Culture and identification of infectious agents

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Culture and identification of infectious agents
Dr. Alvin Fox
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Key Terms

• After culture
• Biochemical (physiological) tests
• Genetic tests
• Sequencing,
• Polymerase chain reaction (PCR)
• DNA-DNA homology
• Restriction enzymes (digests)
• Chemical
• - fatty acid/protein profiling
• Immunological
• Direct detection (i.e. without culture)
• PCR
• Antigen detection
• Staining (e.g. Gram stain)
• Serology (antibody detection)

• Isolation (culture)
• Agar plate plate/colonies
• Liquid media

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

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Taxonomy

• Defines common traits among strains for a
  bacterial species
• Usually genetic
• Allows development of diagnostic kits

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Species versus strains- selecting discriminating
features
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Classification

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

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Identification of infectious agents in the
diagnostic laboratory

• Aids treatment
• Helps antibiotic selection
• General hospital laboratory
• physiological tests
• Reference laboratories
• Genetic (less commonly protein) tests

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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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Sheep blood agar plate culture
Bacillus anthracis
Bacillus cereus.
CDC/Dr. James Feeley
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Mixed colonies
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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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Clinical Microbiology 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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Molecular differentiation

• Genomics
• Gene characterization
• Sequencing
• PCR
• Restriction digests
• Hybridization
• guanine cytosine

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16S rRNA Sequencing

• Differentiates bacterial species
• Development of clinical tests based on sequence
  (e.g. PCR)

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Real-time PCR
ds DNA
Cycle one
Dye
Cycle two
Cycle 30
2 30
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DNA-DNA hybridization
Strain 1
Heat

Strain 2
0 Homology
100 Homology
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Profiles

• Long chain fatty acids
• - structural (e.g. cell membrane)

• Short chain
• - metabolic
• - volatiles
• - Fatty acids/alcohols

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Protein profiling

• M.W. of a few characteristic proteins
• not proteomics

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Rapid diagnosis without culture

• WHEN AND WHY?
• grow poorly
• can not be cultured

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Streptococcal Agglutination 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