Faecal samples: Faecal samples: Examination

Dr.Kedar Karki
Purpose
The demonstration of eggs or larvae in faeces can indicate the presence of
parasitic infection and facilitate the diagnosis of parasitic disease.The eggs and
larvae can be identified and quantified.For this purpose faecal material can be
processed by: making a direct smear on a microscope slide for preliminary egg
identification

N.B. In the examination of poultry, faecal samples cannot be taken directly

from the cloaca. If assessment of individual birds is required they should be
caged separately and the fresh droppings collected. If individual bird
assessment is not required then the fresh droppings can be collected from the
house or pen. a flotation technique to concentrate eggs and separate them
from debris for identification and counting faecal culture to hatch the eggs and
develop the larvae to the third stage for identification
Faecal samples: Principle
Eggs and larvae produced by adult helminths in the host animal are often
passed out in the faeces.

Equipment List

1;Wide mouthed screw capped bottles, minimum 30 ml size

2;Plastic containers with lids, minimum 30 ml size

3:Disposable plastic sleeve-gloves used for collecting samples

4:Plastic bags

5:Pen marker for labelling

6:Cool box for transportation of samples

7;Refrigerator in laboratory suitable for the storage of faecal samples

Faecal Samples collection procedure

Faecal samples for parasitological examination should preferably collected
from the rectum. Appropriate disposable gloves should be worn.

Collection from large animals can be more easily accomplished than from
smaller animals.

Smaller animals such as lambs can be induced to defaecate by inserting a

moistened finger into the rectum and massaging until the external sphincter
relaxes.
If a herd is being examined samples should be collected from several different
randomly selected or worm affected animals, depending on circumstances.

N.B. In the examination of poultry, faecal samples cannot be taken directly

from the cloaca. If assessment of individual birds is required they should be
caged separately and the fresh droppings collected.

If individual bird assessment is not required then the fresh droppings can
be collected from the poultry house. Samples can be collected from the ground
if the animal is seen defaecating.

Faeces collected in this way must be fresh to be suitable for parasitological

examination.

If a herd is being examined several samples, sourced from different animals,

should be collected. Fill the container to capacity before closing the lid or
tighten the sleeve-glove as close to the faeces as possible.

It is important to exclude air from the container as this will retard the
development of the eggs.

Each sample should be clearly labelled with the identification of the animal
from which the faeces were collected, the date and the place of collection.

Samples should be packed in a cool box for transportation to the laboratory.

As soon as the faecal samples arrive at the laboratory they should be stored in
a refrigerator at 4°C until they are processed.

Samples can be kept in the refrigerator for up to three weeks without

significant changes in the egg count and egg morphology.

If it is not possible to store the samples in a refrigerator for some time after
collection, formalin can be added to the faeces to preserve the eggs. The final
concentration of formaldehyde in the mixture should be at least 3% (for
example by mixing 1 ml of concentrated formalin with every 10 mls of faeces).

Warning !!!
THESE PROCEDURES SHOULD ONLY BE UNDERTAKEN BY TRAINED PERSONNEL.
Faeces collected from the ground must be fresh otherwise they may be
contaminated with free-living nematodes. Samples should not be kept in the
freezer. Formalin fixed faeces cannot be used for faecal culture.
Health and Safety

Faeces may contain hazardous pathogens (bacteria, viruses etc). Appropriate

hygiene and safety procedures should be employed. Local health and safety
regulations should be observed. Faeces may contain hazardous pathogens
(bacteria, viruses etc). Appropriate hygiene and safety procedures should be
employed. Local health and safety regulations should be observed. Formalin is
irritant and toxic: use with care in a well ventilated space and avoid inhaling
fumes. Local Health and Safety regulations should be observed.

The faeces are generally processed using the following techniques:

Faecal smear

McMaster

Flotation

Larval culture

Baermann

For information on qualitative and quantitative flotation techniques, or the

flotation fluids used for these purposes, select one of the options below:

Simple flotation

Quantitative flotation

Flotation fluids

The simple test tube flotation technique:

Purpose:
It is a qualitative test for the detection of nematode and cestode eggs.

This is a useful method to use in preliminary surveys to establish which parasite

groups are present.

Principle:
Eggs are separated from faecal material and concentrated by a flotation fluid
of an appropriate specific gravity.
Equipment List
Two beakers or plastic containers
Tea strainer or double layer cheesecloth
Measuring cylinder or container graded by volume
Fork, tongue blades or stirring rod
Test tube
Test tube rack
Microscope
Microscope slides and coverslips
Balance or teaspoon
Flotation fluid

Simple test tube flotation:

Procedure step:
1: Weigh or measure using a precalibrated teaspoon approximately 3g of
faeces and put into container 1.
2: Pour 50 ml of flotation fluid into container 1.
3: Stir or mix faeces and flotation fluid thoroughly with a tongue blade or fork
4: Pour the faecal suspension through a tea strainer or double layer of
cheesecloth into container 2.
5: Pour the faecal suspension into test tube supported in a stand or rack from
container 2.

6: The test tube is gently topped off with the suspension leaving a convex
meniscus at the top of the tube.
7: Carefully place a coverslip on top of the test tube.
8: Leave the test tube to stand for 20 minutes.
9: Carefully lift the coverslip off the test tube together with the drop of fluid
adhering to it.Place the coverlip on a clean slide.
10: Examine using a compound microscope at 10 x 10 magnification.
Faecal Sedimentation Technique:
Purpose:
The sedimentation technique is a qualitative method for detecting trematode
eggs in faeces.
Principle:
The majority of trematode eggs are too large and heavy to float reliably in the
flotation fluids normally used for nematode eggs. They do however sink rapidly
to the bottom of a faecal/water suspension and this is the basis of the faecal
sedimentation technique.
Equipment List:
Two beakers or plastic containers
Tea strainer or double layer of cheesecloth
Measuring cylinder
Fork, tongue blades or stirring rod
Test tubes
Test tube rack
Methylene blue 1% solution or Malachite green 1% solution
Microscope slides and coverslips
Pipettes
Balance or calibrated teaspoon
Microscope
Procedure:
1: Weigh or measure approximately 3 g of faeces into container 1.
(Health and Safety)

Faeces may contain hazardous pathogens (bacteria, viruses etc ). Appropriate

hygiene and safety procedures should be employed. Local health and safety
regulations should be observed
2: Pour 40-50 ml of tap water into container 1.
3: Mix faeces and water thoroughly.
4: Filter the suspension through a tea strainer or double-layer of cheese cloth
into container 2.
5: Pour the filtered material into a test tube. Allow to sediment for 5 minutes.
6: Remove the supernatant with a pipette very carefully.
7: Re-suspend the sediment in 5ml of water.
8: Allow to sediment for 5 minutes.
9: Discard the supernatant carefully.Stain the sediment by adding one drop of
methylene blue or malachite green.The dyes stain the faecal particles a deep
blue or green leaving the trematode eggs unstained.
10: Transfer a small drop of the stained sediment to a microscope slide using a
pipette. Cover droplet with a coverslip.
11:Examine under a microscope at 10 x 10 magnification.

12:Identify eggs.Repeat until all the sediment has been examined.

13:Alternatively, pour the whole amount into a Petri dish and examine
methodically under a stereo-microscope.

Warning!!!

Nematode eggs may be present in the faecal sample but the recovery with this
technique is very low. Nematode eggs should be examined using flotation
techniques or the MacMaster technique.

McMaster Technique:
Purpose:
The McMaster technique is used for demonstrating and counting helminth eggs
in faecal samples.It is the most widely employed method for this purpose.
Principle:
The McMaster technique uses a counting chamber which enables a known
volume of faecal suspension (2 x 0.15 ml) to be examined microscopically.

Thus, if a known weight of faeces and a known volume of flotation fluid are
used to prepare the suspension, then the number of eggs per gram of faeces
(e.p.g.) can be calculated.

The quantities are chosen so that the faecal egg-count can be easily derived by
multiplying the number of eggs under the marked areas by a simple conversion
factor.

The McMaster chamber has two compartments, each with a grid etched onto
the upper surface. When filled with a suspension of faeces in flotation fluid,
much of the debris will sink while eggs float to the surface, where they can
easily be seen and those under the grid counted.

(Warning !!!)

Although it provides a valuable diagnostic aid, care is needed with interpretation of

the data provided as both qualitative and quantitative results can be influenced by a
variety of factors.

Equipment List

Two beakers or plastic containers

Balance

Tea strainer, cheesecloth or dental napkin

Measuring cylinder

Stirring device (fork, spatula, tongue depressor)

Pasteur pipettes and rubber teats

 Flotation fluid (choice of solution dependant on species expected to be
present and availability of reagents)

McMaster counting chamber

Compound microscope.

Procedure:

1: Weigh 4 grams of faeces and place into a container.

(Warning!!!) Faeces must be fresh.

Health and Safety:

Faeces may contain hazardous pathogens (bacteria, viruses etc).

Appropriate hygiene and safety procedures should be employed. Local
health and safety regulations should be observed.

2: Add 56 ml of your chosen flotation fluid.

3: Stir the contents of the beaker thoroughly with a fork, tongue depressor or
spatula.
4: Filter the faecal suspension through a tea strainer or double layer of
cheesecloth or dental napkin into the second container.
5: Stir the filtrate in container two with a Pasteur pipette.Using the pipette
withdraw a sub-sample as the filtrate is being stirred.

Health and Safety:

Faeces may contain hazardous pathogens (bacteria, viruses etc).

Appropriate hygiene and safety procedures should be employed. Local
health and safety regulations should be observed.

6: Stir fluid and fill first compartment of the McMaster counting chamber
with the sub sample.Stir fluid again and fill second chamber with another
sub sample.

7: Allow the counting chamber to stand for 5 minutes. It is important to

leave the chamber to stand to allow the eggs to float to the surface and the
debris to go to the bottom of the chamber.

8: Examine the subsample of the filtrate under the compound microscope at 10

x 10 magnification.
(Warning!!!)

Do not use high power i.e. x 20 / x 40 / x HI 100 oil because the objective
will break the thick upper plate of the McMaster slide.

9: Identify and count all eggs within the engraved area of both chambers.

10: Calculation of Result. The number of eggs per gram can be calculated as
follows: Count the number of eggs within the grid of each chamber, ignoring
those outside the squares .Multiply the total by 50 – this gives the eggs per
gram of faeces (e.p.g.) For example: 12 eggs seen in chamber 1 and 15 eggs
seen in chamber 2 = (12 +15) x 50 = 1350 e.p.g.

Warning !!!

Do not delay reading the count beyond the recommended time as the
flotation fluid may distort or destroy delicate eggs.Therefore it is advisable
to only process a few samples at a time.

11: Interpretation of Results, When interpreting McMaster results, it must be

remembered that a number of factors can influence the occurrence,
recognition or numbers of helminth eggs found in a faecal sample. In
particular, the number of eggs is not necessarily indicative of the number of
worms present. Reasons for this include:

Eggs are produced only by fertile adult female (or hermaphrodite) worms
and will, therefore, be absent in immature or single sex infections

The daily output of eggs by fertile females is influenced by host-

physiological factors such as stress or lactation ( increased ) or immunity
( decreased )

Chemotherapy can also affect egg-production e.g. corticosteroids

( increased ) or sub-lethal anthelmintic doses ( decreased )

Some food-stuffs may have a similar effect e.g. tannin-rich forages

( decreased )

The concentration of eggs (per gram of faeces) is influenced by the daily

volume of faeces being produced by the host, the rate of passage by the
ingesta through the intestine, and the distribution of eggs throughout the
faecal mass
Some types of eggs are heavier than others and may not float well in
solutions of lower specific gravity (e.g. Fasciola)

Some eggs from different species are indistinguishable (particularly

trichostrongylids and strongylids). This complicates clinical interpretation as
some species (e.g. Haemonchus) produce many more eggs per day than
others (e.g. Ostertagia).

Floatation fluid used in Faecal examination:

1. Saturated salt solution

Specific gravity: 1.18 - 1.20

General purpose solution.

Sodium chloride: 400 grams

Water: 1000 ml

Stir thoroughly before use.

May distort eggs.

2. Salt/sugar solution

Specific gravity: 1.28

General purpose solution.

Sodium chloride: 400 grams

Water: 1000 ml

Sugar: 500 grams

Dissolve the salt in water to make a saturated solution.

Add the sugar to the saturated salt solution.

Stir until the sugar is dissolved.

3. Sodium nitrate

Specific gravity: 1.18

This solution is sometimes used for strongyle eggs.

Sodium nitrate: 400 grams

Water: 1000 ml

Add sodium nitrate to water while stirring.

May form crystals and distort eggs if left longer than 20 minutes.