Culture and identification of infectious agents

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

2. Key Terms 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) • Isolation (culture) • Agar plate plate/colonies • Liquid media • Identification & taxonomy • Family • Genus • Species • Type • Strain

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

4. Species versus strains- selecting discriminating features

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

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

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

9. Mixed colonies

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

11. Gram Stain Gram negative Gram positive Heat/Dry Crystal violet stain IodineFix Alcoholde-stain Safranin stain

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

15. شريحة نقيةSLIDE

16. شريحةغير نقيةMIXED SLIDE

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

18. Clinical Microbiology Laboratory Bench

19. Step 4: Antibiotic susceptibility testing

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

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

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

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

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

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

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

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

28. Streptococcal Agglutination Test Streptococcal antigenic extract Antibody Latex beads

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

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

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