This document discusses the process of identifying infectious agents through culture and laboratory testing. It begins by defining key terms related to isolation, identification, and taxonomy of microbes. It then outlines the steps laboratories take to isolate microbes from samples via culture, identify them using staining and biochemical/genetic tests, and determine antibiotic susceptibility. Molecular techniques like PCR, sequencing, and protein/fatty acid profiling can also directly identify microbes or differentiate bacterial strains without requiring culture. The goal is to accurately identify the infectious agent to aid treatment and antibiotic selection.

1. 1
Culture and identification of
infectious agents, Lecture 25
Dr. Alvin Fox

2. 2
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)
• Serology (antibody detection)

3. 3
Taxonomy
• Defines common traits among strains for
a bacterial species
• Usually genetic
• Allows development of diagnostic kits

4. 4
Species versus strains
- selecting discriminating features

5. 5
Classification
• Strain: one single isolate or line
• Type: sub-set of species
• Species: related strains
• Genus: related species
• Family: related genera

6. 6
Streptococcus pyogenes
Both terms define the species name
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

7. 7
Identification of infectious agents
in the diagnostic laboratory
• Aids treatment
• Helps antibiotic selection
• General hospital laboratory
– physiological tests
• More fully equipped laboratories
– Genetic tests

8. 8
Steps in isolation and identification
• Step 1: Streaking culture plates
– colonies on incubation (e.g 24 hr)
– size, texture, color, hemolysis
– oxygen requirement

9. 9
CDC/Dr. James Feeley
Sheep blood agar plate culture
Bacillus anthracis
Bacillus cereus.

10. 10
Mixed colonies

11. 11
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
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

15. 15
Clinical Microbiology
Laboratory Bench

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Step 4:
Antibiotic susceptibility testing

17. 17
No
growth
Antibiotic susceptibility testing
Susceptible Not susceptible
Bacterial
lawn
Growth
Antibiotic disk

18. 18
Molecular
differentiation
• Genomics
• Gene characterization
– PCR
– Sequencing
– Restriction digestion
• Hybridization (probes, arrays)
• % guanine-cytosine

19. 19
16S rRNA Sequencing
• Differentiates bacterial species
• Development of clinical tests based
on sequence (e.g. PCR)

20. 20
Real-time PCR
Cycle one
Cycle two
Cycle 30
2 30
ds DNA
Dye

21. 21
DNA-DNA hybridization
100% Homology
+
Heat
0% Homology
Strain 1
Strain 2

22. 22
Profiles
• Long chain fatty acids
- structural (e.g. cell membrane)
• Short chain
- metabolic
- volatiles
- Fatty acids/alcohols

23. 23
• Protein profiling: defining a species by
characteristic (low molecular weight)
proteins
• Proteomics: defining all proteins
expressed by a species under specific
growth conditions

24. 24
Bacterial DNA sequences can be
amplified directly from human body
fluids

25. 25
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

26. 26
Microscopy
• spinal fluids (meningitis)
• sputum (tuberculosis)
• sensitivity poor

27. 27
Streptococcal Agglutination Test
Latex beads
Streptococcal
antigenic extract
Antibody

28. 28
Serologic identification
• antibody response to the infecting agent
• several weeks after an infection has occurred

29. Epidemics
29
• associated with particular strains/types
• state or federal laboratory system