PARASITOLOGY LABORATORY Prepared by: Chester F.

Ebersole

PARASITOLOGY LABORATORY
REFERENCE BOOK: Clinical Parasitology ( Elizabeth Zeibig)

STOOL FOR OVA AND PARASITE EXAMINATION – Macroscopic and Microscopic Examination
A. Protozoa – Cyst and Trophozoite
B. Helminthes – Eggs, Larvae, Proglottid, Adult
1. Collection and Transport
- Parasites don’t normally seen daily on stool samples
- Stool collection is done on ___ specimens. It is collected every other day or in total of 10 days.
Note: Except in ____________, 6 specimens in 14 days is acceptable
- Patients taking barium, bismuth and mineral oil should be collected _______ to examination or
5 to 7 days after completion of therapy. Antibiotics and Antimalarial are delayed for 5-7 days
- Urine should not contaminate the stool, Not to be retrieved from toilet water and presence of
toilet paper. Water may destroy eggs of Schistosoma and Trophozoite of Amoeba
Proper Collection of Sample
1. It should be collected in a clean, water-tight container with tight fitting lid
2. 2-5g of stool often with the size of walnut
3. The specimen container should be labelled with patient’s name and identification number and date
and time of collection. ZIPLOCK BAG IS NEEDED FOR TRANSPORT
4. PPE should be worn at all times.
5. Time is important in handling fecal parasite
Protozoa Trophozoite - ___________ ( preferably liquid stool and read within ______ after passage
Cyst, Eggs and Larva – can survive longer period of time
Semi-formed specimen may yield a mixture of cyst and trophozoite and should be evaluated 1 hr
Formed stool can be held up to 24hrs after passage
6. If time cannot be done, ____________ may be added ( Fixative) – placed directly upon receiving

ALL SPECIMENS SHOULD BE TREATED AS INFECTIOUS

Fixatives for Preservation
- Substances that preserve the morphology and development of parasites
- Recommended ratio __ parts fixative as to ___ part of stool
- Specimen should be well mixed to ensure thorough fixation
- It should be fixed for at least ____ minutes before processing

Fixative Concentration Method Permanent Stain Antigen Testing

10% Formalin + - +
SAF + + ( Iron Hematoxylin) +
PVA Variable + ( Trichrome or IH) -
Modified PVA ( Zinc) Variable + ( Trichrome or IH) Variable
Single Vial System + + ( Trichrome or IH) Variable

Formalin: all purpose fixative for recovery of protozoans and helminthes. 5% - protozoan cyst and
10% for helminthes and larvae
Advantages Disadvantages
1. Easy to prepare 1. Not suitable for permanent stains
PARASITOLOGY LABORATORY Prepared by: Chester F. Ebersole

2. Preserves for several years 2. Trophozoites cannot be preserved and cyst may
3. Long Shelf Life fade over time.

Polyvinyl Alcohol – with plastic powder that acts as adhesive during staining and often combined
with Schaudinn solution which comprised of zinc sulfate, copper sulfate or mercuric chloride.
Advantages Disadvantages
1. Can be used for helminth eggs and protozoa 1. Mercuric Chloride is injurious to health
2. Suitable for permanent stain
3. Long Shelf Life

NOTE TWO VIAL SYSTEM: Formalin for concentration technique and PVA for stained slide

Sodium Acetate Formalin – alternative for two vial system

Advantages Disadvantages
1. Easy to prepare 1. Albumin is needed during microscopy to ensure
2. Can be used for preparing smears especially adhesion of specimen in the slide
modified acid fast to detect _____ oocyst 2. Protozoa is not as clear as in PVA
3. Long Shelf Life 3. Preferred Stain is Wheatley Trichrome

Modified Polyvinyl Alcohol – same with PVA but without mercuric chloride. ZINC SULFATE is
better than Copper sulfate.
Advantages Disadvantages
1. Can be used for helminth eggs and protozoa 1. Not as good as PVA
2. Suitable for permanent stain 2. Likely to be negative is proper protocol is not
3. Long Shelf Life followed.

NOTE: All of the mentioned above is under ____________ phase

Processing
- The start of ____________ of laboratory testing
Macroscopic Microscopic
1. Color and Consistency 1. Direct Wet Preparation ( Preps)
2. Fresh and Unpreserved 2. Concentration Technique
3. Gross Abnormalities must be checked 3. Permanent Stain
4. Samples containing adult worms may be IF FIXED: Remove ___________
carefully washed through a wire screen.
Charcot Leyden Crystal – disintegration of __________ ( Unstained / Trichrome/ Iron Hematoxylin)
PARASITOLOGY LABORATORY Prepared by: Chester F. Ebersole

QUALITATIVE DETECTION IN STOOL SAMPLE

Direct Wet Preparation
- Small portion of UNFIXED stool added with saline or iodine
- It should be dense enough to read a newspaper print, use the mucus part if present
- Motility can only be demonstrated in ____________ especially liquid or soft consistency
- Should be read in LPO before proceeding to HPO ( light should only be increased when ____)
- Iodine ( Lugol’s or D’ Antoni) is added to enhance detail _______ but destroys _________.
It may also stain the ___________ as brown ( Iodamoeba butschlii).
- Eosin stain (1%) may also be used. Live cysts are unstained in which white objects against
pick background. It does not stain living protoplasm

Concentration Method
- To detect small numbers of parasite and to remove debris
- Not recommended for protozoan trophozoite
- Helminth eggs or larvae are best detected and identified using a concentration technique.
1. Sedimentation – parasites are concentrated 2. Flotation - parasites are less dense than the
in sediment of the tube following centrifugation solutions used and, during centrifugation, they
and the sediment is examined microscopically. float to the surface.
- ___________ is preferred because easier to do and efficient

Formalin-Ethyl Acetate Sedimentation: most widely used sedimentation technique which is based
on specific gravity. Ethyl acetate is added to a saline-washed formalin-fixed sample and the tube
is then centrifuged. Parasites are heavier than the solution and settle in the sediment of the tube,
whereas fecal debris is usually lighter and rises to the upper layers of the test tube.
Advantages Disadvantages
1. Good recovery of most parasites 1. More fecal debris than flotation
2. Easy to perform

Zinc Sulfate Flotation Method ( 1.18-1.20): parasites float to the surface and can be skimmed
from the top of the tube.
Advantages Disadvantages
1. More fecal debris are removed ( cleaner ) 1. Cannot retrieve some helminth eggs such as t
2. Protozoan cyst and Nematode eggs unfertilized roundworm eggs, nematode larvae,
and eggs of most trematodes and large
tapeworms.
A. Saturated Salt Flotation (1.20) – same w/ zinc sulfate but protozoan cyst can also not be recovered
B. Sheather’s sugar floatation technique – for Cryptosporidium parvum
QUANTITATIVE DETECTION FOR STOOL PARASITES
Stoll Dilution Method
- 4g of feces is mixed in sodium hydroxide
- Eggs x 200 = number of eggs per gram of feces
- Multiply by 1 if hard, 2 for mushy, 3 for loose stools and 4 for liquid stools
- Watery stool is unfitted for counting

Thick Smear
A. Kato Katz
- 50mg of stool is taken on a slide and covered with cellophane and soaked in glycerine
containing malachite green Glycerine clears the stool enabling helminth eggs to be seen
- To determine severity of infection
- It is not useful for protozoa and helminths larva
OTHERS TO DETECT STOOL PARASITES
1. Hatching Test for Schistosoma Eggs
- Viability of miracidium
- Specimen is placed in 10 volumes of dechlorinated or spring water. Living miracidia may be
released by hatching within few hours
PARASITOLOGY LABORATORY Prepared by: Chester F. Ebersole

2. Fecal Culture: for Hookworms and Strongyloides stercoralis

2A. Harada Mori Filter Paper Strip Culture
- To detect light infection with hookworm and Strongyloides stercoralis
- Uses a strips of filter paper and feces is smeared in the middle third
- The paper strips are kept in conical centrifuge tubes with water at the bottom in which the
strips dip. Tubes are kept at RT in the dark for 7-10 days which the time the larvae develop
and fall into the water.
B. Baermann Concentration Method
- Small numbers of Strongyloides stercoralis
- Principle: Active larvae migrate from fecal specimen after contact with tap water
C. Charcoal Culture
- Simple and Efficient
- Softened feces is mixed with 5-10 parts of moistened charcoal granules and covered. Larva will
fall down in the bottom of the flask
D. Agar culture for Strongyloides – more sensitive
- 2g of fecal specimens are inoculated onto agar plates. The plates are sealed with tape to prevent
accidental infection and placed in RT for 2 days
- Larvae will crawl over the agar making the visible tracks over it
- Confirmation : Microscopy

Permanent Stain
- To confirm cyst and trophozoite
- To confirm protozoans with trophozoite stages ONLY ( i.e ______________________)
- Preferred sample is PVA preserved sample
- Slides can also be prepared from a fresh stool specimen but must not be allowed to
dry and should be immediately placed into a fixative, such as the Shaudinn fixative.
- READ at OIO and increase light is needed
- 300 fields are reviewed before the slide can be considered negative.

Examples of Permanent Stain

Wheatley Trichrome.
- most widely used
- due to contrasting colors
- long shelf life and easy to do
Iron hematoxylin Modified Acid Fast
- time consuming - detection of oocyst of
- excellent morphology of Cryptosporidium parvum with
intestinal protozoans especially color pink to red.
nuclear details

Stool Screening Methods

 Utilizes monoclonal antibody ( can only detect one or two parasites) per test
PARASITOLOGY LABORATORY Prepared by: Chester F. Ebersole

OTHER SPECIMENS TO DETECT PARASITES

A. Duodenal Material – READ immediately because trophozoite disintegrate

- For parasites that reside the __________________
- May be collected by either nasogastric intubation or enteric capsule ( Enterotest)
- To recover Giardia lamblia, Microsporidium, Strongyloides stercoralis, Fasciola hepatica
- Enterotest:
1. Swallow a gelatin capsule with coiled length yard
2. It dissolves in the stomach and string reaches duodenum
3. After 4 hours, yarn is pulled back and bile stained mucus is examined
B. Sigmoidoscopy
- For colon (Identification of Entamoeba histolytica, Microsporidia and Trichuris trichiura
- Material from ulcers are aspirated or scraped
C. Cellophane Tape Preparation ( Perianal Swab)
- Choice of detection for _______________ ( pinworm eggs), Taenia may also be recovered
- It should be collected at morning before the patient washes or defecates
- To rule out pinworm infection, standard protocol is 5 samples.
D. Blood
- To identify Leishmania, Trypanosoma, Plasmodium, Babesia and microfilaria
- For malaria: collected during paroxysm ( _____ of fever), before antimalarial drugs
- Rule: Smear for suspicion and another smear after bout of fever
- Can be with or without anticoagulant. EDTA is used and prepare within 1hr after collection
- It involves: preparation of thick and thin smear, permanent stain and microscopy.
- It may also involve Knott technique, Buffy coat or reading culture

Thick and Thin Smear Culture Knott Technique

1. Thick - ____________ Novy-Macneal-Nicolle(NNN) Concentrate blood containing
( dehemoglobinized has higher medium; Identification of low amounts of microfilaria
concentration of parasites); Leishmania and Trypanosoma ( 1mL of EDTA blood + 10mL of
cant assess morphology Pencillin:_____________ 2% formalin then centrifuged)
2. Thin - ____________ Negative should held for 1 months then make smear
Permanent Stain Buffy Coat
1. Wright’s White Blood Cell Portion
2. Giemsa - ______________( detailed structure) Identification of Leishmania and Trypanosoma
3. Leishman stain – w/0 alcohol
MALARIA
Reporting Using Thick Smear – 30x times more sensitive and can detect 20 parasites/ uL of blood
1–10 per 100 high power fields +
More than 10 per 100 high power fields ++
1-10 in every high power field +++
More than 10 in every high power fields ++++
Periodicity is important in Microfilaria:
A. Nocturnal Periodic – 10PM to 2AM
B. Nocturnal Subperiodic – 8PM to 10PM
C. Diurnal Subperiodic – 2PM to 6PM
Other Concentration Method For Microfilaria
1. Sedimentation Method – blood is lysed with acetic acid or saponin then centrifuged
2. Membrane Filtration Method – passage through Swinney filter hole and washed with saline.It is not
suitable for Mansonella perstans and ozzardi due to small size
3. Diethycarbamazine Provocation Test – to mobilize microfilaria to the peripheral blood
- Collected 20-50 minutes after drug is given; surveys
- Cannot be used for Onchocerva volvulus
PARASITOLOGY LABORATORY Prepared by: Chester F. Ebersole

CSF and other sterile sites

- For identification Entamoeba histolytica, Trypanosoma, Naegleria fowleri and Acanthamoeba; It
may also include Toxoplasma gondii, microsporidia, Taenia solium larvae and Echninococcus
- the specimen can be cultured on non-nutrient agar seeded with Escherichia coli ( look for
amoeba feeding on bacteria).
Tissue and Biopsy Specimens- histological section and impression smear
- Recovery of Leishmania and Toxoplasma gondii; Trypanosoma, Trichinella spiralis,
Acanthamoeba, Naegleria fowleri
- Hepatic Abscess Material - ___________________________
- Muscle Biopsy - _________________________
Sputum
- Identification of Paragonimus westermani, Strongyloides stercoralis larva, microsporidia,
Entamoeba histolytica, Entameoba gingivalis, Ascris lumbricoides larva and Hookworm larva
- Early morning specimen
- Induced sputum is usually for detection of Pneumocystis jirovecii
- ______________ - viscous and tinged with brownish flecks, which are clusters of eggs (‘iron
filings’) streaked with blood.
Urine and Genital Secretions
- Identification of Schistosoma haematobium eggs, Trichomonas vaginalis trophozoite
- Milky Urine - _______________________
Eye Specimens
- Identification of Acanthamoeba by corneal scrapings; Toxoplasma gondii, Loa loa
- It should be kept with moist sterile saline
- 1.) culture on gram negative bacteria 2.) scrapings to glass slide with calcofluor white with
presence of apple green color 3.) histological method
Mouth Scrapings and Nasal Discharge
Mouth Scrapings: Trichomonas tenax and Entameoba gingivalis
Nasal Discharge: Naegleria fowleri
Skin Snips
- Identification of Onchocerva volvulus ( skin fluid)
-1.) firm (scleral) punch into the skin 2.) razor blade into the skin ( placed on 0.2mL saline)
PARASITOLOGY LABORATORY Prepared by: Chester F. Ebersole

Antigen Detection in Parasitic Disease/ Skin Inoculation

Galactose lectin antigen Entamoeba histolytica
Giardia specific antigen 65 Giardia lamblia
HRP2 antigen Plasmodium falciparum
Vivax specific pLDH Plasmodium vivax
200 KD Ag and OG4C3 antigen Wuchereria bancrofti
Witebsky Klingenstien, and Leishmaniasis
Kuhn (WKK) antigen and rk 39 micro elisa test
Sabin Feldman Toxoplasma Gondii
Skin Test
1. Casoni’s test done in Echinococcus granulosus
2. Montenegro test or Leishmanin test – Leishmania donovani
3. Frenkel’s test – Toxoplasma gondii
4. Fairley’s test – Schistosoma spp.
5. Bachman intradermal test – Trichinella spiralis
Culture Media
Amoeba 1. Boeck and Drbohlav’s diphasic medium
(Axenic or Microorganism - egg slant overlaid with sterile serum or liver extract
Culture) 2. Balamuth Monophasic Liquid Medium
3. Escherichia coli culture if specimen is CSF
Leishmania and Trypanosoma 1. NNN ( Nicole Novy and Macneal)
- defibrinated rabbit blood agar
2. Schneider’s insect tissue culture medium
- Drosophila tissue culture
Plasmodium 1. Roswell Park Memorial Institute (RPMI) 1640 medium

Trypanosoma cruzi 1. Xenodiagnosis

- reduviid bug feed on suspected patient.
- In 4-5 weeks, live flagellate is seen on feces of bugs
SUMMARY OF PARASITES MAY BE ISOLATED FROM OTHER SAMPLES

Duodenal Material Giardia lamblia, Microsporidium, Strongyloides stercoralis, Fasciola hepatica

Sigmoidoscopy Entamoeba histolytica, Microsporidia and Trichuris trichiura

Perianal Swab Enterobius vermicularis; Taenia spp.
Blood Leishmania, Trypanosoma, Plasmodium, Babesia and microfilaria
CSF ( sterile sites) Entamoeba histolytica, Trypanosoma, Naegleria fowleri and Acanthamoeba
Toxoplasma gondii, microsporidia, Taenia solium larvae and Echninococcus
Tissue and Biopsy Leishmania and Toxoplasma gondii; Trypanosoma, Trichinella spiralis,
Acanthamoeba, Naegleria fowleri
Sputum Paragonimus westermani, Strongyloides stercoralis larva, microsporidia, Entamoeba
histolytica, Entameoba gingivalis, Ascris lumbricoides larva and Hookworm larva
Urine and Genital Secretions Schistosoma haematobium eggs, Trichomonas vaginalis trophozoite
Eye Specimens
Acanthamoeba by corneal scrapings; Toxoplasma gondii, Loa loa
Mouth Scrapings and Nasal Mouth Scrapings: Trichomonas tenax and Entameoba gingivalis
Discharge Nasal Discharge: Naegleria fowleri

Skin Snips Onchocerva volvulus

FOR EXAMINATION: READ CHAPTER 2 of the REFERENCE BOOK

PARASITOLOGY LABORATORY Prepared by: Chester F. Ebersole

ARTIFACTS AND CONFUSERS – common in stool and blood samples

-due to disease processes, medications, and/or dietary habits.

Artifact Confused for: Illustration

White Blood Cell Entamoeba histolytica cyst
- Average size of 15um ( Neutrophil)
- with 2-4 nuclear lobes Entamoeba histolytica trophozoite
( Macrophage and Monocyte)

Pollen Grains ( Thick Walled) Taenia spp.

- no notable interior structure Unfertilized egg of Ascaris lumbricoides
- spine like structure outside

Vegetable Cell
- large to roundish oval; Helminth eggs
unorganized interior portion
with large vacuole
Vegetable Spiral
- doesnt have head or tail region Helminth Larva
- Ladder like appearance

Yeast Cyst of Entamoeba hartmani

- oval to spherical that exhibits Cyst of Endolimax nana
budding and without internal Oocyst of Cryptosporidium
structure
Plant Hair
-doesn’t have diagnostic structures Helminth Larva

Epithelial Cell
- does not have internal structure Trophozoite of Amoeba
- smooth cytoplasm
- single nucleus with distinct cell wall
Starch Granules
- no internal structure; dark blue- Cyst of Entamoeba hartmani
black color when stained with iodine. Cyst of Endolimax nana

Stain Precipitate
- bluer in color and varies in size Plasmodium
and shape

OTHER ARTIFACTS ( CDC)

Fungal Spore (Stool) Eggs of Hookworms

Giardia lamblia
Chilomastix mesnili
Mite Eggs ( Stool) Hookworm eggs
Elongated and Degenerating Platelet ( Blood) Trypanosoma
Fungal spore of Helicosporium ( Blood) Microfilaria
Seed ( Intestinal Biopsy) Balantidium coli

FOR OTHER PICTURES AND NOTES ( READ CHAPTER 12 of REFERENCE BOOK) – INCLUDED ON EXAM