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Culture and identification of infectious agents, Lecture 33

This lecture, presented by Dr. Alvin Fox, delves into the principles and techniques used for the culture and identification of infectious agents in diagnostic laboratories. Emphasis is placed on biochemical tests, genetic sequencing methods, and immunological detection for improved diagnosis and treatment decisions. Key steps in isolation and identification, including Gram staining, antibiotic susceptibility testing, and molecular differentiation, are discussed. The lecture also highlights rapid diagnostic methods and serological identification, providing a comprehensive understanding of modern microbial diagnostics.

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Culture and identification of infectious agents, Lecture 33

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  1. Culture and identification of infectious agents, Lecture 33 Dr. Alvin Fox

  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 • Better equipped laboratories • Genetic 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. Step 3:Isolated bacteria are speciated • Generally using physiological tests

  14. Clinical Microbiology Laboratory Bench

  15. Step 4: Antibiotic susceptibility testing

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

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

  18. 16S rRNA Sequencing • Differentiates bacterial species • Development of clinical tests based on sequence (e.g. PCR)

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

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

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

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

  23. Rapid diagnosis without culture • WHEN AND WHY? • The organism grows poorly • The organism can’t be cultured • Speed is of essence

  24. Bacterial DNA sequences amplified directly from human body fluids • Polymerase chain reaction (PCR): • - Rapid diagnosis of tuberculosis • - Many other bacterial species

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

  26. Streptococcal Agglutination Test Streptococcal antigenic extract Antibody Latex beads

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

  28. Epidemics • associated with particular strains • state or federal laboratory system

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