Culture and identification of
infectious agents, Lecture 25
Dr. Alvin Fox
1
Key Terms
Isolation (culture)
• Agar plate
plate/colonies
• Liquid media
Identification & taxonomy
• Family
• Genus
• Species
• Type
• Strain
After culture
• Biochemical (physiological) tests
• Genetic tests
- Polymerase chain reaction (PCR)
- Sequencing
- DNA-DNA homology/arrays
- Restriction enzymes (digests)
• Chemical
- fatty acid/protein profiling
• Immunological
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
3
Species versus strains
- selecting discriminating features
4
Classification
•
•
•
•
•
Strain: one single isolate or line
Type: sub-set of species
Species: related strains
Genus: related species
Family: related genera
5
Both terms define the species name
Streptococcus pyogenes
Genus part
Species part
So Streptococcus pyogenes and Streptococus agalactiae
would be two species in the same genus
In a report
On first usage: Streptococcus pyogenes
On second usage: S. pyogenes
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Identification of infectious agents
in the diagnostic laboratory
• Aids treatment
• Helps antibiotic selection
• General hospital laboratory
– physiological tests
• More fully equipped 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
10
Isolation and identification
• Step 2: Colonies Gram stained
– cells observed microscopically
11
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
14
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
– PCR
– Sequencing
– Restriction digestion
• Hybridization (probes, arrays)
• % 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
+
0% Homology
Strain 2
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 (low molecular weight)
proteins
• Proteomics: defining all proteins
expressed by a species under specific
growth conditions
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Bacterial DNA sequences can be
amplified directly from human body
fluids
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Laboratory diagnosis without
culture
In general, when speed is of essence
Of additional importance:
• The organism grows poorly/slowly
• The organism can’t be cultured
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Microscopy
• spinal fluids (meningitis)
• sputum (tuberculosis)
• sensitivity poor
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Streptococcal Agglutination Test
Streptococcal
antigenic extract
Antibody
Latex beads
27
Serologic identification
• antibody response to the infecting agent
• several weeks after an infection has occurred
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Epidemics
• associated with particular strains/types
• state or federal laboratory system
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