Collection, Preservation and Dispatch of Samples for Laboratory Diagnosis of Animal Diseases

Collection, Preservation and Dispatch of Samples for Laboratory Diagnosis of Animal Diseases

Authors:  Dr. Debasish Behera^1*, Dr. Kanika Kalai² and Dr. Biplab Debroy³

College of Veterinary Sc. and A.H., R.K. Nagar, West, Tripura-799008

Objectives

• Diagnosis of disease or identification of new disease.
• Confirmation of tentative diagnosis.
• To observe the effect of treatment and give direction for further therapy.

General consideration before collection of materials

For correct diagnosis in the laboratory the tissue/ materials must contain the following qualities:

• Material for bacteriological examination should be obtained as for as possible free from contamination.
• Sterile instruments should be used.
• Before primary incision is made, the skin of surrounding area is disinfected
• If tissues are to be cultured the surface of the organ is first smeared with hot iron spatula.

Preservatives:

            No preservatives are used for bacteriological examination. The sample is taken in ice box and preserved. If examination is to be delayed by a few days it should be preserved in 25% glycerin saline.

Equipments required

• Forceps and scissors, Spatula, Sterile test tubes, Sterile cotton swabs, Sterile Pasteur pipette and teats, Spirit lamp and spirit, Alcohol, Sterile filter paper,  Broth media
• For very small sample from peripheral blood – prick the ear tip-press, touch the filter paper and transfer to broth or sterile test tube, Sometimes, direct culturing may be preferred

Shipping specimens for bacterial isolations

• Tissues and organs: in individual sterile polyethylene bags or other suitable leak proof container.
• Portions of the intestine (tied off if appropriate) should be packaged separately.
• Swabs—may be the preferred for specimens from nasal passages, pharynx, tonsil, eye, ear, skin, abscesses, vagina, cervix, etc.
• Avoid drying of specimens after collection and during shipment by using a suitable transport medium.  Swabs in transport medium are available commercially.
• Faeces—faecal specimens should be obtained directly from the rectum avoiding contamination, placed in a leak proof container, and shipped to the laboratory
• Shipping Specimens for Bacterial Isolations
• Milk—specimens should be collected aseptically using sterile screw- capped or stoppered vials (not bags). May be refrigerated          immediately following collection and delivered to the laboratory under refrigerated conditions. Please include a minimum of 1-2 ml of milk
• Urine—Urine must be collected aseptically and refrigerated immediately. Free catch urine samples should be avoided if possible to avoid contamination. Better recovery of organisms is achieved from 1-5 ml     of urine. Urine swabs are not recommended

Routine examination –

• Leucocyte differential count
• Hemoglobin percentage in blood
• Absolute count of RBC and WBC
• Detection of blood parasites and bacteria

Bacteriological and serological tests (diagnostic test)

• Isolation, culture and sensitivity test for micro- organisms
• Detection/ confirmation of presence antibody against many bacterial and viral agents

Biochemical test

• For sugar, bile, cholesterol, minerals & enzymes

Preparation of  blood smear

• Take a drop of blood on one side of a clean dry slide.
• Touch this blood drop with edge of another slide at about 15 degree
• Lift it and place at a little ahead and drag slowly, smoothly over the slide
• It should be so thin that you can read letters through the smear
• After staining examine 100 to 300 leucocytes and the count is expressed in percentage

Collection of stool for culture

• No Antacids, barium bismuth, anti-diarrhoea drugs or oily laxatives prior to collection.
• Do not collect more than one sample per day/animal.
• Collect in a dry, wide-mouth plastic, sterile container. Avoid contamination with urine. No preservative.
• Select an area that may be bloody, slimy, or watery.
• Take with the collection spoon or a wooden stick and put a thumb size sample the dry sterile container.
• Specimens must be transported to the lab immediately.
• If a delay of more than two hours is expected, refrigerate the sample.
• The stool samples should be delivered to the laboratory on the same day of collection for physical ,chemical, macroscopically, biochemical tests and bacteriological test

Urine analysis

• It is quite difficult to collect urine in most animals
• Use of catheters and syringe has been recommended
• In bovine manipulation of bladder per rectum may cause urination
• Nevertheless, it is a discouraging venture
• Urine should be analyzed soon after collection
• One drop of commercial formalin in 30 ml of urine will arrest bacterial growth
• Freezing will destroy the cellular element
• Can be refrigerated few hours before examination
• Voided sample
• Manual compression of bladder
• Catheterization
• Cystocentesis
• Samples should be analyzed within 30 minutes of collection. If the sample is allowed to stand at room temperature, it results in following changes:
• Decreased glucose and bilirubin.
• Disintegration of casts and other cells.
• Increased pH due to bacterial breakdown of urea to ammonia.
• Bacterial proliferation.
• Increased turbidity.
• Storing of samples at 40C or refrigeration does not affect result for 5- 6 hours. Warm the sample to room temperature before examination.
• Following preservatives can be used for preservation of urine samples for several hours:
• 1 drop of 40% formalin in 30 ml urine.
• Toluene in sufficient quantity to cover surface of urine.
• A small crystal of Thymol for 5- 10 ml urine.
• 1 part of 5% phenol to 9 parts of urine.

Histopathology examination of tissue samples

• Establish the pathogenesis and pathology of any disease.
• Disease in which histopathological examination of tissues is the only alternative to diagnose the disease e. g.
• In some cases, the tissues from dead animals are only available material for laboratory diagnosis.
• It is considered as the best reliable technique because histopathological procedures produce permanent slides.
• The histopathological techniques are useful in carrying out the retrospective studies. The unstained slides and blocks can be stored for indefinite period, which can be examined even after many years for further studies.
• The presence of causative agents can also be demonstrated in tissue section using routine histopathological techniques or special staining.
• The detection of chemicals in tissues like enzymes, lipids etc is included in histopathological examination, which not only describe the structural changes but also gives idea about the functional status of the organ.

Fixation of tissue samples

Formalin: It is universally used routine fixative

• Role of a fixative: – 10 % neutral formal-saline
• Confers chemical stability on the tissue.
• Hardens the tissue (helps further handling).
• Halts bacterial putrefaction.
• May enhance later staining techniques.
• The cellular elements are preserved by denaturalization of cellular protein.
• Microorganisms are fixed- no more proliferation.
• Autolysis is prevented – as lysosomal protein is denatured.
• Tissue and cell element remain more or less same as at the time of death / collection.
• 100 ml container with 60 ml solution – ideal for 1-2 tissues
• Often used for ulcerated surface lesions.
• Samples usually from the surface, inflammatory exudates, rather than deeper tissues,

Collection and preservation of tissue samples for Biopsy

• Fixative is also used for biopsies to give an immediate indication of the type of lesion before sending the sample for histopathological interpretation.
• Cut surface of the biopsy sample is blotted to remove surface blood and serum, and then the dried surface is applied to a clean, dry slide with gentle pressure.
• Several areas can be prepared on a single slide.
• The preparations are quickly air dried and then stained as for a fluid sample
• Remove a small piece of tissue (liver, lungs etc.)
• Touch the cut surface lightly on a blotting paper to remove excess blood
• On a glass slide lightly give three to four touches separately in a row.
• Do not rub or spread
• Immediately air dry the slides
• Fix the slides before dispatch

Procedure for the samples for bacteriological, fungal and parasitic examination

• Immediately after taking a smear (also blood smear) it should be air dried by shaking and keeping under fan.
• Fixing can be done by heat – move the slide one or two inches above a spirit lamp (smeared side up) three to four times (for bacteria)
• If you touch the back side of the slide on the back of your hand it gives a mild warm feeling.
• Over heating will cause charring of cells and bad staining.
• Alternatively, flood the slide with methanol for about one minute and dry (you can send unfixed slides also)
• Make several slides and send unstained slides to a lab or stain for examination
• One slide should be stained with Gram stain (for Gm+ve/-ve micro-organism)
• One slide should be stained with ZN stain (for TB, J D)
• One slide should be stained with Geimsa or Leishman stain (for protozoa and general cellular elements)
• Loeffler’s methylene blue staining may be done for Anthrax & Clostridia organisms
• Gridley’s cotton blue stain is used to detect fungal structures
• Other special stains are used to detect various elements like bile, calcium deposition, mucinetc
• Biopsy or PM tissues may be touched on the slide
• Pus materials, Intestine mucosal scraping, skin scraping adhered to the blade etc may be spread over a slide
• Using a 21- 25-gauge needle attached to a 5- 10 ml syringe.
• The resulting sample is often very small and may only be present within the needle, not the syringe.
• The syringe should be detached, filled with air, reattached, and gently depressed to express the sample onto a clean, dry, glass slide
• The force applied should be minimal in order to avoid rupture of the cells.
• Another slide is placed on the top of the sample and pulled lengthwise to spread the sample to a single layer if possible.
• Fine needle aspirate is spread over the slide with a dragger slide to get a monolayer

Packaging of samples for dispatch

• Use clean wide mouth glass/ plastic bottle with stoppers of capacity one liter. Use separate bottle for each material.
• Cut the viscera into small pieces to ensure penetration of the preservative.
• The bottles are filled only up to 2/3^rd and not to the full.
• Apply grease or Vaseline to the stopper and secure by cap and seal the bottle (to prevent leakage).
• Paste the label bearing the identification mark of the animal and viscera.
• A sample preservative used should be kept separately (about 100 ml).
• Keep the bottle in a card board box and seal. All the bottle and packet should be carefully sealed and closed in such a manner that they cannot be opened without destroying the seal.
• Paste the address label and send them by parcel to chief chemical examiner. Better to send it through special messenger.
• The covering letter, detail of material collected, railway receipt (if send by railway parcel), post mortem report along with complete history, detail of seal (sample seal on covering letter) and request and report from police should be send separately by register post to chemical examiner.
• In suspected cases of poisoning whole cadaver of small animals (small dogs, cat, piglet and poultry) may be sent unopened under ice by special messenger to the chemical examiner.

Procedure/ methods to collect samples in large animals during necropsy.

• Stomach and its contents 500- 1000 gm in large animals and all available contents of small animals.
• Upper part of small intestine with its contents about 500- 1000gm.
• Any suspicious substance in stomach and intestine.
• Liver 500- 1000 gms in large animals or whole liver in small animals.
• Kidneys- One.
• Spleen or its portion if large animals.
• Urine (all available) and faeces in preservative kept separately.
• Heart and portion of brain.
• Lung tissue and heart blood without adding preservative.
• Portion of skin and subcutaneous tissue if poisoning by subcutaneous injection.
• Hair 5- 10 gms if poisoning by mineral such as As, Pb etc.
• Portion of long bone- Poisoning by As, Sb.
• Dung without addition of spirit.
• Rectified spirit is normally used as preservative. It is contraindicated in case of suspected poisoning by alcohol, phosphorus, paraldehyde acetic acid, carbolic acid and other drug of phenol group.
• Saturated solution of common salt is employed where rectified spirit is contraindicated.
• Refrigeration temp. If the material is dispatch through special messenger so as to reach the chemical examiner on the same day.
• Describe the clinical signs, lesions, tentative diagnosis and treatment given to animal in letter. Also mention the type of test desired.

Procedure to dispatch of samples

• Write correct address on letter as well as on the parcel preferably with pin code if the materials send by post.
• Mark the parcel ‘Biological material’, ‘Handle with care contain glass material’, ‘Fragile’ etc. in order to avoid damage in parcel.
• Also mark the side to be kept on upper side with arrow.
• Seal the container properly so that it should not leak during transport.
• Try to send the material as soon as after its collection from animal.
• Keep the one copy of cover letter inside the parcel and send another copy by hand or post in a separate cover.
• During dispatch of pathological material the mouth of containers should be sealed with molten paraffin or wax.
• For microbiological examination transport in screw capped water tight bottle is preferred over dry ice.
• For viral examination Mc-cartney bottles with metallic screw cap and rubber lining are suitable and sent on ice packed box.
• The preserved samples are required immediate dispatch to lab on the day of collection by a special messenger.
• Keep adequate material like thermo cool etc. in the parcel which will save the material from out side pressure/ jerks.
• Use dry ice, if available other wise use ice in sealed container
• No spirit is added to dung samples requiring chemical analysis.
• The packing should be done separately in ice box.
• All containers should be serially numbered, properly labelled and kept under safe custody.
• The covering letter should contain the details about specimen and addressed to the Chief Chemical Examiner who is authorized for toxicological examination.

Table Depicting different diseases, etiology and samples to be collected for diagnosis

Bibliography:

1. Benjamin, M.M. (1885). Outline of Veterinary Clinical Pathology. 3^rd, Kalyani Publishers, Rajinder Nagar, Ludhiana, India.
2. Culling, C.F.A (1974). Handbook of histopathological and histochemical technique including museum techniques. 3^rd, Butterworth and Heinemann (Publishers) Ltd., Elsevier, Oxford, UK.
3. Luna, L.G. (1968). Manual of Histologic Staining of the Armed Forces Institute of Pathology, 3^rd, Mc Graw Hill, New York.
4. L. Jackson, (2013) Veterinary Clinical Pathology, An Introduction, Ist Edition, Wiley India Pvt Ltd.
5. Ganti A. Sastry, Veterinary Clinical pathology (Pb-2016), CBS publishers & Distributors Pvt. Ltd.

 Detailed Address and Affiliations of Authors

^1* First and corresponding author: Assistant Professor, Department of Veterinary Pathology, C.V.Sc. and A.H., R.K. Nagar, West, Tripura-799008.

²Second author: Assistant Professor, Department of Veterinary Pathology, C.V.Sc. and A.H., R.K. Nagar, West, Tripura-799008.

³Third author: Assistant Professor, Department of Veterinary Pathology, C.V.Sc. and A.H., R.K. Nagar, West, Tripura-799008.

COLLECTION PRESERVATION AND DESPATCH OF  VETERINARY SPECIMEN FOR LABORATORY DIAGNOSIS

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