Microbial diagnostic testing

• Gram stain:

  • Simplest stain - general go-to
  • Often performed alongside bacterial culture
  • Samples collected using sterile swabs, from patient samples, or use fine-needle aspiration
  • Samples might be blood, urine, CSF, sputum, faeces, tissue or swabs of wounds, throats etc.
  • Allows direct visualisation of bacteria: present or absent, gram-positive or gram-negative
• Gram staining procedure:

  • Crystal violet (purple) 60 seconds
  • Iodine (orange) 60 seconds
  • 95% ethyl alcohol (transparent) 5-10 seconds
  • Safranin (pink) 45 seconds

  Gram negative bacteria stain pink and gram postive bacteria stain purple
• Gram negative bacteria have an outer membrane, whereas gram-positive bacteria have a thicker peptidoglycan layer
• Culture:

  • Microorganisms placed in ideal conditions for replication
  • Blood culture bottles placed in incubators, alarm is triggered if threshold is met
  • Cultured sample is stained and visualised, but also cultured on agar for sensitivity testing
• Culture bottles are placed in incubators with automated monitoring: traceable, rapid workflow, immediate alerts (time to detection)
• Disadvantages/limitations of culture:

  • Time required
  • Low bacterial load in samples - false negatives
  • Difficult-to-culture organisms
• Disc diffusion - a lawn of bacteria grows but may be inhibited if sensitive to certain antibiotics - zones of inhibition indicate sensitivity
• The E-test allows for determination of the minimum inhibitory concentration of a given antibiotic against a given microorganism
• Use of ELISA to diagnose Clostridioides difficile infection

  • Gram positive spore-forming anaerobic bacillus
  • Leading cause of hospital-derived diarrhoea from prolonged use of antibiotics
  • ELISA used to detect glutamate dehydrogenase, a bacteria-produced enzyme
  • May be used alongside latex bead agglutination test to detect toxins
• PCR:

  • Denaturation
  • Annealing
  • Extension

  Multiple cycles
• Gram-staining separates bacteria into Gram-positive and Gram-negative organisms, depending on the thickness of peptidoglycan present in the cell wall; Gram-positive bacteria have a thick layer of peptidoglycan, whereas Gram-negative have a thin layer.

  Not all bacteria can be gram-stained – Mycobacterium tuberculosis is the causative organism of tuberculosis and is considered gram-indeterminate
• • Positively charged crystal violet added - binds to negatively charged cell components.
  • Iodine added, forms complexes with CV. Stain purple.
  • Decolouriser added to remove complexes. If thin peptidoglycan layer, complexes exit, removing blue colouration.
  • Cells stained red with safranin.

  Gram-positive have thick peptidoglycan wall; retain the crystal violet when washed. Safranin retained but obscured by crystal violet. Stain purple.

  Gram-negative have outer lipopolysaccharide layer. Decolouriser dissolves lipids, exposing thin peptidoglycan. Complexes exit - decolourises cell. Stain red.
• Direct ELISA test detects presence of a specific antigen:

  1. monoclonal antibodies bind to bottom of test plate
  2. antigen molecules in sample bind to antibody. rinse excess
  3. mobile antibody with 'reporter enzyme' attached binds to antigens that are 'fixed' on the monoclonal antibodies. rinse excess
  4. add substrate for reporter enzyme. positive result: colour change
• Indirect ELISA test detects presence of an antibody against a specific antigen

  1. antigens bind to bottom of test plate
  2. antibodies in sample bind to antigen. wash away excess
  3. secondary antibody with 'reporter enzyme' attached binds to primary antibodies from the sample
  4. add substrate for reporter enzyme. positive result: colour change
• • The three stages of PCR are:
  • Denaturation – the double-stranded DNA is heated to 95°C which breaks the hydrogen bonds that bond the two DNA strands together
  • Annealing – the temperature is decreased to between 50 - 60°C so that primers (forward and reverse ones) can anneal to the ends of the single strands of DNA
  • Elongation / Extension – the temperature is increased to 72°C for at least a minute, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules
• PCR requires:

  • Target DNA or RNA being amplified
  • Primers (forward and reverse) – short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of DNA or RNA being copied. Define region to be amplified by identifying to DNA polymerase where to begin building new strands
  • DNA polymerase – enzyme used to build new DNA or RNA strand. Taq polymerase - does not denature at high temperature in first stage of PCR, and optimum temperature is high enough to prevent annealing of DNA strands that have not been copied yet
  • Free nucleotides
  • Buffer solution – optimum pH
• • PCR is used to produce large quantities of specific fragments of DNA or RNA from very small quantities (even just one molecule of DNA or RNA). By using PCR scientists can have billions of identical copies of the DNA or RNA sample within a few hours