ZEB 303 (PARASITOLOGICAL TECHNIQUES).22.23.

SESSION
Parasitological techniques involve methods deployed in the diagnosis of parasitic organisms responsible for
disease conditions in other living organisms. In order to understand their applications, let us look at the various
samples needed for specific parasitological techniques.
Diagnostic stages of most parasites can be detected in:

1. Feces: used to diagnose parasite eggs, larvae, oocysts, cysts, Trophozoites, cestode segments adults.

2. Blood: used to diagnose blood parasites: Babesia, Theileria, Trypanosoma, Dirofilaria immitis.
3. Sputum: used to diagnose lung parasite eggs, larvae for example Dictyocaulus species (eggs) is cattle and
sheep.

4. Urine: used to diagnose eggs in urinary system for example: Dioctophyma renale (giant kidney worm),
capillaria species in dogs and cats.

5. Skin: used to diagnose external parasites such as: Mange (Sarcoptes, Psorptes). used for studying the
pathological changes

6. Biopsy from live animals.

Examination of the fecal samples :

There are several procedures commonly used to examine feces for internal parasites:
1. Gross examination of feces:

 Consistency: The condition of the feces that is: soft, watery (diarrheic) or very hard soild, this
description will vary with the animal species, for example, cattle feces are normally softer than those
of horses or sheep.
 Color: Unusual fecal colors should always be reported.

 Mucous: Mucous on the surface of fresh feces may be associated with intestinal parasitism or some
other metabolic diseases .

 Blood: blood may indicate severe parasitemia.

 Age of feces: If the feces appear old and dry, this should be noted in aged sample, parasite eggs have
embryonated or larvated, oocyst may have sporulated or pseudo parasites may be present.
 Gross parasites: Tapeworm segments , round worms , and larval arthopods (bots) may be present .
Microscopic examination of feces:

1. Direct smear:
Procedure of direct smear:

1. Small amount of feces placed directly on the microscope slide, by a stick

2. Dilute this quantity with water or normal saline.

3. Mixed by using applicator stick.

4. A cover slip is applied and the smear is examined under the microscope.

Advantages: short of time and minimal equipment needed

Disadvantages: negative result with this method is not always reliable and the animal may be
incorrectly assumed to be free of parasite. This method also leaves a lot of fecal debris on the slide.

2. Concentration methods for fecal examination:

A. Qualitative methods: these methods used for determination the types of infection
1. Fecal flotation: this procedure based on differences in specific gravity of parasite eggs and larvae and that
of fecal debris.
Specific gravity refers to the weight of an object (for example: parasite eggs) compared with
the weight of an equal volume of pure water. Most parasite eggs have a specific gravity between 1.1 and
1.2, whereas tap water is only slightly higher than 1, therefore, parasite eggs are too heavy to float in tap
water, to make the eggs float, a liquid with a higher specific gravity than the eggs must be used, such
liquid are called flotation solution consist of concentrated sugar or salts solution added to water to
increase its specific gravity. Flotation solution usually have specific gravity between 1.2 and 1.25.
Flotation method is used to diagnose nematode eggs, oocyst and cysts.
Procedure of flotation :

1. put about 2 gm. of the fecal sample in 100 ml glass beaker.

2. Add 15-30 ml of flotation solution

3. Mix the feces solution with solution.

4. Strain the solution through a fine sieve (tea strainer) to remove the layer objects.

5. Pour the mixture in to (10 ml) test tube and fill the tube to the top.

6. Place a glass cover slip gently on the top of the fluid and allow the cover slip to remain for 10 to 20
minutes.

7. Remove the cover slip carefully and immediately place it on the microscope slide.

8. examine the area of the slide under the cover slip with the microscope.
2. Sedimentation:
This method is suitable for trematodes eggs and some cestodes and nematodes whose eggs do
not float readily in common flotation solutions.
Procedure of sedimentation:

1. Place 3-6 gmof the fecal sample in 100 ml glass beaker.

2. Add 30-40 ml of tap water or normal saline

3. Mix the water with the feces.

4. Strain the solution through a fine sieve

5. Pour the strained mixture to the centrifuge tube and centrifuged for 1-2 minutes on 1500 rpm, if a
centrifuge is unavailable, allow the mixture to sit undisturbed for 20-30 minutes.

6. Pour off the liquid in the top of the tube without disturbing the sediment at the bottom.

7. Using the Pasteur pipette, transfer a small amount of the top layer of the sediment to a microscope
slide.

8. Apply a cover slip to the drop and examine the slide microscopically.

3. Baermann method:
 This method used for detection the lung worm larvae and cultural method for specific identification
of the third larval stage of the Strongyles and Trichostrongyles.
 Baermann apparatus consist of:

a. A funnel clamped to a metal stand

b. A short piece of tubing with a clamp is attached to the end of the funnel (Fig. 3).
Procedure of Baermann technique:
1. Apply 5-20 gm of fresh feces or any suspected soil to a gauze and placed it in the funnel.
2. The sample is covered by the warm water.
3. Let the apparatus at room temperature for 8-24 hours.
4. Release the clip and collect the first 3-4 drops of water on a microscope slide and examine the slide,
or collect 10 ml into a centrifuge tube, spin in the centrifuge for several minutes and examine the
sediment.

4. Fecal culture:
This is used in diagnostic parasitology to differentiate parasites whose eggs and cysts can not be
distinguished by examination of fresh fecal sample. For example eggs of some nematodes like Strongylus
species in horses. The feces allowed to incubate at room temperature for several days until the eggs
hatched and the larvae developed to infective third stage (L3).

Procedures of fecal culture :

1. Place 20-30 gm of fresh feces in a jar and moisten slightly with the tap water, until it become soupy.

2. Place the jar on a shelf, away from direct sun light and for 7 days at room temperature if the culture
is dried add few drops of water . .

3. After incubation, concentrate the larvae by means of the Baermann technique and examine .

B. Quantitative fecal examination :

Quantitative procedure indicate the number of eggs or cyst present in each gram of feces
(severity of infection). Several procedures are used to estimate the numbers of parasite eggs per gram
of feces, including:

1. Stoll΄s technique.

2. Mcmaster technique.

1. Stoll technique:

1. Place 5 gm of fresh feces sample in 100 ml graduated measuring cylinder.

2. Add 0.1 N (4%) solution of NaOH (sodium hydroxide) in water up to 75 ml .

3. Shaking the liquid with glass beads .

4. By a graduated pipette, apply 0.15 ml suspension immediately to a microscopic slide and cover the liquid
with a cover slip(22x45) and examine the slide.
It is advisable to check four preparations and take the average number of eggs multiplied by 100,
equals the number of eggs per gram feces (EPG,).
Larvae (LPG)

2. McMaster technique:
Used the counting slide (McMaster slide) procedure of Mc- Master method (Fig 4)

1. Two gm of fresh feces are dissolved in 60 ml saturated solution (flotation solution) such as sodium
chloride.

2. Strain the mixture through a fine sieve.

3. Using a Pasteur pipette, fill one compartment of the counting cell at once
 4. Repeat the same operation to fill the second counting chamber.

5. After a few minutes, the eggs float up to the surface of the concentration solution and stick to the
cover glass.
2 gm 60 ml 1 gm. 60 ml/2 each compartment contains 0.15 ml liquid

EPG = Y X 200 in which Y (number of eggs)

Blood sample

• Collection of blood from animal should be performed aseptically.

1. Swabbing the skin over the vein with alcohol (Ethanol) and using a sterile needle.

2. Blood may be drawn with a standard needle and syringe or a vacuum collected tube.

3. If the blood is used immediately for tests direct smear is made to diagnosis microfilariae and
Trypanosoma .

4. But if the blood is used to obtain serum, it must be allowed to clot .

5. If the tests cannot be performed immediately or if some of the blood must be reserved for further
testing, clotting must be prevent by addition of anticoagulant Ethylene diamine tetra acetic acid (EDTA
)

6. Blood samples should always labeled with owners name and the date of the collection.

• Examination of blood

• Blood smear (Fig 5)

1. Thin blood smear :
This procedure is prepared for white blood cell differential count, protozoa Babesia sp.,
Leucocytozoon and Nematodes (larval stages of Setaria equine and Dirofilaria immitis).

a- Place a clean glass microscope slide on the bench surface and place a small drop of the blood sample
near the short end of the slide.

b- Place the short end of a second slide (the spreader slide) near the middle of the bench surface slide
out the blood drop, and hold it a 35- 45 degree angle.

c- The spreader slide is then smoothly and rapidly slide forward the length of the surface slide, producing
a smear with a feathered edge or tongue shaped.

d- Allow the surface slide to air dry and then stain with Field or Wright's or Leishman or Giemsa΄ s stain
in the following steps.
Giemsa stain

• Fix in absolute methanol for 1-5 minutes.

• Wash in tap water.

• Air dry.
• Dilute stock Giemsa stain 1:20 with distilled water and place slide in staining jar for 30 minutes.

• Wash stain a way gently with tap water.

• Air dry. Examine the slide with the 10X objective for microfilariae or Trypanosoma. The 100X (oil-
immersion) may be used for the intracellular parasite (Babesia, Theleria).
2. Thick blood smear

a- Place 2-4 drops of the blood sample together on a glass slide spread them with a wooden
stick to an area about 1.5-2 cm diameter.

b- Allow the smear to air dry.

c- Wash the smear with distilled water (hypotonic solution).

d- Immerse it for 10 minutes in methyl alcohol. Stain with Giemsa stain for 30 minutes.
Wash excess stain with tap water.
Note: this method cann't be used for the blood of bird because it have a nuclus in R.B.Cs.
• Testing of blood for detecting microfilariae
1- Direct microscopic examination. These procedure for detecting the movement of microfilariae.
• Place a drop of fresh blood or heparinzed blood on the microscopic slide, and then covered with cover
slide.

• Examine at 4X, you can add one drop of 10% formalin at the side of cover slide.

2- Modified knott's test

 • Mix 1ml of blood with 9ml of a 2% formalin (mixed well).

• Centrifuge the mixture at 1500 rpm for 2-5 minutes and discard the supernatant fluid.

• Add 1 drop of 0.1% methylene blue to the sediment to a microscope slide using a Pasteur pipette.

• Examine the slide for the presence of microfilariae using 10X objective lens.

3- Hematocrit method or Buffy coat method

• Fill a hematocrit tube with the whole blood sample.

• Hematocrit centrifuge tube for 5 minutes.

• Observe the location of the buffy coat layer between the red cell layer and the plasma.

• Using a glass cutter, deeply scratch the hematocrit tube at the level of the buffy coat. Immediately take
the part of buffy coat add to a center of a microscope slide.

• Add a drop of normal saline and a drop of (methylene blue stain) and cover with cover slip using 10X
objective lens, examine the slide for the presence of microfilariae

• Other methods for the diagnosis of protozoan infections

• Animal inoculation in the diagnosis of protozoa infection such as (Leishmaniasis, Toxoplasmosis).

• Serological methods for the diagnosis of protozoan infections such as (agglutination,

Immunoflourescence complement fixation, gel diffusion).

• Preparation of tissue smears (impression smear)

• The material obtained by Biopsy (live) Autopsy or Necropsy (postmoterm examination), is an

important method for diagnosing parasitism of the digestive tract or brain, kidney, and muscles.

• The cut surface should be touched with filter paper to remove excess blood.

• The cut surface should be pressed gently on to a slide to leave an impression.

• Treated as a thin blood smear.

• brain smear
• place a small piece of brain near the one end of slide then crush by using the other slide.

• Spread by slipping the slide and then treated as thin blood smear (same procedure).

• Diagnosis of parasitism of urinary system

Collection the urine sample for parasitological examination may be done during normal urination
or catheterization:

• A waxed paper cup 3-5 ml with a lid or other clean container may be used for collection.

• Urine sample should be labeled and refrigerated.

 • Methods for diagnosis.
a- Direct method. b-Urine sedimentation.

• Diagnosis of parasitism of the skin

• Skin scraping for evaluating animals with dermatologic problems Equipment required includes : an
electric clipper with no 40 blade scalpel no 10 or spatula 165 mm stainless steel, mineral oil, and
compound microscope.

• The average area scraped should be 6 to 8 cm square.

• The depth of the scraping varies with the typical location of the parasite in question.

• The skin should be scraped until a small amount of capillary blood oozes from the area (Sarcoptes spp. ,
Demodex spp. ….).

• All of the scraped debris on the forward surface of the blade is then spread in a drop of mineral oil on
glass microscope slide.

• Cover slip is placed on the material.

• Examination using 4X objective lens.

• Gross specimens
Other ecto – parasites like ticks, lice, fleas, chigger mites … are collected from the surface
of the animal's skin by using a pieces of cotton dipped it in 70% of ethyl alcohol and keep in 10%
formalin for shipment to a diagnostic laboratory.

• preparation of the histopathological section

• Fixation of the affected organs in 10% neutral buffered formalin for 4872 hours.

• Dehydration in ethyl alcohol with different concentrations from 70%, 80%, 90%, 100%.

• Cleared in xylol

• Embedded in paraffin.

• Sectioned at 4 µm – 6 µm.

• Stained with Hematoxylin and Eosin stain.

• Tissue sections then examined microscopically.

• Examination of sputum
• The larvae and eggs of respiratory parasites have the same characteristics as those found in the feces.
Dictyocaulus species, lungworms of cattle and sheep, which are usually seen in the sputum as eggs
containing larvae rather than as free larvae in the feces.

• A drop of sputum or nasal discharge on a microscope slide is easily examined.

 • Several slides should be examined

MC Master Slide Baermann Apparatus

TREMATODA

Fasciola hepatica

Egg of Fasciola hepatica

Miracidium

Redia Cercaria

Metacercaria Fasciola gigantica

Egg of Schistosoma haematobium egg Schistosoma japonicum

Egg of Schistosoma mansoni

Cercaria of Schistosoma

CESTODA

Cysticercoid of Hymenolepis nana

Gravid segment of Hymenolepis nana
Egg of Taenia spp Egg of Taenia spp

Mature segment of Spirometra Adult of Spirometra

NEMATODA
 Ova of Parascaris spp Ova of Ascaris spp

Anterior end of Toxocara canis Oxyuris equi

Ova of Oxyuris equi

Anterior end of Ancylostoma caninum

posterior end of Setaria digitata ( Fig : 75) microfilaria of seteria

Trichinella spiralis larvae in striated musculature

Ascaris lumbricoides

Protozoa
 Trypanosma brucei Trypanosma cruzi

Trypanosma evansi Trypanosma equiperdum

 Amastigote of Leishmania spp Trichomonas foetus

Trophozoite of Entamoeba histolytica oocysts Eimeria

Haemoproteus