Faculty: Dr' Alvin Fox

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CLASSIFICATION IDENTIFICATION OF
INFECTIOUS AGENTS
Faculty Dr. Alvin Fox
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Key Terms

• Isolation (culture)
• Agar plate/colonies
• Liquid media test tube - bulk

• Biochemical (physiological) tests
• Molecular tests
• DNA-DNA homology
• 16S rRNA sequencing
• Chemical profiling
• Non culture based detection
• Polymerase chain reaction- (PCR)
• Agglutination (antigen detection)
• Stain
• Serology (antibody detection)

• Identification taxonomy
• Family
• Genus
• Species
• Type
• Strain

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Identification in the diagnostic laboratory

• Aids treatment
• Susceptibility antibiotic selection
• Based on taxonomy
• Simple, low cost, rapid

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Classification

• Strain one single isolate or line
• Species related strains
• Type sub-set of species
• Genus related species
• Family related genera

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Steps in isolation and identification

• Step 1. Streaking culture plates
• colonies on incubation (e.g 24 hr)
• size, texture, color, hemolysis
• oxygen requirement

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Blood Agar Plate
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Isolation and identification

• Step 2. Colonies Gram stained
• cells observed microscopically

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Gram Stain
Gram negative
Gram positive
Heat/Dry
Crystal violet stain
Iodine Fix
Alcohol de-stain
Safranin stain
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Gram stain morphology

• Shape
• cocci (round)
• bacilli (rods)
• spiral or curved (e.g. spirochetes)
• Single or multiple cells
• clusters (e.g. staphylococci)
• chains (e.g. streptococci)
• Gram positive or negative

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Step 3. Isolated bacteria are speciated

• Generally using physiological tests

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Typical Culture Laboratory Bench
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Step 4. Antibiotic susceptibility testing
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Antibiotic susceptibility testing
Susceptible
Not susceptible
Bacterial lawn
Growth
No growth
Antibiotic disk
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Taxonomic characterization of bacteria

• Based on molecular or chemical analysis
• Species comparisons involving multiple strains

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Molecular analysis

• Historical
• guanine (G) cytosine (C) ( GC)
• Now
• Hybridization
• Gene characterization
• sequencing
• other

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DNA-DNA hybridization
Strain 1
Heat

Strain 2
0 Homology
100 Homology
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DNA-DNA hybridization

• Groups bacterial strains into species
• Below species level
• little or no relatedness

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16S rRNA Sequencing

• similarity above species level
• allows relatedness comparisons of all bacteria
• closely related bacterial species may be
  identical
• development of clinical tests based on sequence

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Structural profiles

• long chain fatty acids
• widely used
• carbohydrates
• ubiquinones

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• Metabolic profiles
• volatiles
• alcohols
• short chain fatty acids

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Rapid diagnosis without culture

• WHEN AND WHY?
• grow poorly
• isolation
• slow
• may not be clinically useful
• can not be cultured
• isolation impossible

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Rapid Strep Test
Streptococcal antigenic extract
Antibody
Latex beads
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Bacterial DNA sequences amplified directly from
human body fluids

• Polymerase chain reaction (PCR)
• Great success in rapid diagnosis
• of tuberculosis.

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Microscopy

• spinal fluids (meningitis)
• sputum (tuberculosis)
• sensitivity poor

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Serologic identification

• antibody response to the infecting agent
• several weeks after an infection has occurred