8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

1/26

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:- 08th

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

2/26

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 andtime 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.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

3/26

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.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

4/26

Serum

Serum sample should be collected once early at the time of outbreak andthe another on 14 to 21 days after the first collection for specificdemonstration 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 voidedthrough 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 andculture 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 aspiratedusing aseptic condition and transferred into sterile container.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

5/26

Faeces

Faeces should be collected when freshly voided or directly from rectumand immediately placed in a sterile container.

The faecal sample and swab should be dispatched to lab in a transportmedium at 4c

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 ofphosphate buffered glycerin or Tris buffer tryptose broth.

Vesicular fluid should be collected from unruptured vesicles asepticallyusing 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 sterilesyringe and needle after the area over the infection is disinfected.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

6/26

Genital tract infection

Semen should be collected using artificial vagina.

Vaginal and prepucial washing should be collected as samples in genitaltract 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.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

7/26

Nasal swab

Nasal secretions are collected in sterile cotton or

gauze cloth. It should be transported to the lab intransport media at 4C.

Milk

Milk sample should be collected in sterilecontainer after cleaning and drying of the tip ofthe teat.

The use of antiseptics should be avoided. Theinitial 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.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

8/26

Collection of sample from dead

animals

From dead animals, tissues are collected for isolation of bacteria and forconducting 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 avoidcontamination 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 includingthe 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 formalsaline.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

9/26

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 containersof different volumes and coolantpads 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.

http://www.gophoto.it/view.php?i=http://www.elearnvet.net/moodle/file.php/77/LESSON_1/containers.jpghttp://www.gophoto.it/view.php?i=http://www.elearnvet.net/moodle/file.php/77/LESSON_1/containers.jpg

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

10/26

Commonly used transport sytsem

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

11/26

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

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

12/26

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. Beforeopening the chambers, singe the epicardium over the right ventricle with thehost spatula are required (this is heated on a spirit lamp). Make an incision withthe sterile scalpel, dilate the opening with the sterile forceps and then insert thesterile swab into the incision and collect the blood in the swab into the tubecontaining charcoal transport medium, carefully seal it and forward to thelaboratory.

Organs Should be sent tightly packed in water proof sterile polythene pack and to be

sent in ice pack.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

13/26

Commonly used transfer medium

Transport Medium

Buffered glycerol saline

Stuart transport medium

Charcoal transport medium

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

14/26

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

15/26

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 thecontainer. 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.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

16/26

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 1stcase 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 feedgiven.

Treatment given.

Vaccination status.

Other information considered necessary by Veterinarians

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

17/26

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 andudder .

Blood, esophageal or pharyngeal fliud and throat swab .

Gum scrappings .

Paired serum sample .

50% glycerol saline or Phosphate buffered glycerolsaline.

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.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

18/26

3 IBRT (Infectious bovine rhino tracheitis)

Live animal: Nasal swab or swabs from external genitalia incase 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

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

19/26

5 Peste des petits ruminants-PPR

Live animals: Swabs from conjunctival discharge, nasal and buccalmucosa, 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 materials50 %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

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

20/26

8 Canine Distemper

Blood at high temperature ,swab from conjuntiva,nasaldischarge,scraping from gums,Pustules or fluid fromthe affected animals

Dead animals:Tachea,lung,brain,kidney,mediastinallymphnode,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 overice.

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

21/26

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

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

22/26

3 Anthrax

Peripheral blood smear, swab of blood, exudates fromnatural orifices, Putrified case - ear or muzzle pieces in

boric acid

4 Black quarter

Impression smear from affected muscle, muscle piecein 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

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

23/26

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

24/26

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

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

25/26

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 smearfrom kidney, smear from intestinal mucosa, liver, lung,kidney, brainon ice, Urine (5ml) preserved with thymol

14 Colibacillosis

Cloacal swabs, Intestinal swab and Heart blood swab in transportmedium, intestinal content, mesentric lymphnode, liver, spleen,kidney on ice.

Poultry: Swab from liver, lung, airsac, oviduct, yolk sac

8/12/2019 Collection, preservation and submission of sample to the microbiology laboratory

26/26

MATERIALS FOR FUNGAL DISEASES