Collection of samples for diagnosis of bacterial and viral zoonoses

COLLECION OF SAMPLES FOR BACTERIOLOGICAL AND VIROLOGICAL EXAMINATION

ISOLATION OF BACTERIA

  • Collect specimen before administration of antibiotics.
  • Follow aseptic precautions.
  • Swabs are better transported in transport medium (swabs should never become dry).
  • Organ pieces are collected, stored and transported in chilled conditions till it reaches the laboratory.
  • Ideal method is submission through special messenger in ice-cold condition. 
  • Preservatives
    • Milk: Mix o ne-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 or on ice, if it is to be transported to the distance place.
    • Heart blood swabs: Before opening the chambers of the heart, singe the epicardium over the right ventricle with the hot spatula. Make an incision with the sterile scalpel, dilate the opening with the sterile forceps and then collect the blood swab. Put it into the tube containing the charcoal transport medium, carefully seal it and dispatch to the laboratory.
    • Organs: Should be sent tightly packed in water proof sterile polythene pack and to be sent on ice pack.

Transport medium and its composition

  • Buffered glycerol saline
    • Glycerol: 300mL
    • Sodium chloride: 4.2g
    • Disodium hydrogen phosphate: 10g
    • Phenol red aqueous solution: 15mL (0.02%)
    • Water: 700mL
    • Dissolve sodium chloride in water and add glycerol. Add disodium hydrogen phosphate to dissolve. Add phenol red and adjust pH to 8.4. Aliquot into containers and sterilize by autoclaving. 
  • Stuart transport medium
    • Thioglycolic acid: 2mL
    • Sodium glycerophosphate: 100mL
    • Aqueous calcium chloride: 20%
    • Distilled water: 900mL
    • Mix the ingredients and adjust pH to 7.2 with 1N sodium hydroxide solution. 
  • Charcoal transport medium
    • Sodium chloride: 4g
    • Potassium chloride: 0.2g
    • Dipotassium phosphate: 1.7g
    • Charcoal: 10g
    • Agar: 4g
    • Mix the ingredients in 1000mL of distilled water; adjust pH to 7.2 and autoclave at 121ºC for 15 minutes.

SEROLOGICAL EXAMINATION

  • Collection of blood samples for serum
    • Collect blood in a sterile test tube without anticoagulant, or use vaccutainer. The volume of blood depends on the requirement for the test.
    • Allow the blood to clot by keeping the tube (at room temperature) undisturbed over the plain surface by slanting position so as to make more surface area and oozing of maximum quantity of serum.
    • After 2-3 hours of clotting, separate the serum; or centrifuge the clotted blood at 1500 rpm for 5 minutes and then transfer the serum to a sterile serum storage vial.
    • Despatch the serum sample to the laboratory on ice pack; or store at refrigeration temperature until submission to the lab.
    • Collect paired sera - blood samples from acute and convalescent stage usually at 21 days interval.
    • Points to be remembered
    • Haemolysis of blood should be avoided.
    • Adequate quantity of serum needed for the test should be submitted.
    • Disease suspected should be mentioned.
    • Serum sample from ailing animals, convalescent animals and clinically normal animals is to be followed.
    • Serum should be inactivated at 56ºC for 30 minutes to minimize proteolytic activity.
    • Chemicals such as pinch of sodium azide or sodium fluoride (0.1%), merthiolate (0.01%) can be added to minimize bacterial and/or fungal contamination. Preservatives should not be used, if the serum is used for ELISA.

VIROLOGICAL EXAMINATION

  • Collect clinical specimens in sterile containers from clinically infected animals, mainly at the early stage of infection or at necropsy.
  • Along with clinical materials, also collect blood samples in sterile test tubes from clinically infected animals at the early stage of infection and then at the recovery stage of infection i.e. approximately 3 weeks after the infection (paired sera).  
  • Points to be remembered
    • Clinical specimen should be submitted immediately to the laboratory on ice, if possible through special messenger (as some of the viruses are highly fragile outside the host).
    • Transport medium should be used to prevent inactivation of virus in the clinical specimen.
    • Example: commercially available transport medium or sterile 10% glycerol saline or sterile PBS or sterile normal saline or sterile water.
    • Glycerol saline should not be used as preservative for some viruses.
Last modified: Wednesday, 16 May 2012, 6:18 AM