An

Assignment on
Collection, preservation and submission of sample to
the microbiology laboratory
Submitted to:-
Dr. Arun Patel
Asst. prof. of microbiology
Department of microbiology
Submitted by:
Parth Bubadiya
Roll no : 01
Reg. no.:- 02-00334-09
Sem:- 08
th


Basic concepts of proper specimen
collection

Collection must be with a minimum of contamination from adjacent
tissues, organ or secretions.
Specimen should be collected at optimum time for the best recovery
of microorganisms. Hence the knowledge on history, and patho
physiology of disease is important.
A sufficient quantity of materials to be collected to perform various
diagnostic tests.
Specimen must be collected prior to administration of antimicrobial
therapy.
The specimen must be properly labeled with name, source, date and
time of collection, brief history of diseases and clinical tentative
diagnosis.
After collection, specimen should be transported to laboratory for
examination, if not possible it should be refrigerated immediately.

COLLECTION OF SAMPLES
Blood
Blood samples for culture or direct
examination, appropriate anticoagulants, such as heparin may be used.
Blood must be collected under aseptic conditions. Area over the vein
should be shaved, thoroughly cleaned and sterilized with disinfectant.
The blood is added to a flask containing 50-100 ml of a rich nutrient
medium that will permit growth of fastidious organisms or collected in
specially designed blood culture tubes.
For direct examination of bacteria, it is necessary to make thick and thin
smear from fresh blood.

Serum
Serum sample should be collected once early at the time of outbreak and
the another on 14 to 21 days after the first collection for specific
demonstration of increasing antibody titre. This is known as collection of
paired serum samples.
Urine
Urine secreted in kidney and collected in bladder is sterile, when voided
through urethra it may contaminate with normal urethral micro flora.
In small animals, urine is readily collected by means of cystocentesis.
In large animals, catheterization is the preferred method.
Collected urine should be placed immediately into a sterile test tube and
culture as soon as possible.
If delayed of more than one hour then the urine should be refrigerated.
Fluids
Exudates from the pleural, peritoneal or synovial cavity must be aspirated
using aseptic condition and transferred into sterile container.

Faeces
Faeces should be collected when freshly voided or directly from rectum
and immediately placed in a sterile container.
The faecal sample and swab should be dispatched to lab in a transport
medium at 4

c
Skin
The skin is normally preferred for vesicular diseases. Approximately 2 g of
affected epithelial tissue should be collected and sent to lab in 5 ml of
phosphate buffered glycerin or Tris buffer tryptose broth.
Vesicular fluid should be collected from unruptured vesicles aseptically
using sterile syringe and needle.
Skin scrapings (from periphery of the lesion), and wool samples and hair
(by plucking) are collected for fungal infection.
In birds, feather follicle can be collected for Mareks disease.
Abscesses and wound
Pus may be collected from closed, undrained abscesses with a sterile
syringe and needle after the area over the infection is disinfected.
Genital tract infection
Semen should be collected using artificial vagina.
Vaginal and prepucial washing should be collected as samples in genital
tract infection.
In case of endometritis and cervicitis specimen should be obtained using
cultural swabs through vaginal speculum.
Eye
Suppurative material from an infected eye should be collected from the
lower cul- de-sac or from the inner canthus by gently swabbing the surface.
Ear
Secretions and discharges from internal ear canal should be collected by
gentle swabbing the canal with sterile cotton swab.

Nasal swab
Nasal secretions are collected in sterile cotton or
gauze cloth. It should be transported to the lab in
transport media at 4

C.
Milk
Milk sample should be collected in sterile
container after cleaning and drying of the tip of
the teat.
The use of antiseptics should be avoided. The
initial stream of milk should be discarded
and next streams are collected in the tube.
Milk for serological tests should never be heated,
frozen or subjected to violent shaking.

Collection of sample from dead
animals

From dead animals, tissues are collected for isolation of bacteria and for
conducting histopathological or immuno histochemical techniques. Of late
tissue samples from dead animals are also used for identification of protein
or nucleic acid.
The organs most commonly selected include the kidney, liver, spleen and
heart. While collecting organs during necropsy, care must be taken to avoid
contamination by extraneous microorganisms.
Easily sealed plastic bags are used for collection of organs. If the animal
has been dead for several hours, bacteriologic examination of the tissue will
be less reliable than examination of specimen obtained soon after death.
In large animals sufficient quantity of representative specimens including
the site of lesions should be carefully removed and placed in a container.
In smaller animals the entire organ is to be collected. If immediate
microbial examination is impossible, the specimen should be refrigerated or
frozen.
For histopathlogy, the specimens must be sent in 10% buffered formal
saline.

COLLECTION MATERIALS
Sterile cotton swabs
Microscopic slides
Postmortem knife,
Scalpels,
Forceps,
Scissors,
Syringes and needles
Vacutainers ( with and without anticoagulants)
Small hacksaw for bone specimens
Plastic bags and water proof pens
A polystyrene unbreakable containers of different volumes and coolant
pads for transport of specimens
Specimen should be placed in approve private transport devices to maintain
a buffered and non nutritive environment to prevent metabolic damage to
organisms of interest and overgrowth of contaminants.

Commonly used transport sytsem
PRESERVATION
Preservation of Specimens
In many incidences, there may be a time gap between the collection of
and processing of specimens. During this period, the specimen should be
maintained in an environment favorable for the preservation of organisms
activities.
Various method of Preservation are
Refrigeration
The container containing the specimen should be placed in a larger
container and the place between the smaller and the larger container
should be filled with ice.
Chemical preservation
Certain specimen can be preserved in 50% glycerol saline.
Specimen for histopathological examination should be preserved in
10% formal saline

Preservatives
Serum
Sodium azide (0.1%) or methiolate (0.01%) preservation (Incase if it is used for
ELISA sodium azide should not be added)
Milk
One tenth volume of 5% boric acid as preservative
Intestines
Ligate about 3 inches of the bowel tied at either ends and send the loop
unopened for bacteriological examination of the intestinal flora, at once (in ice
if it is to be transported for some distance).
Heart blood swabs
For this sterile swabs, sterile scalpel, sterile forceps and a spatula. Before
opening the chambers, singe the epicardium over the right ventricle with the
host spatula are required (this is heated on a spirit lamp). Make an incision with
the sterile scalpel, dilate the opening with the sterile forceps and then insert the
sterile swab into the incision and collect the blood in the swab into the tube
containing charcoal transport medium, carefully seal it and forward to the
laboratory.
Organs
Should be sent tightly packed in water proof sterile polythene pack and to be
sent in ice pack.

Commonly used transfer medium
Transport Medium
Buffered glycerol saline
Stuart transport medium
Charcoal transport medium




DESPATCH OF SPECIMENS

The primary concern in the transport of specimen to the nearby laboratory
is to maintain the sample as near its original state as possible with
minimum deterioration and to minimize the hazards.
Adverse environmental conditions such as exposure to cold and heat,
change in pressure, extreme drying should be avoided. If any delay is
expected before the specimen is processed, it is generally preferable to
freeze the specimen at -70C.
The specimen should be properly packed and sent under refrigeration,
should be preferably through a thermoflask or thermocool box . Proper
labeling is important .The specimens must be secured (without any leak or
break) and packed before despatch. They should be delivered within 48 hrs.
Any material before being sent to laboratory for identification, it is always
better to get the consent of the laboratory. In case of shipment to foreign
countries import permit from the authority of the laboratory. This import
permit is called as import license. The permit or license should be placed in
an envelope and pasted on out side of the parcel.

Information to be sent along with sample

Information and case history should always accompany the samples to the
lab. This should be placed in a water proof plastic envelope outside the
container. While writing details, it should be written in waterproof material

The following informations are mandatory
Name and address of the owner with telephone and fax number.
Name, postal ,telephone, fax, E-mail address of the senders.
Disease suspected & diagnostic test requested.
Species, breed, age, sex and identity of the animal.
Date of sample collection and submission.
List of samples submitted and transported.

History of the condition with following points
List of animals examined and PM findings
Length of time of sick animals have been in the farm. If they are
recent animals, state the origin.
The date of report of 1
st
case and subsequent cases previous
occurrences and results provided by the lab.
Description of spread of infection in the herd.
Number of animals in the farm, number died, number showing
clinical signs, age, sex and breed.
Clinical signs observed and duration.
Type of husbandry followed including feeding practices and feed
given.
Treatment given.
Vaccination status.
Other information considered necessary by Veterinarians

MATERIALS FOR VIRAL DISEASES

1 FMD
Vesicular fluid.
Epithelial tissue collected from unruptured or recently ruptured
vesicular lesion in foot, tongue, mouth.gum,iterdigital space,teat and
udder .
Blood, esophageal or pharyngeal fliud and throat swab .
Gum scrappings .
Paired serum sample .
50% glycerol saline or Phosphate buffered glycerol
saline.

2 Bovine viral diarrhoea and Mucosal disease
Defibrinated blood , paired serum samples.
Dead animals: spleen, and mesenteric lymph nodes .
50% glycerol saline or Phosphate buffered glycerol saline.

3 IBRT (Infectious bovine rhino tracheitis)
Live animal: Nasal swab or swabs from external genitalia in
case of vulvo vaginitis or Balanoposthitis. semen, prepucial
washing.
Dead animals : Mucus membrane of Respiratory tract,
portion of tonsil, Lung, Bronchial LN, Fetal Liver, Lung,
spleen, Kidney, cotyledons .
On ice or Cell culture medium containing antibiotics &
fetal bovine serum

4 Blue tongue
Live animal: Blood in an anticoagulent at peak of
temperature, paired serum sample.
In dead animal :Spleen, liver,bone marrow and mesenteric
lymph node on ice.
50% glycerol saline

5 Peste des petits ruminants-PPR
Live animals: Swabs from conjunctival discharge, nasal and buccal
mucosa, whole blood in anticoagulant solution and paired serum
sample .
In dead animals: Lymph node, lung, spleen and intestinal mucosa.

6 Sheep &Goat Pox
Skin papules, scab from affected portion, lung, lymph nodes and
whole blood in EDTA or buffy coat .
Blood in anticoagulant, other materials 50 %glycerol saline.

7 Swine fever
Heart blood,piece of Lymph node, Spleen,heart,distal ileum and
kidney.
On ice or Equal amount of glycerol & 0.4M phosphate buffer with
antibiotics

8 Canine Distemper
Blood at high temperature ,swab from conjuntiva,nasal
discharge,scraping from gums,Pustules or fluid from
the affected animals
Dead animals:Tachea,lung,brain,kidney,mediastinal
lymphnode,liver and urinary bladder scraping.
In Hank's balanced salt solution or in ice.

9 Rabies
Animal head / Brain or impression smear (Thalamus,
pons, medulla, Hippocampus or cerebellum)
50% glycerol saline or portion of brain preserved over
ice.

MATERIALS FOR BACTERIAL DISEASES

1 Actinobacillosis
Smears of pus from deeper portion of the lesions-heat
fixed
Swab of pus from abscess on ice
A piece of affected tongue on ice.

2 Actinomycosis
Smears of pus from deeper portion of the lesions-heat
fixed
Pus in sterile tubes
Swab of pus from abscess on ice

3 Anthrax
Peripheral blood smear, swab of blood, exudates from
natural orifices, Putrified case - ear or muzzle pieces in
boric acid
4 Black quarter
Impression smear from affected muscle, muscle piece
in sterile container on ice, smears of exudate from thigh
5 Bovine genital camphylobacteriosis
Male: Prepucial mucus, smegma, semen, prepucial
washing
Female: Vaginal mucus, aborted fetus, fetal stomach
contents, Lung, Liver and Placenta

6 Brucellosis

Live animal: Serum, blood, Milk, Joint fluid, lymph

node.

Abortion: Placenta, Cotyledons & vaginal discharge

Foetus: Smear from abdominal fluid, fetal stomach

contents, lung, liver on ice.

7 Hemorrhagic septicemia

Auricular vein smear, Oedema fluid smear, Heart blood

smear fixed in methanol.

Swab from Heart blood, pleura, lung in transport

medium

Piece of lung and spleen in sterile container on ice and

long bone in case of putrifaction in charcoal

8 Johnes Disease

Live animal: Rectal pinch smear, Rectal washing,dung samples in
sterile vial.
Dead Animal: Intestinal contents, Lymph nodes on ice, Mucosal
smears from thickened areas of intestine or ileocecal valve.

9 Leptospirosis
Blood, serum, milk and urine
Internal organs: kidney, Liver, Lung, Brain or adrenal gland on ice.

10 Listeriosis
Swab from nasal cavity and heart blood
Foetus: Swab fromheart blood,brain on ice

11 Mastitis
Milk from affected quarter on ice

12 Tuberculosis
Smears from the sputum, lymph node and lesions fixed by heat,
Swab from lesions in transport medium,
TB nodule from any visceral organ and pieces of organs along with
lymphnode on ice.

13 Enterotoxaemia
Intestinal contents(10ml) preserved with 3 to 4 drop of chloroform ,
loop (12") of intestine tied at both ends on ice,Impression smear
from kidney, smear from intestinal mucosa, liver, lung,kidney, brain
on ice, Urine (5ml) preserved with thymol

14 Colibacillosis
Cloacal swabs, Intestinal swab and Heart blood swab in transport
medium, intestinal content, mesentric lymphnode, liver, spleen,
kidney on ice.
Poultry: Swab from liver, lung, airsac, oviduct, yolk sac

MATERIALS FOR FUNGAL DISEASES