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Specimen collection

When collecting a specimen to be analysed by a microbiologist, a clinician must keep the following in mind:

  • The specimen must be representative of the condition- an appropriate site of testing must be determined.
  • Aseptic technique must be used (esp blood and CSF)- this ensures the specimen is not contaminated, and involves swabbing with an antibacterial agent and then making no contact with the swabbed area, the needle/canula or the inoculating bottle.
  • Sterile swabs, containers and needles must be used.
  • Correct transport method for the sample must be determined
    • Blood is injected into a blood culture bottle, which contains an enrichment broth that encourages bacteria multiplication. These bottles are later incubated, also to enhance microbial growth.
    • Specimens collected with a swab have the potential to dessicate (dry out) or experience toxic death from metabolic products of the normal flora of the swabbed area. To prevent this, semi-solid agar is used as a transport media to prevent drying out, and charcoal is sometimes used to inactivate toxic material. Most swabs are transported at 4° or room temperature.
    • Urine must be transported at 4° to prevent the normal flora and the pathogen from multiplying, as a correct measurement of the concentration of the organism in the urine is essential for diagnosis.
    • CSF must be kept at room temperature, as organisms which cause meningitis can be fastidious (fragile, die easily).
  • Correct labelling of the sample and accompanying documentation


In the lab

Once a sample arrives at a microbiology laboratory, firstly the specimen and patient details are registered. The sample will then undergo testing.

Macroscopic observations

This can give an indication of the types of further tests to be run. For example, bloody urine could indicate kidney disease.

Microscopic observations

Microscopy can offer a preliminary identification of a pathogen, but only if the sample is taken from a sterile site (blood, CSF, bladder, tissues, lungs, stomach) or if the pathogen is easily differentiated from the normal flora in the sample.

  • Gram staining shows if bacteria is gram positive or negative, and allows the shape of the bacteria to be seen in microscopy
  • Methylene blue is a stain used to indicate the presence of fungi
  • Electron microscopy is helpful in identifying viruses too small for a light microscope to see

Rapid tests

ELISA (Enzyme-linked immunosorbent assay)- an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme which exhibits a colour change, indicating the presence of antigen.

PCR (Polymerase chain reaction)- a selective replication of a short DNA fragment (primer) can be used to detect the presence of an organism with a specific primer by making the unique DNA replicate to a measurable amount.

Cultures

Bacteria and fungi can be cultured on agar plates to produce colonies which can help to identify them. Most bacteria grow best at pH7 (yeasts pH5-6) at 37°, and many organisms have specific oxygen requirements. There are several types of agar:

  • Basal Medium- nutrient broth
  • Enriched media- blood agar (sheep, horse, chocolate etc)
  • Selective media- allows only certain types of bacteria/fungi to grow, sometimes contains antibiotics to cut back normal flora
  • Differential media- blood agar allows haemolysis pattern to be determined

Identification- The putative pathogen is often subcultured to produce a pure culture from which a single pathogen can be identified by observing gram staining, cell morphology, cultural characteristics, biochemical characteristics and antigenic differences.

Antibiotic resistance and serology testing may be undertaken (if relevant).