Title: Culture and identification of infectious agents, Lecture 25
1Culture and identification of infectious agents,
Lecture 25
Dr. Alvin Fox
2Key Terms
- 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)
- Serology (antibody detection)
- Isolation (culture)
- Agar plate plate/colonies
- Liquid media
- Identification taxonomy
- Family
- Genus
- Species
- Type
- Strain
3Taxonomy
- Defines common traits among strains for a
bacterial species - Usually genetic
- Allows development of diagnostic kits
4Species versus strains- selecting discriminating
features
5Classification
- Strain one single isolate or line
- Type sub-set of species
- Species related strains
- Genus related species
- Family related genera
6Both 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
7Identification of infectious agents in the
diagnostic laboratory
- Aids treatment
- Helps antibiotic selection
- General hospital laboratory
- physiological tests
- More fully equipped laboratories
- Genetic tests
8Steps in isolation and identification
- Step 1 Streaking culture plates
- colonies on incubation (e.g 24 hr)
- size, texture, color, hemolysis
- oxygen requirement
9Sheep blood agar plate culture
Bacillus anthracis
Bacillus cereus.
CDC/Dr. James Feeley
10Mixed colonies
11Isolation and identification
- Step 2 Colonies Gram stained
- cells observed microscopically
12Gram Stain
Gram negative
Gram positive
Heat/Dry
Crystal violet stain
Iodine Fix
Alcohol de-stain
Safranin stain
13Gram 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
14Step 3 Isolated bacteria are speciated
- Generally using physiological tests
15Clinical Microbiology Laboratory Bench
16Step 4 Antibiotic susceptibility testing
17Antibiotic susceptibility testing
Susceptible
Not susceptible
Bacterial lawn
Growth
No growth
Antibiotic disk
18Molecular differentiation
- Genomics
- Gene characterization
- PCR
- Sequencing
- Restriction digestion
- Hybridization (probes, arrays)
- guanine-cytosine
1916S rRNA Sequencing
- Differentiates bacterial species
- Development of clinical tests based on sequence
(e.g. PCR)
20Real-time PCR
ds DNA
Cycle one
Dye
Cycle two
Cycle 30
2 30
21DNA-DNA hybridization
Strain 1
Heat
Strain 2
0 Homology
100 Homology
22Profiles
- Long chain fatty acids
- - structural (e.g. cell membrane)
- Short chain
- - metabolic
- - volatiles
- - Fatty acids/alcohols
23- Protein profiling defining a species by
characteristic (low molecular weight) proteins - Proteomics defining all proteins expressed by a
species under specific growth conditions
24Bacterial DNA sequences can be amplified directly
from human body fluids
25Laboratory diagnosis without culture
- In general, when speed is of essence
- Of additional importance
- The organism grows poorly/slowly
- The organism cant be cultured
-
-
26Microscopy
- spinal fluids (meningitis)
- sputum (tuberculosis)
- sensitivity poor
27Streptococcal Agglutination Test
Streptococcal antigenic extract
Antibody
Latex beads
28Serologic identification
- antibody response to the infecting agent
- several weeks after an infection has occurred
29Epidemics
- associated with particular strains/types
- state or federal laboratory system