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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 thenaked 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 amicroscope.

1.2 Type of MicroscopeGenerally, 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 amicroscope 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

piece

Rotating disc, having holes for fitting the objective lens. It can be

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 fromthe 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. Allowsobserver to look the magnified image through the body tube.

Objective lens Magnifies the specimen (4X, 10X, 40X, 100X)


http://en.wikipedia.org/wiki/Laboratory_equipment

http://en.wikipedia.org/wiki/Microscopy

http://en.wikipedia.org/wiki/Microscopic

http://en.wikipedia.org/wiki/Laboratory_equipment

http://en.wikipedia.org/wiki/Microscopy

http://en.wikipedia.org/wiki/Microscopic




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

Parts FunctionsStage 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





1.4 How to Focus a Microscope?

There are a few simple steps to be followed in focusing a commonmicroscope. 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 andmaintenance 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 thecoatings.

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 forceanything. For example, if increased resistance is encountered when focusing,

then probably a limit will be reached and may be going in the wrong direction.





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 notnecessarily 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 thatthe 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 animalkingdom, 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 thehost without immediately killing it.

2.2 Types of Parasite

Parasites are mostly eukaryotic and distinct from the fungi. They have nochlorophyll 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

lifec) facultative parasite – lives a parasitic life when the opportunity arises which

whenever the immunity is low

d) obligatory parasite – cannot exist without a parasitic lifee) 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.










Protozoa

Plasmodroma Ciliophora

Rhizopoda Mastigophora Sporozoa

Amoebida

Protomonodida

Eg. Trichomonas

Hominis,

Toxoplasma Gondii

Diplomonodida

Eg. Giardia Lamblia

Heterotrichida

Eg. B. Coli

Entamoeba

Eg. Histolitica

Iodamoeba

Eg. Butschilli

Cocadiida

Eg. Plasmodium spp.




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 area 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 anorganized 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 protozoaand the disease it carries.

Entamoeba Histolytica Trichomonas Vaginalis

- causes: diarrhea, dysentry, liver abscess

- transmission: contaminated food/water,fecal-oral

- diagnosis: stool examination

- causes: vaginitis

- transmission: sexual- diagnosis: microscopy of white

discharge with foul odor

Tryponosoma spp. Plasmodium spp.- causes: african sleeping sickness, southamerican chagas disease

- transmission: african tse tse fly, south

american reduvid bug

- diagnosis: blood smears, serology

- causes: malaria- types:

a) vivax - benign malaria

b) falciparum - malignant malaria

c) ovale - quadrant type malariad) malariae - tertiary type malaria

- transmission: female anepheles

mosquitoes bites- diagnosis: blood smears

Cryptosporidium Parvum Toxoplasma Gondii

-causes: diarrhea- transmission: contaminated water, fecal-

oral- diagnosis: stool examination

- causes: miscarriage in pregnant women, blindness & retardation in foetus

- transmission: poorly cooked meat, catstool, 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 liveinside the digestive tract are called intestinal parasites. They can live inside

humans as well as other animals. Approximately 3 billion people globally areinfected with helminths.

Helminths or worm in the other hand is a multicelluar organism. It hasan 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 thefollowing table.


http://en.wikipedia.org/wiki/Eukaryotic

http://en.wikipedia.org/wiki/Protist

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http://en.wikipedia.org/wiki/Movement

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http://en.wikipedia.org/wiki/Organism

http://en.wikipedia.org/wiki/Host_(biology)

http://en.wikipedia.org/wiki/Nourishment

http://en.wikipedia.org/wiki/Nutrient

http://en.wikipedia.org/wiki/Disease

http://en.wikipedia.org/wiki/Digestive_tract

http://en.wikipedia.org/wiki/Intestinal_parasite

http://en.wikipedia.org/wiki/Eukaryotic

http://en.wikipedia.org/wiki/Protist

http://en.wikipedia.org/wiki/Animal

http://en.wikipedia.org/wiki/Movement

http://en.wikipedia.org/wiki/Parasitism

http://en.wikipedia.org/wiki/Organism

http://en.wikipedia.org/wiki/Host_(biology)

http://en.wikipedia.org/wiki/Nourishment

http://en.wikipedia.org/wiki/Nutrient

http://en.wikipedia.org/wiki/Disease

http://en.wikipedia.org/wiki/Digestive_tract

http://en.wikipedia.org/wiki/Intestinal_parasite





Helminths (Worms)

Platyhelminths(Flat worms)

Nematodes

(Round worms)

Hook worms

Cestodes

(Tape worms)

Trematodes

(Flukes)

Whip worms

Thread worms

Pin worms




A. Platyhelminths (Flat worms)

Cestodes (Tape worms) Trematodes (Flukes)

- ribbon like

- no digestive system

eg

- tanea saginata (pork)

- tanea solium (beef)- hymenolepisnana (dwarf)

- echinococcus granuloses (dog)

- diphyllobothrium latum (fish)

(for 1st 2 eg only)

- causes: abdominal discomfort

- transmission: larva in uncooked meat- diagnosis: stool examination (cysts form,

adult segments)

- leaf shaped

eg- paragonium westermani

causes: lung infection, respiratory tract

infection

- schistosoma spp

causes: inflammation, hematuria (blood inurine)transmission: penetration through skin

diagnosis: stool examination, urine analysis

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- ancylostoma duodenale- necator americanus

-causes: blood loss- transmission: penetration through skin

(larval)

- diagnosis: stool examination (larval)

- looks like pin- doesn't affect much, but ↑ number causesdiarrhea

- lay eggs at the anal skin

eg

- enterobium vermicularis





E. Thread worms F. Whip worms

eg

- dracumculus medinensis

eg

- 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 tothe 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 hostover 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 colonieseffectively 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. Withouttreatment, 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 humansand 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 anddestroy 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.


http://www.wisegeek.com/what-is-a-parasite.htm

http://www.wisegeek.com/are-there-still-colonies-in-the-world.htm

http://www.wisegeek.com/what-is-head-lice.htm

http://www.wisegeek.com/what-are-mites.htm

http://www.wisegeek.com/what-is-a-cleanser.htm

http://www.wisegeek.com/what-is-a-parasite.htm

http://www.wisegeek.com/are-there-still-colonies-in-the-world.htm

http://www.wisegeek.com/what-is-head-lice.htm

http://www.wisegeek.com/what-are-mites.htm

http://www.wisegeek.com/what-is-a-cleanser.htm




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 thisis 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 diagnosecertain 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 beexamined in watery liquid stool. This dispersion of stages of parasites can be

explained through the diagram below:

3.1 Type of stool


http://www.webmd.com/hw-popup/digestive-tract-15375

http://www.webmd.com/hw-popup/parasite

http://www.webmd.com/hw-popup/viral-infection

http://www.webmd.com/hw-popup/bacterial-infection

http://www.webmd.com/hw-popup/digestive-tract-15375

http://www.webmd.com/hw-popup/parasite

http://www.webmd.com/hw-popup/viral-infection

http://www.webmd.com/hw-popup/bacterial-infection




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 remainin 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

dysenterye) Liquid – it is mainly of water and readily flow

Such abnormalities can also be categorized according to the diagram given

below:





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 inthe 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 2-3days should be examined.

3.4 Preservatives

Preservation is necessary when stool specimens cannot be examined withinthe 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:





Preservative Advantages Disadvantages

10% Formalin - all purpose fixative

- easy to prepare

- long shelf life- good preservation of morphology of

helminths eggs, larvae, protozoancysts and coccidia- suitable for concentration procedure

and UV fluorescence microscopy

- suitable for acid-fast, safranin and

chromotrope stains- compatible with immunoassay kits

- not suitable for some permanent smears

stained with trichrome

- inadequate preservation of morphologyof protozoan trophozoites

- can interfere with polymerase chainreaction (PCR), especially after extendedfixation time

Merthiolate-iodine-

formaldehyde

(MIF)

- both fix and stain organisms- easy to prepare

- long shelf life

- useful for field surveys

- suitable for concentration procedures

- not suitable for some permanent smearsstained with trichrome

- inadequate preservation of morphology

of protozoan trophozoites

- iodine interferes with other stains andfluorescence

- iodine may cause distortion of protozoa

Low viscosity

polyvinyl

alcohol (LPVA)

- good preservation of morphology of

protozoan trophozoites and cysts

- easy preparation of permanentsmears stained with trichrome

- preserved samples remain stable for

several months


of helminth eggs and larvae, coccidia and

microsporidia- contains mercuric chloride

- difficult and expensive to dispose

- difficult to prepare in the laboratory- not suitable for concentration procedures

-cannot be used with immunoassay kits

- not suitable for acid-fast, safranin andchromotrope stains

Sodium acetate

acetic acidformalin (SAF)

- suitable for concentration procedures

and preparation of permanent stainedsmears

- easy to prepare

- long shelf lifeSuitable for acid-fast, safranin, and

chromotrope stains

- compatible with immunoassay kits

- requires additive (eg. albumin-glycerin)

for adhesion of specimens to slides- permanent stains not as good as PVA or

Schaudinn’s fixative

Shaudinn’s

fixative

- good preservation of morphology of

protozoan trophozoites and cysts- easy preparation of permanent

stained smears

- less suitable for concentration

procedures- contains mercuric chloride


of helminths eggs and larvae, coccidia and

microsporidia- poor adhesion of liquid or mucoid

specimens to slides





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 acoverslip 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 particularlyuseful 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 toconfirm 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 thedigestive 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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