Académique Documents
Professionnel Documents
Culture Documents
infectious agents
Dr. Alvin Fox
1
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
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
Identification of infectious agents
in the diagnostic laboratory
• Aids treatment
• Helps antibiotic selection
• General hospital laboratory
– physiological tests
• Reference laboratories
– Genetic tests
6
Steps in isolation and identification
7
Sheep blood agar plate culture
9
Isolation and identification
• Step 2: Colonies Gram stained
– cells observed microscopically
10
Gram negative Gram positive
Heat/Dry
Iodine Fix
Alcohol de-
de stain
Safranin stain
11
Gram stain morphology
• Shape
– cocci (round)
– bacilli (rods)
– spiral or curved (e.g. spirochetes)
15
Clinical Microbiology
Laboratory Bench
16
Step 4:
Antibiotic susceptibility testing
17
Antibiotic susceptibility testing
Susceptible Not susceptible
Bacterial
lawn
No Growth
growth
Antibiotic disk
18
Molecular
differentiation
• Genomics
• Gene characterization
– Sequencing
– PCR
– Restriction digests
• Hybridization
• % guanine + cytosine 19
16S rRNA Sequencing
20
Real-time PCR
ds DNA
Cycle one
Dye
Cycle two
Cycle 30
2 30 21
DNA-DNA hybridization
Strain 1
Heat
+ Strain 2
22
Profiles
• Long chain fatty acids
- structural (e.g. cell membrane)
• Short chain
- metabolic
- volatiles
- Fatty acids/alcohols
23
• Protein profiling: defining a species by
characteristic proteins
24
Rapid diagnosis without culture
• WHEN AND WHY?
• grow poorly
• can not be cultured
25
Streptococcal Agglutination Test
Streptococcal
antigenic extract
Antibody
Latex beads
26
Bacterial DNA sequences amplified
directly from human body fluids
27
Microscopy
• spinal fluids (meningitis)
• sputum (tuberculosis)
• sensitivity poor
28
Serologic identification
• antibody response to the infecting agent
29
Epidemics
30