Culture and identification of

infectious agents
Dr. Alvin Fox

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 Key Terms After culture
Isolation  Biochemical (physiological) tests
(culture)  Genetic tests
Agar plate Sequencing,
plate/colonies Polymerase chain reaction (PCR)
Liquid media DNA-DNA homology
Identification & taxonomy
 Restriction enzymes (digests)
Family
 Chemical
Genus
Species - fatty acid/protein profiling
 Immunological
Type
Strain
Direct detection (i.e. without culture)
PCR
Antigen detection
Staining (e.g. Gram stain) 2
Serology (antibody detection)
 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 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 cereus. Bacillus anthracis

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 negative Gram positive
Heat/Dry

Crystal violet stain

Iodine Fix

Alcohol de-
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

No Growth
growth

Antibiotic disk

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 Molecular
differentiation
• Genomics

• Gene characterization
– Sequencing
– PCR
– Restriction digests

• Hybridization

• % guanine + cytosine 19
 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 21
 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: defining a species by
characteristic proteins

• Proteomics: defining all proteins

expressed by a species under specific
growth conditions

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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

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 Epidemics

• associated with particular strains

• the responsibility of the state or federal laboratory system

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