formalin
3. Long shelf life  Concentration tech.
4. DISAVANTAGES: does not preserve  Permanent stained smears.
parasite morphology adequately for  Single vial
permanent smears.  Mercury free
5. Trophozoites usally cannot be  Easy to prepare
recovered and morphologic details  Long shelf life
of cysts and eggs may fade with  Preparing smears for staining with modified
time acid fast stain to detect coccidian oocysts
 DIS.A adhesion properties not good
 Albumin to the microscope slide may be Preservatives Concentration Permanent Antigen tests
necessary to ensure adhesion of the specimen stain
to slide. 10% formalin + - +
 Protozoa morphology not clear in permanent SAF + + Iron +
hematoxylin
stain
PVA +, - + Iron -
 Permanent stain smears with Iron hematoxylin
hematoxylin- better result than staining SAF
preserved using Wheatley trichrome. Modifies PVA +,- + Iron +,-
hematoxylin
 Modified Polyvinyl Alcohol other alternative
Single vial + + Iron +,-
to mercury based PVA
system hematoxylin
 ADVANTAGES: concentration methods
 Permanent stain smears  Processing
 Zinc sulfate fixative provide better results  Determine consistency and color of the sample
 More likely negatively afftected if proper  Presence of gross abrnomalities
protocol is not followed (e.g stool to fixative,  Perform: fresh, unpreserved stool
ratio, adequate mixing.  Types of potential parasites present:
 Alternative Single vial system  Soft or liquid - proztoan trophozoites
 Nontoxic fixatives  Fully formed – protozoan cycts
 Free of formalin and mercury
 Liquid or formed stool – helminth eggs and larvae
 Concentration technique
 Brown normal color
 Permanent stained smears
 Gross abnormalities found: adult worms, proglottids, pus
 Perform fecal immuno assay like MPVA
and mucus
fixatives
Consistency Possible colors Gross appearance
 Not provide the same quality of preservation terms tems
as mercury base fixatives Hard Dark brown Conspicuously
 Organism identification more difficult from fibrous
permanent stained slides. Soft Black Fiber scanty
moderate
Mushy Brown Colloidal
(homogeneous)
 Loose Pale brown Scanty mucuous  Defined as slide made by mixing small portion of
Diarecheic Clay Much mucus unfixed stool( no preservatives)
Watery, liquid Yellow Much with scanty  Saline or iodine and subsequent examination –
blood detect presence of motile protozoan
Formed Red brown Other (e.g bloog
trophozoites.
barium)
 Trophozoite motility – demonsrated in fresh
Semi formed Green,other
specimens, especially liquid or soft
2. Microscopic Examination  Direct saline wet prep. Made by placing a drop
 Detect the presence of parasite in stool 0.85% saline glass on a glass slide ( 3- inch size is
 Ova and parasites 3 distinct procedures suggested)
1. Direct wet preparations –  IOI not recommended
 Concentrated tech.  Temporary seal is hot paraffin petroleum jelly
 100x is ability to observe greater detail
 Wet preps.
 Direct iodine wet prep. Made to enhance the
 Permanent stained smear.
detail of protozoan cycts.
All of 3 procedures should performed fresh
 Lugol’s or D’Antoni’s formula using drop iodine
specimen.
 Iodne kills trophozoites present
 Ocular micrometer
 Proper adjustment of the microscope is essential
 Important piece of equipment in the para. Laboratory
to the successfull reading and interpretating of
 Detection of parasite
wet prep.
 Size is important diagnostic feature in para.
 Must be calibrated to ensure accurate measurement
 CONCENTRATION METHODS
 MICRONS - Measured units diagnostic
 O&P examination
 Measuring defined 0.001millimeter
 Concentration tech. – detect small numbers of parasites
 Measure object microscopically accurately
not might detect wet prep.
 Disk inserted eyepiece of the microscope
 Purpose is to aggregate parasites present into small
 Disk is equipped with line evenyly divided into 50 or 100
volume of sample and remove debris
units
 Performed: fresh or preserved stools
 Direst wet preparation
 Detect: protozoan cysts, occyst, helminth eggs and
 Direct wet amount- primary purposed of DWP
larvae
 Protozoan trophozoites: not usually survive in the  Specific gravity is 1.18 to 1.20
procedure.  When add to the specimen the parasite float
 Two types concentration methods: Sedimentation and to the surface and can be skimmed from the
floatation. top.
 Use to diffrences in specific gravity and  Advantage more fecal debris is removed and
centrifugation to seperate the parasites it yields a cleaner preps. , easier for
from fecal debris and increase their microscopic examination
recovery  Disadvantage some helminth eggs are very
 Sedimentation – tube follw. Centrifugation; dense and not float
recommended to be used, easier to  Therefore some parasite will be missed
perform  RECOMMENDED: SALINE AND IODINE PREPS
 Floatation- less dense than the solutions, MADE FROM SEDIMENT MICROSCOPICALLY
during centri they float to the surface so that not to miss any parasite
 Formalin – Ethyl acetate Sedimentation  REFERRED: CONCENTRATED SALINE WET
Procedure PREPS. AND CONCENTRATED IODINE WET
 Most wide used PREPS.
 Principle is based specific gravity  PERMANENT SRAINS.
 ethyl acetate added to a saline washed  Final procedure in the O&P examination.
formalin fixed sample and tube centrifuged  Fixed sample that has been allowed to dry and
 the tube is decanted subsequently stained.
 advantage provides recovery of most  Presence confirm: protozoa cysts and trophozoites
parasites and is easy to perform  Observe detailed features of protozoa by staining
 disadvantage more fecal debris than intracellular organelles
floatation tech. And more challenging to the  Dientamoeba fragilis example of permanent stain
microscopist.  Appear distorted and stain too dark
 Zinc Sulfate Floatation Technique  Common stain used in O&P :
 Differences specific gravity btw the sample  Trichrome – Wheatley Trichrome
debris  long shelf life
 Heavy, sinks to the bottom of the test tube  procedure: easy to perform
 Lighter and float toward the top of the tube.
  a suggested procedure slide made from PVA fixed
specimen
APPEARANCE OF SELECT
PROTOZOAN STRCTURE AND
BACKG MATERIAL ON
HEMATOYXLIN STAIN
APPEARANCE OF SELECT
STRUCTURE OF MATERIAL APPEARANCE
PROTOZOAN STRCTURE AND
Protozoa cytoplasm Blue to purple
BACKG MATERIAL ON
Protozoa nuclear material Dark blue to dark purple
TRICHROME STAIN.
Debri’s and background Light blue, sometimes with pink
STRUCTURE OF MATERIAL APPEARANCE material tint
CYTOPLASM OF Entamoeba Light pink or blue green
histolytica trophozoites and
 Special stains
cysts
Cytoplasm of Entamoeba coli Purple tink  Disadvantages:
cysts  Do not detect oocysts of the coccidian parasites or
Nuclear kayosomes Bright red ro red purple spores of microsporidia
Degenerated parasites Light green  Modified acid fast stain detection: Oocyts
Background Green Crytosprodium; isospora and clyclospora
APPEARANCE OF PROTOZOAN,
 Iron Hematoxylin STRUCTRES, YEAST AND BCKG.
 Time consuming MODIFIED ACID FAST STAIN.
 Shorter tech. Using stain STRUCTURE OR MATERIAL APPEARANCE
Oocyts of cryptosporodium and Pink to red
 Reveals excellent morphology of intesinal protozoa
Isospors
 Nuclear detail of organisms considered to be Oocysts of cyclospora Variable: clear to pink to red
stained clearer and sharper Yeast Blue
 Carbol fuchsin Background Blue or light red
APPPEARANCE MICROSPORIDIA
ON MODIFIED TRICHROME
STAIN
STRUCTURE OR M. A
Spores of microsporida Pink to red with clear interior
Polar tubule Red horizontal or diagonal bar
Bacteria, yeast, debris Pink to red
Background Green

STOOL SCREENING METHODS

 Monoclial antibody
 This commercial antibody is to detect:
 Antigens , EIA, direct flourescent antibody , membrane
flow cartridge tech.
 Detection: protozoa; Entamoeba histolytica, Giardia
intestinalis; Cryptosporidium spp.
 Sensitve and specific
 Detect one or two pathogen at a time
PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1

PARASITOLOGICAL SPECIMENS
SPECIMEN PARASITE & PARASITE STAGE SPECIMEN PARASITE & PARASITE STAGE
STOOL Ova of INTESTINAL HELMINTHS like SPUTUM Ova of Paragonimus westermani
Ascaris lumbricoides; Trichuris trichiura
Capillaria philippinensis ; Necator americanus Trophozoite of Entamoeba histolytica
Ancylostoma duodenale
Schistosoma mansoni and Schistosoma japonicum Filariform larva of
Diphyllobothrium latum Necator americanus and Ancylostoma duodenale
Taenia solium and Taenia saginata
Larva of Ascaris lumbricoides
Rhabditiform Larva of Strongyloides stercoralis

Cyst & Trophozoite stages of

Entamoeba histolytica
Giardia lamblia and Balantidium coli

Oocyst of Cryptosporidium parvum

URINE Ova of Schistosoma haematobium BLOOD Ring form trophozoites & gametocytes of
Plasmodium spp and Babesia microti
Trophozoite of Trichomonas vaginalis
Microfilaria of Filarial worms like
Wuchereria bancrofti ; Brugia malayi ; Loa loa
Duodenal aspirate Trophozoite of Giardia lamblia CSF Naegleria fowleri and Acanthamoeba spp.

STOOL SPECIMEN
COLLECTION , TRANSPORT & SPECIMEN PROCESSING
• parasites are often shed (i.e., enter and subsequently passed in the stool) intermittently, they may not appear in a stool specimen on a daily basis;
therefore, multiple specimens are recommended for adequate detection.
• The typical stool collection protocol consists of three specimens, one specimen collected every other day or a total of three collected in 10 days. One
exception is in the diagnosis of amebiasisin which up to six specimens in 14 days is acceptable
• medications and substances may interfere with the detection of parasites. Stool samples from patients whose therapy includes barium, bismuth, or
mineral oil should be collected prior to therapy or not until 5 to 7 days after the completion of therapy. If the samples are taken during the course of
therapy, these interfering substances may mask possible parasites during examination.
• Collection of specimens from patients who have taken antibiotics or antimalarial medications should be delayed for 2 weeks following therapy.

PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 1

PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1
• Stool specimens should be collected in a clean, watertight container with a tight-fitting lid. The acceptable amount of stool required for parasite study is
2 to 5 g, often referred to as the size of a walnut.
• Urine should not be allowed to contaminate the stool specimen because it has been known to destroy some parasites. Stool should not be retrieved from
toilet bowl water because free-living protozoa and nematodes may be confused with human parasites. In addition, water may destroy select parasites,
such as schistosome (eggs and amebic trophozoites.
• Toilet paper in the stool specimen may mask parasites or make examination of the sample difficult
• The specimen container should be labeled with the patient’s name and identification number, the physician’s name, and the date and time of sample
collection
• To demonstrate the motility of protozoan trophozoites, a fresh specimen is required. The trophozoite stage is sensitive to environmental changes and,
on release from the body, disintegrates rapidly.
• Other parasite stages (e.g., protozoan cysts, helminth eggs and larvae) are not as sensitive and can survive for longer periods outside the host. Because
trophozoites are usually found in liquid stool, it is recommended that liquid specimens be examined within 30 minutes of passage.
• In keeping with stool consistency, semiformed specimens may yield a mixture of protozoan cysts and trophozoites and should be evaluated within 1 hour
of passage.
• Formed stool specimens are not likely to contain trophozoites; therefore, they can be held for 24 hours following collection. If these guidelines cannot be
met, the specimen should be placed into a preservative.

PRESERVATION
• If the stool is to be processed within 1 hour, it may be stood at room temperature. Beyond one hour, the stool must be refrigerated (3-5degC for 4 hours).
Hookworm eggs mature and hatch if allowed to remain at room temperature & may be confused with Strongyloides stercoralis larvae. Formed stools
may be refrigerated 1-2 days if examination is delayed although this will not guarantee recovery of all parasites. Never Freeze the sample. Trophozoites
from a refrigerated stool can regain motility in warm saline on a warm slide. Never keep stool samples in the incubator. 37 degC beyond 30 minutes
destroys ameba

STOOL PRESERVATIVE/FIXATIVES:
• The ratio of fixative to stool is important for the successful recovery of parasites and, whatever fixative is used, the recommended ratio is three parts
fixative to one-part stool.
• The specimen must be fixed in the preservative for at least 30 minutes before processing begins

FORMALIN POLYVINYL ALCOHOL

✓ Formalin has been used for many years as an all-purpose fixative ✓ is comprised of a plastic powder that acts as an adhesive
for the recovery of protozoa and helminths. for the stool specimen when preparing slides for staining.
✓ Two concentrations of formalin are commonly used; a 5% PVA is most often combined with Schaudinn solution,
concentration ideally preserves protozoan cysts and a 10% which usually contains zinc sulfate, copper sulfate, or
concentration preserves helminth eggs and larvae. mercuric chloride as a base.
✓ Formalin may be routinely used for direct examinations and ✓ Trophozoites and cysts of the protozoa, as well as most
concentration procedures, but not for permanent smears. helminth eggs, may be detected using this fixative.
✓ There are three primary advantages for the use of formalin: (1) it is ✓ it can be used for preparation of a permanent stained
easy to prepare; (2) it preserves specimens for up to several years; smear. PVA-preserved specimens have a long shelf life
and (3) it has a long shelf life. when stored at room temperature.
✓ biggest disadvantages of formalin is that it does not preserve
parasite morphology adequately for permanent smears.
PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 2
PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1
✓ Other disadvantages include the fact that trophozoites usually ✓ Suited for concentration techniques but the recovery of
cannot be recovered and morphologic details of cysts and eggs certain parasites is not as effective as when formalin is
may fade with time used.
✓ The biggest disadvantage of the use of PVA is that
Schaudinn solution contains mercuric chloride. Because of
the potential health problems caused by mercury
SODIUM ACETATE FORMALIN MODIFIED PVA
✓ can be used for performing concentration techniques and ✓ Can be used for concentration methods and permanent
permanent stained smears. stained smears. However, this will not provide the same
✓ SAF is easy to prepare, has a long shelf life, and can be used for quality of preservation for adequate protozoan morphology
preparing smears for staining with the modified acid-fast stain to on a permanent stained slide as the mercury-based
detect coccidian oocysts. fixatives. Therefore, parasite identification will be more
✓ Disadvantages is that protozoa morphology from SAF-preserved difficult.
specimens is not as clear in permanent stains as when mercury-
containing preservatives are used.

MACROSCOPIC EXAM MICROSCOPIC EXAM

Stool specimens submitted for parasitic study should first be examined • Smears can be prepared using NSS (direct wet mount/unstained) or
macroscopically to determine the consistency and color of the sample. by using iodine
• The specimen should be screened and examined for the presence of • use of iodine will better preserve protozoan cyst however it will cause
gross abnormalities. disappearance of protozoan trophozoites
• The consistency or degree of moisture in a stool specimen may serve • during microscopy, scan all microscopic fields, Helminth eggs & larvae
as an indication of the types of potential parasites present. For example, are reported under LPO. Protozoan cyst and trophozoites are best seen
soft or liquid stools may suggest the presence of protozoan under HPO. RBCs and Pus cells are to be observed under HPO.
trophozoites. Protozoan cysts are more likely to be found in fully formed • Normal microscopic structures like vegetable hair, cells, spirals, starch
stools. Helminth eggs and larvae may be found in liquid or formed granules are not reported.
stools. • All abnormal microscopic findings should be reported
• The color of a stool is important because it may indicate the condition
of the patient, such as whether a patient has recently had a special
procedure (e.g., a barium enema) or if the patient is on antibiotic
therapy. The range of colors varies, including black to green to clay, and
colors in between. The color of normal stool is brown
• Gross abnormalities possibly found in stool include adult worms,
proglottids, pus, and mucus.
• Other macroscopic abnormalities in the specimen may have parasitic
indications. blood and/or mucus in loose or liquid stool may suggest the
presence of amebic ulcerations in the large intestine. Bright red blood
on the surface of a formed stool is usually associated with irritation and
bleeding

PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 3

PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1

PERMANENT STAINS
IRON HEMATOXYLIN WHEATLY TRICHROME
✓ Historically, this procedure was ✓ most widely used
considered to be time- permanent
consuming. However, a shorter ✓ uses reagents with a
technique using this stainis now relatively long shelf life
available. and the procedure is
✓ It reveals excellent morphology easy to perform.
of the intestinal protozoa.
✓ In some cases, the nuclear detail
of these organisms is considered
to be stained clearer and sharper
than when stained with
trichrome.
✓ The color variations among
specific parasitic structures and
background material however
are not as distinct as with
trichrome

PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 4

PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1
DIAGNOSTIC PROCEDURES THAT REQUIRE STOOL AS SAMPLE:

KATO THICK SMEAR EGG COUNTING TECHNIQUE CULTURE

CELLOPHANE COVERED THICK SMEAR
GLYCERINE MALACHITE GREEN METHOD
• It is different from the standard direct smear • A quantitative procedure • Culture methods are not a common means of
procedure in that a larger amount of fecal • Carried out to (1) determine detecting parasites. There are a few techniques
sample is employed and cellophane strips are degree of infection (2) to assess available but they are not usually performed in the
used as cover slips instead of glass. effectiveness of anti- helminthics routine laboratory.
• Glycerine acts as clearing agent while • STOLL DILUTION – uses 0.1 N • Parasites that can be isolated with culture include
malachite green will provide green background NaOH E. histolytica, T. vaginalis, Leishmania spp., T.
and will reduce brightness of microscopic field • Kato-Katz technique cruzi, and T. gondii.
• Advantages: economical; applicable for thick • ALL FIELDS SHOULD BE USE AND ALL • HARADA MORI / TEST TUBE CULTURE
shelled eggs like Ascaris & Trichuris EGGS SEEN SHOULD BE COUNTED METHOD and COPRO CULTURE – these are
Procedure is simple • NUMBER OF EGGS IS PROPORTIONAL culture methods for Hookworms & Strongyloides
• Disadvantages: not suited for watery /liquid TO THE SEVERITY OF INFECTION stercoralis
stools, not for cyst & trophozoites; not for thin- • NOT SUITED FOR PROTOZOA
shelled eggs like those of hookworms
THIS IS QUALITATIVE USES 20-50 G STOOL

PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 5

PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1
CONCENTRATION TECHNIQUES
The purpose of concentrating feces is to increase the possibility to finding protozoan cyst, helminth eggs, and larvae by decreasing the amount of
background material in the preparation and by an actual concentration of organisms. Direct examination, however, should be done first before proceeding to
fecal concentration. Some infections may be light. Concentration techniques will increase the number of organisms detected, compared with direct microscopy.
Motile protozoan trophozoites are not found in concentrated preparations. Concentration procedures may be performed on fresh or preserved specimens.
Concentration techniques can be performed on fresh or preserved stool specimens.
USES 1 GRAM OF STOOL
PRINCIPLE BEHIND ALL CONCENTRATION TECHNIQUES:
SEDIMENTATION FLOTATION
In this method, parasites have higher specific gravity that is parasites are lighter and float toward the top of the tube. the parasites float to the surface and
why after centrifugation all parasites that might be present in the can be skimmed from the top of the tube.Higher specific gravity is reagent.
sample will all settle at the bottom of the tube.
ACID ETHER CONCENTRATION TECHNIQUE ZINC SULFATE CENTRIFUGAL FLOTATION
✓ Method of choice if the specimen is from animal source ✓ Uses zinc sulfate with a specific gravity of 1.18 to 1.20, is used as the concentrating
✓ Recommended for the recovery of Trichuris, Capillaria & solution. When the zinc sulfate is added to the specimen and centrifuged, the parasites
Schistosoma eggs float to the surface and can be skimmed from the top of the tube.
✓ HCl is used as clearing agent while ether is used to ✓ advantage of this technique is that more fecal debris is removed and it yields a cleaner
remove fats preparation, making it easier for microscopic examination.
✓ Hcl and Ether ✓ disadvantage of this method is that some helminth eggs are very dense and will not
float; therefore, some parasites will be missed
FORMALIN ETHYL ACETATE (better preservation of parasite) ✓ recommended for G. lamblia cyst & H. nana ova
✓ most widely used sedimentation technique. SHEATHER’S FLOTATION
✓ Ethyl acetate is added to a saline-washed formalin-fixed ✓ Uses sugar solution preserved in phenol
sample and the tube is then centrifuged. The advantage ✓ Recommended for concentrating oocyst of Cryptosporidium & Isospora
of this technique is that it provides good recovery of most
parasites and is easy to perform. The disadvantage of this BRINE’S FLOTATION
technique is that the preparation contains more fecal ✓ Uses salt solution, stool is directly mixed with brine, no centrifugation required
debris than a flotation technique ✓ Procedure is simple, economical and suited for mass stool exam
✓ Useful in the recovery Giardia lamblia cyst & cestode ✓ Not suited for Trematode eggs since their eggs do not float in Brines
eggs ✓ Hookworm eggs & Schistosome eggs becomes badly shrunken

FAUST MALONEY EGG HATCHING - quantitative test, a miracidial hatching test for Schistosoma

NOTES
Egg counting, faust maloney and kato katz are quantitative
PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1

HELMINTH EGGS MICROSCOPICALLY SEEN IN FECES

BLOOD SPECIMEN

✓ Several parasites may be detected thru examination of blood. This include the Plasmodium species, Babesia microti, the filarial worms i.e. W. bancrofti
A. THICK & THIN BLOOD SMEAR
✓ Regarded as the gold standard for Malaria detection
✓ Malaria is caused by Plasmodium species (P. falciparum, P. malariae, P. vivax & P. ovale)
✓ Purpose of Thick smear to indetify if there is a present parasite Thin smear identify the parasite, what species.
✓ Part of the smear that needs to be dehemoglobinized (using distilled water) is the thick smear
✓ Part of the smear that needs to be fixed with alcohol is thin smear
✓ Usual Stain giemsa stain (blood parasita stain)
✓ Best time to collect blood for malaria detection at a high fever or paroxysm
✓ Plasmodium stages seen microscopically in the blood are gametocyte, ring form trophozoites, schizonts

PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 7

PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1

Crescent /sausage/banana PLASMODIUM FALCIPARUM Malignant tertian malaria PLASMODIUM FALCIPARUM

shaped gametocytes Benign Tertian malaria PLASMODIUM VIVAX
Band form trophozoites PLASMODIUM MALARIAE Quartan Malaria PLASMODIUM MALARIAE
Ovale Malaria PLASMODIUM OVALE
Aplique/accole forms PLASMODIUM FALCIFARUM The most severe form of PLASMODIUM FALCIPARUM
malaria is due to
Amoeboid trophozoite PLASMODIUM VIVAX Characteristic symptom of PAROXYSM
Malaria
Rosette merozoites PLASMODIUM MALARIAE

SPECIES PERIODICITY of MICROFILARIA

W. bancrofti NOCTURNAL PERIODIC
B. KNOTT’S CONCENTRATION TECHNIQUE 8pm-2am or 10 pm -4am
✓ Concentration technique for the diagnosis of Filariasis B.malayi Nocturnal subperiodic
✓ In the collection of blood, we consider the PERIODICITY of MICROFILARIA *microfilaria goes to the blood both at day and
✓ PERIODICITY RYTHMICAL APPEARANCE OF MICROFILARIA IN THE BLOOD night time but the greatest number of
✓ Diagnostic stage: MICROFILARIA (LARVA) _ microfilariae in the blood is at night
✓ Reagent used: 2%FORMALIS _ L. loa DIURNAL PERIODIC
11am-1pm
O. volvulus NON-PERIODIC
M. ozzardi
PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 8
M. perstans
PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1

Presence /absence Location of body

of sheath nuclei
Wuchereria bancrofti Sheathed With nucleus NOT
extending to tip of
tail
Brugia malayi With 2 terminal
nuclei
Loa loa With nucleus
extending to tip of
tail
Mansonella ozzardi Unsheathed With nucleus NOT
extending to tip of
tail
Mansonella perstans With nucleus
extending to tip of
tail

SHEATH
HH

PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 9

PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1

PERIANAL SWAB / SCOTCH TAPE SWAB

✓ Diagnostic procedure for ENTEROBIUS VERMICULARIS
✓ Collected at night or early in the morning, since laying of eggs happens at night(NUCTURNAL)
✓ Can detect embryonated eggs of Enterobius vermicularis

NOTES
_
__
_
_
PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 10
PARASITOLOGY LABORATORY - DIAGNOSTIC PROCEDURES
NOTES # 1

PREPARED BY: DR. MA. CRISTINA SJ LIWANAG pg. 11

Direct Fecal Smear
Introduction:
This is a routine procedure of stool examination useful in the detection of motile
protozoan trophozoites. In this preparation, the trophozoites appear very pale and
transparent. Trophozoites can be stained to demonstrate the nuclear morphology using
Nair’s buffered methylene blue solution (BMB). Entamoeba cytoplasm will stain pale
blue and the nucleus, darker blue. Protozoan cysts can also be identified in a direct
saline fecal smear. A weak iodine solution can be used as a temporary stain to
demonstrate the nuclei. Helminth eggs and larvae can also be detected using his
preparation. Ideally, one fecal smear should contain about 2 mg of stool. Because this
amount is very small, light infections may not be detected.
Procedure:

1. Place a drop of NSS in the middle of a clean glass slide.

2. With an applicator stick, poke at various portions of the fecal specimens
especially on mucoid and bloody areas.
3. Spread the specimen over the NSS to form a homogenous suspension.
Never allow fecal suspension to run down the edges of the slide.
4. Put on coverglass avoiding bubble formation.
5. Check thickness of preparation by placing the slide over the printed material.
If the prints are barely readable, the preparation is correct.
6. Draw and label.

Kato Technique
Introduction:
Kato technique (Kato Katz and Kato Thick) is used to enhance the morphologic details
and increase the chances of isolating the ova of the different parasites. Glycerin is used
to clear all the fecal debris so that the ova become more visible during the microscopic
analysis. Kato katz is the quantitative method that requires a template for a more
accurate reporting while Kato thick is the qualitative method.
Procedure:

1. Place approximately 50-60 mg of stool ( the size of a soy bean ) at the center
of a glass slide and cover with a square piece of pre-treated cellophane.

2. By means of a cork stopper, press the cellophane gently to spread the stool
specimen does not spread beyond the cellophane cover. The cellophane thus
serves as a coverslip.

3. Keep the slides at room temperature for 30 minutes to 1 hour. The glycerine
clears the specimen. Allowing the slides to stand for a long period of time
will cause drying, and shells of hookworm ova will dissolve in the glycerine
solution

Sedimentation and Flotation

Techniques
Introduction:
Direct examination of stool may not always be able to reveal the presence of
small numbers of parasites; hence, these will be missed during direct examination. The
purpose of concentrating feces is to increase the possibility to finding protozoan cyst,
helminth eggs, and larvae by decreasing the amount of background material in the
preparation and by an actual concentration of organisms. Direct examination, however,
should be done first before proceeding to fecal concentration. Some infections may be
light. Concentration techniques will increase the number of organisms detected,
compared with direct microscopy. Motile protozoan trophozoites are not found in
concentrated preparations. Concentration procedures may be performed on fresh or
preserved specimens.

As concentration methods do not work well on liquid stools, direct microscopy may also
be necessary. In general, two methods are used : sedimentation and flotation.

1. Sedimentation

The procedure requires suspension of feces in a fluid that is lighter that the parasitic
forms. The latter sink to the bottom of the suspension by gravity. The process of
sedimentation can be expedited by centrifugation. However, the preparation is not as
clean as the flotation method and it contains more fecal debris. Acid-Ether
sedimentation technique is useful for concentration of most helminthic eggs, especially
for Schistosoma, but it is not satisfactory for protozoan cysts, eggs larvae, including
operculated and Schistosome eggs. Cysts are less distorted and more effective in
formalinized specimens.

1. Flotation

The aim is to suspend washed sediment in a solution having a slightly greater specific
gravity that parasite elements so that when centrifuged, the cysts, eggs, and larvae
concentrate on the surface film where they can be removed to a slide for examination,
while heavier elements are thrown to the bottom of the tube. The best single method is
zinc sulfate. Although it destroys all trophozoites of protozoa, cysts are unaffected. It is
efficient for helminthic ova except those of Schistosome and operculated eggs.
The brine flotation is one of the oldest concentration techniques still in use. It is simple
and efficient for recovery of all eggs other than operculated and Schistosome eggs. It is
especially recommended for concentration of hookworm eggs and is probably the most
efficient single technique for the recovery of these eggs. Protozoan cysts, however, will
be unrecognizably shrunken.
Procedure (Sedimentation Technique - Acid Ether Concentration Technique)

1. Obtain about 1 gram of feces by scrapping the outer surface of the fecal
specimen

with applicator stick

2. Comminute in 10 ml 15% HCl

3. Strain through two layers of wet gauze
4. Place 5 ml of the filtrate in a centrifuge tube and add equal amount of ether
5. Put a stopper and shake vigorously for 1 minute.
6. Remove stopper and allow to stand for a while; spin at 1500 rpm for 1 minute.
7. Observe the following layers from top to bottom
8. ethereal layer
9. plug of fecal debris
10. acid layer
11. sediment
12. Carefully insert an applicator stick along the sides of the test tube through the
second layer and ring to loosen the plug

9. Carefully discard the upper three layers but do not disturb the sedimennts
10. Keep the tube in a slanting position. Get a sample of the sediment and place
it on a slide.

11. Add a drop of iodine solution, mount with a clean coverslip, and perform
microscopy.

12. Draw and label the four layers and all parasites rcoveered after microsocopy.
Procedure (Floatation - Zinc Sulfate Floatation):

1. Place about 1 gram (size of a pea) of feces in a test tube and comminute with
ten times its volume of water.
2. Strain through two layers of wet gauze and spin about 5 mL of the filtrate at
2500 rpm 1 minute.
3. Decant and add about 1ml of water and shake to break off the sediments; fill
up with water to the original volume, and spin at the same speed and time.
4. Repeat this procedure three times or until the supernatant is clean.
5. After the last centrifugation, decant completely then add 5 ml of zinc sulfate
and break off sediments; add more zinc sulfate up to about 1/2 inch from the
rim of the tube and spin at 2500 rpm for 1 minute.
6. Place preparation in a test tube rack and allow to stand for 2 minutes without
disturbing.
7. Obtain material from surface film by means of wire loOp and transfer to a
clean glass slide.
8. Add one drop of iodine solution and mount preparation with coverslip.
9. Get ready for microscopy.

Thick and Thin Smear for Malaria and Knott's Concentration

Technique
Peripheral blood samples for the diagnosis of malaria can be taken from a finger prick
or preferably from a bottle with EDTA anticoagulant. The slides must be made
immediately. If the blood os left for several hours in anticoagulant, the following effects
may be seen:

1. Male gametocyte may develop and exflagellate, releasing microorganisms

which may be mistaken for other organisms such as Borrelia.

2. “Accole” forms, which are characteristic of Plasmodium falciparum, may be

seen in Plasmodium vivax infections because of reinvasion of the RBC by

merozoites, which cannot enter the cell and are retained on the membrane.

3. The morphology of the RBC may be altered by shrinkage or crenation.

4. Collection of samples should be done as soon as malaria is suspected. If the
slide is negative, the sample should be repeated just after or during fever
when the parasites are present at their highest density. During the apyrexial
phase, the parasites disappear from the peripheral blood and may not be
seen at this time.
 5. It should be taken prior to anti-malarial therapy.
6. Blood taken during the primary stage of infection, i.e., during the first 2-3
days, may not show parasites.
7. Repeat samples should be taken, preferably at 4-hour intervals or just after a
fever when slides have been negative but malaria is still suspected.
8. Repeat samples should also be taken at regular intervals during therapy to
check the parasitemia of Plasmodium falciparum, particularly in cases of
initial high parasitemia.

Materials: Blood extraction kit, EDTA tube, glass slides, applicator stick, microscope,
cedar wood oil, Wright's or Giemsa stain
Procedure:

1. Clean ring finger with alcohol and puncture the tip.

2. Discard the first drop.
3. Place a small drop at the center of a clean slide and 2 or 3 on the same slide
about an inch from one end.
4. Spread the drop of blood at the center as in ordinary differential smear
moving towards the other end of the slide. This is the thin smear.
5. Using one corner of another slide, spread the larger drops of blood to form a
circular smear about the size of a ten centavo coin. Air dry and label. This is
the thick smear.
6. Dehemoglobinize thick smear as follows:
7. flood the thick drop of blood with clean distilled water avoiding any droplet of
water to come in contact with the thin smear and stand for a few minutes.
8. drain off the water by tipping off slide slowly.

9. Repeat a and b until the washing is nearly clear and only a white film is left out
of the thick smear.
10. Dry slide by standing vertically, making sure that the thin smear is up.
11. Fix and stain with Giemsa or Wright’s stain.
12. Do microscopy.
13. Draw and label the whole preparation.
Nematodes
Introduction:
The members of the class Nematoda may assume three basic morphologic forms:
eggs, larvae and adult. The eggs vary in size and shape. In the appropriate
environment, developing larvae located inside the eggs emerge and continue to mature.
These larvae are typically long and slender. The growing larvae complete the
maturation process, resulting the emergence of adult worms. Sexes are separate. The
adult female worms are usually larger than the males. The adults are equipped with
complete digestive and reproductive systems. Specific features may vary with the
individual species.

Trematodes
Parasites belonging to this group are generally leaf-like with few cylindrical forms and
inhabiting various organs of the body.

The life cycles of fluke are very similar. A complete life cycle is composed of the
following stages:

• Eggs are laid by the adults.

• Miracidium is the ciliated larva which hatches from the egg.
• Sporocyst is non-ciliated, sac-like larva developed from the miracidium.
Found in snails.
• Cercaria is a larva developed within either a sporocyst or a redia. It has a
mouth, digestive tract, and a tail which is forked in the Schistosomes and
straight in the hermaphroditic flukes. The cercaria emerges from the snail.
• Metacercaria is a cercaria which has lost its tail upon entrance into a second
host.

Trematodes infecting man are grouped according to the portal of entry of the infective
stage and the location of the parasites in man.

1. Fork-tailed cercaria penetrate the skin:

Blood flukes or Schistosomes (separate sexes)

Schistosoma japonicum
 Schistosoma mansoni
Schistosoma haematobium

2. Straight tailed cercaria encyst, depending on the species, vegetables,

grass, fish, crabs. The encysted cercaria, i.e., the

1. Intestinal Flukes (hermaphroditic)

Fasciolopsisbuski
Heterophyesheterophyes
Echinostomailocanum
Metagonimusyokogawai

1. Liver Flukes (hermaphroditic)

Clonorchis sinensis
Opistorchisfelineus
Fasciola hepatica

1. Lung Fluke (hermaphroditic)

Paragonimuswestermani

Cestodes
Introduction:
This activity and discussion focuses on the class of mutlicellular organisms noted for
their flat or ribbon like appearance known as Cestoda (or cestodes). The characteristic
appearance of the cestodes forms the basis for the common names associated with this
group: flatworms or tapeworms.
Introduction
Protozoa are unicellular animals consisting of a nucleus, or nuclei and cytoplasm. The
nucleus of some species is merely a mass of chromatin. In others, it consists of a
nuclear membrane containing the nuclear sap in which the karyosome is found. The
nucleus is concerned with multiplication; the morphological structure of the nucleus is
used in the identification of a number of protozoa.
The cytoplasm is differentiated into an inner portion, the endoplasm, and an outer layer,
the ectoplasm. The endoplasm is of syrupy consistency and represents the viscera of
the organism. It is concerned with nutrition. It may contain ingested materials which may
be found within food vacuoles. Contractile vacuoles, which occur in some protozoa, are
believed to eliminate waste products.
The endoplasm is a dense, resilient structure. It performs the function of the skin
(protection), the limbs (locomotion), the mouth (ingestion of food), and excretory organs
of the larger animals. Locomotion is accomplished by ectoplasmic organelles.
The amoebas move by means of ectoplasmic protrusions, i.e., pseudopodia.The
flagellates move by means of long, thread-like filaments, i.e. flagella. The ciliates move
by means of hair-like filaments, i.e. cilia. The sporozoans are protozoans which have a
sexual stage in their life cycle. Some species of protozoa encyst, i.e., the ectoplasm, is
modified into resistant cyst wall.
Protozoa Parasitic in Man

Intestinal/Atrial Protozoa Blood Protozoa

Entamoeba histolytica
Entamoeba coli
AMOEBA Endolimax nana
Iodamoebabutschlii
Entamoeba gingivalis

Giardia lamblia Leishmania donovani

Dientamoeba fragilis Leishmania braziliensis
FLAGELLATES
Chilomastixmesnili Leishmania tropica
Trichomonas hominis Trypanosoma cruzi
 Enteromonas hominis Trypanosoma brucei gambiense
Retortamonas intestinalis Trypanosoma brucei rhodesiense
Trichomonas tenax
Trichomonas vaginalis

CILIATE Balantidium coli

Plasmodium falciparum
Toxoplasma gondii
Plasmodium vivax
Cyclospora cayetanensis
Plasmodium ovale
SPOROZOA Cryptosporidium parvum
Plasmodium malariae
Cytoisospora belli
Plasmodium knowlesi
Sarcocystis hominis
Babesia microti

Amoeba and Ciliate

Introduction:
The most important feature that separates amoebas from the group from the other
groups of unicellular Protozoa is the means by which they move. Amoebas are
equipped with with the ability to extend their cytoplasm in form of pseudopods (often
referred to as false feet), which allows them to move within the environment. With one
exception, there are two morphologic forms in the amoebic life cycle - trophozoites, the
form that feeds, multiplies, and possesses pseudopods, and cysts the nonfeeding stage
characterized by a thick protective wall designed to protect the parasite from the harsh
outside environment when deemed necessary.
Atrial & Luminal Flagellates and
Hemoflagellates
Introduction:
Flagellates belong to the phylum Protozoa and members of the subphylum
Mastigophora. The flagellates can be categorized into two: intestinal and atrial.
Intestinal are those found/resides in the intestine while atrial are those that can be found
in other areas other than the blood (oral cavity, vagina, etc.)
Members of the clinically significant group of parasites located in the blood and tissue
that move by means of flagella belong to the genera Leishmania and Trypanosoma.
Transmission of all hemoflagellates are vector borne. The difference between the two
genera lies on the diagnostic stage that can be detected in the blood of infected
patients. Amastigote is the diagnostic stage for Leishmania while trypomastigotes
for Trypanosoma (except Trypanosoma cruzi in which amastigote can also be found).

Sporozoa
Introduction:
Malaria and Babesiosis refers to the disease process resulting from the infections of
parasites belonging to the phylum Apicomplexa. Their respective genera are
Plasmodium and Babesia. Both genera of parasites belong to the class of parasites that
have no obvious structures for the purpose of motility, known as sporozoa. The most
clinically relevant organisms belonging to this genera of this discussion are Plasmodium
vivax, Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium
knowlesi and Babesia microti.
 EXAMINATION OF BLOOD FOR DETECTION OF MICROFILARIA

Variety of methods can be used to detect microfilariae in venous blood samples.

Direct Blood Smear (wet film examination)

Procedure

• Place one drop of heparinized or EDTA mixed blood on a slide, add a droplet of
physiological saline, mix and cover with a coverslip.
• Examine directly under low power (10X) of a microscope for live microfilariae. Larvae
can be immobilized by placing a drop of 10% formalin or Lugol’s iodine at the edge of
the coverslip.

KNOTTS CONCENTRATION METHOD

This is the standard test used to screen microfilariae in blood which includes lysis of
erythrocytes, fixation and staining of larvae.
Nematodes
Ascaris lumbricoides
• Ova
• Fertilized (45-70 um X 35-50um) vs. Unfertilized (88-94 um X 39-44
um)
• 2-3 layers (Mammillary Albumin coat, Glycogen layer, Vitelline
lipoidal layer)
• Adult
• Long with 3 oval lips
Trichuris trichiura/Trichocephalus
trichiurus
• Ova
• Barrel or football shaped with BIPOLAR MUCOUS PLUGS (50-
54um X 23um)
• “JAPANESE LANTERN OVA”
• Adult
• Slender anterior and fleshy posterior
Enterobius vermicularis
Oxyuris vermicularis
• Ova
• D shaped, double walled/layered, flattened on one end (50-60um
X 20-30um)
• Thin transparent shell and occasionally contains the LARVA
• Adult
• Female worms have the distinct “cephalic alae”
Capillaria philippinensis
• Ova (36-45um x 20um)
• Peanut shaped, double walled, striated with bipolar plugs
• Adult
• Delicate tiny worms, male have chitinized spicules
Stronglyoides stercoralis
• Adult
• Parasitic female is longer than free living
• Parthenogenesis
• Male are smaller with two spicules
• Egg (50-58um x 30-34um)
• “Chinese lantern ova”
Hookworms
• Adult:
• Male: Copulatory bursa
• Female : straight tail
• Eggs:
• Hookworm eggs are IDENTICAL (reported only as “hookworm
egg”)
• Thin walled with 2-8 germ cells
Rhabditiform larva

Hookworms S. stercoralis
• Long buccal cavity • Short buccal cavity
• Small genital primordium • Large genital primordium
Filariform Larva

Hookworms S. stercoralis
• Short esophagus • Long esophagus
• Pointed and sheathed tail • Notched and unsheathed tail
 Morphology of the Diagnostic Stage
• DIAGNOSTIC STAGE OF FILARIAL WORMS: MICROFILARIA
• Basis: Presence of sheath, Terminal nuclei (tail)
SHEATH TERMINAL NUCLEI

Brugia malayi 2 distinct terminal nuclei

Wuchereria bancrofti SHEATHED No terminal nuclei
Loa loa Extended terminal nuclei
Dipetalonema (Mansonella)
Extended terminal nuclei
perstans
Onchocerca volvulus UNSHEATHED No terminal nuclei
Mansonella ozzardi No terminal nuclei
Trematodes
Fasciola spp.

Fasciola hepatica Fasciolopsis buski

• Adult: • Adult:
• Fleshy, 1 x 3 cm • Fleshy, 1.5 x 5 cm
• Very distinct shoulders • No shoulder
• Ova: • Ova:
• Oval, small operculum • Oval, small operculum
• 128-150um x 60-90um • 128-140 x 78-85um
Laboratory Diagnosis
• Eggs are almost indistinguishable
• May recover adult to further differentiate
• Check signs and symptoms
• Travel history of patient
• Enterotest, ELISA and Gel Diffusion
Clonorchis sinensis
• Adults:
• Flat, fleshy and the anterior portion is narrower than the body
• 2 x 0.5 cm
• Egg
• Resmebles old fashioned light bulb
• 30 x 15 um
• With opercular shoulders and abopercular knob
• Embryonated
Heterophyid flukes
Heterophyes heterophyes Metagonimus yokogawai
• Adult: • Adult:
• 1 x 0.5 mm, pyriform in shape • 1 x 0.5 mm, pyriform
• Fine spines • Scaly spines
• With gonotyl/3rd sucker • No gonotyl
• Egg: • Egg:
• 30 x 15um, less prominent • 30 x 15um, less prominent
opercular shoulders, no opercular shoulders, no
abopercular knob abopercular knob
• Shell: thick • Shell: thin
Laboratory Diagnosis
• Ova of Clonorchis, Metagonimus and Heterophyes are almost
indistinguishable
• Recovery of adult may be used for differentiation
• Upon seeing the egg, you may report “Heterophyd egg”
Paragonimus westermani
• Adult:
• Fleshy, oval, red-brown color
• Tegument/cuticle contains spines
• 1 x 0.7 cm
• Egg:
• Similar with D. latum
• 78-120 um, with opercular shoulders, VERY LARGE OPERCULUM
• Opposite the operculum: abopercular thickening without knob
Schistosoma mansoni
• Adult:
• Male tegument: tuberculated, Testes: 6-9 in cluster
• Female uterus: short
• Ova:
• 112-182um X 40-75um
• Developed miracidium
• Oval shaped with prominent lateral spine
Schistosoma haematobium
• Adult:
• Male tegument: tuberculated with smooth granulations
• Testes: 4-5 in cluster
• Female uterus: 20-100 eggs
• Ova:
• 110-170um X 38-70um
• Developed miracidium
• Oval shaped with prominent terminal spine
Schistosoma japonicum
• Adult:
• Male tegument: smooth, Testes: 7, linear
• Female uterus: up to 500 eggs
• Ova:
• 50-85um X 38-60um
• Developed miracidium
• Round with small lateral knob