Download bacteria identification

Culture and identification of
infectious agents
Dr. Abdullatif Neamatallah
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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)
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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 (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 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-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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‫شريحة نقية‬
‫‪SLIDE‬‬
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‫شريحةغير نقية‬
‫‪MIXED SLIDE‬‬
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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
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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
• 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
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