ISSN 2398-2977      

Bacteriology

pequis
Contributor(s):

Richard Walker


Bacterial Culture

  • Bacteria differ in their growth requirements.
  • Most pathogenic bacteria are heterotrophs, ie they need organic materials for growth.

Specimen collection

  • Collect a representative sample of bacteria and examine the material promptly.
  • Collect specimens from areas where organisms most likely to be found, eg edge of spreading skin lesions, incised pustules, contaminated cavities.
  • Collect specimens prior to use of antibiotics, if possible.
  • Avoid contamination of the specimen; use aseptic technique.
  • Cystocentesis is the most reliable method of avoiding contamination of urine samples.
  • Label specimens clearly.
  • Remember that an isolated bacterium is not necessarily responsible for the disease.

Constituents of culture media

  • Water.
  • Sodium chloride and other electrolytes.
  • Peptone - protein digest.
  • Meat or yeast extract, used to enrich media.
  • Blood - usually defibrinated horse or sheep blood.
  • Agar - carbohydrate derived from seaweed: melts at 90°C but does not solidify until cooled to 40°C.
  • Most media are prepared from commercial dry ingredients which are reconstituted and sterilized before use.

Solid media

  • Blood agar, chocolate agar   Taylorella equigenitalis: culture - chocolate agar  , MacConkey agar,etc.
  • These are dispensed in petri dishes.
  • Contain 1.5% agar to give the consistency of a firm jelly.
  • Selective media contain ingredients which inhibit unwanted contaminants but allow certain pathogens to grow.

Plating technique

  • Streak specimen out on the medium, in a quadrant pattern, with a wire loop to ensure a reducing inoculum.
  • Sterilize loop in a Bunsen flame or an electronic sterilizing device between each quadrant.
  • Gives isolated colonies.

Advantages

  • Allows separate colony formation.
  • Can identify bacteria from colonial morphology.
  • Can estimate proportion of different bacterial species in a mixed inoculum.
  • Can obtain pure cultures by picking isolated colonies onto fresh medium.

Liquid media

  • Nutrient broth, Selenite F broth, etc.
  • Dispensed in tubes.
  • Growth is recognized by turbidity in the medium.

Uses

  • Some bacteria grow only in fluid media.
  • Used to test biochemical activities of bacteria for identification.
  • Facilitate isolation of fastidious organisms or those present in small numbers in the sample.
  • Enrichment media encourage preferential growth of a particular species and contain inhibitors for contaminants.
  • Used to grow up bacteria for molecular biologic techniques.

Disadvantages

  • Impossible to identify bacteria by colonial morphology.
  • Cannot estimate relative proportion of different bacterial species.

Blood culture media

  • Blood cultures   Blood: microbiology  should be taken as fever begins, and often need to be repeated in 1-3 hours.
  • Two bottles with rubber seals and perforated metal cap.
  • Inoculated through hole in cap.
  • Contain:
    • Nutrient broth for aerobic culture, sometimes containing an agar slope.
    • Nutrient broth with sodium thioglycollate (reducing agent) for anaerobic culture.

Transport media

  • Used to protect bacteria during transit to the laboratory.
  • Usually buffered.
  • Non-nutritive to prevent overgrowth by contaminants.
  • May contain charcoal to absorb toxic metabolites from the bacteria to ensure survival.
  • Common types include Amies' and Stuart.

Incubation

  • Most pathogens are aerobic.
  • Some require addition of 5 or 10% carbon dioxide.
  • Anaerobic bacteria are incubated in sealed jars or a specialized anaerobe chamber.
  • Most pathogens are incubated at 37°C.

Bacterial Identification

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Microscopy

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Antibiotic Susceptibility Testing

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Further Reading

Publications

Refereed papers

  • Recent references from PubMed and VetMedResource.
  • Scarff D H (2004) Microscopy in practice. UK Vet (8), 77-81.
  • Salmon S A, Watts J L, Walker R D & Yancey R J (1995) Evaluation of a commercial system for the identification of gram-negative, non-fermenting bacteria of veterinary importance. J Vet Diagn Invest (1), 161-164 PubMed.
  • Rosenblatt J E (1991) Laboratory tests used to guide antimicrobial therapy. Mayo Clinic Proc 66 (9), 942-948 PubMed.

Other sources of information

  • Balows A, Hausler W J, Herrmann K L, Isenberg H D & Shadomy H J (1991) Manual of Clinical Microbiology. 5th Edn. American Society for Microbiology. ISBN 1-55581-030-6.
  • Jones R L (1990) Laboratory diagnosis of bacterial infections.In: Infectious diseases of the Dog and Cat. Ed: Greene C E. W B Saunders, USA. pp 453-460.
  • Sleigh J D & Timbury M C (1986) Notes on Medical Bacteriology. Churchill Livingstone. ISBN 0-443-033327-7.

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