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 than 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, 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
- Stroke specimen out on the medium with a wire loop to ensure a reducing inoculum.
- Sterilize loop in Bunsen flame between each group of strokes.
- Gives separate 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 biological techniques.
Disadvantages
- Impossible to identify bacteria by colonial morphology.
- Cannot estimate relative proportion of different bacterial species.
Blood culture media
- Blood cultures 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 specialized incubators.
- Most pathogens are incubated at 37°C.
Sources
Publications
Refereed papers
- Rosenblatt J E (1991) Laboratory tests used to guide antimicrobial therapy. Mayo Clinic Proceedings 66 , pp.942-8.
- Salmon S A, Watts J L, Walker R D, and Yancey R J (1995) Evaluation of a commercial system for the identification of gram-negative, nonfermenting bacteria of veterinary importance. Journal of Veterinary Diagnostic Investigation 7 , pp.161-4.
Other sources of information
- Balows A, Hausler W J, Herrmann K L, Isenberg H D, and Shadomy H J (1991) Manual of Clinical Microbiology 5th Edn. American Society for Microbiology. ISBN 1-55581-030-6.
- Sleigh J D, and Timbury M C (1986) Notes on Medical Bacteriology Chruchill Livingstone. ISBN 0-443-033327-7.
- Jones R L (1990) Laboratory diagnosis of bacterial infections. In: Infectious Diseases of the Dog and Cat Ed Greene C E. Philadelphia: W B Saunders. pp. 453-460.
