Diagnostic techniques in medical microbiology

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Diagnostic techniques in medical microbiology
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The diagnosis of infectious diseases

• Clinical
• Radiological
• Laboratory investigation
• microbiological
• haematological and other

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Diagnostic microbiology

• Patient
• Diagnosis

Microbiology Laboratory
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Diagnostic microbiology

• Microscopic methods
• Cultural methods
• Serological methods
• Molecular methods

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Direct microscopy

• Light microscopy
• Unstained preparations
• Simple stains - Gram, Giemsa
• Special stains - Ziehl-Nielson, India ink
• Immunoflourescence
• Electronmicroscopy

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Gram stain

• Staining of micro-organisms dependent on cell
  wall structure
• Sensitivity low (gt100,000 organisms / ml
  necessary to detect bacteria)
• Useful for examination of sterile specimens

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Ziehl-Neilson

• Identification of organisms that are acid and
  alcohol fast (AAFB)
• Mycobacteria possess a lipid rich wall and retain
  pink dye
• limited number of organisms are AAFB

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Immunofluorescence

• Direct
• detection of antigen by use of fluorescence
  labelled antibodies
• sensitive and specific
• used for diagnosis of viral infection, Chlamydia,
  legionella, Treponema pallidum, Giardia,
  Pneumocystis
• Indirect
• detection of antibody

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Electron-microscopy
Important diagnostic technique for many viral
infections
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Culture

• Used in bacterial, fungal, viral, parasitic
  diagnosis
• Mainstay of bacterial diagnosis
• Liquid media based culture
• Solid agar based culture
• Ensures organism available for
• susceptibility testing
• epidemiological studies
• Types of specimen
• blood, CSF, swabs, fluid sputum, urine

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Blood cultures

• Continuous bacteraemia
• Low level (1-30 bacteria/ml)
• Volume is the major determinant of yield
• Contamination

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Serology

• Depend on the detection of either antibody and/or
  antigen in patients serum
• Antigen detection useful in acute diagnosis
• Antibody detection may be delayed
• IgM vs IgG
• Types of tests
• Precipitation
• Agglutination
• Complement fixation (CFT)
• Radioimmunoassay
• Enzyme immunoassays (EIA)

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Molecular techniques

• Allows the detection of either RNA or DNA
• Western blotting - protein detection
• Southern blotting - DNA detection
• Polymerase chain reaction - DNA or RNA detection

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Collection of specimens

• Differential diagnosis
• Sterile Vs non sterile sites
• Sterile e.g. Blood, CSF,
• Non sterile e.g. throat swabs, skin swabs, sputum

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Diagnosis of urinary tract infection

• Blood
• Urine
• specimen type mid stream urine (MSU), catheter
  specimen urine(CSU), early morning urine(EMU),
  supra-pubic aspirate(SPA)
• Investigation
• urinalysis
• microscopic examination
• culture

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Diagnosis of pneumonia

• Blood culture
• Respiratory tract specimens
• sputum, bronchoalveolar lavage
• Serology
• acute/convalescent
• Molecular techniques

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Diagnosis of skin/soft tissue sepsis

• Blood cultures
• Skin / soft tissue swabs
• Biopsy

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Diagnosis of bacteriaemia

• Blood culture
• liquid system
• sensitivity of 1 CFU/ml
• level bacteraemia greater in children
• problems with contamination
• advent of molecular techniques