MLS-409-LAB | CLINICAL PARASITOLOGY – LABORATORY

WEEK 1: Biosafety and Quality Assurance in Parasitology

LECTURER/S: Mr. Christian John Villahermosa, RMT, MSMT
SOURCE/S: Onsite Discussion | Belizario, V.Y., De Leon., W. U. “Medical Parasitology in the Philippines”
3rd ed.
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CLINICAL PARASITOLOGY transmission of biologic agents to workers, other

● This course deals with the study of human parasites persons, and the environment
which are of medical importance especially those
commonly found in the Philippines. ● The laboratory should work together with the
Institutional Biosafety Committee (IBC) Involves:
● Emphasis is given on the epidemiology, ○ Risk assessment
pathogenicity, distribution, life cycle, and laboratory ○ Engineering technology
identification of each parasite. ○ Use of PPE
○ Development of policies
● Preventive measures against infection and control ○ Promotion of safe lab practices and
are also given emphasis. procedures
○ Proper use of containment equipment and
BIOSAFETY IN MEDICAL PARASITOLOGY facilities

● Laboratory workers are exposed to a wide variety of

potential health risks RISK ASSESSMENT
○ Chemicals, radiation, mechanical hazards,
electrical hazards, and biological hazards ● Pathogenicity of the infectious agent
● Biological hazard associated with laboratory ● Route of transmission
infection: MOST hazardous agent in the laboratory ● Stability of biological agents
● Biohazard: potential danger posed by a living or ● Infectious dose or volume
biologically derived material ● Concentration, origin, and processing of the agents
○ Any organism, vectors, biological toxin, ● Presence and availability of effective therapeutic
infectious substance, or any naturally intervention or prophylaxis
occurring, bioengineered, or synthesized
component capable of causing disease,
death, or other biological malfunction BIOSAFETY LEVELS
● Laboratory workers are at risk of
laboratory-acquired infection (LAI) and of
accidentally spreading infectious materials to the
community and environment LVL MICROBE DEFINITION

1 Low Risk Not known to consistently cause

Microbes disease in healthy adult humans,
and of minimal potential hazard to
laboratory personnel and the
environment.

Examples: Saccharomyces
cerevisiae, E. coli K-12, and
non-infectious bacteria

↓
2 Moderate potential hazard to
personnel and the environment.
Includes bacteria and viruses that
cause mild disease to humans, or
are difficult to contract via aerosol
in a lab setting.

Examples: Hepatitis A virus,

Streptococcus pyogenes,
Borrelia burgdorferi (Lyme
disease), Salmonella species

3 High Risk Microbes there can either

Microbes indigenous or exotic, and they can
BIOSAFETY – DEFINITION
cause serious or potentially lethal
disease through respiratory
● Biosafety is the development and implementation of
transmission.
administrative policies, work practices, facility
design, and safety equipment to prevent

Edited/Transcribed by: Astronomo, M. and Budo, R. D. | Page 1 of 3

 performance is performed by an outside
Examples: Yersinia pestis party or agency
(plague), Mycobacterium
tuberculosis, SARS, rabies TYPES OF EXTERNAL QUALITY ASSESSMENT
virus, West Nile Virus,
hantaviruses

4 High Risk Dangerous and exotic, posing a Proficiency → Unknown samples are sent to the
Microbes high risk of aerosol-transmitted Testing laboratory for testing and the results are
infections. Infections caused by compared to the standard
these microbes are frequently → National Quality Assessment
fatal and without treatment or Scheme (NEQAS)
vaccines.
Rechecking → Slides that have been read are
Examples: Ebola virus, or Retesting rechecked or samples that have been
smallpox virus analyzed are retested by the reference
laboratory
RELATION OF RISK GROUPS TO BIOSAFETY LEVELS,
On-site
PRACTICES AND EQUIPMENT
Evaluation

Risk BSL Lab Type Lab Safety

THREE STAGES OF QUALITY ASSURANCE
Group Practices Equipment
● Pre-analytical
1 Basic - Basic GMT None; open ● Analytical
1 teaching, bench work ● Post-analytical
research
ROLE OF THE LAB SUPERVISOR
2 Basic - Primary GMT plus Open bench
2 health protective plus BSC for ● Should ensure that
services, clothing, potential ○ A procedure manual is available
diagnostic biohazard aerosols ○ Records are properly kept
services, sign
○ Controls are available for diagnostic
research
procedures
○ Equipment and instruments are properly
3 Contain Special As Level 2 BSC and/or
ment - 3 diagnostic plus special other primary functioning and calibrated
services, clothing, devices for ○ Clerical and analytical errors are corrected
research controlled all activities ○ Unusual laboratory results (e.g.,
access, uncommon parasites) are checked
directional ○ Standardized procedures are being
airflow followed in the laboratory

4 Maximu Dangerous As Level 3 Class III PROCEDURE MANUAL

m pathogen plus airlock BSC, or
contain units entry, positive
ment shower exit, pressure ● Should contain the following information:
special suits in ○ Instructions for proper collection and
waste conjunction handling of samples
disposal with Class II ○ Information on when to reject samples
BSCs, ○ Preparation of reagents and solutions
double-
ended
○ Detailed description of techniques
autoclave ○ Criteria for identification of parasites
(through the ○ Quality control procedures
wall), filtered ○ Reporting and interpretation of results
air ○ General safety precautions

INSTRUMENTS AND EQUIPMENT

QUALITY ASSURANCE IN A PARASITOLOGY
LABORATORY
● Preventive maintenance must be routinely done
● Quality assurance: continuous improvement in ● Most important instrument in diagnostic
reliability, efficiency, and utilization of laboratory parasitology: MICROSCOPE
services ○ Protect from dust, vibration, and moisture
● How to attain quality assurance? ○ Calibrate using an ocular and stage
○ Internal quality control (IQC): a set of micrometers
procedures is utilized by lab personnel in ○ Availability of stereoscopic microscopes to
the assessment of their laboratory work examine adult worms and worm segments
■ In the Philippines, all clinical ● Centrifuges for concentration techniques
laboratories are required to ● Fume hoods when volatile or toxic chemicals are
provide a quality assurance plan needed
for the procurement of license to
operate
○ External quality assessment: an objective
and periodic assessment of the laboratory

Edited/Transcribed by: Astronomo, M. and Budo, R. D. | Page 2 of 3

 MICROSCOPES

REAGENTS

● Antigens, fixatives, and stains should be checked

prior to use
● All reagents must have proper labels and dates of
preparation and expiration
● Refer to MSDS before using a chemical

CURRENT PROBLEMS

● Lack of political commitment to support the

development and expansion of lab services
● Poor quality of microscopy, especially at the
peripheral level
● Logistic problems and high costs of maintaining
adequate supplies and equipment
● Lack of adequate training and retraining of
laboratory staff
● Lack of quality assurance and supervision of
laboratory services
● People's beliefs

Edited/Transcribed by: Astronomo, M. and Budo, R. D. | Page 3 of 3

 MLS-409-LAB | CLINICAL PARASITOLOGY – LAB
MIDTERMS - LESSON 2: Direct Fecal Smear, Specimen Handling, Fecal Fixatives and Storage
LECTURER/S: Mr. Christian Villahermosa, RMT, MSMT | Mr. Niel John Maravilla, RMT, MLS(ASCP)
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SPECIMEN COLLECTION AND HANDLING

COLLECTION OF STOOL SPECIMENS

● Use clean, dry, wide-mouthed, leak-proof containers
with a screw cap
○ No dirt, urine, or water contamination.

● Bring or send the specimen to the laboratory as

soon as it is voided to prevent deterioration of
protozoans and morphological alterations
○ Note the patient's name/identifier and
date/time of collection on the container FACTORS TO CONSIDER
○ The request form should include the
patient's name, age, sex, date/ time of ● Intake of drugs/medicinal substances
collection, requesting physician, requested ○ Antacids, anti-diarrheals, barium, bismuth,
procedure, presumptive diagnosis, prior and laxatives
infections, and travel history ■ Leave crystalline residues that
can interfere with fecalysis
● Collect freshly passed stool ■ Collection must be done a week
○ 5 ml or 5-6 tbsp if liquid after the last intake or
○ 20-40g if solid (thumb size) administration of any of these
drugs
SPECIMEN REJECTION ● Intake of antibiotics usually decreases the number
of protozoans for several weeks
1. Contaminated with water or urine or collected from
the soil SPECIMEN TRANSPORT
2. With evidence of barium
3. Liquid fecal samples greater than 60 minutes after ● Store at 4-8°C and maintain it in cool temperature
passage before processing or preservation (inside a box with coolant) during transport
4. Formed stool samples greater than 24 hours after
passage before processing or preservation SENDING SPECIMENS TO A REFERENCE LABORATORY

● Preservatives used: 10% formalin (for wet mount

BARIUM ENEMA examination) and polyvinyl alcohol/PVA (for
specimens that require staining)
● Ratio: 1 part stool to 3 parts preservatives

MACROSCOPIC EXAMINATION OF SPECIMENS

● Color: brown, black, yellow, green

● Consistency: formed, soft, or watery (some labs
include 'loose')
○ Note the presence of mucus and blood
streaks
● Prioritize examination of liquid stools especially
those with mucus or blood as they may contain
motile amoeba

CONSISTENCY GUIDE FOR ENTAMOEBA HISTOLYTICA

Edited/Transcribed by: Astronomo, M. | Page 1 of 7

 ● The water sediment is screened daily under a low
magnification for living larvae, which should be
differentiated from those of hookworm.

EXAMINATION OF SPUTUM

PROCESSING OF STOOL SAMPLES ● First morning specimen is the best specimen

○ If the patient cannot expectorate, 10%
sodium chloride or hydrogen peroxide may
be used
WATERY must be examined ideally within 30 ● Examine consistency (serous, mucoid, purulent,
SPECIMENS minutes after passage bloody) and color
● Test using wet mount (saline or iodine for
SOFT must be examined ideally within 1 trophozoites) or concentrate by adding an equal
SPECIMENS hour after passage amount of 3% NaOH, mix, centrifuge, and perform
wet mount on the sediment)
FORMED must be examined within 12 hours ● For the detection of migrating larvae of Ascaris
SPECIMENS after passage lumbricoides, Strongyloides stercoralis, and
hookworms; Paragonimus ova; pulmonary hydatid
cysts of Echinococcus granulosus; and trophozoites
STOOL CULTURE METHODS from pulmonary amebic abscess by Entamoeba
histolytica
1. Copro Culture
● Positive stools are EXAMINATION OF URINE
mixed with
moistened soil or ● First morning urine is the best specimen
granulated charcoal ● May be used to detect Trichomonas vaginalis and
● Harvest larvae Schistosoma haematobium
using the
Baermann EXAMINATION OF TISSUE ASPIRATES
technique
● Liver aspirates for the demonstration of Entamoeba
histolytica trophozoites in amebic liver abscess
cases
○ Aspirate on the margins or the walls of the
abscess, not on the center
● Duodenal aspirates for Giardia lamblia and
Strongyloides stercoralis
○ Intestinal intubation or Entero test/String
test
■ A capsulated yarn is swallowed
by the patient and reach the
duodenum
■ Retrieve after 4 hours and
examine the mucoidal material
clinging to the yarn

AMEBIC LIVER ABSCESS

2. Harada-Mori or Test Tube Culture Method

● Increases the chances of recovering Strongyloides

from samples
● Positive stool is applied to the filter paper and
placed into a test tube with 7 ml of boiled or distilled
water
● The tube is incubated at 24-28°C for up to 10 days.
● Strongyloides rhabditiform larvae migrate to the
water and transform into filariform larvae.

Edited/Transcribed by: Astronomo, M. | Page 2 of 7

 DIRECT FECAL SMEAR

PURPOSE

● The Direct Fecal Smear

(DFS) is a routine
ENTERO TEST OR STRING TEST
method of stool
examination primarily
useful in the detection of
motile protozoan
trophozoite.
● Small amount of fecal
material is added to the
NSS and lodine Solution.

EXAMINATION OF TISSUE ASPIRATES

SPECIMEN
● Cutaneous or skin aspirates
○ Cutaneous ulcerations in leishmaniasis Recommended specimens are FRESH STOOL SPECIMENS
○ Aspirate below the ulcer bed using a sterile and samples that HAS NOT BEEN refrigerated nor
needle preserved.
○ Prepare a smear and stain with Giemsa
stain About 2 mg of stool Amount forming a cone at the tip of an
applicator stick)
EXAMINATION OF CEREBROSPINAL FLUID
IMPORTANCE
● May demonstrate the presence of the
trypomastigotes of Trypanosoma spp. and the - To detect motile trophozoites
trophozoites of Naegleria - To detect ova and cysts present in moderate
● Immediate examination is needed within 20 minutes number
● CSF must be centrifuged at 7,000 g for 10 minutes, - To detect erythrocytes, leukocytes, cellular debris or
discard the supernatant, and examine the sediment excess fat

EXAMINATION OF TISSUE BIOPSY MATERIAL

● Muscle biopsy
○ Useful in the diagnosis of Trichinella
spiralis infection (presence of
encapsulated larvae)
○ Small pieces of muscles are pressed
between two glass slides
● Rectal biopsy
○ Can reveal the presence of deposited
Schistosoma japonicum eggs

DIRECT FECAL SMEARS - DFS

SALINE AND IODINE WET PREPARATIONS

MATERIALS

● applicator sticks

Edited/Transcribed by: Astronomo, M. | Page 3 of 7

 ● microscope glass slide
● cover slips
● 0.85% NSS
● Lugal's lodine Solution
● Pipette
● beaker
● permanent marker

DFS PROCEDURE

1. Wear the prescribed PPE.

2. Prepare the needed materials. Transfer an ample
amount of reagents into separate beakers. Ensure
that the stool sample is fresh and is not refrigerated.
3. On the frozen edge, label the microscope slide with
the patient's name or laboratory code and date
using a permanent marker.

REMEMBER: PROPER PATIENT IDENTIFICATION IS

IMPORTANT BEFORE DOING ANY EXAMINATION!
● MAKE SURE TO WORK ON A FLAT AND STEADY
SURFACE.

4. Using a pipette, add a drop of 0.85% NSS (reagent

A) on the LEFT center-portion and a drop of Lugal's
lodine solution (reagent B) on the RIGHT side of the
slide as shown below.

6. Slowly roof each mixture with a cover slip. Avoid

dropping the cover slip quickly since this will cause
formation of bubbles and spaces which may impede
in the microscopic examination. Leakage of the
solution and/or the mixture should also be avoided.
Do not attempt to wipe any residue on the edge of
the cover slip.

5. With the aid of an applicator stick, amass about 2

mg of the stool and mix it thoroughly with reagent A
until a homogenous mixture is achieved. Using
another applicator stick, repeat the same procedure
but this time, mix the stool sample with reagent B.
Make sure to mix the sample thoroughly to avoid
fecal lumps from forming.

Edited/Transcribed by: Astronomo, M. | Page 4 of 7

7. Examine the DFS preparation under a microscope.
Do not delay since the preparation is prone to
drying.
8. Report your results. PRESERVATION OF FECAL SPECIMENS

Portions of the specimen should be preserved for future

examination and for staining.

Kits containing vials of preservatives are for:

1. Performing direct examinations

2. Concentration procedures
3. Preparation of stained smears

CHARACTERISTICS OF AN IMPERFECT SMEAR

1. Improper patient identification;

2. Mismatched sample and patient; WHO ARE THESE KITS FOR?
3. Insufficient and/or excessive stool sample;
4. Insufficient and/or excessive reagents; These kits are especially helpful for :
5. Incomplete mixing of specimen and reagent; 1. Those patients who are unable to bring in a fresh
6. Presence of fecal debris; sample in timely fashion
7. Bubble formation; 2. Those who will be collecting several specimens
8. Uneven distribution of mixture; over the course of several days.
9. Crooked/ Offset/ Slanted Cover Slips;
10. Leakage of mixture on the edge of the slip and TWO-VIAL TECHNIQUE
slide;
Note: Most laboratories use a
MICROSCOPIC EXAMINATION two-vial technique.

● Aside from parasites, we may also see the With the two-vial technique,
following: one portion of specimen is
○ White blood cells/pus cells: PMNs or fixed in three parts of 5-10%
eosinophils buffered formalin and another
○ Red blood cells portion in three parts of
○ Fat globules polyvinyl alcohol (PVA)
○ Macrophages which may be mistaken for fixative.
amoebic trophozoites
○ Charcot-Leyden crystals 1:3 Specimen to Fixative
○ Epithelial cells from the intestinal tract Ratio
○ Eggs of arthropods, plant nematodes, and
spurious parasites
○ Fungal spores
○ Plant elements: fibers, cells, pollen grains,
starch granules, vegetable spirals

Edited/Transcribed by: Astronomo, M. | Page 5 of 7

 DIFFERENT FIXATIVES USED IN FECAL SPECIMEN
STORAGE procedures and albumin-glycerin) for
preparation of adhesion of
permanent stained specimens to slides
smears 2. Permanent stains not
10% FORMALIN 2. Easy to prepare as good as with PVA
3. Long shelf life or Schaudinn's
ADVANTAGES DISADVANTAGES 4. Suitable for acid-fast, fixative
safranin, and
1. All-purpose fixative 1. Not suitable for some chromotrope stains
2. Easy to prepare permanent smears 5. Compatible with
3. Long shelf life stained with trichrome immunoassay kits
4. Good preservation of 2. Inadequate
morphology of preservation of
helminth eggs, morphology of
larvae, protozoan protozoan
cysts, and. coccidia trophozoites SCHAUDINN’S FIXATIVE
5. Suitable for 3. Can interfere with PC
concentration especially after ADVANTAGES DISADVANTAGES
procedures and UV extended fixation time
fluorescence 4. lodine interferes with 1. Good preservation of 1. Less suitable for
microscopy other stains and morphology of concentration
6. Suitable for acid-fast, fluorescence protozoan trophozoites procedures
safranin, and 5. lodine may cause and cysts 2. Contains mercuric
chromotrope stains distortion of protozoa 2. Easy preparation of chloride
7. Compatible with permanent stained 3. Inadequate
immunoassay kits smears preservation of
and UV fluorescence morphology of
microscopy helminth eggs and
8. Components both fix larvae, coccidia, and
and stain organisms microsporidia
9. Useful for field 4. Poor adhesion of
surveys liquid or mucoid
10. Suitable for specimens to slides
concentration

MODIFIED PVA WITH COPPER OR ZINC

LV-PVA (LOW VISCOSITY POLYVINYL-ALCOHOL)
ADVANTAGES DISADVANTAGES
ADVANTAGES DISADVANTAGES
1. Permanent smears 1. Staining not
1. Good preservation of 1. Inadequate can be made and consistent
morphology of preservation of stained with trichrome 2. Organism
protozoan morphology of 2. Zinc is preferred over morphology may be
trophozoites and helminth eggs and copper poor
cysts larvae, coccidia, and 3. No mercuric chloride 3. Copper-morphology
2. Easy preparation of microsporidia of cysts and
permanent smears 2. Contains mercuric trophozoites is poor
such as stained with chloride 4. Zinc-better
trichrome solution 3. Difficult and morphology but not
both preserves expensive to dispose comparable to
organisms and of LIV-PVA
makes them adhere 4. Difficult to prepare in
to slides) the laboratory
3. Preserved samples 5. Not suitable for
remain stable for concentration ONE-VIAL FIXATIVES (ECOFIX, PARASAFE, UNIFIX,
several months procedures PROTO-FIX, STF, AND OTHERS THAT MAY BE
6. Cannot be used with AVAILABLE)
immunoassay kits
7. Not suitable for ADVANTAGES DISADVANTAGES
acid-fast, safranin
and chromotrope
1. Concentrate and 1. Certain one-vial
stains
permanent smear can fixatives must use
be made out of one certain stains
vial 2. Color difference of
2. Immunoassays can be stain
SAF (SODIUM ACETATE ACETIC ACID-FORMALIN) done on most 3. Staining not always
3. No mercuric chloride consistent
ADVANTAGES DISADVANTAGES 4. Sometimes more
expensive than
1. Suitable for both 1. Requires additive formalin and LV-PVA
concentration (e.g.,

Edited/Transcribed by: Astronomo, M. | Page 6 of 7

WHAT DID WE LEARN? 1. Fold together sticky surfaces of a piece of
Because 10% formalin and PVA have complementary cellophane tape 1×8 cm, for about 1 cm at each
advantages, it is recommended that the specimen be divided end.
and preserved in both types of preservatives. 2. Stretch tape, sticky side out, over butt end of a test
tube or a wooden tongue blade, holding nonsticky
Commercial two-vial kits are available for this purpose. ends firmly with thumb or forefinger.
Preserved specimens can be stored for several months 3. Apply tape to anal area, rocking back and forth to
(Center for Disease Control, 2004). cover as much as the mucosa and mucocutaneous
area as possible.
4. Remove tape and apply to microscope slide, sticky
TAKE NOTE slide down. Press firmly into position.
5. Examine for eggs under low power objective, then
shift to high power objective when Enterobius eggs
Examination of three specimens collected every other day is are suspended.
considered the minimum necessary to perform an adequate
O & P evaluation (Garcia, 2003; Clinical and Laboratory
Standards Institute, 2005).

This procedure ensures an optimum interval for recovery of

those parasites that are known to shed diagnostic forms
intermittently, especially Giardia lamblia and Stronguloides
stercoralis.

Additional sensitivity may be achieved in detecting these

parasites, as well as Entamoeba histolytica, using purgation.
The laboratory must be notified prior to initiation of purgation.

CELLOPHANE TAPE SWAB

● Pinworm infections are acquired by ingestion of the

pinworm ova
● The female pinworm migrates out of the anus to
deposit eggs in the perianal region during the night.
● CTS is essential in diagnosing pinworm infection.

MATERIALS

● Wooden tongue blade

● Cellophane tape (1 x 8 cm)
● Glass slide
● Test tube

PROCEDURE

Edited/Transcribed by: Astronomo, M. | Page 7 of 7

 MLS-409-LAB | CLINICAL PARASITOLOGY – LABORATORY
MIDTERMS - LESSON 3: Kato-Katz Method
LECTURER/S: Mr. Christian Villahermosa, RMT, MSMT | Ms. Lorraine Mission, RMT
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Kato-Katz Method - Introduction DISADVANTAGE OF KATO-KATZ AND KATO THICK

(p.177)
Kato-Katz or Kato Thick-Smear technique for feces is
satisfactory for detecting medium and large helminth eggs in ● Rapid clearance of hookworm eggs after 30-60
feces but is not good for small trematode eggs. minutes with the use of glycerine as a clearing
agent.
● Its chief advantage is that a larger quantity of feces
can be placed on the slides than the usual DFS. KATO-KATZ AND TREMATODE INFECTIONS
This technique is of little value or no value in detecting ● Definitive diagnosis is by detection of eggs in the
protozoa infections. stool using the modified Kato thick method, which
- In cases of amoeba where the mesh is hard to has a higher sensitivity compared to
sieve, it is very disadvantageous. formalin-ether/ethyl acetate concentration technique
(31.0% vs. 13.6%).
● The Kato-Katz Technique is a quantitative method
that employs egg counting to determine the DFS and KATO KATZ
intensity of helminth infection.
○ This technique can be used to assess the ● DFS (DIrect Fecal Smear) is less sensitive
efficacy of anthelminthic drugs in terms of compared to Kato thick Smear and Kato-Katz
cure rate (CR) and egg reduction rate techniques
(ERR). ● A number of intestinal helminths are better
○ This technique can also be used for demonstrated using modified Kato thick smear
epidemiological surveys for the monitoring method than by direct fecal smear.
of a helminth control program.
PURPOSE

SENSITIVITY for Trichuris 91.4% Kato-Katz is a diagnostic technique for the detection of
helminth eggs in stool using a light microscope.
SPECIFICITY for Trichuris 94.4%
● This test is based on the detection of helminth
eggs in small amounts (41.7 mg) of fresh stool.
The Kato-Katz technique is the preferred egg-counting
technique and is considered the most suitable for Kato thick smear method is an alternative diagnostic
quantification of eggs. technique tha uses about 20-60 mg of stool sample.
It is the most commonly used stool examination technique for ● Useful in schistosomiasis surveillance and
evaluating epidemiology, effect of control measures, and Epidemiologic investigations
drug trials.
DEFINITION OF TERMS
The Kato-Katz preparation can be kept for at least 2 weeks
for later examination depending on the workload.
KATO-KATZ
There is practically no loss of eggs during storage and ● Kato-Katz technique facilitates the detection and
processing which makes the technique satisfactory for quantification of helminth eggs that infected
determining fecal egg density. subjects pass in their stool.

Specimens with less than 20 eggs per gram of feces require ● A thick smear is prepared on a microscopic slide
examination of at least three Kato-Katz preparations to have and helminth eggs are enumerated under a light
92% sensitivity. microscope and recorded separately for each
helminth species.

KATO-KATZ AND HOOKWORMS ● Subsequently, the prevalence and intensity of

helminth infections can be determined.
● Kato-Katz method may increase detection rates
since more stools are examined using these - If hookworm takes too long to view, it can be
techniques. damaged or has a distorted appearance.
● The Kato-Katz method may also provide - This explains why it is not for thin shells.
quantitative diagnosis by determining the intensity - Applicable to thick shells only (ascaris,
of infection in terms of the number of helmith eggs trichuris)
per gram of feces.
● Useful in mass stool examinations
● Economical

Edited/Transcribed by: Astronomo, M. | Page 1 of 3

PARASITE INTENSITY

● The number of eggs counted for each

Soil-Transmitted Helminth (STH) species per 1
gram of stool

● A Kato-Katz smear consists of 41.7 mg of stool

CALCULATING THE NUMBER OF EGGS PER 1g OF

STOOL (EPG)

● Multiply the egg count from the slide by a factor

of 24 (24 x 41.7 mg ≈ 1g)
WHY IS MALACHITE GREEN PREFERRED OVER
How is it quantified? METHYLENE BLUE?
- The doctor can diagnose if the patient is heavily
infected with soil-transmitted helminthes (STH). ● In Malachite green, the parasite is much more
visible.
● Malachite green absorbs visible light, while
EPG (EGG PER GRAM) methylene blue absorbs ultraviolet light.

● EPG is the estimated number of eggs per gram of

stool excreted by the examined infected individual. PROCEDURE (BASED ON LAB MANUAL)
STEPS 1-6 LANG INCLUDED SA PPT
KATO KATZ METHOD OR THE CELLOPHANE COVERED
THICK SMEAR (DIAGNOSTIC PARASITOLOGY)
1 Place a small mound of fecal material on
This procedure uses a measured amount of stool which has newspaper or scrap paper, and press the small
been sieved through a wire mesh and pressed under screen on top so that some of the feces are
cellophane paper soaked in glycerine-malachite green sieved through the screen and accumulate on
solution. top.

A uniform amount of stool is examined through the use of a

template with a uniform-sized hole in the middle. All eggs
seen in the whole preparation are counted. The total egg
count is multiplied with a factor depending on the amount of
stool used.

The procedure is useful for assessing the intensity of

infection with Schistosoma and common soil-transmitted
helminths like Ascaris, Trichuris, and hookworm.

Consistency of the stool is the main determinant for the 2 Place a small mound of fecal material on
sensitivity of this technique, since well-formed stools yield newspaper or scrap paper and press the small
higher egg counts than moist ones. The technique can only screen on top so that some of the feces are
be done on fresh formed stools and not on liquid and sieved through the screen and accumulate on top
preserved samples.

For the identification of Schistosoma ova, 1% eosin solution

can be layered over the cellophane paper. This method can
help in the visualization of the miracidium.

MATERIALS AND REAGENTS

● Applicator sticks, wooden

● Screen, stainless steel, nylon or plastic: 60 - 105
mesh 3 Scrape the flat-sided spatula across the upper
● Template, stainless steel plastic, or cardboard; surface of the screen to collect the sieved feces
● spatula, plastic, microscopic slides
● Hydrophilic cellophane, 40-50 um thick
○ Strips 25 x 30 or 25 x 35 mm in size
● Flat-bottom jar with lid
● Forceps

● Toilet paper or absorbent tissue, newspaper

● Glycerol-malachite green;
● or Glycerol methylene blue solution (1mL of 3%
aqueous malachite green or 3% methylene blue is
added to 100mL of distilled water and mixed well.)
4 Place template with hole on the center of a

Edited/Transcribed by: Astronomo, M. | Page 2 of 3

 microscope slide and add fece from the spatula several months but it is preferable in a
so that the hole is completely filled. schistosomiasis-endemic area to examine the
slide preparations within 24 hours.
Using the side of the spatula, pass over the
template to remove excess feces from the edge
of the hole (the spatula and screen may be NOTE
discarded, or if carefully washed, may be reused)
The smear should be examined in a systematic manner and
the number of eggs of each species reported.

Later, multiply by the appropriate number to give the number

of eggs per gram of feces (by 20 if using a 50 mg template;
by 50 for a 20 mg template; and by 24 for a 41.7 mg
template)

5 Remove the template carefully so that the With high egg counts, to maintain a rigorous approach while
cylinder of feces is left on the slide. reducing reading time, the Stoll quantitative dilution
technique with 0.1 mol/liter NaOH may be recommended
6 Cover the fecal material with the pre-soaked (Basic Laboratory Methods in Medical Parasitology, WHO,
cellophane strip (Figure 6-5). The strip must be 1991.)
very wet if the feces are dry and less so if the
feces are soft (if excess glycerol solution is STOLL QUANTITATIVE DILUTION / STOLL EGG COUNT
present on upper surface of cellophane, wipe ● 0.1 mol/L NaOH
with toilet paper). In dry climates, excess glycerol ● diluent
will retard but not prevent drying. ● saponifies fat
○ Lyses fecal debris to release egg
7 Invert the microscope slide and firmly press the
fecal sample against the hydrophilic cellophane This technique makes use of 0.1 N NaOH and a stool
trip on another microscope slide or on smooth displacement flask calibrated at 56 mL and 60 mL.
hard surface such as a piece of tile or flat stone.
The fecal material will be spread evenly between The sodium hydroxide acts as a stool diluent. It saponifies fat
the microscope slide and the cellophane strip and frees eggs from fecal debris. The amount of diluted stool
(Figure 6-6). It should be possible to read used for egg counting is measured by Stoll pipettes
newspaper print through the smear after calibrated at 0.075 mL and 0.15 mL.
clarification (Figure 6-7).
The constant used to multiply the total egg count depends on
the amount of stool examined.

Like the Kato-Katz method, sensitivity is determined by the

consistency of the stool since formed stool can displace
more sodium hydroxide than liquid stool.

Aside from the constant, there may be a need for a

correction factor in computing for the egg count taking into
consideration stool consistency.

RESULTS

Table 7.1. WHO classification of intensity of infections with

soil-transmitted helminths and Schistosoma spp.

Organism Light Moderate Heavy

intensity intensity intensity
8 Carefully remove slide by gently sliding it
sideways to avoid separating the cellophane strip Ascaris 1 - 4,999 5,000 - ≥ 50,000
or lifting it off. Place the slide on the bench with lumbricoides epg 49,999 epg epg
the cellophane upwards. Water evaporates while
glycerol clears the feces.
Trichuris 1 - 999 1,000 - ≥ 10,000
trichiura epg 9,999 epg epg
9 For all except hookworm eggs, keep slide for one
or more hours at ambient temperature to clear
Hookworm 1 - 1,999 2,000 - ≥ 4,000 epg
the fecal material prior to examination under the
epg 3,999 epg
microscope. To speed up clearing and
examination, the slide can be placed in a 40°C
Schistosoma 1 - 99 epg 100 - 399 ≥ 400 epg
incubator or kept in direct sunlight for several
japonicum/ epg
minutes.
Schistosoma
mansoni
10 Ascaris and Trichuris eggs will remain visible and
recognizable for many months in these
preparations. Hookworm eggs clear rapidly and
will no longer be visible after 30-60 minutes.
Schistosome eggs may be recognizable for up to

Edited/Transcribed by: Astronomo, M. | Page 3 of 3

 MLS-409-LAB | CLINICAL PARASITOLOGY – LAB
MIDTERMS - LESSON 4: Formalin-Ether Concentration Technique
LECTURER/S: Mr. Christian Villahermosa, RMT, MSMT
Check out https://bit.ly/masterli_st for the Full Transes Masterlist.
CORRECTIONS? https://bit.ly/correctio_ns

STOOL CONCENTRATION METHODS

2. SEDIMENTATION TECHNIQUES
● Separate parasites from fecal debris and increase
the chances of detecting parasitic organisms when ● Use solutions of lower specific gravity than the
these are in small numbers parasitic organisms, thus concentrating the latter in
the sediment
TWO TYPES OF STOOL CONCENTRATION METHODS ○ Parasites will not float and instead, sink at
1. Sedimentation Techniques the bottom (They are at the
2. Floatation techniques precipitate/sediment)

1. FLOATATION TECHNIQUES ● Easier to perform and less prone to technical errors

● Disadvantage: Appears dirtier compared to
● Use solutions that have higher specific gravity floatation.
than the organisms, so that the organisms rise to ○ But when it comes to the isolation of
the top and the debris sinks to the bottom. parasite, this is the better option.
● ADVANTAGES: produces a cleaner material than
the sedimentation technique EXAMPLES OF SEDIMENTATION TECHNIQUES
● DISADVANTAGES: the walls of eggs and cysts will - FECT (Formalin-Ether Concentration Technique)
often collapse, thus hindering identification + some - Merthiolate-iodine-formalin concentration technique
parasite eggs do not float - Exclusive for Schistosoma

EXAMPLES OF FLOATATION TECHNIQUE FORMALIN-ETHER CONCENTRATION TECHNIQUE

- zinc sulfate and Sheather sugar solution
● If you add zinc sulfate or sugar solution with the
stool sample → centrifuged
○ The fluid should be up to the brim or the
opening of the test tube (Letter B)
- Since chemicals have higher specific gravity than
the parasites, in this test tube (B) the parasites are
found at the chemical.
- We expect the parasites to float

HOW TO COLLECT SAMPLES? (FLOATATION)

- Since the tube is full of zinc sulfate ● Higher sensitivity compared to DFS and Kato-Katz
- Get a cover slip, touch it with the tip of the fluid → ● A sedimentation technique that concentrates
place into the slide → microscope parasitic elements to enhance their recovery
- You should not decant because debris will be left ● Used to separate or recover intestinal helminth
eggs/larvae (nematodes, cestodes, trematodes),
protozoan cysts, coccidian from fecal material
○ Protozoan trophozoites will not be seen
as they are usually destroyed during the
concentration procedure
■ If you are looking for amoeba
trophozoites, DFS is the
recommended procedure.

● Helpful in cases where few or scanty parasites are

present in stool sample (recommended test for food
handlers)

HOW TO COLLECT SAMPLES? (FECT)

A - sedimentation ● Decant and leave the sediment
B - floatation ● Pipette
C - sedimentation ● Put in the slide and place coverslip

Edited/Transcribed by: Astronomo, M. | Page 1 of 2

 FECT PROCEDURE

1. With an applicator stick, add 1.0-1.5 g of feces

(marble size) to 10 mL formalin in a centrifuge tube
and stir to form a suspension

2. Strain the suspension through the 400-micrometer

mesh sieve or 2 layers of wet surgical gauze in a
glass funnel directly into a different centrifuge tube
or a small beaker.

3. Add more 10% formalin to the suspension in the

tube to bring the total volume to 10 mL

4. Add 3 mL ether (or ethyl acetate or gasoline) to the

suspension. Insert screw cap, mix well, and shake
vigorously for 10 seconds

5. Remove the stopper and place the tube in the

centrifuge.

6. Centrifuge for 2-3

minutes.

7. Remove the tube and

observe the formation
of 4 layers
a. Top layer:
ether
b. Second layer:
plug of fatty
debris that adhered to the wall of the tube
c. Third layer: formalin
d. Bottom layer: sediment

8. Gently loosen the second layer with an applicator

stick and pour off the top 3 layers in a single
movement

9. Mix the residual fluid with the sediment (or add a

drop of saline to have sufficient fluid to suspend the
sediment) with a disposable pipette

10. Transfer a drop of the suspension to a slide for

examination under a coverslip.
a. An iodine-stained preparation can also be
made.

Edited/Transcribed by: Astronomo, M. | Page 2 of 2