Laboratory Manual in Parasitology 2021

Department of Medical Laboratory Science
School of Natural Sciences

Saint Louis University
Laboratory Manual
in
PARASITOLOGY
Arlene A. Mangiduyos, RMT

P R E F A C E
Parasitic diseases are responsible for considerable morbidity and mortality

throughout the world, and often present with nonspecific symptoms and signs.
Most parasitic diseases cannot be diagnosed by physical examination, and
laboratory investigation is necessary to decide whether or not the patient is
infected with a parasite and if so, what species of parasite is present. Thus, the
laboratory plays an important role in establishing the diagnosis of parasitic
diseases and is therefore the key to the selection of appropriate drug for
treatment. Laboratory tests must be accurate and reliable if the results are to
help the physician and benefit the patient.
ABOUT THE MANUAL
This manual has been developed to provide information for a one-semester course
in Parasitology to address the needs of students in the area of Medical Laboratory
Science. This sought to introduce Medical Laboratory Science students to various
medically relevant parasites from different clinical specimens and to assist in
training in the parasite detection and identification.
The laboratory class will provide the students with an opportunity to identify and
study the parasites. As emphasis is placed on examination of stool specimen,
procedures for collection, preservation, and processing of stool specimen are
included. It also contains schematic diagrams illustrating the appearance and
diagnostic features of parasites known to infect humans. The examination of fixed
and stained material is an essential first step to deciphering the morphology of the
parasites. In an effort to fully appreciate the biology of parasites, it is also
important to examine them in their natural states. Thus, periodically, students
shall be required to prepare specimen mounts on the slides for examination.
In order to use the laboratory manual most effectively, it is important that

students read assigned exercises before coming to class and that instructions for
each laboratory activity are meticulously followed.
i
LABORATORY SAFETY
Laboratorians working with stool and other biological specimens face potential
risks including ingestion of eggs or cysts, skin penetration by infective larvae, and
infection by nonparasitic agents found in the specimen. These risks can be
minimized by adopting universal precautions as well as standard good laboratory
practices. These include:
▪ Always wear a laboratory gown when inside the laboratory and in
performance of the exercises.
▪ Do not eat, drink, smoke, apply cosmetics or manipulate contact lenses in
work area.
▪ Contaminated work surfaces shall be decontaminated with an appropriate
disinfectant after completion of procedures; immediately or as soon as
feasible when surfaces are overtly contaminated or after any spill of
potentially infectious materials.
▪ If you have cuts or abrasions on the skin of your hands, cover them with
adhesive dressing.
▪ If you use any sharp instruments, dispose of them in a “sharps” container for
decontamination.
▪ Wash your hands after completing any task involving the handling of fecal
material.
▪ Always wash the hands with disinfectant soap after completing the
laboratory exercises and remove protective gown before leaving the
laboratory.
Note: These precautions should be taken even with stool specimens that have been
fixed in preservatives because they may still be infectious.
ii
TABLE OF CONTENTS
Exercise No. Title Page No.
1 THE COMPOUND MICROSCOPE 1
2 OCULAR MICROMETER 5
3 COLLECTION and PRESERVATION OF STOOL SPECIMEN 8
4 ROUTINE STOOL EXAMINATION 10
5 STOOL CONCENTRATION TECHNIQUES 17
6a PROTOZOA 21
General Characteristics
6b PROTOZOA 23
Phylum Sarcomastigophora – Subphylum Sarcodina
6c PROTOZOA 25
Phylum Sarcomastigophora – Subphylum Mastigophora
6d PROTOZOA 29
Phylum Ciliophora
6e PROTOZOA 31
Phylum Apicomplexa – Class Sporozoea
7a PHYLUM NEMATODA 34
7b PHYLUM NEMATODA 36
Class Adenophorea (Aphasmidia)
7c PHYLUM NEMATODA 39
Class Secernentea (Phasmidia)
7d PHYLUM NEMATODA 47
Filaria Parasites
8a PHYLUM PLATYHELMINTHES 49
Class Cestoidea
8b PHYLUM PLATYHELMINTHES 54
Class Trematoda – Monoecious Flukes
8c PHYLUM PLATYHELMINTHES 59
Class Trematoda – Dioecious Flukes
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Name: __________________________________________ Date: ______________________
Instructor: _______________________________________ Score: _____________________
Exercise No. 1
THE COMPOUND MICROSCOPE
I. Objectives:
After completion of this exercise, the student should be able to:
1. Identify the component parts of a compound microscope, and explain the function of
each.
2. Using correct technique, focus a prepared smear, with both dry and oil immersion
objectives.
3. Demonstrate proper care and storage of the compound microscope.
II. Materials:
Compound Microscope
Prepared smear
III. Activity:
A. Familiarization:
1. First, familiarize yourself with all the parts of a microscope so that you can
easily move from one part to another during operation.
Locate the following component parts of the compound microscope:

Eyepiece/Ocular Mechanical stage
Observation tube Stage control knobs ( X- and Y-axis)
Observation tube clamping knob Main switch
Objective lenses Light source/Illuminator
Revolving nosepiece Light intensity adjustment knob
Coarse focus adjustment knob Filter
Fine focus adjustment knob Arm
Condenser with an iris diaphragm Base
Condenser focus knob
Additional features in a binocular microscope:

Interpupillary adjustment and scale
Diopter ring adjustment
1
2. Label the component parts of the microscope:
The COMPOUND MICROSCOPE

(Olympus CX21)
2
B. Focusing the Microscope:
1. Connect the microscope to the power supply.
2. Turn on the light source and adjust the brightness by rotating the light intensity
adjustment knob.
3. Place the specimen on the stage: open the bow-shaped lever outward, slide
the specimen glass plate from the front toward the rear, and return the bow-
shaped lever gently. Center the specimen over the aperture on the stage.
4. Using the low-power objective

a. Hold and rotate the revolving nosepiece until the 10x objective lens is
“clicked” into position (i.e.,directly above the stage and in the light
path).
b. Rotate the condenser focus knob to bring the condenser down to the
bottom and partially open the iris diaphragm.
c. While looking at the objective lens and the stage from the side, turn the
coarse focus adjustment knob so that the stage moves upward toward
the objectives. Move it as far as it will go without touching the slide.
d. Now, look through the eyepiece and slowly turn the coarse focus
adjustment knob so that the stage moves down (away from the slide).
Continue until the image comes into broad focus. Then, turn the fine
focus adjustment knob, as necessary, for precise focus.
e. Move the microscope slide until the image is in the center of the field of
view. Then readjust the illuminator or diaphragm in order to attain
maximum, comfortable level of light.
.
• When using a monocular microscope, the correct technique is to
look through the eyepiece with one eye and keep the other eye
open.
• When using a binocular microscope adjust the interpupillary

distance and diopter, when necessary.
Adjusting the Interpupillary Distance:

While looking through the eyepieces, move both eyepieces
until the left and the right fields of view coincide completely.
Adjusting the Diopter:

While looking through the right eyepiece with the right eye,
rotate the coarse and fine focus adjustment knobs to bring
the specimen into focus.
Then, while looking through the left eyepiece with the left
eye, rotate the diopter adjustment ring to focus on the
specimen.
Once a clear image is attained under LPO, one should be able to change to
a higher power objective lens with only minimal use of the focusing
adjustment.
3
5. Using the high-power objective
a. Hold and rotate the revolving nosepiece until the 40x objective lens is
“clicked” into position.
b. Rotate the condenser focus knob to bring the condenser half-way up
and open the iris diaphragm to attain the clear image.
c. While looking through the eyepiece, rotate the fine focus adjustment
knob to bring the specimen into precise focus. (DO NOT USE THE
COARSE FOCUS ADJUSTMENT KNOB !)
6. Using the oil-immersion objective:

a. Before engaging the oil-immersion objective in the light path, place a
drop of immersion oil on the specimen at the area to be observed.
b. Hold and rotate the revolving nosepiece in such a direction that the 10x
and 40x objective lenses never come in contact with the oil on the
slide, until the 100x objective lens is “clicked” into position. The oil-
immersion objective lens should dip into the oil slightly.
c. Rotate the condenser focus knob to bring the condenser up and open
the iris diaphragm fully to attain the clearest image.
d. While looking through the eyepiece, rotate the fine focus adjustment
knob to bring the specimen into precise focus. (DO NOT USE THE
COARSE FOCUS ADJUSTMENT KNOB !)
e. After use, remove the oil from the objective front lens by wiping with
lens paper or gauze slightly moistened with 70% alcohol [or
ether(70%)-alcohol(30%) mixture].
7. Steps in putting away the microscope:

a. Rotate the lowest power objective into position.
b. Remove the slide.
c. Clean the microscope surfaces free of dust, debris and/or oil.
d. Coil the power cord around the base of the microscope.
e. Place the microscope back in its proper place in the cabinet.
IV. QUESTIONS FOR RESEARCH:
4
Name: __________________________________________ Date: ______________________
Exercise No. 2
OCULAR MICROMETER
I. Objectives:
1. Determine the width in micrometers of each ocular scale division, when calibrated
against the stage micrometer scale.
2. Calculate the size of an object using an ocular micrometer after calibration for each
set of oculars and objectives.
II. Materials:
Ocular micrometer
Stage micrometer
Compound microscope
III. Activity:
A. Calibration of Ocular Micrometer
Ability to measure accurately the size of parasitic forms is often necessary in

making species identification. This measurement can be made with a calibrated
scale called micrometer. Ocular micrometers are flat glass discs etched with a
fixed scale, usually consisting of 50 or 100 small divisions. These divisions will
have different measurement values depending on the power of the microscope
objective used. The measurement values are calculated using a stage
micrometer etched with a known calibrated scale of 0.1 mm divisions subdivided
into 0.01 mm divisions.
1. Remove the eyepiece (10x or other) from the microscope and unscrew the top or
bottom lens, depending on its construction. Place the ocular scale on the
diaphragm within the eyepiece with the etched surface on the undersurface of
the reticule. Screw back the lens and re-insert the eyepiece into the microscope.
2. Place the stage micrometer on the microscope stage and focus the low-power
objective on some portion of the scale with the 10x eyepiece.
3. Adjust the stage micrometer by moving the stage so that the 0 line of the ocular
micrometer is exactly superimposed on the 0 line of the stage micrometer.
5
4. Without moving the stage micrometer, find another point at the extreme right
where two other lines are exactly superimposed. This second set of
superimposed lines should as far to the right as possible from the 0 lines. This
distance will vary with the objective used. At higher magnifications, the
thickness of the etched lines may be great that you need to look for
superimposition of either the left or right edge of the individual lines.
5. Count the number of division lines on the ocular micrometer between the 0 line
and the point where the second set of lines is superimposed. In the example
provided in the figure, this number, indicated by the dotted line, equals 33 ocular
units.
6. Then count the number of 0.1 mm division lines between the 0 line and the
second superimposed line on the stage micrometer; in the figure, this number,
indicated by the arrowhead, equals 0.22 mm.
7. Calculate the length represented by one ocular unit:
Example: 33 ocular units = 0.22 mm
1 ocular unit = 0.22 mm = 0.0066 mm = 6.6 µm

33
Thus, 1 ocular unit = 6.6 µm for this specific objective.
Each objective on the microscope must be calibrated separately.
8. When all the objective have been calibrated, prepare a simple chart that displays
the calibration factor for each one.
6
B.USE OF THE OCULAR MICROMETER:
1. With the ocular micrometer in place after calibration, focus on the object to be
measured and determine the size in ocular units.
2. Calculate the size of the object by multiplying the ocular units by the calibration
factor for that specific microscope, objective and ocular micrometer.
Example:
A helminth egg was measured using an ocular micrometer in the eye piece of a
compound microscope and its low-power objective.
The helminth egg was 10 ocular micrometer units long.
The calibration factor for that specific micrometer used on the phase scope with
the low-power objective is 6.6 µm
10 ocular micrometer units x 6.6 µm = 66 µm
The helminth egg is 66 µm long.
REMEMBER:
The units of the micrometer disc are arbitrary and a calibration procedure must
be done to determine the calibration factor for each different objective and each
different microscope.
7
Name: __________________________________________ Date: ______________________
Exercise No. 3
COLLECTION and PRESERVATION OF STOOL SPECIMEN
I. Objectives:
1. Describe proper stool specimen collection for the laboratory diagnosis of parasitic
infections with emphasis on quality assurance.
2. Describe various stool preservation methods and cite the advantages and
disadvantages of each.
II. Materials:
Specimen container (a glass jar or plastic cup with tight-fitting lid)
Applicator sticks / scoop
III. Activity:
Stool specimens are examined for the presence of intestinal parasites. Because of the
fragile nature of many intestinal parasites and the need to maintain their morphology for
accurate identification, reliable microscopic diagnosis cannot be made unless the fecal
specimen is collected properly.
1. Give the patient a clean, dry specimen container (a glass jar or plastic cup with tight-
fitting lid and applicator sticks/scoop).
2. Tell the patient to pass the stool specimen directly into the container, or to pass the stool
on a clean pan and use the applicator sticks/scoop to transfer it into the container.
The stool specimen should be large enough for satisfactory examination (i.e., about the
size of one’s thumb for soft or formed specimen, or ½ teaspoon of watery specimen)
The stool specimen must not be mixed with urine and/or toilet water.
3. The container with the specimen should be labeled clearly with the following information:
- Patient’s name
- Date of collection
- Time of collection
4. The specimen must reach the laboratory very soon (i.e., within half an hour) after
passage. (If this is not possible, the specimen must be treated with preservative.)
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Name: __________________________________________ Date: ______________________
Exercise No. 4
ROUTINE STOOL EXAMINATION
I. Objectives:
1. Perform routine stool examination (macroscopic, chemical and microscopic) for the
diagnosis of parasitic infections with emphasis on good laboratory practices and
quality assurance.
2. Record and report results of examination accurately.
II. Materials:
Wooden applicator sticks
Hema-Screen guaiac slide test kit
Glass slides
Coverslips
Pen or marker for indelible labeling
Normal saline solution
1% Lugols’s iodine solution
Cellophane strips (pretreated with glycerine-malachite green solution)
III. Activity:
A. MACROSCOPIC / PHYSICAL EXAMINATION

Stool samples are submitted to the laboratory in the fresh state or as
preserved samples.
a. If the fecal samples are fresh, as soon as the specimen is received in the
laboratory, the color and the consistency (degree of moisture) must be
checked.
Color: light to dark brown

variations in color:
bright red
black / tarry
pale yellow, white, gray, clay / putty
green
9
Consistency:
Formed
Semi-formed
Soft
Watery
b. Observe also for the presence of mucus and blood.
c. If several specimens are received at the same time, those containing

blood and mucus should be examined first followed by the watery
specimens.
These specimens are most likely to contain amoebic trophozoites
and must be examined within one (1) hour after passage. Formed
specimens may be examined at any time during the day (but must
NOT be left overnight).
B. CHEMICAL EXAMINATION
OCCULT BLOOD DETERMINATION

Hema-Screen Guaiac Slide Test
Reagents:
1. Hema-Screen Slides with on-slide monitors
Hema-Screen Slides are made of quality-controlled paper
impregnated with guaiac resin. The positive monitor contains an
impregnated substance which will turn blue if product is
functioning properly. The negative monitor consists of guaiac
impregnated paper.
2. Hema-Screen Developer
Hema-Screen developer contains <6% hydrogen peroxide and
denatured alcohol.
Procedure:
1. Using an applicator stick, pick a small amount of the fecal sample
from the specimen container, then, apply a very thin smear in box 1.
2. Re-use the applicator stick to obtain a second sample from a different
part of the fecal specimen. Apply a very thin smear in box 2.
10
Discard the used applicator stick into a container with disinfectant
[1:20 dilution (5%) of Lysol].
3. Allow the specimen to air-dry, then close the cover.
4. Open the perforated window on the back of the slide.
5. Apply two (2) drops of Hema-Screen developer to the back sides of
boxes 1 and 2.
6. Wait for 30 seconds, and read results within 2 minutes.
Results:
Positive: Any trace of blue on or at the edge of the smear
Negative: No detectable blue on or at the edge of the smear
C. MICROSCOPIC EXAMINATION
PREPARATION OF FECAL SMEARS:
1. DIRECT FECAL SMEAR (Saline and Iodine Wet Mounts)
The saline wet mount is used for the initial microscopic examination of fecal
specimen. It is employed primarily to demonstrate worm eggs, larvae, protozoan
trophozoites, and cysts. This type of mount can also reveal the presence of red
blood cells and white blood cells.
The iodine wet mount is used mainly to stain glycogen and the nuclei of cysts, if
present. Cysts can usually be specifically identified in this mount.
a. With a pen, write the specimen number at the right-hand end of the slide.
b. Place a drop of saline in the center of the left half of the slide and place a
drop of iodine solution in the center of the right half of the slide.
11
c. With an applicator stick, pick up a small portion of the stool specimen (size of
a match head) and mix with the drop of saline. Make a smooth uniform
emulsion in the drop of saline by rotary motion of the applicator stick, starting
from the center going to the periphery.
Note:
Formed stool: Poke at various positions of the stool specimen to obtain a

representative sample from both the surface and within the specimen. Use
applicator sticks to break up the stools as necessary.
Stool with mucus and/or blood: Make sure the applicator stick touches these
portions.
Watery stool: If mucus is not present, pick up a small portion of the stool (any
part).
It is at this step when the specimen is examined macroscopically to

determine color and consistency. Also observe for the presence of adult
worms, proglottids, scolices and other abnormal conditions.
d. Similarly, pick up a small amount of the stool specimen and mix with the drop
of iodine, to prepare an iodine mount.
Discard the used applicator stick into a container with disinfectant [1:20
dilution (5%) of Lysol].
e. Without delay, cover the saline and iodine emulsions with the coverslip. Hold
the coverslip at an angle, touch the edge of the drop, and lower gently onto
the slide. An applicator stick may be used to hold the coverslip while lowering
into the emulsion. This will reduce the chance of including bubbles on the
mounts.
Check the saline and iodine mounts for the following:

• They should be smooth and homogenous without fecal clumps,
vegetable peels or seeds, or other similar materials.
• They should be completely filled with fecal emulsion.
12
• They should be of correct density. One should be able to read small
print through it (but NOT too clearly). If it is too thick or too thin,
observation of the elements in the mount may be difficult.
•
Thickness of the wet mount should be as the image below illustrates.
2. KATO THICK SMEAR
The Kato thick smear has proved to be an efficient means of diagnosis of

intestinal schitosomiasis and intestinal helminths. This technique is NOT suitable
for examining larvae, cysts or eggs from certain intestinal parasites.
a. Place approximately 50-60 mg of stool specimen (the size of 2 monggo

beans) at the center of a glass slide and cover with a piece of cellophane
pretreated with glycerine-malachite green solution. Discard the used
applicator stick into a container with disinfectant [1:20 dilution (5%) of Lysol].
If the fecal specimen is watery, KTS preparation is NOT indicated.
The cellophane strip should be moist (not too wet). The cellophane strip may
be dabbed onto a paper so that the paper absorbs some of the moisture, or
may be allowed to adhere onto the upper inner side of the container to allow
excess fluid to flow down. However, the cellophane should not be allowed to
become overly dry.
b. With the aid of a rubber stopper (about 3/4 inch in diameter) or test tube,
press the cellophane gently in order to spread the stool specimen evenly,
taking care that the specimen does NOT spread beyond the cellophane
cover. The cellophane thus serves as the coverslip.
c. Leave the prepared slide at room temperature. During this time the
microscopic field becomes clear due to the action of glycerine to the fecal
constituents.
d. The slide is examined after 10-20 minutes or within 1 hour after preparation.
Allowing the slides to stand for a long period of time will cause drying and
shells of hookworm ova will become too transparent and will be difficult to
see.
13
EXAMINATION OF STOOL MOUNTS
a. Put the slide with the mounts in the microscope stage and focus with the 10x
LPO.
b. Regulate the light in the microscope field with the substage diaphragm. The
objects in the field must be seen distinctly. Too much or too little light is NOT
good. the condenser should be racked down.
c. Examine the entire coverslip area with the 10x LPO; focus the objective on
the top left-hand corner and move the slide systematically backwards and
forwards, or up and down, and manipulating the fine focus knob from time to
time to observe it in different focal planes.
d. When parasites or suspicious materials are seen, switch to 40x HPO and
increase the light by opening the substage diaphragm to observe detailed
morphology.
To continue scanning, revert back to 10x LPO and regulate the light in the
microscopic field.
At least one-third of the coverslip should be examined with the 40x HPO,
even if nothing suspicious has been seen.
This is systematic examination. If mounts are examined in this way, any

parasites present will be found. If the mount is not examined systematically,
parasites may be missed. Examine each microscopic field carefully, focusing
up and down, before going to the next field..
REMEMBER:
EXAMINE MOUNTS SYSTEMATICALLY.
14
REPORTING OF TEST RESULTS
The following details should be given when reporting results of stool

examinations:
1. Physical Examination
- Consistency (formed, semi-formed, soft, or watery)
- Color
- Abnormal features seen with the naked eye such as, but not
limited to:
flakes of mucus present
gross streaks of blood
blood-stained mucus
2. Chemical Examination
- Occult blood test: Positive or Negative
3. Microscopic Examination
a. If no parasites are found, state:
“NO OVA and/or PARASITES SEEN”, or
“NO INTESTINAL PARASITES SEEN”.
b. Parasites found by microscopic examination specifying:

- Parasite species
- Stage of development
- Quantity reported as average number (range) of parasites
present per field of view (FOV) using low-power
magnification for helminths and high-power magnification
for protozoa.
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Name: __________________________________________ Date: ______________________
Exercise No. 5
STOOL CONCENTRATION TECHNIQUES
I. Objectives:
1. Perform stool concentration techniques for the diagnosis of parasitic infections with
emphasis on good laboratory practices and quality assurance.
2. Record and report results of examination accurately.
II. Materials:
Centrifuge
Centrifuge tubes, 15 ml, conical
Wooden applicator sticks
400 µm mesh sieve or surgical gauze
Glass slides
Coverslips
Funnels
Pasteur pipettes with rubber bulbs
Rubber stopper for centrifuge tubes
Test tube rack
Formalin, 10%
Ether, or ethyl acetate
Zinc sulfate solution, sp. gr. 1.180
Normal saline solution
1% Lugol’s iodine solution
III. Activity
Stool concentration technique allows the detection of small numbers of parasites that
may be missed by using only a direct fecal smear. It is indicated when the direct wet
mount examination is negative despite the clinical symptoms indicating parasitic
infection of a patient. Helminth eggs and larvae, and protozoan cysts may be recovered
by concentration but protozoan trophozoites will not be seen as they are usually
destroyed during the concentration procedure.
16
A. FORMALIN-ETHER CONCENTRATION TECHNIQUE
1. With an applicator stick, add 1.0-1.5 g of feces to 10 ml of formalin in a

centrifuge tube and stir to form a suspension.
2. Strain the fecal suspension through 2 layers of gauze (fitted into a funnel)
directly into a different centrifuge tube. Discard the gauze.
3. Add more 10% formalin to the suspension in the tube to bring the total
volume to 10 ml.
4. Add 3 ml of ether (or ethyl acetate) to the suspension in the tube and mix well
by putting a rubber stopper in the tube and shaking vigorously for 10
seconds.
5. Remove the rubber stopper (to release pent-up aerosol of ether after
shaking) and centrifuge for 2-3 minutes.
6. Remove the tube from the centrifuge: the contents consists of 4 layers: (a)
top layer of ether (or ethyl acetate), (b) a plug of fatty debris that is adherent
to the wall of the tube, (c) a layer of formalin, and (d) sediment.
7. Gently loosen the plug of debris with an applicator stick by a spiral

movement and pour off the top 3 layers in a single movement, allowing the
tube to drain inverted for at least 5 seconds. When done properly a small
amount of residual fluid from the walls of the tube will flow back onto the
sediment. Mix the fluid with the sediment.
17
8. Using a pipette, place a drop of sediment into a glass slide and prepare either
unstained or iodine-stained mount of the concentrated sediment for
microscopic examination.
CAUTION !
Ether is a highly flammable compound and will ignite and explode quickly
if there is a flame or spark nearby.
B. ZINC SULFATE FLOTATION TECHNIQUE
1. Prepare a fecal emulsion by mixing fecal specimen (about half size of a

thumb) and water to produce about 10 ml of suspension.
2. Strain the fecal emulsion through 2 layers of gauze (fitted into a funnel) and
collect into the centrifuge tube. Remove the gauze and discard.
3. Centrifuge the emulsion at 2,000 rpm for 12 minutes.
4. Decant and throw away the supernatant.

Repeat the process several times using water to wash the fecal sediment,
and centrifuge until the supernatant fluid is clear. (Three washings and
centrifugations are usually sufficient.) The last supernatant fluid is poured off.
5. Add about 2 ml of 33% zinc sulfate (sp.gr. 1.180 for fresh stool, 1.20 for
formalin-preserved stool),the sediment is broken up, and add additional zinc
sulfate to fill the tube. Then, centrifuge.
6. With the aid of a flame-sterilized inoculating loop fish out 5 loopfuls of the top
portion of the preparation and transfer into a clean glass slide. Also, make an
iodine-stained preparation.
7. Cover the preparation with coverslip and examine under the microscope.
18
MICROSCOPIC EXAMINATION:
Mounts of concentrated material should be examined in the same way as
described for direct fecal smears. The saline (or unstained) mount should be
examined systematically, looking forhelminth eggs and larvae, and protozoan
cysts. If protozoan cysts, or structures resembling cysts, are seen, you should
examine the iodine mount for more details.
19
Name: __________________________________________ Date: ______________________
Exercise No. 6a
PROTOZOA
I. Objectives:
1. List and describe the parts of the protozoan cell and state the function of each.
2. Outline classification scheme for medically important groups and species of protozoa.
3. Appreciate the public health importance of protozoa.
II. Materials:
Chart
III. Activity:
1. Label the parts of the protozoan cell.
Schematic diagram of a protozoan cell

that has a vesicular type of nucleus.
20
2. Complete the chart below:
Structure Description Function
1. NUCLEUS
• Nuclear membrane
• Nucleoplasm
• Chromatin
2. ENDOPLASM
3. ECTOPLASM
4. CELL MEMBRANE
IV. Questions for Research:
21
Name: __________________________________________ Date: ______________________
Exercise No. 6b
PROTOZOA
Phylum Sarcomastigophora – Subphylum Sarcodina
I. Objectives:
1. Identify and differentiate the various amoebae according to genus and species.
2. Describe laboratory techniques for the diagnosis of amoebic infections with
3. Appreciate the public health importance of the amoebae.
II. Materials:
Compound microscope
Prepared microscopic slides
III. Activity:
1. Using the schematic diagrams in the following pages, study the appearance and
distinguishing morphologic features of the amoebae.
2. Examine microscopically utilizing the 100× oil objective the following prepared
smears; then draw and label the parts.
Trophozoite and cyst stages of:

a. Entamoeba histolytica
b. Entamoeba coli
c. Endolimax nana
d. Iodamoeba butschlii
22
Entamoeba histolytica
A – Trophozoite containing red blood

cells undergoing digestion
B – Precystic amoeba devoid of

cytoplasmic inclusions
C – Immature, uninucleate cyst
D – Binucleate cyst
E – Mature, quadrinucleate cyst
ect – ectoplasm n – nucleus c – chromatoidal bodies r.b.c. – red blood cell

end – endoplasm k – karyosome g – glycogen vacuole
Other Amoebae of Man
Entamoeba Entamoeba Endolimax Iodamoeba

coli gingivalis nana butschlii
Trophozoites
Cysts
No Cyst Stage
ect – ectoplasm n – nucleus f – food vacuoles i – inclusion nuclei

end – endoplasm k – karyosome g – glycogen vacuole
23
Name: __________________________________________ Date: ______________________
Exercise No. 6c
PROTOZOA
Phylum Sarcomastigophora – Subphylum Mastigophora
I. Objectives:
1. Identify and differentiate the various flagellates according to genus and species.
2. Describe laboratory techniques for the diagnosis of infections caused by the various
flagellates with emphasis on good laboratory practices and quality assurance.
3. Appreciate the public health importance of the flagellates.
II. Materials:
Compound microscope
III. Activity:
distinguishing morphologic features of the flagellates.
smears; then, drawn and label the parts.
a. Trophozoite and cyst stages of

• Giardia lamblia
• Chilomastix mesnili
b. Amastigotes of Leishmania donovani
c. Trypomastigotes of Trypanosoma cruzi
24
Giardia lamblia
Trophozoite Cyst
14 x 7 µm 8 -12 µm
2 Sucking discs
Blepharoplasts
2 Nuclei (thin nuclear Thick cyst wall
membrane without granules; Axostyle
centralkaryosome)
2 Flagella (anterior) 2 – 4 nuclei
2 Axostyles Granular
Parabasal body cytoplasm
4 Flagella (midporUon) Remnants of
Blepharoplasts of locomotor
apparatus
2 Flagella (posterior)
Chilomastix mesnili
Trophozoite Cyst
15 x 7 µm 7 – 10 µm
6 Flagella
(3 free anteriorly; 1 in mouth;
2 surrounding mouth)
Blepharoplast Anterior projecUon
Single nucleus Thick, unstained
cyst wall
Cytostome
Cytostome and remains
Spiral groove of locomotor apparatus
Single nucleus
25
Dientamoeba fragilis
Trophozoite
5 – 12 µm
NO CYST STAGE
2 Nuclei
with large cluster of 4 – 8 granules
Highly vacuolated endoplasm

with ingested bacteria, yeast, and other debris
Trichomonasspecies
Trophozoites NO CYST STAGE
Trichomonas vaginalis Pentatrichomonas hominis Trichomonastenax
a = axostyle c.g. = chromaUn granules p.f. = posterior flagellum

a.f. = anterior flagella f.v. = food vacuole p.fib = parabasal fiber
b = blepharoplast i.a.f. = inferior anterior flagellum r = rhizoplasts
c = cytostome k = karyosome u.m. = undulaUng memb.
c.b.r. = chromatoidal basal rod n = nucleus
26
Developmental Forms of Trypanosomatidae
27
Name: __________________________________________ Date: ______________________
Exercise No. 6d
PROTOZOA
Phylum Ciliophora
I. Objectives:
1. Identify and differentiate the ciliates according to genus and species.
2. Describe laboratory techniques for the diagnosis of infections caused by the ciliates
with emphasis on good laboratory practices and quality assurance.
3. Appreciate the public health importance of the ciliates.
II. Materials:
Compound microscope
III. Activity:
1. Using the schematic diagrams in the following page, study the appearance and
distinguishing morphologic features of the trophozoite and cystic stages of
Balantidium coli.
2. Examine microscopically utilizing the 40× high-dry objective the prepared

smear of Balantidium coli trophozoite; then, draw and label the parts.
28
Balantidium coli
Trophozoite
50-100 x 40-70 µm
Cytostome
Food vacuole
Micronucleus
Macronucleus
2 ContracUle vacuoles
Cytopyge Cilia
Cyst
45 – 65 µm
Double wall
Cilia
Macronucleus
29
Name: __________________________________________ Date: ______________________
Exercise No. 6e
PROTOZOA
Phylum Apicomplexa – Class Sporozoea
I. Objectives:
1. Identify and differentiate the various malaria parasites according to genus and
species.
2. Describe laboratory techniques for the diagnosis of malaria infections with
3. Appreciate the public health importance of the malaria parasites.
II. Materials:
Compound microscope
III. Activity:
distinguishing morphologic features of the malaria parasites.
smears; then, draw and label.
a. Plasmodium falciparum (ring-form and gametocyte stages)
in thin and/or thick blood smears
b. Plasmodium vivax (various developmental stages)
c. Plasmodium malariae (various developmental stages)
d. Plasmodium species exo-erythrocytic stages in the liver cells
e. Plasmodium species oocyst in the gut of Anopheles mosquito
30
The Malaria Parasites
Plasmodium Plasmodium Plasmodium Plasmodium
falciparum vivax malariae ovale
Ring Forms
(Early
Trophozoites)
Size 1/5 of rbc 1/3 of rbc Up to 1/3 of rbc 1/3 of rbc
Shape Very delicate ring Delicate ring Compact ring Dense ring
Chroma:n Fine dots, Fine dot, 1 mass Dense,

Frequently 2 Occasionally 2 oden inside ring Well defined mass
Accole’ (Applique) Frequent Occasional None None
Mul:ple infec:on Frequently with high SomeUmes Rare Rare

of rbc parasitemia
Pigment None at this stage None at this stage May be present None at this stage
Trophozoites
Size Small Large Small, but larger Small

relaUve to size of rbc
Shape Compact, Very irregular, Compact, Compact

With cytoplasmic Amoeboid oden band forms
vacuolaUon
Chroma:n Dots or threads Dots or threads Prominent, oden as a Large, irregular

band clumps
Pigment Few coarse, black, Few fine, yellow- Abundant coarse, Few coarse, dark
aggregated in 2 brown, scagered dark brown, scagered yellow-brown,
clumps scagered
Schizonts
(mature)
Size Nearly fills rbc Fills rbc Nearly fills rbc Fills ¾ rbc
Merozoites 12-30 (ave: 24), 12-24 (ave:16), 6-12 (ave; 8), 4-12 (ave; 8),
in compact cluster In irregular cluster In rosege (daisy head)
formaUon
31
Plasmodium Plasmodium vivax Plasmodium Plasmodium
falciparum malariae ovale
cont.
Schizonts
(mature) Aggregated in center Aggregated in center Aggregated in center Aggregated in center
Pigment (black) (yellow-brown) (dark brown} (dark yellow brown)
Gametocytes
Microgametocytes
Size Larger than rbc Fills ¾ rbc Fills 1/2 to 2/3 rbc Fills 1/2 to 2/3 rbc
Shape Crescent-, sausage-, Round or oval, Round, Round,

or banana-shaped, Compact Compact Compact
bluntly rounded ends
Chroma:n Fine granules Single, Single, Single,

scagered throughout Well defined Well defined Well defined
Pigment Dark granules Abundant brown Abundant brown Abundant brown

throughout the granules throughout granules throughout granules throughout
cytoplasm the cytoplasm the cytoplasm the cytoplasm
Macrogametocyte
Size Larger than rbc Fills rbc Smaller than rbc Size of rbc
Shape Crescent-, sausage-, Round or oval, Round, Round,

or banana-shaped, Compact Compact Compact
sharply rounded or
pointed ends
Chroma:n Compact near center Compact peripheral Compact peripheral Compact peripheral
mass mass mass
Pigment Black, rod-like (rice Small masses around Small masses around Small masses around
grain-like) around the periphery periphery periphery
chromaUn
32
Name: __________________________________________ Date: ______________________
Exercise No. 7a
PHYLUM NEMATODA
I. Objectives:
1. List and describe the parts of the nematodes and state the function of each.
2. Outline classification scheme for medically important groups and species of
nematodes.
3. Appreciate the public health importance of nematodes.
II. Materials:
Chart
III. Activity:
distinguishing morphologic features of the nematodes.
2. List and classify the medically important groups and species of nematodes in the table
below.
Class Adenophorea Class Secernentea

(Aphasmidia) (Phasmidia)
33
Phylum Nematoda
General Morphology
Long, cylindrical, unsegmented, generally tapering at each end.
Possesses a body cavity and alimentary tract.
Sexes generally separate
Mouth
CUTICLE- non-nucleated SUBCUTICULAR TISSUE
- generally smooth - with somaUc muscular bands
- may have : sensory papillae in four groups
bosses
spines, or
tubercles
ALIMENTARY SYSTEM EXCRETORY SYSTEM

MOUTH may have: lips MOUTH
papillae
buccal capsule with teeth or CENTRAL EXCRETORY PORE
cunng plates
EXCRETORY CANALS
ESOPHAGUS may be: cellular or muscular in thickenings of
bulbous posteriorly subcutaneous Ussues
ALIMENTARY CANAL - a simple tube NERVOUS SYSTEM

MOUTH
ANUS in RING OF GANGLIA
CLOACA in NERVE TRUNKS (6)

with transverse commisures
REPRODUCTIVE SYSTEM
VULVA UTERUS becomes

coiled, full of ova
VAGINA or larvae filling TESTES
body cavity
UTERI (paired) SEMINAL VESICLE
SPICULE
OVARIES SEMINAL
(usually paired) RECEPTACLE
OVIDUCT PAPILLAE, pre-anal and post-anal,
ANUS OVARY someUmes present
(Basis for species May have
idenUficaUon in some cases) copulatory BURSA
34
Name: __________________________________________ Date: ______________________
Exercise No. 7b
PHYLUM NEMATODA
Class Adenophorea (Aphasmidia)
I. Objectives:
1. Identify and differentiate the various aphasmid nematodes according to genus and
species.
2. Describe laboratory techniques for the diagnosis of the various aphasmid nematodes
3. Appreciate the public health importance of the various aphasmid nematodes.
II. Materials:
Compound microscope
III. Activity:
distinguishing morphologic features of the various aphasmid nematodes.
2. Examine microscopically utilizing the LPO and/or HPO the following prepared slides;
then, draw and label the parts.
a. Trichuris trichiura
• ova
• male adult
• female adult
b. Capillaria philippinensis
• ova
c. Trichinella spiralis
• encysted larva in muscle biopsy
35
Trichuris trichiura
(a) Female Adult. (b) Male Adult (c) Egg.
30 – 45 mm
30 – 45
Barrel-shaped egg
35 - 50 mm Smooth, thick, yellow-brown shell
Unembryonated
Hyaline plug at each pole

(c)
50 x 22 µm
36
Trichinella spiralis
a) Male Adult. (b) Female Adult. (c) Larva.
1.5 x 0.045 mm
3-4 x 0.06 mm
3-4 x 0.06 mm
(c)
90-100 x 6.0 µm
37
Name: __________________________________________ Date: ______________________
Exercise No. 7c
PHYLUM NEMATODA
Class Secernentea (Phasmidia)
I. Objectives:
1. Identify and differentiate the various phasmid nematodes according to genus and
species.
2. Describe laboratory techniques for the diagnosis of the various phasmid nematodes
3. Appreciate the public health importance of the various phasmid nematodes.
II. Materials:
Compound microscope
Formalin-preserved Ascaris lumbricoides adult
III. Activity:
distinguishing morphologic features of the various phasmid nematodes.
2. Examine a formalin-preserved Ascaris lumbricoides adult; then, draw and label.
a. Ascaris lumbricoides
• fertilized ova
• unfertilized ova
b. Enterobius vermicularis
• ova
• male adult
• female adult
c. Hookworm species
• ova
• rhabditiform larva
• filariform larva
• male adult (Ancylostoma)
38
• female adult (Ancylostoma)
d. Strongyloides stercoralis
• rhabditiform larva
• filariform larva
39
Ascaris lumbricoides
(a) Male and Female Adults. (b) Eggs.
200-350 x 4-6 mm
150-200 x 2-4 mm
Broadly ovoid
Golden-brown in color
Unembryonated at oviposiUon
(b) Fertilized Egg Ovoid mass of coarse lecithin granules
Thick-shelled
Inner, non-permeable, lipoidal vitelline membrane
Middle, thick, transparent, glycogen membrane
Outer, coarsely mammillated, albuminous layer
60 x 45 µm
Longer and narrower than ferUlized egg
Completely filled with disorganized, highly

Unfertilized Egg refracUle granules
Thinner shell, and

irregular mammillated, albuminous layer
88-94 x 39-44 µm
40
Enterobius vermicularis
(a) Male Adult. (b) Female Adult. (c) Egg
2-5 x 0.1 mm
8-13 x 0.3-0.5 mm
Thick-walled, colorless shell

Shell flagened on one side
Coiled larva developing in egg
(c)
50-60 x 20-30 µm
41
Hookworm species
(a) Male Adult. (b) Female Adult. (c) Egg.
Ovoidal in shape
Thin, smooth, colorless shell
Two- to eight-cell stage when passed in feces

(c)
65 x 40 µm
42
Hookworm species
Necator Ancylostoma Ancylostoma Ancylostoma

americanus duodenale caninum braziliense
Buccal Capsule
A pair of semilunar Two ventral pairs of Three ventral pairs of Two ventral pairs of
cunng plates. fused teeth. teeth. unfused teeth.
Median teeth.
Copulatory Bursa
Longer than broad. Short and broad. Large, flame-shaped. As broad as long.
Dorsal rays – deep cled Dorsal rays – shallow Rays – long and slender. Rays – stunted.
and Ups biparUte. cled and Ups triparUte.
Two spicules – fused Two spicules – unfused

and barbed. and NOT barbed.
43
Strongyloides stercoralis
(a) Parasitic Female. (b) Free-living Male. (c) Free-living Female.(d) Egg.
Similar to Hookworm egg
Two- to four-cell stage
(d)
55 x 30 µm
44
Rhabditiform Larvae
Hookworm Strongyloides
Filariform Larvae
Hookworm Strongyloides
45
Name: __________________________________________ Date: ______________________
Exercise No. 7d
PHYLUM NEMATODA
Filaria Parasites
I. Objectives:
1. Identify and differentiate the various filaria nematodes according to genus and
species.
2. Describe laboratory techniques for the diagnosis of the various filaria nematodes with
3. Appreciate the public health importance of the various filaria nematodes.
II. Materials:
Compound microscope
III. Activity:
distinguishing morphologic features of the various filaria nematodes.
a. Microfilaria of Wuchereria bancrofti
b. Microfilaria of Brugia malayi
46
Characteristics of Human Microfilariae
47
Name: __________________________________________ Date: ______________________
Exercise No. 8a
PHYLUM PLATYHELMINTHES
Class Cestoidea
I. Objectives:
1. Identify and differentiate the various cestodes according to genus and species.
2. Describe laboratory techniques for the diagnosis of the various cestodes with
3. Appreciate the public health importance of the various cestodes.
II. Materials:
Compound microscope
III. Activity:
distinguishing morphologic features of the various cestodes.
a. Diphyllobothrium latum
• ova
• gravid segment
b. Taenia species ova
c. Taenia solium
• scolex
• Cysticercuscellulosaein muscle biopsy
d. Taenia saginata
• immature segment
• mature segment
• gravid segment
e. Hymenolepis nana
• ova
f. Hymenolepis diminuta
• mature segment
• gravid segment
g. Dipylidium caninum
• ova
• scolex

48
49
50
51
Name: __________________________________________ Date: ______________________
Exercise No. 8b
Class Trematoda – Monoecious Flukes
I. Objectives:
1. Identify and differentiate the various monoecious flukes according to genus and
species.
2. Describe laboratory techniques for the diagnosis of the various monoecious
flukes with emphasis on good laboratory practices and quality assurance.
3. Appreciate the public health importance of the various monoecious flukes.
II. Materials:
Compound microscope
III. Activity:
distinguishing morphologic features of the various monoecious flukes.
a. Fasciola hepatica
• ova
• adult (whole mount)
b. Clonorchis sinensis
• ova
• adult (whole mount)
c. Paragonimus westermani
• ova
52
53
54
55
56
Name: __________________________________________ Date: ______________________
Exercise No. 8c
Class Trematoda - Dioecious Flukes
I. Objectives:
1. Identify and differentiate the various dioecious flukes according to genus and
species.
2. Describe laboratory techniques for the diagnosis of the various dioecious
flukes with emphasis on good laboratory practices and quality assurance.
3. Appreciate the public health importance of the various dioecious flukes.
II. Materials:
Compound microscope
III. Activity:
distinguishing morphologic features of the various dioecious flukes.
a. Schistosoma japonicum
• ova
• miracidium
• cercaria
• male adult
• female adult
• male and female adult in copula
b. Schistosoma mansoni
• ova
c. Schistosoma haematobium
• ova
57
The BLOOD FLUKES
58

Laboratory Manual in Parasitology 2021

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Laboratory Manual in Parasitology 2021

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Department of Medical Laboratory Science

School of Natural Sciences

Arlene A. Mangiduyos, RMT

Parasitic diseases are responsible for considerable morbidity and mortality

ABOUT THE MANUAL

In order to use the laboratory manual most effectively, it is important that

Exercise No. Title Page No.

1 THE COMPOUND MICROSCOPE 1

3 COLLECTION and PRESERVATION OF STOOL SPECIMEN 8

4 ROUTINE STOOL EXAMINATION 10

5 STOOL CONCENTRATION TECHNIQUES 17

THE COMPOUND MICROSCOPE

Locate the following component parts of the compound microscope:

Additional features in a binocular microscope:

The COMPOUND MICROSCOPE

4. Using the low-power objective

• When using a binocular microscope adjust the interpupillary

Adjusting the Interpupillary Distance:

Adjusting the Diopter:

6. Using the oil-immersion objective:

7. Steps in putting away the microscope:

IV. QUESTIONS FOR RESEARCH:

A. Calibration of Ocular Micrometer

Ability to measure accurately the size of parasitic forms is often necessary in

7. Calculate the length represented by one ocular unit:

Example: 33 ocular units = 0.22 mm

1 ocular unit = 0.22 mm = 0.0066 mm = 6.6 µm

Thus, 1 ocular unit = 6.6 µm for this specific objective.

Each objective on the microscope must be calibrated separately.

10 ocular micrometer units x 6.6 µm = 66 µm

The helminth egg is 66 µm long.

IV. QUESTIONS FOR RESEARCH:

COLLECTION and PRESERVATION OF STOOL SPECIMEN

IV. QUESTIONS FOR RESEARCH:

ROUTINE STOOL EXAMINATION

A. MACROSCOPIC / PHYSICAL EXAMINATION

Color: light to dark brown

b. Observe also for the presence of mucus and blood.

c. If several specimens are received at the same time, those containing

OCCULT BLOOD DETERMINATION

PREPARATION OF FECAL SMEARS:

1. DIRECT FECAL SMEAR (Saline and Iodine Wet Mounts)

Formed stool: Poke at various positions of the stool specimen to obtain a

It is at this step when the specimen is examined macroscopically to

Check the saline and iodine mounts for the following:

2. KATO THICK SMEAR

The Kato thick smear has proved to be an efficient means of diagnosis of

a. Place approximately 50-60 mg of stool specimen (the size of 2 monggo

If the fecal specimen is watery, KTS preparation is NOT indicated.

This is systematic examination. If mounts are examined in this way, any

The following details should be given when reporting results of stool

b. Parasites found by microscopic examination specifying:

IV. QUESTIONS FOR RESEARCH:

STOOL CONCENTRATION TECHNIQUES

1. With an applicator stick, add 1.0-1.5 g of feces to 10 ml of formalin in a

7. Gently loosen the plug of debris with an applicator stick by a spiral

B. ZINC SULFATE FLOTATION TECHNIQUE

1. Prepare a fecal emulsion by mixing fecal specimen (about half size of a

3. Centrifuge the emulsion at 2,000 rpm for 12 minutes.

4. Decant and throw away the supernatant.

IV. QUESTIONS FOR RESEARCH:

1. Label the parts of the protozoan cell.

Schematic diagram of a protozoan cell