Maizatul Mohd.

MLT/DP/10-3/0027

1. Microscope
1.1 Introduction
A microscope is an instrument used to see objects that are too small for the
naked eye. Microscope helps the observer to see the magnified image of an
object. The science of investigating small objects using such an instrument is
called as ‘microscopy’. Microscopic means invisible to the eye unless aided by a
microscope.

1.2 Type of Microscope

Generally, there are 3 main types of microscope which are the light
microscope, electron microscope, and X-ray microscope. The common
microscope that is usually used in the laboratory falls under the light microscope
type. Light microscope has a few other types which are the simple, binocular,
phase contrast, interference, polarizing, dark field, ultraviolet, fluorescent and
cinematography microscope. In the other hand, the electron microscope varies
into two types which are the scanning and transmission electron microscope.

1.3 Parts and Functions

There are mainly 3 systems that combine together to form the mechanism of a
microscope which are the supporting system, illumination system and the
magnification system.

A. Supporting System
Parts Functions
Body tube It’s a tube with objective lens at the lower end and eyepiece at the
upper end.
Revolving nose Rotating disc, having holes for fitting the objective lens. It can be
piece rotated to change the power of magnification.
Stage For the placement of slide. Have hole in the center to allow light
to fall on the specimen through the hole.
Arm Holds the body tube and coarse adjustment.
Coarse adjustment Moves the stage up and down rapidly to correct the distance from
the specimen (focal length).
Fine adjustment To obtain the exact focusing.
B. Illumination System
Parts Functions
Illuminator The light source. An electric light provided by a lamp built inside
the microscope beneath the stage.
Reflecting Mirror Reflects the light on the specimen.
Condenser Focuses the reflected light on the specimen.
Iris/Diaphragm Allows the required amount of light to pass through.
C. Magnification System
Parts Functions
Eyepiece/Ocular lens Magnifies the image produced by the objective lens. Allows
observer to look the magnified image through the body tube.
Objective lens Magnifies the specimen (4X, 10X, 40X, 100X)

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Apart from that, there are also some other parts that help the microscope to
function well. The parts and functions are as follows:

Parts Functions
Stage clips Hold the slide
Coxial stage control To adjust the stage left/right and up/down (horizontally)
Brightness control To adjust the brightness of the light
Switch On/off the instrument

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1.4 How to Focus a Microscope?

There are a few simple steps to be followed in focusing a common
microscope. Those steps are as follows:
a) Clean the microscope by muslin cloth or soft cloth.
b) Turn the light source.
c) Focus the microscope by starting with the low power objective lens first.
Bring the lens down as close to the specimen as possible without touching it.
d) Look through the eyepiece and focus upward only until the image is sharp.
e) Once the image is sharp with the low power lens, change to the high power
objective and do fine adjustment with the focus knob.
f) Put one drop of cedar wood oil on the slide and then turn the oil immersion
lens.

1.5 Care of the Microscope

Microscope is unbelievably expensive. Thus this device must be always
handled with serious care. A good quality microscope depends on the care and
maintenance of the device. Below are some of the guidelines on how to care the
microscope:
a) Hold a microscope firmly by the stand, only. Never take it by the eyepiece
holder, for example.
b) Hold the plug (not the cable) when unplugging the illuminator.
c) Since bulbs are expensive and have a limited life, turn the illuminator off
when it is not in used.
d) Always make sure that the stage and lenses are clean before putting away the
microscope. Never use a paper towel, a kimwipe, any shirts, or any other
materials to clean the optical surfaces. Be gentle. Appropriate lens cleaner can
be used or distilled water to help to remove any dried materials. Orgnanic
solvents in the other hand may separate or damage the lens elements or the
coatings.
e) Cover the instrument with a dust jacket when it is not in used.
f) Focus smoothly; do not try to speed through the focusing process or force
anything. For example, if increased resistance is encountered when focusing,
then probably a limit will be reached and may be going in the wrong direction.

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2. Classification of Parasite
2.1 What is a parasite?
When people hear the word ‘parasite’, one of the first ideas or images that
probably comes to mind is that of disease. Though many parasites do carry
diseases, including some extremely deadly ones, "disease-carrying" is not
necessarily a defining characteristic of a parasite. Rather, a parasite can be
identified as any organism that depends on another organism, the host, for food,
shelter, or some other benefit and which receives these benefits in such a way that
the host experiences detrimental effects as a consequence.
Theoretically, organisms from all across the kingdoms of living things can be
characterized as parasites; in practice, however, the realm of organisms studied by
parasitologists is confined to protozoa and various species within the animal
kingdom, mostly worms and arthropods. Included among these organisms are
countless varieties of tapeworm and roundworm as well as a parade of insects that
have plagued humankind since the dawn of time: cockroaches, lice, bedbugs,
flies, fleas, ticks, mites, and mosquitoes.
In other words, a parasite is an organism that spends a significant portion of
its life in or on the living tissue of a host organism and may causes harm to the
host without immediately killing it.

2.2 Types of Parasite

Parasites are mostly eukaryotic and distinct from the fungi. They have no
chlorophyll and a mixed group of organism from protozoa to helminths. There are
a few terms that can be used to describe a parasite based on their behavior. Those
terms are as follows:
a) temporary parasite – visits its host for a short period of time
b) permanent parasite – leads to parasitic life throughout the whole period of its
life
c) facultative parasite – lives a parasitic life when the opportunity arises which
whenever the immunity is low
d) obligatory parasite – cannot exist without a parasitic life
e) occasional/accidental parasite – attacks an unusual host
f) wandering/aberrant parasite – happens to reach a place where it cannot live

Generally, there are 2 main classes of parasite which are the endoparasite and
the ectoparasite.

2.2.1 Endoparasite
Endoparasite or also known as entozoa are organisms that live inside the
body of the host. Protozoa and helminths are the examples of endoparasite.
However, protozoa can be further down into several subgroups as shown in
the diagram below.

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Protozoa

Plasmodroma Ciliophora

Heterotrichida
Rhizopoda Mastigophora Sporozoa
Eg. B. Coli

Protomonodida
Eg. Trichomonas Cocadiida
Amoebida
Hominis, Eg. Plasmodium spp.
Toxoplasma Gondii

Entamoeba Diplomonodida
Eg. Histolitica Eg. Giardia Lamblia

Iodamoeba
Eg. Butschilli

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Protozoa, as shown in the above diagram has a few important subgroups

that divide it into classes according to their shape, features and behavior. They
can also be divided into subgroups of flagellates, ciliates and etc. Protozoa are
a diverse group of single cell eukaryotic organisms, many of which are motile.
Historically protozoa were defined as single cell protists with animal-like
behaviour, e.g. movement. As protozoa are a single celled organism, it has an
organized cellular structure. It may ingest solid particles. Most of protozoan
require aquatic or watery environment to live. They reproduce by binary
fission at some point in their life cycle. Below are some of important protozoa
and the disease it carries.

Entamoeba Histolytica Trichomonas Vaginalis

- causes: diarrhea, dysentry, liver abscess
- transmission: contaminated food/water,
fecal-oral
- diagnosis: stool examination
Tryponosoma spp.
- causes: african sleeping sickness, south
american chagas disease
- transmission: african tse tse fly, south
american reduvid bug
- diagnosis: blood smears, serology
Cryptosporidium Parvum
-causes: diarrhea
- transmission: contaminated water, fecal-
oral
- diagnosis: stool examination
- causes: vaginitis
- transmission: sexual
- diagnosis: microscopy of white
discharge with foul odor
Plasmodium spp.
- causes: malaria
- types:
a) vivax - benign malaria
b) falciparum - malignant malaria
c) ovale - quadrant type malaria
d) malariae - tertiary type malaria
- transmission: female anepheles
mosquitoes bites
- diagnosis: blood smears
Toxoplasma Gondii
- causes: miscarriage in pregnant women,
blindness & retardation in foetus
- transmission: poorly cooked meat, cat
stool, contaminated water
- diagnosis: serology

Helminths are also subdivision of eukaryotic parasites that live inside

the host (endoparasite/entozoa). They are worm-like organisms that live and
feed off living hosts, receiving nourishment and protection while disrupting
their hosts' nutrient absorption, causing weakness and disease. Those that live
inside the digestive tract are called intestinal parasites. They can live inside
humans as well as other animals. Approximately 3 billion people globally are
infected with helminths.

Helminths or worm in the other hand is a multicelluar organism. It has

an organized internal structure. Helminths can be divided into several groups
or classes according to their features. Those classes are shown in the diagram
below as well as the examples and the diseases they are carrying in the
following table.

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Helminths (Worms)

Platyhelminths
(Flat worms)

Cestodes Trematodes
(Tape worms) (Flukes)

Nematodes
(Round worms)

Hook worms

Pin worms

Thread worms

Whip worms

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A. Platyhelminths (Flat worms)

Cestodes (Tape worms) Trematodes (Flukes)
- ribbon like - leaf shaped
- no digestive system
eg
eg - paragonium westermani
- tanea saginata (pork) causes: lung infection, respiratory tract
- tanea solium (beef) infection
- hymenolepisnana (dwarf)
- echinococcus granuloses (dog) - schistosoma spp
- diphyllobothrium latum (fish) causes: inflammation, hematuria (blood in
urine)
(for 1st 2 eg only) transmission: penetration through skin
- causes: abdominal discomfort diagnosis: stool examination, urine analysis
- transmission: larva in uncooked meat
- diagnosis: stool examination (cysts form,
adult segments)
B. Nematodes (Round worms)
- separate sexes
- present in GI tract

eg
- ascaris lumbricoides
causes: abdominal pain, discomfort in the gut
transmission: fecal-oral
diagnosis: stool examination (adult worm)

- wuchereria bancrofti
causes: filariasis, elephantiasis (chronic)
transmission: mosquito bites
diagnosis: blood smear
C. Hook worms D. Pin worms
eg - looks like pin
- ancylostoma duodenale - doesn't affect much, but ↑ number causes
- necator americanus diarrhea
- lay eggs at the anal skin
-causes: blood loss
- transmission: penetration through skin eg
(larval) - enterobium vermicularis
- diagnosis: stool examination (larval)

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E. Thread worms F. Whip worms

eg eg
- dracumculus medinensis - trichuris trichuria

2.2.2 Ectoparasite
Ectoparasite or also known as ectozoa are organisms that live on the
surface of the body of a host organism to the detriment of this host. It does not
live within the body of the host it invades. Ectoparasites attach themselves to
the outer layer of skin of their hosts. There, they feed and thrive for the entire
life cycle of the particular parasite. They are completely dependent on the host
for nourishment, a fact that can seriously impair the general health of the host
over time. There are examples of these types of parasites that actively feed off
humans as well as different types of animals.

It is not unusual for ectoparasites to exist in colonies. These colonies

effectively create nests on the outer layer of skin, creating a breeding ground
where the parasites can multiply. When this happens, the host is weakened at
an accelerated rate, sometimes to the point of incapacitation. Without
treatment, there is the possibility of death at some point.

There are a number of different human ectoparasites. Body and head lice
are two prime examples. Various types of mites are also part of the
ectoparasite family, including nasal mites. Animals may experience these
unwelcome parasites in the form of nest mites or feather mites. Both humans
and animals may be plagued by an ectoparasite infestation that involves fleas
or mosquitoes.

Fortunately, there are ways to exercise ectoparasite control. In the way

of preventive measures, it is a good idea to fumigate interior spaces as soon as
any evidence of mites, lice, and other forms of ectoparasites are found. This is
true whether the parasites are currently nesting on a human or animal host.
Removing the general threat will help to minimize the changes of the parasites
spreading to others living in the household.

At the same time, each individual within the home should be treated to
remove any possible traces of mites or lice from the body. This includes
washing with specialized cleansers that are designed to kill the parasites and
destroy their colonies. Many forms of ectoparasites will thrive in areas of the
skin where body hair is present. This means that the individual must use the
cleanser from head to toe in order to get rid of the pests. Depending on the
severity of the infestation, it may be necessary to undergo several treatments
before the external parasites are removed.

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All types of parasites, including ectoparasites, are capable of doing a

great deal of physical damage over time. In addition to draining the energy of
the host, the pests can also transmit bacteria and viruses to the host. When this
is the case, the host will weaken at a faster rate, making it necessary to address
some sort of bodily ailment as well as get rid of the parasites in order to regain
health.

3. Stool Examination
A stool analysis is a series of tests done on a stool (feces) sample to help diagnose
certain conditions affecting the digestive tract. These conditions can include infection
(such as from parasites, viruses, or bacteria), poor nutrient absorption, or cancer.

Most cysts can be found in the hard formed stool while trophozoites can be
examined in watery liquid stool. This dispersion of stages of parasites can be
explained through the diagram below:

3.1 Type of stool

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An ideal stool is medium brown, the color of plain cardboard. It leaves the
body easily with no straining or discomfort. It should have the consistency of
toothpaste, and be approximately 4 to 8 inches long. Stool should enter the water
smoothly and slowly fall once it reaches the water. There should be little gas or
odor.

However, there are a few type of stools that differ from the normal condition
which is also indicates infection from parasites. Such abnormalities are as
follows:
a) Dry and hard – may due to constipation and dehydration
b) Ribbon like – may due to the inflammation of intestine (colon) and
obstruction of the anus or rectum
c) Mushy – it is an unformed stool and does not flow readily but does not remain
in the bowel cast. This is due to excessive carbohydrate fermentation. Trapped
gas may also be formed.
d) Semi-liquid – this type of stool is slowly flowing and may due to diarrhea or
dysentery
e) Liquid – it is mainly of water and readily flow

Such abnormalities can also be categorized according to the diagram given

below:

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3.2 Stool Collection

Stool should be pure and free from contamination. Hence, stool must be
collected in a dry, clean, leak proof container. There should be no urine, water,
soil or any other material gets in the container. Fresh stool should be examined,
processed and preserved immediately. This will ensure that the specimen is
contamination-free. An exception is made where the specimen need to be kept in
the refrigerator when the preservatives are not available. This situation is
applicable where the specimens are suitable for antigen testing only.

Usually, the specimen is preserved according to the instruction given with the
commercial kit. However, if the kit is not available, the specimen should be
divided and stored in preservatives using a suitable container. The ratio used is
one part of preservatives is added to 3 parts of stool.

It is strictly advised to ensure that the stool is mixed well with the
preservative. Formed and hard stool in the other hand should be broken up into
pieces first. The containers should be sealed well with parafilm, for example, or
any other suitable material to avoid any contamination. Container is then kept in a
plastic bag.

3.3 Unsatisfactory Collection of Stool

In some cases, certain drugs and compounds will somehow render the stool.
Thus, the specimen should be collected before these substances are administered,
or collection must be delayed until after the effects have passed. Such substances
include antacids, kaolin, mineral oil and any other oily materials. Non-absorbable
anti-diarrheal preparations are also affect the stool consistency while barium or
bismuth takes about 7-10 days for clearance of the effect. Antimicrobial agents
usually take about 2-3 weeks for the effect to be cleared and gallbladder dyes
need around 3 weeks.

Hence, specimen collection may need to be repeated if the first examination is

negative or unsatisfactorily. If possible, three specimens passed at intervals of 2-3
days should be examined.

3.4 Preservatives
Preservation is necessary when stool specimens cannot be examined within
the prescribed time interval apart from ensuring the specimen is pure and
contamination-free. The two most commonly used are the 10% aqueous formalin
and polyvinyl alcohol (PVA). A few other preservatives are also used in
preserving the stool. However, there are advantages and disadvantages of using
different preservatives. Table below shows the advantages and disadvantages of
using different types of preservatives:

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Preservative Advantages Disadvantages

10% Formalin - all purpose fixative - not suitable for some permanent smears
- easy to prepare stained with trichrome
- long shelf life - inadequate preservation of morphology
- good preservation of morphology of of protozoan trophozoites
helminths eggs, larvae, protozoan - can interfere with polymerase chain
cysts and coccidia reaction (PCR), especially after extended
- suitable for concentration procedure fixation time
and UV fluorescence microscopy
- suitable for acid-fast, safranin and
chromotrope stains
- compatible with immunoassay kits
Merthiolate- - both fix and stain organisms - not suitable for some permanent smears
iodine- - easy to prepare stained with trichrome
formaldehyde - long shelf life - inadequate preservation of morphology
(MIF) - useful for field surveys of protozoan trophozoites
- suitable for concentration procedures - iodine interferes with other stains and
fluorescence
- iodine may cause distortion of protozoa
Low viscosity - good preservation of morphology of - inadequate preservation of morphology
polyvinyl protozoan trophozoites and cysts of helminth eggs and larvae, coccidia and
alcohol (LPVA) - easy preparation of permanent microsporidia
smears stained with trichrome - contains mercuric chloride
- preserved samples remain stable for - difficult and expensive to dispose
several months - difficult to prepare in the laboratory
- not suitable for concentration procedures
-cannot be used with immunoassay kits
- not suitable for acid-fast, safranin and
chromotrope stains
Sodium acetate - suitable for concentration procedures - requires additive (eg. albumin-glycerin)
acetic acid and preparation of permanent stained for adhesion of specimens to slides
formalin (SAF) smears - permanent stains not as good as PVA or
- easy to prepare Schaudinn’s fixative
- long shelf life
Suitable for acid-fast, safranin, and
chromotrope stains
- compatible with immunoassay kits
Shaudinn’s - good preservation of morphology of - less suitable for concentration
fixative protozoan trophozoites and cysts procedures
- easy preparation of permanent - contains mercuric chloride
stained smears - inadequate preservation of morphology
of helminths eggs and larvae, coccidia and
microsporidia
- poor adhesion of liquid or mucoid
specimens to slides

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3.5 Microscopic Examination of Stool

The unstained wet film is the standard preparation and is made by emulsifying
a small quantity of feces in a drop of saline placed on a slide and applying a
coverslip on top, avoiding air bubbles. A proper preparation should be just dense
enough for newspaper print to be read through it. If the faces contain mucus, it is
advisable to prepare films using the mucus part. Wet saline mount are particularly
useful for detecting live motile trophozoite of E. Histolytica, B. Coli and G.
Lamblia. Eggs of helminth are also readily seen.

Eosin, 1% aqueous solution can be used for staining wet films. Eosin stains
everything expect living protoplasm. Trophozoites and cysts of protozoa as well
as helminth larvae and thin walled eggs stand out as pearly white object against a
pink background and can be easily detected. Chromatoid bodies and nuclei of
amoebic cysts can be seen prominently. Eosin also indicates the viability of cysts,
live cysts are unstained an dead ones stained pink.

Iodine staining of wet mounts is another standard method of examination.

Either Lugol’s iodine diluted 1to 5 or Dobell and O’Connor iodine solution (1g
iodine, 2gm potassium iodide, 100 ml distilled water) is used. Iodine helps to
confirm the identity of cysts, as it stains prominently the glycogen vacuoles and
nuclei.

By following a few simple steps, the presence of parasite that affects the
digestive tract can be detected easily. Materials needed for microscopic
examination of stools are as follows:
a) microscope slides
b) cover slips
c) sodium chloride
d) wooden stick
e) fresh stool
f) gloves

The simple procedures of microscopic examination to detect the presence of

parasite in the stool are as follows:
a) Place a drop of saline solution onto the microscope slide. The slide should be
grease-free clean slide.
b) Take a small amount of stool with a clean wooden stick and mix it with the
saline. Too much stool should be avoided.
c) Place a clean cover slip onto the mixture. Air bubbles must be avoided to
prevent difficulties when observing through the microscope.
d) Observe the slide under 10x and 40x objective lens.

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