SOP for Parasitology Sample collection and

Examination

Prepared by : Dr Kedar Karki Parasitology Unit

Central Veterinary Laboratory

1
Table of Content;

1. Scope

2. Introduction

3. Code of Conduct
3.1.1. Purpose

3.1.2. Requirements

3.1.3. Procedure

3.2. Occupational Health and Safety

3.2.1. Purpose

4.0 General Procedure

4.1.1Simple test tube flotation:Procedure:
4.1.2. . Sedimentation method Procedure:
4.1.3. McMaster Technique: Procedure:
5.Health and Safety:

6.Handling and disposal procedures for contaminated materials and

wastes

7.Contaminated (potentially infectious) materials for autoclaving and

reuse

8.Contaminated (potentially infectious) materials for disposal

9.If a broken containers and spilled infectious substances

10.Breakage of tubes inside sealable buckets (safety cups)

11.Receiving the specimen
12. Recording the specimen
13. Physical Examination of the Specimen
14. Test results

2
 15. Referring the test reports to report delivering desk of the laboratory
16. Record keeping

17. Ensuring the receipt of test results by the clients

18. Feedback
SOP for Parasitology Sample collection and
Examination

1. Scope

This document would work as national reference method for faecal sample

submission, processing technique in the laboratory using standard

procedure. The various methods and the specific procedures for a method

outlined herein this manual shall be applied for the parasitological sample

collection and carry out various test methods in Parasitology Unit of central

veterinary laboratory.

2. Introduction

To diagnose gastro-intestinal parasites of ruminants, the parasites or their

eggs/larvae must be recovered from the digestive tract of the animal or from
faecal material. These are subsequently identified and quantified. This SOP
presents diagnostic techniques within the reach of laboratories to identify and
quantify parasite infections from the examination of faecal material.

The following are the main tasks involved in this process:

1.Collection of faecal samples

2. Separation of eggs/larvae from faecal material, and their concentration

3. Microscopical examination of prepared specimens.

3. Code of Conduct
3.1.1. 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

3
and larvae can be identified and quantified. For this purpose faecal material
can be processed by: McMaster, Flotation, Sedimentation methods.
3.1.2. Requirements
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

8;Two beakers or plastic containers

9:Tea strainer or double layer cheesecloth
10:Measuring cylinder or container graded by volume
11:Fork, tongue blades or stirring rod
12:Test tube
13:Test tube rack
14:Microscope
15:Microscope slides and coverslips
16:Balance or teaspoon
17;Flotation fluid
18:Methylene blue 1% solution or Malachite green 1% solution
19: McMaster counting chamber

20 :Compound microscope.

3.1.3. 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 defecated by inserting a

moistened finger into the rectum and massaging until the external sphincter
relaxes.

3.2. Occupational Health and Safety

4
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.

3.2.1. 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. The sedimentation technique is a qualitative method for detecting
trematode eggs in faeces. The McMaster technique is used for demonstrating
and counting helminth eggs in faecal samples.It is the most widely employed
method for this purpose.

4.0 General Procedure

4.1.1.Simple test tube flotation:Procedure:
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.
4.1.2.Sedimentation method 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.

5
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.
4.1.3.McMaster Technique: Procedure:

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

(Warning!!!) Faeces must be fresh.

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.

6
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.

5.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.

6.1.1.Handling and disposal procedures for contaminated materials and

wastes

Identification and separation system for infectious materials and their

containers should be adopted. National and international regulations must be
followed. Categories should include:

1. Non-contaminated (non-infectious) Plastic sample bottle ,plastic sample

bags, Tea strainer, plastic beakers, plastic tubes, glass slides that can be reused
or recycled or disposed of as general, “household” waste.

2. Contaminated (infectious) “sharps” – hypodermic needles, scalpels, knives

and broken glass; these should always be collected in puncture-proof
containers fitted with covers and treated as infectious.

3. Contaminated material for decontamination by autoclaving and thereafter

washing and reuse or recycling.

4. Contaminated material for autoclaving and disposal.

5. Contaminated material for direct incineration.

7
Sharps

After use, hypodermic needles should not be recapped, clipped or removed

from disposable syringes. The complete assembly should be placed in a sharps
disposal container. Disposable syringes, used alone or with needles, should be
placed in sharps disposal containers and incinerated, with prior autoclaving if
required. Sharps disposal containers must be puncture-proof/-resistant and
must not be filled to capacity. When they are three-quarters full they should be
placed in “infectious waste” containers and incinerated, with prior autoclaving
if laboratory practice requires it. Sharps disposal containers must not be
discarded in landfills.

7.Contaminated (potentially infectious) materials for autoclaving and reuse

No pre-cleaning should be attempted of any contaminated (potentially
infectious) materials to be autoclaved and reused. Any necessary cleaning or
repair must be done only after autoclaving or disinfection.

8.Contaminated (potentially infectious) materials for disposal

Apart from sharps, which are dealt with above, all contaminated (potentially
infectious) materials should be autoclaved in leakproof containers, e.g.
autoclavable, colour-coded plastic bags, before disposal. After autoclaving, the
material may be placed in transfer containers for transport to the incinerator.
If possible, materials deriving from healthcare activities should not be
discarded in landfills even after decontamination.
9.If a broken containers and spilled infectious substances
Broken containers contaminated with infectious substances and spilled
infectious substances should be covered with a cloth or paper towels.
Disinfectant should then be poured over these and left for the appropriate
amount of time. The cloth or paper towels and the broken material can then be
cleared away; glass fragments should be handled with forceps. The
contaminated area should then be swabbed with disinfectant.
If dustpans are used to clear away the broken material, they should be
autoclaved or placed in an effective disinfectant. Cloths, paper towels and
swabs used for cleaning up should be placed in a contaminated-waste
container. Gloves should be worn for all these procedures. If laboratory forms
or other printed or written matter are contaminated, the information should be
copied onto another form and the original discarded into the contaminated-
waste container.
All broken tubes, glass fragments, buckets and the rotor should be placed in a
non-corrosive disinfectant known to be active against the organisms concerned.
Unbroken, capped tubes may be placed in disinfectant in a separate container
and recovered.
The centrifuge bowl should be swabbed with the same disinfectant, at the
appropriate dilution, and then swabbed again, washed with water and dried.
All materials used in the clean-up should be treated as infectious waste.

8
10.Breakage of tubes inside sealable buckets (safety cups)
All sealed centrifuge buckets should be loaded and unloaded in a biological
safety cabinet. If breakage is suspected within the safety cup, the safety cap
should be loosened
and the bucket autoclaved. Alternatively, the safety cup may be chemically
disinfected.

• Park your vehicle in designated parking lane.

• Don't enter inside lab with samples or dead carcasses.
• Meet the personnel in help desk or inquiry point.
• Submit sample there in accordance with laboratory requirement.
• Make record of samples
• Wear apron and shoes and submit samples as per test required or
investigation requested for.
• Properly examination of samples, throw it in a biological pit.
• Materials remaining and equipment used for sample collection and
processing should be autoclave.
• Collection of the sample is done on normal office hours.
• Samples may be collected at any time in outbreak of infectious diseases
and zoonotic diseases.
• When samples will be in out of office time, one has to put it in particular
freezer. Recording can be done in normal office hour following day.

11.Receiving the specimen

The sources of specimen to CVL are private practitioners and government

veterinarians, hatcheries, farms, farmers, government as well as non-

government agencies and the other stakeholders, and PMLU (Post mortem

laboratory unit) of its own.

12. Recording the specimen

• Any kind of specimen brought to or received by CVL shall be initially

registered in the main entry of the laboratory in help desk.

• The main entry register shall code the specimen.

• The specimens shall be distributed to the relevant laboratory unit of CVL.

9
13. Physical Examination of the Specimen

A specimen shall be examined against the following criteria:

• Condition of the specimen; any departure of the specimen from the normal
condition shall be conveyed to the test requesting party.

• Accompanying documents

• Information of the documents in line with the specimen.

• Preservation technique employed

• Sufficiency of the specimen

14. Test results

• The test report shall be duly signed by technician and in-charge veterinarian
and result interpreted by technical manager of the laboratory.

15. Referring the test reports to report delivering desk of the laboratory

16. Record keeping

A back up (official copy) of the test reports prepared and handed over to the

report delivery desk shall be maintained in the form of record allotting a

dispatch number.

17. Ensuring the receipt of test results by the clients

• It would be rather a duty of respective lab to ensure the fact that the client
has received the test report from the report delivery desk of the laboratory.

• The respective lab shall maintain the dispatch number and date of delivery
of the test report to the clients from the laboratory.

18. Feedback

10
• The respective lab shall collect, study, process and make corresponding
improvement in its routine work to address the feedback and critical
comments of the clients. It would be considered as an endeavor in the way
of quality improvement of the each and every units of CVL.

• The respective lab shall let it know to its clients the necessary corrective
actions taken to improvement in its works.
Reference: The epidemiology, diagnosis and control of helminth parasites of
ruminants:
A Handbook Jørgen Hansen, DVM, PhD Animal Production and Health Division Food
and Agriculture Organization Rome, Italy Brian Perry, BVM&S, DTVM, MSc, DVM&S,
MRCVS International Laboratory for Research on Animal Diseases Nairobi, Kenya ILRAD
1994 Published by the International Laboratory for Research on Animal Diseases, P.O.
Box 30709, Nairobi, Kenya Printed by the International Livestock Centre for Africa
Addis Ababa, Ethiopia ISBN 92-9055-703-1 .Chapter. 3. Techniques for parasite assays
and identification in faecal samples,3.1-3.8

11