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Culture and identification of infectious agents

Learn about culture, genetic tests, PCR, identification steps, molecular differentiation, and rapid diagnosis methods used in diagnostic labs to identify infectious agents. Understand classification, taxonomy, and species versus strains concept.

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Culture and identification of infectious agents

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  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

  13. شريحة نقيةSLIDE

  14. شريحةغير نقيةMIXED SLIDE

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

  16. Clinical Microbiology Laboratory Bench

  17. Step 4: Antibiotic susceptibility testing

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

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

  20. 16S rRNA Sequencing • Differentiates bacterial species • Development of clinical tests based on sequence (e.g. PCR)

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

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

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

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

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

  26. Streptococcal Agglutination Test Streptococcal antigenic extract Antibody Latex beads

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

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

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

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