sample collection and storage method

1. Overview of Sample collection, Storage
and transportation
by
Daniel Gizaw (DVM,MSc)
May 2021
Sebeta Ethiopia

2. General information
samples can be collected from all livestock, equines,
poultry, native and feral animals and birds, fish and bees.
A clearly dated sample submission form accompany
sample or set of sample
Separate forms must be sent for each owner, each farm
or peasant association if necessary
Ensure that the form is fully completed and contains all
relevant case details.
 Before sending live animal it is necessary to inform the
laboratory.

3. Successful laboratory investigations
depends on
 Advance planning
 Collection of adequate and appropriate specimens
 Sufficient documentation
 Biosafety and decontamination
 Correct packaging
 Rapid transport
 Timely communication of results

4. Key issue
Consider differential diagnoses
Decide on test(s) to be conducted
Decide on clinical samples to be collected to
conduct these tests
consultation between microbiologist, clinicians
and epidemiologist

5. Samples may not be useable if:
• Mismatch of information on the label and the
request
• No accompanying document exist
• Inappropriate transport temperature maintained
• Inappropriate transport medium used
– specimen received in a fixative for virology
– dry specimen virus may die
– sample with questionable relevance
• Insufficient quantity
• Leakage

6. Labelling of sample
sample should be labeled or marked with the following information:
 The type of materials e.g. Serum, whole blood, liver, brain, urine,
etc. If preservative used, mention it if not write preservative not
used.
 An identification of the animal (e.g. ID number, name, colors of hair
coat, etc.)
 The names of the owner, Date of collection, Diseases suspected
• Unique identifier or combination of identifiers
• Firmly affixed to the container
• Number or bar code
• Information system tracks bio sample from collection through
processing, storage and distribution
• Data used for clinical and epidemiological study
Clearly and legibly marked
• Able to endure storage conditions

7. Labelling of sample
 Label able to withstand multiple freeze-thaw cycles, water-baths,
solvents, abrasion and long-term storage in liquid nitrogen and
ultra-low temperature freezers, even down to -196°C, without
detaching, cracking or fading
 Samples must be labelled serially (e.g. from 1 to 30) with a water
proof pen, preferably on an adhesive label. Keep a key list which
correlates sample numbers with animal identification.
 Do not label the stopper, which is removed during testing.

8. Handling and Transport of Samples
• The parcels should be very securely packed in order to
withstand rough handling.
• The samples should be packed in order to protect transporter
and the environment
• Great care should be taken to ensure that the containers with
samples do not leak! (a sticky tape may be used)
• Use cotton wool, newspaper or other absorbent materials to
soak up any leakage that, despite all that precautions, might
occur.
• Small cooler boxes are preferred for transporting refrigerated
samples.

9. Toxicology samples
The submission form accompanying samples for toxicological analysis
should provide the following information:
Chemical toxins
• A full record of history, clinical signs, and number died post
mortem findings and, particularly, the results of a search in the
environment of the poisoned animals for access to a poisonous
substance.
• The drugs that were used for treatment as well as the dates of
administration. The poison or group of poisons suspected.
Poisonous Plants
• A full record of history, clinical signs, post mortem findings.
• The results of a search for poisonous plants in the area where
animals normally graze or browse
• The plant (s) suspected to have caused the poisoning.
• The treatment that was applied as well as its effects.

10. Preservation and storages of sample
Equipment designed for samples storage has to meet two
major requirements:
1. It must ensure optimum temperature conditions for
storage
2. It must be large enough to hold the maximum samples to
be stored at the level of temperature where it will be
used.
Regular temperature monitoring is essential for all types
equipment

11. Anticoagulants
• EDTA and Heparin used to collect whole blood for
Haematology, virology.
• After collection of blood with anticoagulants mix
thoroughly by gently inverting tubes several times.
• Do not use formalin for virological samples

12. Storage Temperature
• Vapor phase nitrogen –170ºC
• Ultra-low –70ºC to –80ºC
• Frozen –20ºC
• Refrigerated 2ºC to 8ºC
• Ambient 20ºC to 25ºC

13. Storage temperature
1. Serum samples can be stored in the refrigerator (2 – 6.0° C)
for up to one week.
Serum samples can be stored frozen (≤ -20° C).
2. Swab samples can be stored in the refrigerator (2 – 6.0° C)
for up to one week.
Swab samples can be stored frozen (≤ -70.0 ° C).
3. Tissue samples
Tissue samples can be stored in the refrigerator (2 – 6.0° C) for up
to one week.
Tissue samples can be stored frozen (≤ -70.0 ° C)
4 whole blood
Whole blood samples can be stored in the refrigerator (2 - 6.0° C)
for up to one week. Do not freeze.

14. Transport Media
• Virus transport media (VTM) contains antibiotics
• Viral transport media can be used in line with of cold
storage.
• Sample to medium proportion should be 1:5 to 1:10.
• Medium contain
– pH buffer.
– Protein to stabilise.
– Antibiotics
• Preserve fluid or semi-fluid samples for virological
examination.
• Swabs can also be used for tissues of internal organs
• Dry swabs are not ideal for virological examination

15. COLLECTION OF SAMPLES
• The best sample to send to the laboratory is a live
animal affected with the disease in question.
• The next best sample would be an animal that died
recently showing signs of disease in question.
• It is, of course, not always feasible to submit entire
animals for laboratory examination.
• The third choice of samples is therefore those that are
collected in the field from either live animals or during a
necropsy.

16. COLLECTION OF SAMPLES
The following basic rules can be followed for proper
collection
• Sample diseased animals if possible.
• Sample lesions of suspect animals.
• Sample animals in early stages of disease.
• Sample animals again 2-3 weeks later for a “convalescent”
serum sample.
• Identify sampled animals properly.

17. Collection of Samples
In general the under mentioned samples are routinely
collected from categories of animals as indicated.
From live animals:
• Whole blood in EDTA, Heparin
• Paired serum samples
• Blood smears
• Faeces
• Swabs
• Skin scrapings
• Biopsies and impression smears

18. Collection of Samples
From dead animals or during authopsy
• Smears from blood , brain, spleen, liver, lung etc
• Liver, spleen, kidney, lung, lymph node and brain on ice and Virus
transport medium( VTM)
• Liver, spleen, kidney, lung, lymph node and brain in 10% buffered
formalin for histopathology
1 WHOLE BLOOD
Aseptically bleed directly in to vacutainers containing EDTA or
heparin preservatives, Mix gently by rolling the tube, Do
not freeze but keep cool on ice.
2. SERUM
• Use red topped vacutainer tubes for serology and mineral
analysis
• After collecting the blood sample, the tube is left untouched
at room temperature till clotting is complete
• The clot is then carefully removed

19. Avoiding haemolysis of sera or samples
1. Haemolysis occurs as a result of poor collection techniques,
contaminated equipment or poor handling of the sample once
it is collected.
2. Common causes of haemolysis include:
• Use of non sterile containers for collection or storage.
• Contamination by faecal and other material due to faulty aseptic
techniques.
• Contamination of the sample by water.
• A slow flow from the needle, obstruction of the needle, or failure to
insert into mid-vein.
• Forcibly expelling blood through a needle.
• Heating of samples, usually in car boots or through back windows of
car, or after prolonged exposure to direct sunlight during collection.
• Freezing.

20. SKIN BIOPSY
• Cut about 1 cm square and deep enough to
reach subcutaneous tissue.
• Preserve in 10% buffered formalin for
histopathology
• Use ice for skin biopsies to be screened for
viruses e.g. LSD and Orf, sheep and goat pox.

21. FLUIDS
• This refers to fluids from joint, peritoneal, pleural,
pericardial, cerebrospinal fluid etc
• Collect asceptically using a sterile needle and
syringe/vacatainers.
• Should be preserved on ice.

22. MILK
• Ensure NO antibiotic treatment was given prior to
sampling
• Wash the teats with water
• Wash your hands and disinfect with 70% alcohol
• Dry off teats and disinfect with 70% alcohol
• Expel the foremilk then milk into a sterile bottle
held almost horizontally to avoid dirt particles
falling inside
• Submit within a short time while fresh
• Keep cool on ice
• Recent used for diagnosis of FMD

23. ABORTED FOETUS, PLACENTA, FOETAL
FLUIDS
• Preferably submit whole foetus or placenta
preserved on ice
• Do not freeze but keep cool on ice
• If not possible, submit foetal stomach contents
in a sterile universal bottle and foetal, lung, liver,
spleen on ice and brain in transport medium
(VTM)

24. Transportation of Samples
• The viability of microbes is adversely affected by exposure
to temperature, extremes of pH, most common
disinfectants, light, irradiation, and desiccation.
• It is therefore necessary to protect the sample during the
interval between its collection and processing.
• Specimens taken in small amounts, viz., swabs and skin
scrapings, are most likely to be affected by ambient
conditions and should be placed in a suitable medium.
• Each container should consist of a vial, made up of strong
neutral glass, and have a screw top or cap complete with a
rubber liner or washer so that no leakage occurs.

25. Transportation of Samples
• After putting the material in the bottle/vial with the
medium, and closing of the top, the outside of the
vial should be disinfected either with 4% sodium
carbonate or with 0.2% citric acid solution.
• The screw cap should be sealed by winding around
with an adhesive tape.
• Another piece of adhesive tape should be put on the
vial on which are recorded the identification
particulars of the sample.

26. Thank you for
your attention