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 selecting discriminating features Species versus strains - selecting discriminating features

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

6. 6 Identification of infectious agents in the diagnostic laboratory Aids treatmentAids treatment Helps antibiotic selectionHelps antibiotic selection General hospital laboratoryGeneral hospital laboratory –physiological tests Reference laboratoriesReference laboratories –Genetic (less commonly protein) tests

7. 7 Steps in isolation and identification Step 1: Streaking culture plates 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 anthracisBacillus cereus.

9. 9 Mixed colonies

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

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

12. 12 Gram stain morphology ShapeShape –cocci (round) –bacilli (rods) –spiral or curved (e.g. spirochetes) Single or multiple cellsSingle or multiple cells –clusters (e.g. staphylococci) –chains (e.g. streptococci) Gram positive or negativeGram positive or negative

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15. 15 شريحة نقية SLIDE

16. 16 شريحةغير نقية MIXED SLIDE

17. 17 Step 3: Isolated bacteria are speciated Generally using physiological testsGenerally 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 lawn Growth Antibiotic disk

21. 21 Molecular differentiation GenomicsGenomics Gene characterizationGene characterization –Sequencing –PCR –Restriction digests HybridizationHybridization % guanine + cytosine% guanine + cytosine

22. 22 16S rRNA Sequencing Differentiates bacterial speciesDifferentiates bacterial species Development of clinical tests based on sequence (e.g. PCR)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 Long chain fatty acids - structural (e.g. cell membrane) Short chain Short chain - metabolic - volatiles - Fatty acids/alcohols

26. 26 Protein profiling M.W. of a few characteristic proteinsM.W. of a few characteristic proteins not “proteomics” 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) Polymerase chain reaction (PCR) Great success in rapid diagnosis Great success in rapid diagnosis of tuberculosis. of tuberculosis.

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

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