SECTION 1: INTRODUCTION TO PARASITOLOGY Flashcards by Arriane Cyrel (MTI)

Organisms may develop unique relationships due to their (?) with one another. These relationships are very important to their survival.

habitual and long associations

How well did you know this?

Not at all

Perfectly

is the living together of unlike organisms

Symbiosis

How well did you know this?

Not at all

Perfectly

may involve protection or other advantages to one or both partners

Symbiosis

How well did you know this?

Not at all

Perfectly

Different forms of symbiosis may be distinguished on the basis of whether or not the association is (?) to one of the two partners.

detrimental

How well did you know this?

Not at all

Perfectly

is a symbiotic relationship in which two species live together and one species benefits from the relationship without harming or benefiting the other.

Commensalism

How well did you know this?

Not at all

Perfectly

is a symbiosis in which two organisms mutually benefit from each other.

Mutualism

How well did you know this?

Not at all

Perfectly

is a symbiotic relationship where one organism, the parasite, live in or on another, depending on the latter for its survival and usually, at the expense of the host.

Parasitism

How well did you know this?

Not at all

Perfectly

Majority of animal parasites are (?) which are harmful and which frequently cause mechanical injury to their hosts.

pathogens

How well did you know this?

Not at all

Perfectly

A (?) harbors a particular pathogen without manifesting any signs and symptoms.

carrier

How well did you know this?

Not at all

Perfectly

is the process of inoculating and infective agent

Exposure

How well did you know this?

Not at all

Perfectly

connotes the establishment of the infective agent in the host

infection

How well did you know this?

Not at all

Perfectly

is the period between infection and evidence of symptoms. It is sometimes referred to as the clinical incubation period.

incubation period

How well did you know this?

Not at all

Perfectly

also known as the biologic incubation period

pre-patent period

How well did you know this?

Not at all

Perfectly

is the period between infection or acquisition of the parasite and evidence of demonstration of infection

pre-patent period

How well did you know this?

Not at all

Perfectly

results when an infected individual becomes his own direct source of infection.

Autoinfection

How well did you know this?

Not at all

Perfectly

happens when the already infected individual is further infected with the same species leading to massive infection with the parasite

superinfection or hyper infection

How well did you know this?

Not at all

Perfectly

There are various sources of parasitic infections. The most common sources are

contaminated soil and water

How well did you know this?

Not at all

Perfectly

Lack of (?) and the use of (?) or human excreta as fertilizer allow the eggs to get in contact with the soil.

sanitary toilets
night soil

How well did you know this?

Not at all

Perfectly

Another possible source of infection is (?), which contain the infective stage of the parasite.

water and food

How well did you know this?

Not at all

Perfectly

Consumption of (?) can result in several intestinal and liver fluke infections.

undercooked or raw fresh water fish

How well did you know this?

Not at all

Perfectly

can also transmit infection

Arthropods

How well did you know this?

Not at all

Perfectly

are vectors of malaria and filaria parasites

Mosquitoes

How well did you know this?

Not at all

Perfectly

Other animals, whether (?), may also harbor the parasite.

wild or domesticated

How well did you know this?

Not at all

Perfectly

Other sources include (?).

another person, his beddings and clothing, the immediate environment he has contaminated, or even one’s self

How well did you know this?

Not at all

Perfectly

An (?) may be transmitted from its natural reservoir to a susceptible host in different ways.

infectious agent

There are different classifications for modes of transmission:

1. Direct 2. Indirect

contact w/ an infected person or animal, directly from the source to the susceptible host without involving an intermediate object

Direct

a) Droplet spread

Direct

b) Sexual intercourse

Direct

c) Kissing

Direct

d) Holding hands

Direct

e) Transplacental / Vertical - mother to fetus

Direct

a) Ingestion of contaminated food & drink

Indirect

b) Contact w/ contaminated soil

Indirect

c) Bite of an infected arthropod (vector)

Indirect

d) Through fomites

Indirect

Since the most common source of parasitic infection is contaminated food and water, the most likely portal of entry is the (?).

mouth

Majority of infections among cestodes, trematodes, and intestinal protozoans are

foodborne

from eating food harboring the infective larval stages

Taenia solium, Taenia saginata, and Diphyllobothrium latum

from drinking water contaminated with cysts

Entamoeba histolytica and Giradia lamblia

through ingesting raw or improperly cooked freshwater fish containing the parasitic larvae

Clonorchis, Opistorchis and Haplorchis

is another route of transmission

Skin penetration

enter via exposure of skin to soil

Hookworms and Strongyloides

species enter skin via water.

Schistosoma

also serve as vectors and transmit parasites through their bites

Arthropods

Examples are agents of malaria, filariasis, leishmaniasis and trypanosomiasis.

Arthropods

can cross the placental barrier during pregnancy

Toxoplasma gondii trophozoites

In transmammary infection with (?), the parasites may be transmitted through the mother’s milk.

Ancylostoma and Strongyloides

Other ways of acquiring the infection include inhalation of airborne eggs of (?), and sexual intercourse as in the case of (?).

Enterobius Trichomonas vaginalis

Although parasitic life cycles range from simple to complex, they all have three common components

mode of transmission a morphologic form one (or more) forms

invades humans, known as the infective stage

a morphologic form

can be detected via laboratory retrieval methods, known as the diagnostic stage

one (or more) forms

Some parasites require only a (?), whereas others also require one or more (?).

definitive host intermediate hosts

A parasitic life cycle consists of two common phases

One phase involves the route a parasite follows when (?).

in or on the human body

This information provides an understanding of the symptomatology and pathology of the parasite.

in or on the human body

Insights about the best the method of diagnosis and selection of appropriate antiparasitic medication may also be determined.

in or on the human body

The other phase, the route a parasite follows (?), provides crucial information pertinent to epidemiology, prevention, and control.

independently of the human body

A (?) may affect the entire body or body areas associated any of its parts.

parasitic disease

The major with such processes include the following:

(1) the gastrointestinal (GI) and urogenital (UG) tracts; other (2) blood and tissue; (3) liver, lung, and major organs; and (4) miscellaneous locations, eye, skin, and extremities

A wide variety of representative (?), may occur when a parasite infects a human host.

symptoms

Some persons remain (?), whereas other parasites produce severe symptoms and may result in death.

asymptomatic

The most commonly observed symptoms include (?).

diarrhea, fever, chills, abdominal pain, and abdominal cramping

Other symptoms, such as (?), may develop.

elephantiasis, anemia, vitamin deficiency, bowel obstruction, edema, enlargement of major organs, skin lesions, and blindness

an enlargement of areas such as the breast, leg, and scrotum caused by a parasite’s presence

elephantiasis

may be taken against every parasite infective to humans.

Prevention and control measures

designed to break the transmission cycle are crucial for successful parasite eradication.

Preventive measures

Examples of such measures include the following: (?).

education programs, use of insecticides and other chemicals, protective clothing, protective netting, proper water treatment, good personal hygiene, proper sanitation practices, proper handling and preparation of food, and avoidance of unprotected sexual relations

The vast capital expenditures required to accomplish these measures are not available in many endemic countries in the world. The problem of eradicating parasites is an ongoing process and is a key goal of international health groups such as the

World Health Organization (WHO)

The following are the specimens needed to properly diagnose the presence of parasites inside the human body.

1.Stool 2.Urine 3.Sputum 4.Blood 5. Cerebrospinal fluid 6. Tissue aspirates 7. Orifice swab 8. Tissue Biopsy

- for intestinal protozoans, nematodes and helminthes

Stool

- for the recovery of Trichomonas vaginalis and Schistosoma haematobium

Urine

Urine Collection:

mid-stream catch

- Paragonimus westermani, larvae of nematodes

Sputum

Sputum Must be digested using

4-5% sodium hydroxide

- for malarial parasites, filarial worms, Leishmania and Trypanosoma

Blood

- Acanthamoeba species

Cerebrospinal fluid

Cerebrospinal fluid Collection:

lumbar tap

Tissue aspirates:

a) Liver aspirate b) Duodenal aspirate c) Broncho-alveolar lavage

hydatid cyst and liver amoebic abscess

a) Liver aspirate

Giardiasis and Strongyloidiasis infection

b) Duodenal aspirate

Paragonimus westermani

c) Broncho-alveolar lavage

Duodenal aspirate Collection:

endoscopy

: duodenal contents collected for Giardia and Strongylodes

Duodenal drainage or “String test”

: Schistosomiasis, Amoebiasis, Balantidiasis and Shigellosis (Large intestines)

Sigmoidoscopy

Orifice swab

a) Vaginal swab b) Perianal swab

Trichomonas vaginalis

a) Vaginal swab

Enterobius vermicularis and Taenia

b) Perianal swab

Tissue Biopsy

a) Muscle b) Rectal

Trichinella spiralis

a) Muscle

granulomas secondary to Schistosomiasis

b) Rectal

There are parasites where the method of isolation and identification is through processing of (?).

blood

❖ Blood Films

1. Fresh water smears 2. Thin Dry smears 3. Thick Dry smears

for diagnosis of Trypanosomes and microfilaria

1. Fresh water smears

for the study of the morphology of the parasites and the blood cells

2. Thin Dry smears

used for malaria survey among patients with chronic infections or who are undergoing anti-malaria therapy

3. Thick Dry smears

❖ Stains

1. Giemsa 2. Rapid stains 3. Permanent stains & Other stains

Rapid stains:

a) Wright’s stain b) Leishmann stain c) Field’s stain d) Acridine orange e) Jaswant Singh Battacharya (JSB) Stain for thick and thin films

Permanent stains & Other stains:

a) Iron Hematoxylin Stain b) MIF Fixative Stain (Merthiolate iodine formaldehyde) c) Chlorazol Black E d) Modified Kohn’s e) Wheatley Trichrome f) Methenamine Silver g) Fluorescent Staining

most preferred

Giemsa

Giemsa Composition

Stock solution 1 ml Buffered water (pH 7.0 – 7.2) 49 ml

Giemsa Staining time:

30 minutes

Too dark :

acidic pH

Too red:

alkaline pH

– It is used to stain blood smears in the detection of blood parasites.

Wright’s stain

The stain distinguishes easily between blood cells and became widely used for performing differential white blood cell counts, which are routinely ordered when infections are expected.

Wright’s stain

The stain contains a fixative, methanol, and the stain in one solution.

Wright’s stain

Thin films of blood are fixed with methanol to preserve the red cell morphology so that the relationship between parasites to the red cells can be seen clearly.

Wright’s stain

Wright’s stain ✓ Fix with 1-2 drops of (?) ✓ Cover the film with (?): 5 minutes ✓ wash with (?), drain, dry and examine

methanol 10% Giemsa stain distilled water

It is a mixture of Methylene blue, and Eosin dye, prepared in Alcohol medium and diluted with buffer or distilled water during staining procedure.

Leishmann stain

is a differential stain that is used to variably stain the various components of the cells and it can be used to study the adherence of pathogenic bacteria to the human cells

Leishmann stain

It differentially stains the human and bacterial cells and appeared as purple and pink colored bodies respectively.

Leishmann stain

is one of the best stains for routine blood stain to stain the Peripheral blood smear for the examinations of blood film under the microscope and is satisfactory for malaria and other blood parasites

Leishmann stain

Leishmann stain ✓ Add (?) of the stain: (?) ✓ Add (?) of buffered distilled water ✓ Mix thoroughly, let stand (?) ✓ Rinse, drain, dry and examine

7-8 drops; 1-2 minutes 12-15 drops 4-8 minutes

It is a histological method for staining of blood smears.

Field’s stain

It is used for staining thick blood films in order to discover malarial parasites.

Field’s stain

Field's stain consists of -

two parts

Field's stain A is

methylene blue and Azure 1 dissolved in phosphate buffer solution

Field's stain B is

Eosin Y in buffer solution.

is used as a fluorescent staining agent to detect the presence of malaria parasite in blood cultures and other bodily fluids

Acridine orange

is a fluorochrome dye that can interchalate into nucleic acid.

Acridine orange

standard method

Jaswant Singh Battacharya (JSB) Stain for thick and thin films

laboratories under the National Malaria Eradication Programme in India

Jaswant Singh Battacharya (JSB) Stain for thick and thin films

- used for most of the original morphological descriptions of intestinal protozoa found in humans

Iron Hematoxylin Stain

- diagnosis of Trichomonas

MIF Fixative Stain (Merthiolate iodine formaldehyde)

- An acid dye, used as a fat and general tissue stain, and to stain protozoa in fecal smears or in tissues.

Chlorazol Black E

- modification of the chlorazol black E staining technique

Modified Kohn’s

- all-purpose (amoebae, flagellates)

Wheatley Trichrome

- cyst

Methenamine Silver

- Microsporidium

Fluorescent Staining

Infected erythrocytes are counted in relation to a predetermined number of WBCs

Thick Blood Film

Standard: average of 8000/µl

Thick Blood Film

All parasite species and forms (sexual and asexual) are counted

Thick Blood Film

Thick Blood Film Formula:

If parasites are ≥10, 200 leukocytes are counted:

# of parasites/µl =# of parasites counted x 40

If parasites are <10, 500 WBCs should be counted:

# of parasites/µl = # of parasites counted x 16

Earle and Perez method

# of asexual parasites/ 5µl

thick film is used

Thick Blood Film

used only in research studies

Thick Blood Film

% of parasitemia: P. falciparum

Thin Blood Film

# of infected red cells in 1000 RBCs

Thin Blood Film

smear is scanned carefully, one 'row' at a time

Thin Blood Film

total number of red cells and the number of parasitized red cells are tabulated separately

Thin Blood Film

If 1000 red cells are counted:

• divide the number of parasitized red cells by 10 to get the percentage • less than 1000 red cells counted • # of parasitized cells/# of RBCs X 100

less precise

"plus system"

variation in the thickness of the film

"plus system"

results in variation in parasite count

"plus system"

= 1–10 per 100 thick fields

= 11-100 per 100 thick fields

= 1–10 per thick field

+++

= >10 per thick field

++++

(?) of fecal sample is essential for the isolation and proper diagnosis of infection.

Proper collection

The following should be considered:

1. Container 2. Avoid contamination with urine, water and soil 3. Label 4. Handle carefully

Container (for physical examination of the specimen)

sterile, disposable, wide mouth with tight-fitting lid, transparent

Handle carefully because it is a potential source of infection unsuitable samples from patients receiving:

a. Barium b. Oil c. Bismuth d. Kaolin e. Antibiotics

TYPES of FECAL SPECIMEN

1. Liquid 2. Soft 3. Formed

Liquid –

either diarrheic or saline-purged

PRESERVATION a) Physical:

1. Room temperature 2. Refrigeration

PRESERVATION b) Chemical

1. Polyvinyl Alcohol (PVA) 2. 5-10% formalin 3. Merthiolate-Iodine-Formalin (MIF) or Thimerosal Iodine Formalin (TIF) 4. Schaudinn’s Fixative

used for the preservation of stained fecal+ smears; preservation of trophozoites

Polyvinyl Alcohol (PVA)

▪ For concentration techniques or direct fecal smear

5-10% formalin

▪ For cysts, helminthes and larvae

5-10% formalin

▪ Not sufficient for preparing permanent stained fecal smears

5-10% formalin

▪ Good for all stages

Merthiolate-Iodine-Formalin (MIF) or Thimerosal Iodine Formalin (TIF)

▪ For liquid stools

Merthiolate-Iodine-Formalin (MIF) or Thimerosal Iodine Formalin (TIF)

▪ For bulk feces, the solution is mixed in the proportion of 9.4 ml MIF and 0.6 ml Lugol’s for a gram of fecal material

Merthiolate-Iodine-Formalin (MIF) or Thimerosal Iodine Formalin (TIF)

▪ For fresh materials and those recovered from intestinal mucosal linings

Schaudinn’s Fixative

▪ For permanent staining

Schaudinn’s Fixative

IMPORTANCE OF FECALYSIS

1. To detect the presence of intestinal parasites 2. For the detection of evidence of malfunction of some parts of the GIT, liver and pancreas 3. For the detection of GIT bleeding 4. For the detection of excessive fats in stool (steatorrhea) 5. Used as a clue in medical and surgical diagnosis.

1. Physical Examination ❖ Form and Consistency: Normal:

soft to formed

– seen in diarrhea and administration of saline cathartic

a) Very soft and watery

– constipation due to lack of mucus

b) Excessively hard and scybalous

– cholera

c) Rice water stool

– early typhoid

d) Pea soup stool

– syphilis, spastic colitis and obstruction at the lower portion of the colon

e) Flattened or ribbon like

- fibrocystic disease of the pancreas

f) Butter like

– excessive carbohydrate fermentation

g) Gaseous and fermentative

Color Normal:

light brown to dark brown due to stercobilin

- due to administration of santonin and senna

a) Yellow

– seen after ingestion of Barium meals

b) Light clay or putty color

- bleeding in the lower GIT; undigested beets and tomatoes

c) Reddish or bloody

- bleeding in the upper GIT

d) Dark red/chocolate brown

- associated with digestion of blood due to bleeding in the upper GIT

e) Black/tarry

– amoebiasis

f) Greenish

Odor Normal:

foul to offensive due to indole, skatole and butyric acid

– found in ulcerative and malignant tumor of the lower bowel

a) Putrid odor

- indicates gas formation, fermentation of carbohydrate, unabsorbed fatty acids

b) Sour/rancid odor

– usually in alkaline stools, putrefaction of undigested protein.

c) Extremely foul odor

– blood which is not visible to the naked eye and can only be detected by chemical means only

Occult blood

Laboratory Examination for Occult Blood:

a) Benzidine test b) Guaiac;s test c) Hematest

The (?) (due to peroxidase contained in the heme portion of hemoglobin) decomposes (?) to produce (?).

peroxidase-like activity of blood H2O2 water and oxygen

The (?) causes the oxidation of the colorless chromogen into a colored compound (blue)

liberation of oxygen

Hematest Patient Preparation:

meat free diet for 3-5 days prior to the test

The following structures may be seen microscopically:

a) Trophozoites and cysts of amoeba b) Helminth eggs and larvae c) RBC d) Macrophages present in bacterial or parasitic infection e) WBC f) Fungi g) Plant cells, pollen grain and spores h) Epithelial cells i) Calcium oxalate and triple phosphate crystals j) Bacteria k) Plant fibers, root hairs and animal cells ( similar to helminth ova) l) Charcot-layden crystals

indicative of inflammation

WBC

- due to hemorrhagic disorder, ulcers and contamination

RBC

TECHNIQUES

– simplest and most frequently used

DIRECT FECAL SMEAR

- for the observation of the motility of trophozoites

a) 0.85% sodium chloride/NSS

– for protozoan cysts and helminth ova

b) D’antonis solution/Lugol’s Iodine

KATO THICK SMEAR (KTS) ▪ Reagents:

Distilled water 100 ml Glycerin 100 ml 3% malachite green 1 ml

Use cellophane as cover slip

KATO THICK SMEAR (KTS)

- used in the detection of a small number of parasites which are not detected using DFS

CONCENTRATION TECHNIQUES

– uses either specific gravity or centrifugation

Sedimentation Method

– concentrates helminth eggs, larvae and protozoan cysts

Formalin-Ether techniques

– used for formed stools; applicable for helminth eggs and larvae

Acid-Ether Sedimentation

– time-consuming

Simple sedimentation

- for concentration of microfilariae’ utilizes venous blood as specimen

Knott Concentration Technique

Allows the separation of helminth eggs, protozoan cysts and larvae (1.05 – 1.15) using chemical solutions of higher specific gravity (1.12-1.21)

Flotation method

Eggs and cysts float to the surface of the solution while fecal materials sink to the bottom

Flotation method

Superior to sedimentation for concentrating cysts and eggs other than operculated, schistosomal and infertile Ascaris eggs

Flotation method

- valuable method for concentrating cysts and eggs

Zinc Sulfate centrifugal flotation technique

- specific gravity: 1.18 (hydrometer); 1:20 formalinized feces

Zinc Sulfate centrifugal flotation technique

- Cryptosporidium (rounded oocysts)

Sugar Flotation Technique/Sheather’s Sugar Flotation

- uses sodium chloride solution

Wilie’s Brine

Lane’s Direct Centrifugal Technique Magnesium Sulfate Techniques

• D’Antonis

Temporary Staining

1% Isotonic Eosin Solution & 2% Brilliant Cresyl Blue

Temporary Staining

Eosine in saline

Temporary Staining

Buffered Methylene Blue (ex. Nairs Methylene Blue)

Temporary Staining

1% Isotonic Eosin Solution & 2% Brilliant Cresyl Blue - Trophozoites- - Background-

blue green pink

Permanent Staining

Gomori’s Trichrome Stain Lawless’ Permanent Mount Stain Modified Acid-Fast stain Iron Hematoxylin Stain MIF Fixative stain ( Merthiolate iodine formaldehyde) Chlorazol Black E Modified Kohn’s

The trichrome technique of Wheatley for fecal specimens is a modification of Gomori's original staining procedure for tissue.

Gomori’s Trichrome Stain

It is a rapid, simple procedure which produces uniformly well stained smears of the intestinal protozoa, human cells, yeast cells, and artifact material in about 45 min or less.

Gomori’s Trichrome Stain

- Wheatley’s modification

Gomori’s Trichrome Stain

- For intestinal protozoa

Gomori’s Trichrome Stain

A rapid permanent-mount stain technic for the diagnosis of the intestinal protozoa

Lawless’ Permanent Mount Stain

Rapid method of staining trophozoites and cysts

Lawless’ Permanent Mount Stain

Protozoa – blue to purplish color

Lawless’ Permanent Mount Stain

- Cryptosporidium, Isospora and Cyclospora

Modified Acid-Fast stain

- Two separate stains: Carbol fuchsin and methylene blue

Modified Acid-Fast stain

- lacks specificity

Modified Acid-Fast stain

- Use of the modified Ziehl-Neelsen stain for faecal smears has already been established for coccidian protozoa, in particular, oocysts of Cryptosporidium species, but it is also useful to confirm the presence of oocysts of Isospora belli and Cyclospora cayetanensis

Modified Acid-Fast stain

- used for most of the original morphological descriptions of intestinal protozoa found in humans.

Iron Hematoxylin Stain

- On oil immersion power (1,000 x ), one can examine the diagnostic features used to identify the protozoan parasite.

Iron Hematoxylin Stain

can be used with either fresh, SAFpreserved, or PVA-preserved specimens.

Iron Hematoxylin Stain

diagnosis of Trichomonas

MIF Fixative stain ( Merthiolate iodine formaldehyde)

An acid dye, used as a fat and general tissue stain, and to stain protozoa in fecal smears or in tissues.

Chlorazol Black E

modification of the chlorazol black E staining technique

Modified Kohn’s

Enterobius vermicularis and Taenia

Cellulose Tape Preparation/Graham Scotch Tape Method

Cellulose Tape Preparation/Graham Scotch Tape Method Collection:

morning before the patient washes or defecates

Commercial collection kits are available

Cellulose Tape Preparation/Graham Scotch Tape Method

provides an estimate of worm burden

Egg Count Technique

Determines the degree of infection (light, moderate or heavy)

Egg Count Technique

For recovery of hookworms, Ascaris and Trichuris

Egg Count Technique

Egg Count Technique Methods:

1. Direct Smear Egg Count (Method by Beaver) 2. Dilution Egg Count 3. Thick Smear Egg Count 4. Dilution-Filtration Egg Count

Method by Beaver

Direct Smear Egg Count

= eggs per gram (epg)

- 1.5 mg feces (667)

= epg

- 2.0 mg feces (500)

– uses 0.1 N NaOH

Dilution Egg Count

- Stoll’s Egg Counting Technique

Dilution Egg Count

– KTS for schistosomes

Thick Smear Egg Count

– Schistosomes

Dilution-Filtration Egg Count

is a laboratory tool used to determine a given parasite's resistance to extant drug therapy.

Egg hatch assay (EHA)

Fresh eggs are incubated from the parasite of interest and serial dilutions of the drug of interest are applied.

Egg Hatching Technique

The percentage of eggs that hatch or die is determined at each concentration and a drug response curve may be plotted.

Egg Hatching Technique

The data can then be transformed and analysed to give further statistics such as an ED50.

Egg Hatching Technique

This technique is labour intensive, expensive and can take some time, however an egg hatch assay will give and accurate and reliable result.

Egg Hatching Technique

✓ For accurate detection of the organisms as a supplement to other methods

CULTURE METHODS

✓ For obtaining a rich yield of organisms to be used as antigen in immunologic diagnosis

CULTURE METHODS

✓ For in-vitro screening of drugs

CULTURE METHODS

✓ For investigating the physiology of organisms

CULTURE METHODS

✓ As a source for inoculating susceptible experimental animals

CULTURE METHODS

CULTURE METHODS for PROTOZOA

a) Balamuth’s Monophasic Medium b) Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw c) Cleveland & Collier’s d) Trussel and Johnson’s medium e) Axenic Culture Method

- All liquid medium commonly used for the isolation and maintenance of Entamoeba histolytica and some other intestinal protozoa.

Balamuth’s Monophasic Medium

- It is satisfactory for Balantidium coli, although the quality and amount of starch may need to be adjusted

Balamuth’s Monophasic Medium

- Use of egg yolk

Balamuth’s Monophasic Medium

- Egg slant

Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw

- E. histolytica

Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw

- Concentrate from saline centrifugal sedimentation of feces rather than the feces is recommended.

Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw

- Use of whole hen’s eggs

Boeck & Drbohlav’s Diphasic as Modified by Dobell and Laidlaw

- This medium is highly specific for E. histolytica , which grows luxuriantly on this medium.

Cleveland & Collier’s

Other intestinal amoebae do not grow readily on this medium.

Cleveland & Collier’s

The technique of overlaying the medium with fresh sterile horse serum-saline mixture, as reported by Cleveland and Collier, was reported to be the best method of isolation of E. histolytica .

Cleveland & Collier’s

Proteose peptone and infusion from liver provide amino acids and other nitrogenous substances that support growth of E. histolytica .

Cleveland & Collier’s

Sodium chloride maintains the osmotic balance of the medium and sodium a-glycerophosphate acts as a phosphorous source.

Cleveland & Collier’s

Isolation of Trichomonas vaginalis

Trussel and Johnson’s medium

- histolytica

Axenic Culture Method

- Axenic cultivation has been an essential component of numerous immunological and biochemical studies

Axenic Culture Method

- Monophasic clear liquid for rapid growth of dense populations of amebae

Axenic Culture Method

CULTURE METHOD for LEISHMANIA & TRYPANOSOMES

a) Novy, MacNeal and Nicole (NNN) Diphasic b) Cellular Media

- For Trypanosoma cruzi and Toxoplasma

b) Cellular Media

- Use of sterile defibrinated rabbit blood

Novy, MacNeal and Nicole (NNN) Diphasic

- Best medium

Novy, MacNeal and Nicole (NNN) Diphasic

IMMUNOLOGIC or SEROLOGIC TESTS

a) Indirect Hemagglutination (IHA) b) Flocculation or Agglutination c) Indirect Fluorescent Antibody (IFA) d) Gel Diffusion (Ouchterlony) and Countercurent Electrophoresis (CEP) e) Enzyme -linked immunosorbent assay (ELISA) f) Sabin-Feldman Dye Test g) Circumoval Precipitin Test (COPT) h) Radioimmunoassay (RIA) i) Radioallergosorbent Test (RAST) j) Immunoblotting/Western Blot k) Complement Fixation (CF)

involves antigen-antibody reactions

IMMUNOLOGIC or SEROLOGIC TESTS

PRINCIPLE: Patient serum and commercial sheep blood cells coated with the appropriate antigen are required. The serum and cells are combined and allowed to react. The test sample is then examined for the presence of red blood cell agglutination

Indirect Hemagglutination (IHA)

USES: amoebiasis, Chaga’s disease, malaria, toxoplasmosis, cystisercosis, hydatid cyst, filariasis, schistosomiasis

Indirect Hemagglutination (IHA)

PRINCIPLE: The patient serum is added to known cultured parasites. This mixture is observed for the presence of parasite clumping.

Flocculation or Agglutination

USES: Leishmaniasis, Chaga’s disease

Flocculation or Agglutination

PRINCIPLE: Microscopically visible parasites fixed on a slide are allowed to react with test serum. The slide is washed and treated with anti-human globulin with fluorescein (sandwich method)

Indirect Fluorescent Antibody (IFA)

PRINCIPLE: Patient serum and known antigens are placed in adjacent wells located on the agar gel. The antigen and antibody are allowed to migrate across the agar towards one anther. Specific patterns of precipitate ( precipitin bands) result between the inoculated wells and are then interpreted.

Gel Diffusion (Ouchterlony) and Countercurent Electrophoresis (CEP)

USES: Detect amebiasis and fascioliasis infections

Gel Diffusion (Ouchterlony) and Countercurent Electrophoresis (CEP)

PRINCIPLE: Test serum is allowed to react with an antigen (coat the surface of plastic well). Anti-human IgG antibody conjugated with enzyme is added. If specific antibody was present in the test serum, the anti-human IgG antibody labeled with an enzyme attaches to the antigen-antibody complex. The addition of a suitable substrate generates a visible color reaction.

Enzyme -linked immunosorbent assay (ELISA)

USES: T. gondii, T. vaginalis, leishmaniasis, malaria, schistosomiasis etc.

Enzyme -linked immunosorbent assay (ELISA)

✓ Modification of the standard ELISA for increased sensitivity and specificity in detecting antibodies of the IgM class to Toxoplasma