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
Isolation (culture)
Agar plate plate/colonies
Liquid media
Identification & taxonomy
Family
Genus
Species
Type
Strain
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)
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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 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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CDC/Dr. James Feeley
Sheep blood agar plate culture
Bacillus anthracis
Bacillus cereus.
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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
Safranin stain
Alcohol de-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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No growth
Antibiotic susceptibility testing
Susceptible
Not susceptible
Bacterial
lawn
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
Cycle one
Cycle two
Cycle 30
2 30
ds DNA
Dye
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DNA-DNA hybridization
100% Homology
+
Heat
0% Homology
Strain 1
Strain 2
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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
Latex beads
Streptococcal
antigenic extract
Antibody
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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
Epidemics
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associated with particular strains

the responsibility of the state or federal laboratory system