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Investigation strategies and methods. Overview of Microbiology Methods. May 2007. Learning objectives. At the end of the presentation, participants should: Understand what the laboratory does with samples that arrive Have an understanding of the range of test methods used to analyse samples.

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Investigation strategies and methods

Overview of Microbiology Methods

May 2007

learning objectives
Learning objectives

At the end of the presentation, participants should:

  • Understand what the laboratory does with samples that arrive
  • Have an understanding of the range of test methods used to analyse samples
managing the sample administrative
Managing the sample: administrative
  • When an outbreak sample is received it is assigned:
    • laboratory identification number
    • an outbreak identification label
managing the sample technical
Managing the sample: technical
  • Macroscopic evaluation
  • Split sample for different laboratory disciplines
  • Two possible approaches:
    • perform only those tests requested by sender
    • perform diagnostics for syndromes/clinical description (laboratory initiative)
  • Storage of samples
    • refrigerator or freezer
types of laboratory methods
Types of laboratory methods
  • Direct methods
    • look for/detect the agent
  • Indirect methods
    • detect host response to the agent
direct methods
Direct methods
  • Macroscopic evaluation
  • Direct microscopy
  • Electron microscopy
  • Staining
  • Rapid tests
  • Molecular methods
  • Propagate the agent

No propagation required

1 macroscopic evaluation
1. Macroscopic evaluation
  • Consistency
    • rice water stools for Cholera
  • Blood
  • Visible parasites
    • helminths
    • segments
2 direct microscopy
2. Direct microscopy

Wet mount technique

hanging drop

Dark background microscope

fragile organisms (e.g. spirochetes)

Viability maintained

mobility may be observed


white blood cells (denotes invasion)

red blood cells





moving bacteria

3 electron microscopy
3. Electron microscopy

Referral laboratories

Examination of viruses

e.g. Rotavirus in stool sample

Being replaced by antigen detection

Ebola virus

Photo: WHO

4 staining
4. Staining
  • Aspecific staining
    • Gram staining
  • Specific staining with chemicals
    • Ziehl Neelsen staining (Mycobacteria)
    • Modified Ziehl Neelsen staining (Cryptosporidium)
  • Specific staining with labelled antibodies
    • Immunofluorescence - used when gram stain cannot help in diagnosis (e.g. Legionella too small to be visible in a Gram stain)
5 rapid tests
5. Rapid tests
  • Goals
    • bacterial, viral or parasite antigen (surface antigen, soluble antigen)
    • toxin in biological fluids (e.g. cerebrospinal fluid, blood, urine)
  • Main techniques
    • direct agglutination: slides, cards
    • latex agglutination: slides, cards
    • immuno-chromatography: dipsticks
6 molecular methods
6. Molecular methods
  • Direct blotting
    • no amplification (enough DNA)
      • DNA of the agent is released
        • gets spotted onto a membrane and fixed
        • is recognized by labelled probes (hybridization)
          • radio-labelling
          • electro-luminescent labelling
  • Polymerase chain reaction (PCR)
    • amplification (not enough DNA)
no propagation advantages disadvantages
No Propagation: advantages/disadvantages
  • Advantages
    • fast (<1 hour)
    • inexpensive
    • no major laboratory infrastructure needed
  • Disadvantages
    • limited sensitivity
      • high concentrations needed
    • limited specificity
7 direct methods propagation required
7. Direct Methods - propagation required
  • Bacteriology and mycology
    • most agents can be propagated on culture media
  • Virology
    • most agents can be propagated on cells
  • Parasitology
    • monocellular organisms can be propagated in culture media
propagation advantages disadvantages
Propagation: advantages/disadvantages
  • Advantages
    • allows anti-microbial susceptibility testing
    • allows typing of the micro-organism
    • allows storage of the strain
  • Disadvantages
    • depends upon the viability/condition of the agent
    • takes time
types of laboratory methods1
Types of laboratory methods
  • Direct methods
    • look for/detect the agent
  • Indirect methods
    • detect host response to the agent
indirect methods
Indirect methods


  • antibodies against the agent
  • T-cell response against the agent
  • interferon
1 detecting antibodies
1. Detecting antibodies
  • Precipitation
  • Agglutination
  • Haemagglutination and haemagglutination inhibition
  • Viral neutralization test
  • Radio-immunoassays
  • Immunoflourescence
  • Immunoblotting
  • Immunochromatography
antibody detection advantages disadvantages
Antibody detection: advantages/disadvantages
  • Advantages
    • inexpensive
    • easy to perform
    • allows identification of
      • IgM (acute infection)
      • IgG (past infection)
  • Disadvantages
    • delayed response (false negative results during sero-conversion window)
    • time of infection not always clear
2 detection of t cell response
2. Detection of T-cell response
  • Intra-dermal injection of antigen (e.g. Tuberculin skin test)
    • some don’t consider this a laboratory test
t cell response advantages disadvantages
T-cell response: advantages/disadvantages
  • Advantages
    • very specific and sensitive assay for tuberculosis
    • easy to perform
  • Disadvantages
    • delayed response (few days)
    • patient has to be seen twice
  • Sequence analysis of nucleic acid fragment after PCR amplification
  • Compares alignment of nucleotides with other sequences present in different data bases for the identification of an agent
  • Confirmatory analysis
    • the final DNA fingerprint is molecular signature of the micro-organism

Investigation strategies and methods

Developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from: European Program for Intervention Epidemiology Training Canadian Field Epidemiology Program Thailand Ministry of Health Institut Pasteur