Collection of sample,presevation and shipment

SAMPLE COLLECTION, PRESERVATION AND SHIPMENT
AUTHOR:SHAREEF NGUNGUNI 1
Email:shareefngunguni@gmail.com
Contents
INTRODUCTION....................................................................................................2
DISCUSSION...........................................................................................................2
1. Foot and Mouth Disease:.............................................................................2
2. East Coast Fever: ............................................................................................................................2
3. Heart water......................................................................................................................................3
4. African Swine Fever: ......................................................................................................................3
5. Newcastle .........................................................................................................................................4
6. Hemonchosis;...................................................................................................................................4
7. Gumboro:.........................................................................................................................................4
8. Babesiosis;........................................................................................................................................5
9. Rabies:..............................................................................................................................................5
10. Tuberculosis.................................................................................................................................5
11. Coccidiosis:..................................................................................................................................5
12. Black quarter: .............................................................................................................................6
13. Mange...........................................................................................................................................6
14. Tumor: .........................................................................................................................................6
15. Brucellosis:...................................................................................................................................6
CONCLUSION ...........................................................................................................................................7
REFERENCE..............................................................................................................................................7

INTRODUCTION
One of the major goals of microbiology laboratory is to isolate or detect
clinically significant organisms from the affected site or any other site to
determine a possible infectious etiology. Samples can be collected from
different organisms depending on the aims that microbiologists have at that
particular time. Most of the clinical samples are collected from animal
orifices like mouth, anus, and nose and on skin. Realistically, the time between sample collection
and processing may range from minutes to hours to days but it is recommended that after sample
collection, the sooner the specimen is processed in the microbiology laboratory, the better. (Scott
et al, 2011). According to Markey et al (2013), drying all microorganisms and exposure to
anoxious atmosphere (oxygen for obligate anaerobes) are the major factors that compromise the
specimens and for this reason the samples must be kept moist. Moistness is maintained by
placing the sample in transport (holding) medium composed of balanced salt solution in gelled
matrix because this does not contain any nutrient material, microorganisms in the sample
multiply poorly if at all and there by relative numbers and ratios are preserved. Swabs should
always be placed in transport media regardless of the time elapsed between processing and
collection
DISCUSSION
1. Foot and Mouth Disease:
This is a viral infection and suitable clinical specimens are vesicular fluid, affected mucous
membranes, pharyngeal and esophageal fluid (obtained with a probang), blood, and serum. The
affected epithelium (mucous membrane) is placed into phosphate buffer saline (PBS) or in
glycerol with Ph 7.2-7.6. Blood is collected in plain tubes to obtain serum in the laboratory
(Markey et al,2013). Fluid samples are collected by using a swab or a probang cup as in case of
pharyngeal and esophageal fluid. The samples are preserved by refrigeration (for serum less than
-150
C for longer storage) and the swabs/tissues are stored at 4O
C for 72-96hrs or at -76 o
C in
freezer for long storage(Markey et al,2013). The blood samples tubes are wrapped in absorbent
towel and placed into biohazard plastic bag. The biohazard plastic bags are placed in cardboard
shipping box. Then it is shipped at ambient temperature, priority overnight. The swabs are
placed in envelopes and sealed. The envelopes are placed in zip lock bag, then zip lock bag is
placed in cardboard shipping box and shipped at room temperature. Shipment of infectious
substance is regulated by WHO, and infectious substances are classified in category A and
Shipment of substance in this categories require packaging of the specimen or samples in a
component consisting of a leaky-proof primary receptacle, a leaky-proof secondary receptacle
and outer covering with maximum strength. If the samples are liquids, additional absorbent is
placed between primary and secondary leaky proofs so that to absorb and prevent the liquids to
reach the outer packaging. This is simply called Basic Triple Packaging System. FMD virus it’s
in category A and shipment FMD virus cultures follow Basic triple packaging system with
frozen cold packs.
2. East Coast Fever:
In live animals Blood smears, biopsy smears from swollen lymph nodes are collected and fixed
in methanol and whole blood in plain tubes. From dead animals’ emphsymetous lungs, trachea,
lymphocytic infiltrated kidneys, spleen, lymph nodes such as mesenteric, bronchial; and liver are

collected since schizonts are found there. The impression smears from the
collected tissues are air dried and preserved by fixation in methanol. The
tissue samples such as spleen, lymph nodes, liver , kidney are also preserved
by fixation in Formalin for histopathology to demonstrate schzonts. Whole
blood is preserved by refrigeration (2o
C-6o
C) for short storage. The blood
samples tubes are wrapped in absorbent towel and placed into biohazard
plastic bag. The biohazard plastic bags are placed in cardboard shipping box. Then it is shipped
at ambient temperature, priority overnight. The tissues are shipped on shipping containers in
sealed cardboard shipping box with frozen cold packs. East coast fever is in category B and
samples are not shipped as infectious agents.
3. Heart water;
It is caused by E. ruminantium originally Cowdria ruminantium, in ruminants.. Amblyoma ticks
are the main vectors.Organisms replicate in the reticuloendothelial cells particularly
macrophages, and in the endothelial cells of capillaries, especially those of the CNS. Clinical
specimens include well vascularized portions of brain (such as hippocampus, the cerebral
cortex,or cerebellum), spinal cord, the intima of large veins, spleen whole blood from live
animals, Amblyomma ticks. Whole blood is preserved by refrigeration (2o
C-6o
C) for short
storage and the tissues by fixation in 10% Buffered formalin (Mazza et al, 1997). The organisms
are also heat labile and lose their viability within 12-38hrs at room temperature hence their
viability is maintained or cryopreserved in dimethyl sulphide(DMSO) or sucrose-pottasium
glutamate medium(SPG)(Scott et al,2013). The blood samples tubes are wrapped in absorbent
towel and placed into biohazard plastic bag. The biohazard plastic bags are placed in cardboard
shipping box. Then it is shipped at ambient temperature, priority overnight. The tissues are
shipped on shipping containers in sealed cardboard shipping box with frozen cold packs.
Heartwater is in category B and samples are not shipped as infectious agents.
4. African Swine Fever:
This is a high contagious disease of domestic and some species of wild swine. Causes similar
symptoms and lesions as that of hog cholera which include hemorrhages(Mazza et al,1997).
From sick animals blood is collected in heparin tubes and from dead animal’s pieces of spleen,
liver, lymph nodes, kidney, and tonsils. Illeoceal region of intestine in 50% glycerol saline for
isolation; and pieces of brain, lung, intestine in 10% Formalin for histopathology. The virus is
stable in the tissues and excretions; withstand a range of ph 4-13 can be inactivated by heating at
20 degree celcius for 20 minutes ,lipid solvents and disfectants such as paraphenylphenolic hence
the samples mainly the tissues are preserved by fixation in Formalin and refrigeration(2.0 – 6.0°
C) or freezing(≤ -60.0 ° C)(Markey et al,2013). The blood samples tubes are wrapped in
absorbent towel and placed into biohazard plastic bag. The biohazard plastic bags are placed in
cardboard shipping box. Then it is shipped at ambient temperature, priority overnight. African
Swine Fever Virus is in Category A, and shipment of cultures follow Basic triple packaging
System. The tissues are shipped on shipping containers in sealed cardboard shipping box with
frozen cold packs.

5. Newcastle:
It is a highly contagious disease which is characterized by respiratory
distress, diarrhea and neurological signs. Samples from live birds should
include tracheal and cloacal swabs. Suitably specimen for recently dead bird
include spleen, lung, brain, intestine and its contents, feaces. Fecal samples
are collected in gloves, and The virus is inactivated by heat, ultraviolet light,
oxidation, Ph,detergents and butylated hydroxytoluene and survive long in frozen
carcases(Markey et al,2013). From that point the samples are kept refrigerated and the samples
may be stored at 4 degrees Celsius for not less than 4 days; except fecal sample, the other
samples are also preserved by fixation in formalin solution. The swabs are placed in envelopes
and sealed. The envelopes are placed in zip lock bag, then zip lock bag is placed in cardboard
shipping box and shipped at room temperature. Newcastle Virus grouped in category A of
infectious substances and shipment of cultures follow basic triple packaging system with frozen
cold packs. The tissues are shipped on shipping containers in sealed cardboard shipping box with
frozen cold packs.
6. Hemonchosis;
This is a tropical and subtropical disease caused by Hemonchus species. The nematode sucks
blood in the abomasum cattle, sheep and goats. Preferred samples are feaces to detect the eggs,
whole blood to detect packed cell volume and at necropsy gastric/abomasamal mucosa, bone
marrow to expansion of medullary cavity, abomasamal lymph nodes are collected. The tisues
such as abomasamal mucosa, lymph nodes are preserved by fixation in 10% Formalin for
histopathology. Feacal cultures and whole blood preserved by refrigeration (2o
C-6o
C). Tissues
are preserved by fixation in Formalin and refrigeration (2.0 – 6.0° C) or freezing (≤ -60.0 ° C).
The blood samples tubes are wrapped in absorbent towel and placed into biohazard plastic bag.
The biohazard plastic bags are placed in cardboard shipping box. Then it is shipped at ambient
temperature, priority overnight. The tissues are shipped on styrofoam containers in sealed
cardboard shipping box with frozen cold packs. Hemonchosis is in category B and samples are
not shipped as infectious agents.
7. Gumboro:
The virus replicates in immature B-lymphocytes of Bursa of fabricus and impair immunological
response. Suitable samples from sick birds is paired serum and in dead birds include Bursa of
Fabricious in 50% buffered Glycerine saline for isolation; liver, spleen, kidney, lungs and Bursa
Fabricious in Formalin for Histopathology. The virus is extremely stable and resist inactivation
by acid(stable at Ph 3), disfectants and heat(survives 60 degress for 30 mins).Serum samples are
stored at- ≤ 15o
C and the tissues by fixation in Formalin. tissues are preserved by fixation in
Formalin and refrigeration(2.0 – 6.0° C) or freezing(≤ -60.0 ° C). The serum samples tubes are
wrapped in absorbent towel and placed into biohazard plastic bag. The biohazard plastic bags are
placed in cardboard shipping box and shipped with ice packs.The tissues are shipped in shipping

containers on sealed cardboard shipping box with frozen cold packs.
Hemonchosis is in category B and samples are not shipped as infectious
agents.
8. Babesiosis;
The preferred sample is whole blood, and Thin Blood smears fixed in methanol. Whole blood is
preserved by refrigeration; and the blood samples tubes are wrapped in absorbent towel and
placed into biohazard plastic bag. The biohazard plastic bags are placed in cardboard shipping
box. Then it is shipped at ambient temperature, priority overnight. The fixed smear is shipped in
Styrofoam container with one cold pack on sealed cardboard shipping box. Babesiosis is in
category B and samples are not shipped as infectious agents
9. Rabies:
The disease is manifested by rabies virus which causes severe and invariable disease of central
nervous system(Mazza et al,1997). The rabies virus is abundant in pons, medulla and thalamus
of brain hence head or brain is taken on ice from dead animals .The virus is inactivated by
heating at 56 degrees celcius for 30 mins, chemical agents such as formalin(1%),cresol (3%) and
beta-propiolactone(0.1%) hence the sample is placed into 50% glycerol in phosphate buffered
saline and transported in ice box(Markey et al,2013). The sample is kept refrigerated ( 40
C for
many weeks) to prevent decomposition and provide reliable results, autolyzed samples reduces
specificity and sensitivity of results. The tissues are shipped in shipping containers on sealed
cardboard shipping box with frozen cold packs. Rabies virus in category A of infectious
substances and shipment of cultures follow basic triple packaging system on cold packs.
10. Tuberculosis.
This is a chronic granulomatous disease caused by Mycobacteriam species. Suitable clinical
specimen in live animals include: aspirates from cavities, lymph nodes, biopsies,
tracheobrancheaol lavages, 50ml of milk in sterile vials on ice in case of suspected mastitis
tuberculosis. From dead animals include: fresh or fixed samples of lesion or selected lymph
nodes from a tuberculin reactor with no visible lesion are taken, lymph glands and lung lesions
are collected in sterile container with 50%Glycerol phosphate buffer (Scott et al, 2013). The
bacteria is resistant to heat( can be killed at 72 degrees for 15 seconds),some disinfectants such
as sulphulic acid,5% oxalic acid, even 4% KOH or NaOH and inactivated by disinfectants such
as 2% phenol, 1% lysol. The affected tissues are preserved by fixation in 10% Formalin and by
refrigeration (2.0 – 6.0° C) or freezing (≤ -60.0 ° C). TB virus is in category A of infectious
substances and shipment of cultures only follow basic triple packaging system (Stockhalm and
Scott, 2008). The tissues are shipped on shipping containers in sealed cardboard shipping box
with frozen cold packs.
11. Coccidiosis:
This is a protozoon disease caused by species of genus Eimeria. Most of coccidian are
intracellular parasites of intestinal tract but few are located in liver and kidney. The samples to
be taken include intestinal tracts such as (duodenum, ileum ,cecum) or scrapings from those

intestinal surface , feaces; if the animal is geese kidneys may be taken and
liver in rabbits. The intestines, liver and kidney are transported in 10%
Formalin for histopathology, the feaces are collected in gloves or containers
and preserved by refrigeration.Coccidiosis is in category B and samples are
not shipped as infectious agents. The tissues are shipped on shipping
containers in sealed cardboard shipping box with frozen cold packs.
12. Black quarter:
This is an emphysematous, necrotizing myositis caused by clostridium chauvoie(Scott et
al,2013). Samples to be collected should be fresh and from affected muscle because clostridia
invaders may spread from intestine to tissues leading difficulties in interpreting laboratory
results. Impression smears from that affected muscle, exudate from the swelling, piece of
affected muscle is collected. The samples are placed in transport media or quickly cultured after
collection. The clostridia grow better on blood agar rich in yeast extract, vitamin K and haemin.
The samples are left in an anerobic conditions such as anaerobic jars containing hydrogen
supplemented with 5% CO2. The samples are preserved by refrigeration (2.0 – 6.0° C) or
freezing (≤ -60.0 ° C). Black quarter is in category B and samples are not shipped as infectious
agents(Stockhalm and Scott,2008). The impression smears, exudate and muscle tissues are
shipped on shipping containers in sealed cardboard shipping box with frozen cold packs.
13. Mange:
The preferred sample is skin scrapings. The sample is collected in escape proof containers or in
ointment tins. The samples are preserved by freezing and they are shipped on ice
packs(Stockhalm and Scott,2008).
14. Tumor:
Suspected Tumor tissues are collected and they are placed in containers or tubes. The containers
or tubes are labeled for easy identification. The samples are preserved by freezing or fixation in
10% Formalin for Histopathology (Kumar et al, 2005). The samples are shipped on dry ice to
interne national locations. The slides are stored in shipping containers while fixed blocks can be
left in Styrofoam containers with one cold pack. The shipping containers and Styrofoam
containers are placed in cardboard shipping box which is securely closed.
15. Brucellosis:
This is a bacterial infection which affect animals e.g. cattle and humans. The bacteria is highly
infectious, has public health and economic significance (Mazza et al, 1997). Samples collected
include blood, milk, paired serum sample, vaginal mucus, uterine fluid, stomach contents of
fetus, aborted fetus and culture on blood agar, tryptose etc. All species of Brucella are readily
killed by common disinfectants and pasteurization but some of them will survive for 4-5hrs to
direct sunlight, 5 days in cloth at room temperature and 75days in aborted fetus during cool
weather. Tissue samples are preserved/fixed in 10% Formalin for histopathology, serum by
freezing at- ≤ 15o
C (Markey et al, 2013); furthermore tissues and cultures are preserved by

refrigeration (2.0 – 6.0° C) or freezing (≤ -60.0 ° C). Brucella is in category
A of infectious substances and shipment of cultures follow basic triple
packaging system. The tissues are shipped on shipping containers in sealed
cardboard shipping box with frozen cold packs (Stockham and Scott, 2008)
CONCLUSION
All in All, sample collection is a tool used to diagnose diseases. Proper sample collection and
preservation help microbiologists to isolate the agents since the agents are more labile to
harsh/external conditions. Infectious substances should be shipped with proper procedures and
care.
REFERENCE
Kumar ,V.,Abbas,K.,Fausto, N.(2005).Robbins and Cotran Pathologic Basis of Diseases. 7th
Edition. Elsevier,Inc.Philadelphia.
Markey,B.K.,Leornad,F.C.,Archambault,M.,Cullinane,A.,& Maguire,D.(2013).Clinical
veterinary Microbiology.2nd Edition.Elsevier Ltd.London.
Mazza,M.L.,Pezzlo,T.M.and,Baron,E.J.(1997).Color Atlas For Diagnostic Microbiology.Mosby-
Year Book,Inc.St Louis-Missouri 63146.
Scott D.,Melisa,K.,and Chengappa M.M. (2013).Veterinary microbiology.3rd
.Edition.John
Willey and Sons,inc. lowa.
Stockham,L.S.,and Scott M.A.(2008). Veterinary clinical pathology.2nd
Edition.Blackwellpublishing .Lowa 50014.

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