micro

• Clinical Microbiology


  Hospital Laboratory Discipline
• Objectives of Clinical Microbiology

  • Identification of disease causing microbe (Pathogen)
  • Diagnosis of Infection
  • Appropriate treatment initiated
  • Antibiotic V Non-antibiotic chemotherapy
  • Promotes the recovery of the patient
  • Data can be gathered about the prevalence of pathogens and disease in the population
  • Epidemiological data
  • Control of future outbreaks
• Very simple strategy


  1. Collect specimen from patient

  2. Examine specimen for evidence of pathogen

  3. Find pathogen in specimen

  4. Find evidence of immune reaction to pathogen

  5. Arrive at a diagnosis
• Clinical Specimens


  • Material has to be appropriate to the clinical condition
  • Aseptic collection
  • Collect an appropriate quantity
  • Collect specimen before treatment is initiated
  • Fast transport from collection to laboratory analysis
• Specimen Collection Methods


  • Swabs (skin, wounds, nose, ear etc.)
  • Needles (Blood, tissue fluids, Cerebrospinal Fluid)
  • Sterile cups (mucus, stool)
  • Catheter (urine, blood)
  • Intubation (extraction of fluids from hollow organs)
• Then follow appropriate work-flow scheme and SOPs
• Clinical Microbiology Laboratories


  • Continue to rely heavily on traditional methods (culture, phenotypic, immunological and biochemical tests)
  • Due to the complexity and variability of specimens received
• New methods must be developed and validated by companies for approval in hospital labs. Then clinical labs will have to train their staff in the appropriate SOP(s).
• Sensitivity


  A test's ability to designate an individual with disease as positive. A highly sensitive test means there are few false negative results, and thus fewer cases of disease are missed. A sensitive test for an infectious disease should also be able to work with relatively low numbers of pathogens present in the specimen.
• Specificity


  The ability of a test to designate an individual who does not have a disease as negative. A highly specific test means there are few false positive results.
• It is desirable to have a test that is both highly sensitive and highly specific. This is frequently not possible. Typically there is a trade-off.
• Sensitivity


  Ability of a test to detect a true positive
• Specificity


  Ability of a test to exclude a true negative
• Is/was a pathogen present in the specimen?

  1. Specimen subjected to a number of treatments

  2. Microscopy

  3. Laboratory Culture of microorganisms

  4. Direct detection: Immunology, nucleic acid technology, analytical chemistry
• Microscopy Techniques


  • Bright Field Microscopy
  • Phase-Contrast Microscopy
  • Dark-Field Microscopy
  • Fluorescence Microscopy
  • Electron Microscopy
• Bright Field Microscopy


  • Can be used directly on specimen or on samples after further processing. Examples: Gram Stain and Acid Fast Stain.
• Phase-Contrast Microscopy


  • Contrast provided by difference in refractive indices of components of the sample. Staining NOT required. Use with living samples, particularly useful for fungal diagnosis.
• Dark-Field Microscopy


  • Light hits slide at an angle. If an object present light it deflected into lens. Staining NOT required. Use with difficult to culture pathogens e.g. Treponema pallidum [Syphillis]
• Fluorescence Microscopy


  • UV light excites a dye to produce a fluorescent signal. Fluorochromes: dyes interact directly with a microbial component. Immunofluorescence: dyes conjugated to antibodies.
• Electron Microscopy


  • High resolution but relatively complex sample preparation. Expensive equipment.
• Advantages of Microscopy


  • Rapid
  • Cheap
  • Adaptable according to sample
  • Specificity possible (antibody/nucleic acid staining)
• Disadvantages of Microscopy


  • Limited specificity
  • No recovery of organism for further analysis
  • Poor sensitivity
  • Expensive for viruses
• Why are Ziehl-Neelson and antibody staining of TB useful for diagnosis using Microscopy?
• Bacterial Diagnosis using Laboratory Culture

  1. Inoculation of specialised medium with specimen

  2. Incubate for specified time

  3. Recover individual colonies (possibly grow a culture from a single colony)

  4. Look at morphology, gram reaction, biochemistry to obtain an ID
• Laboratory Culture Strategy


  • Selective Medium
  • Sample
  • Gram Stain
  • Biochemical tests
  • Immunological tests
  • Molecular tests
  • Enrichment: general or selective
• Bacteriological Media


  • General purpose media (Tryptone Soy Agar, Brain-Heart Infusion Agar)
  • Selective media (Vogel and Johnston Agar)
  • Differential media (MacConkey Agar, Blood agar)
  • Chromogenic media (Most recent development, Species-specific chromogens, Beta-galactosidase, Beta glucuronidase)
• Chromogen


  A chemical compound, itself without colour, that can be transformed into a coloured compound, or can react with another material to form a coloured compound
• Chromogenic Media


  Chromogen transformed by a specific enzyme. Enzyme only found in one particular species.
• Reasons to use Chromogenic Media


  • Improve overall specificity of a diagnostic medium
  • Positive colonies are easy to identify
  • Reduce number of sub-cultures required
• Advantages of Culture


  • Unequivocal demonstration of presence of pathogen
  • Pathogen is available for antimicrobial susceptibility testing
  • Established technology and standards
  • Relatively inexpensive
  • Low tech
  • Automation of primary sample processing and plating has reduced time spent on mundane tasks (plating) associated with the initial steps of clinical bacteriology and improved laboratory efficiency
• Molecular methods are often more sensitive than the culture methods to which they are being compared. This can be problematic during validation of new molecular tests when specimens are nucleic acid positive but culture negative.
• Disadvantages of Culture


  • Inherently slow and laborious
  • Useless in certain instances (Chlamydia spp., Rickettsia spp., Mycobacterium leprae)
  • Difficult to culture pathogens (Mycobacterium tuberculosis, protozoan parasites)
  • Culturing methods assume that all pathogens are culturable all of the time and does not take into account either injured organisms or viable but non-culturable organisms
• Bacterial Diagnosis using Laboratory Culture

  1. Selective Medium

  2. Sample

  3. Gram Stain

  4. Biochemical tests

  5. Immunological tests

  6. Molecular tests

  7. Enrichment: general or selective
• Biochemical Tests for Bacteria

  • Catalase test
  • Sugar fermentation tests
  • API: Miniaturised biochemical tests on a strip
• Catalase Test


  2 H2O2 → 2 H2O + O2. S. aureus positive, Streptococcus negative.
• Sugar Fermentation Tests


  Specific sugar and pH indicator in the broth. Purple: no fermentation. Yellow: fermentation occurs and acid produced. Yellow with gas: fermentation occurs and both acid and gas produced.
• API: Miniaturised biochemical tests on a strip. Can easily see that these three species are different based on the different patterns. ID based on database.
• Bacterial Confirmation: Step by Step Approach

  1. Selective Medium

  2. Sample

  3. Gram Stain

  4. Biochemical tests

  5. Immunological tests

  6. Molecular tests

  7. Enrichment: general or selective
• Immunodiagnostics


  Takes advantage of the specificity of antibodies. Recognition of species. Fast assays. Very flexible. Direct analysis of sample to detect presence of pathogen. Used to confirm identification when pathogen isolated.
• Immunodiagnostic Techniques


  • Agglutination
  • ELISA