Culturing and infections

1. 1
Culture and identification of
infectious agents
Dr. Abdullatif Neamatallah

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
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)

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
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

7. 7
Steps in isolation and identification
• Step 1: Streaking culture plates
– colonies on incubation (e.g 24 hr)
– size, texture, color, hemolysis
– oxygen requirement

8. 8
CDC/Dr. James Feeley
Sheep blood agar plate culture
Bacillus anthracis
Bacillus cereus.

9. 9
Mixed colonies

10. 10
Isolation and identification
• Step 2: Colonies Gram stained
– cells observed microscopically

11. 11
Gram negative Gram positive
Heat/Dry
Crystal violet stain
Iodine Fix
Safranin stain
Alcohol de-stain

12. 12
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

13. 13

14. 14

15. 15
‫نقية‬ ‫شريحة‬
SLIDE

16. 16
‫نقية‬ ‫شريحةغير‬
MIXED SLIDE

17. 17
Step 3:
Isolated bacteria are speciated
• Generally using physiological tests

18. 18
Clinical Microbiology
Laboratory Bench

19. 19
Step 4:
Antibiotic susceptibility testing

20. 20
No
growth
Antibiotic susceptibility testing
Susceptible Not susceptible
Bacterial
lawn
Growth
Antibiotic disk

21. 21
Molecular
differentiation
• Genomics
• Gene characterization
– Sequencing
– PCR
– Restriction digests
• Hybridization
• % guanine + cytosine

22. 22
16S rRNA Sequencing
• Differentiates bacterial species
• Development of clinical tests based
on sequence (e.g. PCR)

23. 23
Real-time PCR
Cycle one
Cycle two
Cycle 30
2 30
ds DNA
Dye

24. 24
DNA-DNA hybridization
100% Homology
+
Heat
0% Homology
Strain 1
Strain 2

25. 25
Profiles
• Long chain fatty acids
- structural (e.g. cell membrane)
• Short chain
- metabolic
- volatiles
- Fatty acids/alcohols

26. 26
Protein profiling
• M.W. of a few characteristic proteins
not “proteomics”

27. 27
Rapid diagnosis without culture
• WHEN AND WHY?
• grow poorly
• can not be cultured

28. 28
Streptococcal Agglutination Test
Latex beads
Streptococcal
antigenic extract
Antibody

29. 29
Bacterial DNA sequences amplified
directly from human body fluids
• Polymerase chain reaction (PCR)
• Great success in rapid diagnosis
of tuberculosis.

30. 30
Microscopy
• spinal fluids (meningitis)
• sputum (tuberculosis)
• sensitivity poor

31. 31
Serologic identification
• antibody response to the infecting agent
• several weeks after an infection has occurred