Collection of blood ,microbial and toxicological samples

COLLECTION OF MATERIALS FOR DISEASES DIAGNOSIS

  • Materials are collected from dead animals for microbiological, histopathological, parasitological and chemical examination to arrive at a correct diagnosis.

Collectionof blood smear

  • Peripheral blood smear is prepared from ear vein and then air dried and fixed in methanol for one minute. Blood smears can also be prepared from heart blood however peripheral blood smear is preferred.)

Collection of materials for microbiological examination

  • Heart blood if not clotted can be drawn using sterile Pasteur pipette for microbiological examination.
  • Material (swabs or tissues or contents of lesion and tubular organs) for microbiological examination should be collected in aseptic conditions.
  • Collect material from areas showing lesions and from sites where the organisms are concentrated.
  • e.g Vesicles in the mouth in FMD skin lesions in pox; Lymph nodes in theileriosis, strangles or glanders.
  • Always collect materials from fresh carcasses. In case of poultry, birds in advanced stage of aliment are sacrificed and materials are collected.
  • Use freshly sterilized equipment and containers.
  • Collected materials are sent in refrigerated condition (+4 ° C) or on ice.
  • Materials may also be sent using preservative.

Bacteriology

Amies transport medium with charcoal or Stuart transport medium without charcoal is used to transport the swabs.

Preparation of charcoal swabs

  • Prepare swabs by rolling absorbent cotton-wool on wooden sticks.
  • Boil the swabs in a phosphate buffer solution pH7.4 of the following composition.
  • Disodium hydrogen phosphate 0.81 g
  • Potassium dihydrogen phosphate 0.18 g
  • Distilled water 100 mL
  • Immediately dip the swabs into a 1% suspension of Charcoal (Bacteriological). Place in cotton- wool plugged test tubes, dry at 100 ° C and sterilize in the hot air oven for 1 hour at 160 ° C

Transport of swabs

  • After the collection of specimen, place the swab in the middle of Stuart’s transport medium and store in a cool place.

Virology

  • Storage of virus specimens
  • Samples for virological investigation can be collected in Charcoal virus transport medium
  • Phosphate buffered saline (pH 7.3) 50 % Glycerol saline
  • Phosphate buffered saline is a very useful general diluent and suspending fluid
  • Glycerol saline is prepared by mixing equal amounts of glycerine and saline (o.85% Na Cl)
  • Media or collection solution may be autoclaved at 121 ° C for 15 min.
  • Keep the specimens in tightly capped container to avoid deleterious effect of CO2.

Despatch of materials

  • Properly pack and despatch the materials within 24 hours under refrigerated condition ( + 4 ° C) or on ice in a thermos flask. Dry ice (solid CO2) can also be used. If delay is expected store at -70 ° C (-20 to -25 ° C is also satisfactory but infectivity is reduced in certain cases).
  • Paste the label containing specimen number, date of collection and nature of specimen
  • Enclose detailed history sheet containing specimen number, date of collection, name of the owner and address, species, age, sex, particulars of illness, symptoms, treatment and lesions, herd or flock health, disease suspected etc.

Materials to be collected in abortion cases

  • Placenta
    • Impression smears prepared from at least 2-3 cotyledons after blotting the cotyledons with filter paper. Air dry and gently heat fix (2 slides)
    • Small pieces from 2-3 cotyledons in a sterile bottle on ice. Pieces from 2-3 cotyledons and intercotyledonary membrane preserved in 10% formal saline
  • Foetus
    • Smears from thoracic and abdominal fluid, air dried and gently heat fixed
    • Thoracic and fluid (5ml) in a sterile container on ice for cultural isolation
    • Small pieces from lung, liver, spleen, kidney and brain in a sterile bottle on ice
    • Foetal stomach after tying both the ends and kept in a sterile bottle on ice or 10-20 ml of stomach content in sterile vials on ice for isolation of bacteria
    • Small pieces of lung, liver, spleen, thymus, stomach, kidney, brain, heart, intestine and any gross lesion from foetus, preserved in 10% formal saline
  • Dam (aborted)
    • Vaginal (deeper part) and uterine mucosal smears are to be prepared within 3 days of abortion, and air dried and gently heat fixed
    • Swab from uterine discharge
    • Milk in sterile container in case of brucellosis
    • Collect serum on the day of abortion and again 3 weeks after abortion
    • If leptospiral abortion is suspected, 20 ml of midstream urine from the dam preserved in 1.5 ml of 10 % formalin
  • Sire
    • Preputial washing in a sterile container on ice
    • Serum sample

Collection, packing and despatch of materials in poisoning cases for chemical examination

  • Materials required 
    • Clean wide mouthed, colourless glass bottles fitted with glass stoppers of one litre capacity
    • Rectified spirit- used as preservative. It is contraindicated in cases of suspected poisoning by alcohol, phosphorus, paraldehyde, acetic acid or carbolic acid and other drugs of phenol groups. The amount of spirit added to each vessel should be measured and the amount so added has to be written on the label affixed to the container. Methylated spirit may be used if rectified spirit is not available.
    • Saturated solution of common salt is employed where rectified spirit is contraindicated.
    • A postmortem examination kit
    • Cloth
    • Seal
  • Samples to be collected
    • Hair in suspected cases of subacute or chronic poisoning by minerals (Most minerals are eliminated through hair)
    • Portion of skin and subcutaneous tissue in suspected poisoning by subcutaneous injection or absorption
    • Portion of liver not less than 500 g or the whole liver Spleen or its portion weighing not less than 500 g One kidney
    • Stomach and its contents- one kilogram
    • Upper part of the small intestine with its contents (About one kilogram)
    • Any suspicious substance in the stomach and intestine
    • Faeces in preservative
    • Dry dung without addition of spirit
    • Urine in preservative (Thymol is used for preserving urine if rectified spirit is contraindicated)
    • Genitalia –uterus and its appendages and upper part of vagina in suspected case of criminal abortion
    • Blood from heart chamber without adding preservative in cases of suspected poisoning of carbon monoxide, coal gas, hydrocyanic acid, alcohol or chloroform
    • Heart, if poisoning by nuxvomica or strychnine is suspected
    • Lung tissues without adding preservative in cases of suspected poisoning of carbon monoxide, coal gas, hydrocyanic acid, alcohol or chloroform
    • CSF in alcohol
    • Brain or portion of brain, if poisoning by nuxvomica or strychnine is suspected
    • Portion of long bones in suspected cases of subacute or chronic poisoning by arsenic and antimony (especially in case of extreme putrefaction or in exhumed body after long burial)
  • Packing
    • Paste the label bearing the name of the owner, identification mark of the animal viz. Number, name and markings, type of tissue collected, type of preservative used and its quantity. The label should bear the office forwarding letter number, date and signature of the officer and examination requested and disease or poisoning suspected should also be written
    • Apply vaseline or any other suitable grease to the stopper (to prevent sticking) and secure by tape or string and seal the bottle
    • A sample of preservative (About 125 ml ) used should be kept separately
    • All the bottles and packets should be carefully sealed and closed in such a manner that they cannot be opened without destroying the seal. The containers preferably packed with cloth and sealed with sealing wax.
    • Keep the bottles in card board box and seal it
    • Keep the cardboard box in wooden box. Lock the wooden box and seal it
    • Paste the address label covering the keyhole
    • Use separate box for different cases
    • All these should be done in the presence of a Veterinary Officer
  • Despatch
    • The samples collected should be despatched to Forensic Science Laboratory as early as possible to enable toxicologic analysis done early and to avoid artefactual changes.
    • The forwarding letter consists of A covering letter with reference stating the name of the officer from whom the order has been received to forward the materials and the number and date of such order, the body of letter requesting type of examination needed, an impression of the seal used in closing the containers and the information as to how the articles have been forwarded (by person, regiatered post with acknowledgement due or courier)
    • Details of materials collected including quantity of materials collected, type of preservative used and type of packing container used.
    • Railway receipt
    • Postmortem report along with complete history giving details of signs, treatment etc.
    • Details of seal
    • A copy of First Information Report
    • A copy of request from the police.
    • One copy of the forwarding letter should be sent along with samples and one copy of the same sent separately by registered post to the Chemical Examiner and one copy should be kept in file for future reference. The forwarding letter should bear the office number and date.
    • The address of the Forensic Laboratory should be clearly written
    • The packed material may be sent through messenger for safer transport (to avoid any breakage in transit) or send them by railway parcel to the Chemical Examiner or Forensic Science Laboratory

Precautions

  • In vetrolegal cases, all samples should be collected in the presence of police
  • Collect the tissues as early as possible after death of animal
  • Collect the tissues directly in fixative / preservative
  • Cut the viscera into small pieces to ensure penetration of preservative Hard organs like liver, kidneys etc. should be collected along with capsule
  • Stomach and intestinal contents should be sent after proper ligation at both the ends
  • The bottles are filled only up to two-thirds of the height, and not to full height ( to avoid bursting due to gas formation or decomposition)
  • Tissues for histopathology must be collected in 10 % formalin or formol saline. This can be sent to Pathology Laboratory under normal temperature
  • All the packets and bottles should be carefully sealed by the officer making the examination and closed in such a manner that they cannot be opened without destroying the seal
  • The seal used should be the same through out, either a private seal or an official seal which is always kept in safe custody
  • When an officer forwards articles to the Chemical Examiner for examination, he should at the same time address and forward separately a letter to the Chemical Examiner advising their despatch
  • If the samples are not forwarded to Chemical Examiner, the viscera and other articles are preserved for a period of six months and destroyed with the consent of District Magistrate

The address of the forensic science laboratory at chennai 

The director and chemical examiner,
Forensic sciences department,
Goernment of Tamil Nadu,
Forenic House,
Kamarajar salai,
Chennai - 600 004.

 
Last modified: Friday, 23 March 2012, 10:55 AM