Macroscopic and Microscopic

Examination of Feces
What is stool or feces?
Macroscopic Examination
Physical Description
Physical Description

Color
Loose or watery specimens
trophozoites
 Formed or semiformed specimens
cyst stages.
Reporting
 Report the Color and consistency of stool
 Report the presence of blood on or in the
fecal specimen.
Example:
Stool Color: Reddish-Brown
Stool consistency: Semi-formed
Fresh blood seen on stool specimen.
Microscopic examination
Fecal Elements
Epithelial
cells
White Blood Fungal spores
cells Parasite
Red blood cells Food remnants
Macrophages
Charcot leyden
crystals
Techniques
Unstained
Direct fecal smear
0.85% Normal saline solution
Iodine
Kato-Thick
Concentration Techniques
Stained
Unstained/Wet mount
Microscopic examination
Direct Fecal Smear

Routine method
2 mg stool
0.85% NSS and Iodine
Motile Trophozoites
Kato-Thick
50 to 60 mg of stool
10 to 20 minutes
Cellophane paper
Glycerine
Malachite green
Reporting results
Report:
adult helminths or portions of helminths.
Morphology and size
Developmental stage
Proglottids
Number of parasite seen/field
Examples:
Ascaris lumbricoides adult worm 0-1/lpo
Entamoeba histolytica cysts 2-9/hpo
Taenia saginata gravid proglottid 0-8/lpo
Concentration Techniques
Microscopic examination
Formalin-Ethyl Acetate
Sedimentation

• 10 % Formalin
• Ether/Ethyl
Acetate
 Zinc Sulfate Flotation

• 33 % Zinc Sulfate
• S.G. :1.18-1.20
Quality control

Zinc sulfate: specific gravity of 1.18.

Using the hydrometer
Stained smear
Microscopic examination
Permanent Stained Smear (Trichrome)

Trichrome technique of Wheatley

modification of Gomori’s staining
It is a rapid, simple procedure
Results
The cytoplasm of protozoan trophozoites:
blue-green, with sometimes a tinge of
purple.
 Cyst : slightly more purple
Nuclei and inclusions : red, sometimes
tinged with purple.
Background material : stains green,
providing a nice color contrast with the
protozoa
Iron Hematoxylin Stain (Modified Spencer-
Monroe Method)

Permanent stained slide

detecting and quantitating parasitic
organisms.
Morphological descriptions
intestinal protozoa found in humans
Results

Background material: blue-gray.

Cells and organisms: various intensities of
blue-gray.
Inclusions, chromatoidal bodies, and
nuclear structures : darker than the
surrounding cytoplasm.
Calcofluor White

Acanthamoeba cysts
disodium salt of 4,4-bis-(4-anilino-
bis-diethylamino-5-triazin-2-
ylamino)-2,2-stilbene disulfonic
acid
 Positive control
1. Acanthamoeba cysts
doubled walled
outer wall is wrinkled
cysts will fluoresce.
Negative control
1. E. coli will not fluoresce.
2. Most of the bacteria and other debris will
not fluoresce. However, there will still be
some yeast and debris that may also
fluoresce.
Special Stains for Microsporidia

Modified Trichrome-Weber
Green
Modified Trichrome-Ryan Blue
 Special Stains for Coccidia

Modified Kinyoun’s Acid-Fast Stain

(Cold)
Modified Ziehl-Neelsen Acid-Fast
Stain (Hot)
Culture techniques
“Culture” of Larval-
Stage Nematodes
Baermann Technique

principle
active larvae will migrate out of
a fecal specimen that has been
placed on a wire mesh covered
with several layers of gauze.
Harada-Mori Technique

 Principle
filterpaper
Moisture is provided by adding water
to the tube.
hatching of ova and/or development
of larvae
Petri Dish-Filter Paper Slant
Additional Techniques for
diagnostic parasitology
Examination for Pinworm
Cellulose Tape Preparation
Cellulose Tape Preparation

the most widely used

procedure for the detection of
human pinworm infections
Quality Control

 If opaque tape is submitted by mistake, a drop of

immersion oil on the top of the tape will clear it
enough to proceed with the microscopic
examination.
Duodenal Contents
String Test (Entero-Test Capsule)

 specimens from the duodenum

 gelatin capsule lined with silicone rubber
that contains a spool of nylon string
 4 hours
Duodenal Aspirate

Strongyloides stercoralis larvae

Giardia lamblia trophozoites
Cryptosporidium parvum
Isospora belli oocysts.
Urogenital Specimens
Direct Saline Mount
 Trichomonas vaginalis
 Specimens
Vaginal discharge
Urethral discharge
Penile discharge
Urethral-mucosa scrapings
First-voided urine with or without prostatic
massage
Permanent Stained Smear (Giemsa)

Trichomonas vaginalis
Giemsa and Papanicolaou stains
Urine Concentration: Centrifugation

 Helminthic larval stages and eggs

 some protozoa
 Filariasis
 Trichomonas vaginalis
 Schistosoma haematobium eggs
 Microfilariae may be detected
 Microsporidia: Encephalitozoon (Septata)
intestinalis
SPECIMENS
A. T. vaginalis
first-voided urine
prostatic massage
B. S. haematobium
midday urine specimen or a 24-hours
Peak egg excretion: noon and 3 p.m
C. Filariasis
 Microfilariae
patients with chyluria
patients with heavy filarial infections
patients treated with diethylcarbamazine
D. Microsporidia
 Microsporidial spores
concentrated urine of patients who are
immunosuppressed
Expectorated Sputum
Direct-Mount and Stained Preparations
 Directsmear
detect large or motile organisms from the
lung
Parasites which can be detected
pneumonia, pneumonitis, or Loeffler’s
syndrome
Entamoeba histolytica, Paragonimus spp.,
Strongyloides stercoralis, Ascaris
lumbricoides, and hookworm.
Trichrome stains
aid in differentiating E.
histolytica from Entamoeba
gingivalis
Giemsa stain
larvae and juvenile worms
Aspirates and Bronchoscopy
Specimens
• Useful when routine specimens and methods have
failed to demonstrate the organisms.
• Aspirates include liquid specimens
• collected from a variety of anatomic sites
Fine-needle aspirates

 Giemsa and methenamine-silver nitrate

Pneumocystis carinii
 Giemsa
Toxoplasma gondii
 Trichrome
amebae,
 Modified acid fast
Cryptosporidium parvum.
Aspirates of cysts and abscesses

concentration by
centrifugation,
Digestion
microscopic examination
motile organisms
Bone marrow aspirates

Leishmania amastigotes
Trypanosoma cruzi
amastigotes,
Plasmodium spp.
Fluid specimens collected by
bronchoscopy

concentrated by centrifugation
P. carinii
T. gondii
C. parvum,
Microsporidia
Biopsy Specimens
 Diagnosis of tissue parasites.
 histological preparations
impression smears
teased
squash preparations
 Specimen
skin, muscle, cornea, intestine, liver,
lung, and brain.
Examination of Blood
Anticoagulants
 EDTA
0.020 g/10 ml of blood
 Heparin
2 mg/10 ml of blood
 Sodium citrate
0.050 g/10 ml of blood
Methods
 Capillary
blood
Wet/fresh preparation
Stained smear

 Venousblood
Knott’s Concentration
Membrane Filtration
Capillary blood
Wet/fresh preparation
Stained smear
 Wet/fresh preparation
 Microfilariae
 Trypomastigote
Stained smear

Thick films
Demonstration of parasite
Thin Films
Specie identification
Giemsa Stain
 Prepare and stain films from “normal”
blood, and microscopically evaluate the
staining reactions of the RBCs, platelets,
and WBCs.
1. Macroscopically
 blood films appear purplish
If blue
if pink to red
2. Microscopically
RBCs: pinkish gray
Platelets: deep pink
WBCs: purple-blue nuclei and lighter
cytoplasm.
Eosinophilic granules : bright purple-red
neutrophilic granules: purple
Basophilic stippling within uninfected
RBCs: blue.
Quality control

 Prepared within 1 h
 Correct pH for all buffered-water and
staining solutions
 Stain a QC slide each time patient blood
films are stained
Wright’s Stain
 Prepare and stain films from “normal”
blood, and microscopically evaluate the
staining reactions of RBCs, platelets, and
WBCs.
1. Macroscopically,
 blood films appear pinkish purple.
If blue,
if pink to red
1.Microscopically
 RBCs: tan to pinkish red
Platelets: deep pinkish red
WBCs: light blue nuclei and lighter
cytoplasm.
Delafield’s Hematoxylin Stain

Detailed structure of Microfilariae

Main stain
Hematoxylin
Ammonium alum
1.Macroscopically
the films appear bluish purple.
2. Microscopically,
the nuclei: blue or purplish
Sheath: light purple.
Cytoplasm: reddish.
inner body : whitish structure.
Venous blood
 Knott’s Concentration
 Membrane Filtration
Knott’s Concentration

Lowmicrofilariae
1 ml : 10 ml 2% formalin
Sediment
Membrane Filtration
 Membrane filtration recovers most species
of microfilariae
Mansonella perstans
Mansonella ozzardi
 Low microfilariae
Swinney Filter holder
1 ml blood
10 ml distilled water
 Clinical
Parasitology
Morphological characteristics
Laboratory Diagnosis
PROTOZOA
 simple, single-celled animals
 absorb food through their cell membrane
 digest their food in stomach-like compartments
called vacuoles
 reproduces by splitting in half
DEVELOPMENTAL STAGES:

TROPHOZOITE CYST
 active stage  inactive/dormant stage
 shows motility/motile forms  non-motile
 vegetative stage
 non-feeding
 feeding stage
 no reproduction
 procurement of food happens
 resistant stage
 non-resistant stage
 non-infective but responsible for  infective stage/transfer
signs and symptoms stage
 pathogenic stage
Phylum Sarcomastigophora
Subphylum Sarcodina
Subphylum Mastigophora
Subphylum Sarcodina
Parasitic Amoeba
Free-living, Pathogenic Amoeba
Entamoeba histolytica
Entamoeba dispar
Entamoeba hartmanni
Entamoeba coli
Entamoeba gingivalis
Endolimax nana
Iodamoeba butschlii

Parasitic Amoeba
Entamoeba
histolytica
Morphology

Trophozoite
Precystic stage
Cystic stage
Trophozoite
 12 to 60 micrometer
 Motility: Crawling or Gliding motility
 Karyosome
 small, compact, centrally located
 Cytoplasm
 “ground glass” appearance
Cyst
 Spherical
 10 to 20 micrometer
 Quadrinucleated cyst
 chromatoidal bodies
 cigar-shaped
Laboratory Diagnosis

 Definitive diagnosis
 Demonstration of E. histolytica trophozoite or
cysts
 Specimen
 Stools
 Tissues
 Discharges from lesions
 Culture
 Not employed for routine diagnosis
 Immunological test
 Not helpful for intestinal Amoebiasis
 Extraintestinal amoebiasis
Laboratory Diagnosis: Intestinal amoebiasis

 Macrosopic
 Stool
 Copious, semiliquid. Brownish black
 Foul smelling
 Blood streaked mucus intermingled
Microscopic

 Cellular exudate
 Charcot leyden crystals
 E. histolytica: ingested RBC

Sigmoidoscopy
 Amoebic ulcers
Biopsies
Laboratory Diagnosis: Extraintestinal
Amoebiasis
 Stool exam : negative
 Serologic test
 Pulmonary amoebiasis: Sputum
Entamoeba dispar

 Morphologically similar to E. histolytica

 Different in DNA, rRNA and isoenzymes
 no ingested erythrocytes in its trophozoite
Entamoeba hartmanni

”small race” E. histolytica

Trophozoite

4 to 12 micrometer
 Do not ingest RBC
 Motility: Less vigorous
Cyst

5 to 10 micrometer
 chromatoidal bars
slightly smaller and more numerous
Entamoeba coli

 Trophozoite
 20 to 50 micrometer
 Sluggish motility
 Cyst
 10 to 30 micrometer
 Chromatoid bodies
splinter like or irregular
Entamoeba polecki

 Pigs and monkeys

 Rarely infects humans
 Trophozoite
 Resembles that of the E. coli
 Cyst
 Uni nuclear
Entamoeba gingivalis

 Gingival tissues
 Unhygeinic mouth
 Transmitted by direct oral contact
 Saliva or fomites (utensils)
 Trophozoite
 10 to 20 micrometer
 Pulmonary suppuration
 Bronchial washing
Trophozoite
 progressive motility
 nucleus
 Karyosome
 small, well-defined, usually centrally located

 Cytoplasm
 finely granular and vacuolated
 Endolimax nana

 Human intestine
 Common commensal amoeba and it is widely
distributed
Trophozoite

 6 to 15 micrometer
 Sluggish motility
 Nucleus
 eye of a bird
 Cytoplasm
 Granular

 small, well-defined vacuoles

Cyst

6 to 12 micrometer
 Immature
 Binucleate cyst
 Mature
 Tetranucleate cysts

“ Crossed Eye “
Iodamoeba butschlii

 Trophozoite
9 to 14 micrometer long
 Large vesicular nucleus
 Large endosome surrounded with achromatic granules
 Cyst
 oval
”basket
 uninucleated
nucleus”
Laboratory Diagnosis:

 cysts
and/or trophozoites in stool
specimens
 Trophozoites
 scrapings of the gums and teeth
 Sputum: rare
Free-living, Pathogenic Amoeba
Acanthamoeba species
Naegleria species
Acanthamoeba species

 Small free living amoeba

 Active trophozoite stage and dormant cyst
stage
 Trophozoite stage
 Sluggish motility
Naegleria species

 Free living amoeba –flagellate

 Amoeba : Trophozoite form
 Flagellate : Swimming form
Naegleria gruberi

 Non-pathogenic
 Morphogenetic studies
 Induction and differentiation flagellation
 Cytoplasmic origin
Naegleria philippinensis

 Isolated from
 Thermally polluted stream
 Artificially heated swimming pool
 Brain aspirate of a young patient
Naegleria fowleri

 Pathogenic
3 stages
 Dormant cyst
 Amoeboid trophozoite (10-20 um)
 flagellate
 Subphylum Mastigophora
Intestinal and Atrial Flagellates
Blood and Tissue Flagellates
Intestinal/Luminal Flagellates

 Giardia lamblia
 Dientamoeba fragilis
 Chilomastix mesnili
 Pentatrichomonas hominis
Giardia lamblia
 most common flagellate of the intestinal tract
 flagellate of world-wide distribution
 humans are the only important reservoir of the
infection
Trophozoite
 pear or teardrop-shaped
 ”falling leaf” or “flip flop”
 bilaterally symmetrical
Cyst

 8 -12mmin length
 Ellipsoid in shape
 4 nuclei
 axonemes and parabasal bodies
Laboratory diagnosis

 Demonstration of parasite in stool

 Cysts: Asymptomatic carriers
 Duodeno-jejunal aspiration or biopsy
 Entero test/String test
String Test (Entero-Test Capsule)

 specimens from the duodenum

 gelatin capsule lined with silicone rubber
that contains a spool of nylon string
 4 hours
 Antigen detection
 ELISA & Immunochromatographic strip test
 Antibody detection
 Direct fluorescence Antibody
Dientamoeba fragilis
 amoeba-flagellate with a cosmopolitan
distribution
 livesin the lumen of the cecum and upper
colon
 cysts have not been identified
Trophozoite
3 -22mm
 no peripheral chromatin
 Cytoplasm
 Vacuolated
 ingested debris
Laboratory Diagnosis

1.Demonstration of parasites
a. DFS
b. Permanent staining

*HAKANSSON PHENOMENON????
Chilomastix mesnili
 cosmopolitan in distribution
 non-pathogenic
 largest flagellate found in man with an incidence
of 1-10% being in the large intestine
Trophozoite
 pear shaped : 6-20mm in length
 1 large nucleus with a small karyosome
 3 flagella that extend from the nucleus at the
anterior end of the parasite
 distinct oral groove or cytosome
 “cork-screw” motility
Cyst

 6-9mm
 prominent side knob: lemon shape
 Cytostome : Curved shepherds crook fibril
LABORATORY DX
1.Demonstration of parasites
a. DFS
b. Permanent staining
Pentatrichomonas hominis

 most commonly found flagellate next to G.

lamblia and D. fragilis
 cosmopolitan in its distribution
 non-pathogenic
 no cystic stage
Trophozoite
 5-15mm in length by 7-10mm in width
 -shape is pyriform
 Axostyle
 runsfrom the nucleus down the center of the body and
extends from the end of the body
 undulating membrane: same as the body length
 wobbly movement
Laboratory Diagnosis

1.Demonstration of parasites
a. DFS
b. Permanent staining

*In a fresh stool, the flagellates move very rapidly in

a jerky, non-directional manner
Atrial Flagellates (body cavities)

 Trichomonas vaginalis (GUT)

 Trichomonas tenax (oral cavity)
Trichomonas vaginalis
 infects the urogenital tract
 cosmopolitan in distribution
 largest among trichomonads
 no cystic stage
Trophozoite
 rapid, jerky motility
 single nucleus on anterior end
 5 flagella
 undulating membrane: half the length of parasite
 cytoplasm has large amounts of siderophil
granules
Lab Dx

1.Demonstration of parasites
a. unstained wet mounts
b. permanent stained smears:
 -Papanicolou, Giemsa, Romanowsky, Acridine orange
c. culture (Diamond’s Feinberg and
Whittington)
2. Serology

 Recombinant DNA technology

 specificity with this method can be less than with wet mount due to false-positive tests
 Dot blot hybridization
 employs a 2.3 kbT vaginalis DNA fragment as a probe
 Monoclonal antibodies
 derived from specific proteins have been effective in identifying T vaginalis from
clinical specimens
 Rapid antigen testing and transcription-mediated amplification
Trichomonas tenax
 commensal of the human oral cavity
 no cystic stage
 known to feed on bacteria and therefore is not
able to cause pulmonary disease by itself
Trophozoite
 5um to 16um long 2-15um wide
 4 anterior free flagella, fifth curving around cell
 undulating membrane: 2/3 the length of the body
Lab Dx

1.Demonstration of parasites
 unstained wet mounts
 permanent stained smears:
2. Serology
Blood and tissue flagellates

 Leishmania spp
 Trypanosoma spp
Leishmania spp

 Leishmania tropica
 Leishmania braziliensis
 Leishmania donovani
Leishmania tropica

other names
 Old world leishmaniasis
 Oriental sore
 Aleppo button
 Jericho boil
 Baghdad boil
cutaneous leishmaniasis
 Delhi boil

 Vector: Phlebotomus papatasi, P. sergenti

Leishmania braziliensis
other names:
 New world leishmaniasis
 Espundia
 Chiclero ulcer
 Uta

 Vector:P.intermedius,
Lutzomyia, Psychodopygus
mucocutaneous leishmaniasis
Leishmania donovani
other names:
Kala-Azar
Dumdum fever
Death fever
visceral leishmaniasis
Vector: P. papatasi, P. argentipes,Lutzomyia
LABORATORY DX

1.Demonstration of parasites
a. L. tropica
 material from cutaneous lesion or skin ulcer
 fluid aspirates
 biopsy
b. L. braziliensis
samewith L. tropica
mucosal scrapings
c. L. donovani
 tissue aspirates: spleen, liver, bone marrow
 blood film
 Buffy coat
Direct microscopy
dx stage:
Culture
Novy-MacNeal-Nicolle (NNN)
dx stage:
Skin Test
a.Montenegro skin test/Leishmanintest
 Intradermal injection of suspension of killed promastigote
 (+) reddening and thickening at the site of injection
 L. tropica(+)
 L. braziliensis(95% positivity)
 L. donovani(+) 2 months after cure; (-) in active visceral
leishmaniasis
Non-specific Tests

based on increased gamma-globulins

(from 11% to 60-70%)
Aldehyde Test of Napier

a. 1-2mL of pt. serum in test tube

 1-2 drops of 40% formalin
 (+) jellification resembling the egg white
 (+)
in visceral leishmaniasis of atleast3
months duration
Trypanosoma spp

 African Trypanosomiasis
 Trypanosoma brucei gambiense
 Trypanosoma brucei rhodesiense
 American Trypanosomiasis
 Trypanosoma cruzi
Mode of Transmission

 bite blood-sucking invertebrate

 ingestion of infected feces of intermediate host
by definitive host
 entrance of parasite through any break in the skin
Trypanosoma brucei

 Trypanosoma brucei gambiense

 Disease: West African or Gambian African trypanosomiasis
 Geographical Dist: Central & West Africa
 Trypanosoma brucei rhodesiense
 Disease: East African or Rhodesian African trypanosomiasis
 Geographical Dist : Central and East Africa
Morphology

spindle –shaped elongated body

 Size
 nucleus
 kinetoplast
 undulating membrane
 Anterior flagellum
 Volutin granules
Lab Dx

Microscopic demonstration of trypanosome

 acute
stage : blood, lymph nodes and bone
marrow
 chronic stage : CSF
Serological

detect anti-trypanosome AB
 sensitive
tests but cannot differentiate
between species of Trypanosoma
 Immunoflourescence test / indirect
agglutination test
CSF Analysis

WBC CHON
early : normal <3 cells <45mg/100ml
intermediate 3-10 cells ≤45mg/100ml
late >40 cells >45mg/100ml
Trypanosoma cruzi
Synonyms
 South American Trypanosomiasis
 Chagas’ disease
Geographical Distribution
 Central & South America
Morphology

 spindle –shaped body

 has free flagellum about 1/3 of body length
 nucleus centrally located and a large kinetoplast
 the undulating membrane
Insect Vector

Reduviid bug/ Triatomid bug/ Assassin bug

Kissing bug/ Cone-nosed bug
 Species:
 Panstrongylus megistus
 Triatoma infestans
 Rhodnius prolixus
Mode of transmission

 Biteof reduviid bug which defecate during

the process of feeding
 Accidental ingestion of bug
 Blood transfusion
Stages of development of parasite

 vertebrate host
 trypomastigote

 amastigote

 invertebrate host (reduviid bug)

 Trypomastigote

 epimastigote
Lab Dx

 Microscopic demonstration of parasite stained blood smear or

lymph node aspirate
 Xenodiagnosis
 Culture
 Serological
 Complement fixation test
 Biopsy
BALANTIDIUM COLI

Trophozoite
large, oval
covered with short cilia
50 to 150 μm long and 40 to 70 μm
wide
 The anterior end
pointed and has a cytostome
 The posterior
broadly rounded.
 The cytoplasm
vacuoles with ingested bacteria and debris.
The trophozoite has two nuclei:
macronucleus and micronucleus
rapid, rotatory motion
cyst
Two nuclei
macronucleus and micronucleus.
50 to 70 μm in diameter
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7 8 9 10 11 12

13 14 15 16 17 18
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4 5 6