Culture and identification of infectious agents Dr. Alvin Fox
Key Terms <ul><li>Isolation (culture) </li></ul><ul><ul><li>Agar plate plate/colonies </li></ul></ul><ul><ul><li>Liquid me...
Taxonomy <ul><li>Defines common traits among strains for a bacterial species </li></ul><ul><li>Usually genetic  </li></ul>...
Species versus strains -  selecting discriminating features
Classification <ul><li>Strain: one single isolate or line </li></ul><ul><li>Type: sub-set of species </li></ul><ul><li>Spe...
Identification of infectious agents in the diagnostic laboratory <ul><li>Aids treatment </li></ul><ul><li>Helps antibiotic...
Steps in isolation and identification <ul><li>Step 1:  Streaking culture plates  </li></ul><ul><ul><ul><li>colonies on inc...
CDC/Dr. James Feeley   Sheep blood agar plate culture Bacillus anthracis Bacillus cereus .
Mixed colonies
Isolation and identification <ul><li>Step 2: Colonies Gram stained  </li></ul><ul><ul><li>cells observed microscopically <...
Gram negative Gram positive Heat/Dry Crystal violet stain Iodine   Fix Safranin stain Alcohol   de - stain
Gram stain morphology <ul><li>Shape </li></ul><ul><ul><li>cocci (round) </li></ul></ul><ul><ul><li>bacilli (rods) </li></u...
 
 
Step 3:   Isolated bacteria are speciated   <ul><li>Generally using physiological tests </li></ul>
Clinical Microbiology Laboratory Bench
Step 4:   Antibiotic susceptibility testing
Antibiotic susceptibility testing No growth Susceptible Not susceptible Bacterial lawn Growth Antibiotic disk
Molecular differentiation <ul><li>Genomics </li></ul><ul><li>Gene characterization  </li></ul><ul><ul><li>Sequencing </li>...
16S rRNA Sequencing <ul><li>Differentiates bacterial species  </li></ul><ul><li>Development of clinical tests based on seq...
Real-time PCR Cycle one Cycle two Cycle 30 2  30 ds DNA Dye
DNA-DNA hybridization 100% Homology + Heat 0% Homology Strain 1 Strain 2
Profiles <ul><li>Long chain  fatty acids </li></ul><ul><ul><li>- structural (e.g. cell membrane) </li></ul></ul><ul><li>Sh...
Protein profiling <ul><li>M.W. of a few characteristic proteins </li></ul><ul><li>not “proteomics”  </li></ul>
Rapid diagnosis without culture <ul><li>WHEN AND WHY? </li></ul><ul><ul><li>grow poorly </li></ul></ul><ul><ul><li>can not...
Streptococcal Agglutination Test Latex beads Streptococcal antigenic extract  Antibody
Bacterial DNA sequences amplified directly from human body fluids <ul><li>Polymerase chain reaction (PCR)  </li></ul><ul><...
Microscopy <ul><li>spinal fluids (meningitis)  </li></ul><ul><li>sputum (tuberculosis) </li></ul><ul><li>sensitivity poor ...
Serologic identification <ul><li>antibody response to the infecting agent </li></ul><ul><li>several weeks after an infecti...
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  1. 1. Culture and identification of infectious agents Dr. Alvin Fox
  2. 2. Key Terms <ul><li>Isolation (culture) </li></ul><ul><ul><li>Agar plate plate/colonies </li></ul></ul><ul><ul><li>Liquid media </li></ul></ul><ul><li>Identification & taxonomy </li></ul><ul><ul><li>Family </li></ul></ul><ul><ul><li>Genus </li></ul></ul><ul><ul><li>Species </li></ul></ul><ul><ul><li>Type </li></ul></ul><ul><ul><li>Strain </li></ul></ul><ul><li>After culture </li></ul><ul><li>Biochemical (physiological) tests </li></ul><ul><li>Genetic tests </li></ul><ul><ul><li>Sequencing, </li></ul></ul><ul><ul><li>Polymerase chain reaction (PCR) </li></ul></ul><ul><ul><li>DNA-DNA homology </li></ul></ul><ul><ul><li>Restriction enzymes (digests) </li></ul></ul><ul><li>Chemical </li></ul><ul><ul><li>- fatty acid/protein profiling </li></ul></ul><ul><li>Immunological </li></ul><ul><li>Direct detection (i.e. without culture ) </li></ul><ul><ul><li>PCR </li></ul></ul><ul><ul><li>Antigen detection </li></ul></ul><ul><ul><li>Staining (e.g. Gram stain) </li></ul></ul><ul><ul><li>Serology (antibody detection) </li></ul></ul>
  3. 3. Taxonomy <ul><li>Defines common traits among strains for a bacterial species </li></ul><ul><li>Usually genetic </li></ul><ul><li>Allows development of diagnostic kits </li></ul>
  4. 4. Species versus strains - selecting discriminating features
  5. 5. Classification <ul><li>Strain: one single isolate or line </li></ul><ul><li>Type: sub-set of species </li></ul><ul><li>Species: related strains </li></ul><ul><li>Genus: related species </li></ul><ul><li>Family: related genera </li></ul>
  6. 6. Identification of infectious agents in the diagnostic laboratory <ul><li>Aids treatment </li></ul><ul><li>Helps antibiotic selection </li></ul><ul><li>General hospital laboratory </li></ul><ul><ul><li>physiological tests </li></ul></ul><ul><li>Reference laboratories </li></ul><ul><ul><li>Genetic (less commonly protein) tests </li></ul></ul>
  7. 7. Steps in isolation and identification <ul><li>Step 1: Streaking culture plates </li></ul><ul><ul><ul><li>colonies on incubation (e.g 24 hr) </li></ul></ul></ul><ul><ul><ul><li>size, texture, color, hemolysis </li></ul></ul></ul><ul><ul><ul><li>oxygen requirement </li></ul></ul></ul>
  8. 8. CDC/Dr. James Feeley Sheep blood agar plate culture Bacillus anthracis Bacillus cereus .
  9. 9. Mixed colonies
  10. 10. Isolation and identification <ul><li>Step 2: Colonies Gram stained </li></ul><ul><ul><li>cells observed microscopically </li></ul></ul>
  11. 11. Gram negative Gram positive Heat/Dry Crystal violet stain Iodine Fix Safranin stain Alcohol de - stain
  12. 12. Gram stain morphology <ul><li>Shape </li></ul><ul><ul><li>cocci (round) </li></ul></ul><ul><ul><li>bacilli (rods) </li></ul></ul><ul><ul><li>spiral or curved (e.g. spirochetes) </li></ul></ul><ul><li>Single or multiple cells </li></ul><ul><ul><li>clusters (e.g. staphylococci) </li></ul></ul><ul><ul><li>chains (e.g. streptococci) </li></ul></ul><ul><li>Gram positive or negative </li></ul>
  13. 15. Step 3: Isolated bacteria are speciated <ul><li>Generally using physiological tests </li></ul>
  14. 16. Clinical Microbiology Laboratory Bench
  15. 17. Step 4: Antibiotic susceptibility testing
  16. 18. Antibiotic susceptibility testing No growth Susceptible Not susceptible Bacterial lawn Growth Antibiotic disk
  17. 19. Molecular differentiation <ul><li>Genomics </li></ul><ul><li>Gene characterization </li></ul><ul><ul><li>Sequencing </li></ul></ul><ul><ul><li>PCR </li></ul></ul><ul><ul><li>Restriction digests </li></ul></ul><ul><li>Hybridization </li></ul><ul><li>% guanine + cytosine </li></ul>
  18. 20. 16S rRNA Sequencing <ul><li>Differentiates bacterial species </li></ul><ul><li>Development of clinical tests based on sequence (e.g. PCR) </li></ul>
  19. 21. Real-time PCR Cycle one Cycle two Cycle 30 2 30 ds DNA Dye
  20. 22. DNA-DNA hybridization 100% Homology + Heat 0% Homology Strain 1 Strain 2
  21. 23. Profiles <ul><li>Long chain fatty acids </li></ul><ul><ul><li>- structural (e.g. cell membrane) </li></ul></ul><ul><li>Short chain </li></ul><ul><li>- metabolic </li></ul><ul><li>- volatiles </li></ul><ul><ul><ul><li>- Fatty acids/alcohols </li></ul></ul></ul>
  22. 24. Protein profiling <ul><li>M.W. of a few characteristic proteins </li></ul><ul><li>not “proteomics” </li></ul>
  23. 25. Rapid diagnosis without culture <ul><li>WHEN AND WHY? </li></ul><ul><ul><li>grow poorly </li></ul></ul><ul><ul><li>can not be cultured </li></ul></ul>
  24. 26. Streptococcal Agglutination Test Latex beads Streptococcal antigenic extract Antibody
  25. 27. Bacterial DNA sequences amplified directly from human body fluids <ul><li>Polymerase chain reaction (PCR) </li></ul><ul><li>Great success in rapid diagnosis </li></ul><ul><li>of tuberculosis. </li></ul>
  26. 28. Microscopy <ul><li>spinal fluids (meningitis) </li></ul><ul><li>sputum (tuberculosis) </li></ul><ul><li>sensitivity poor </li></ul>
  27. 29. Serologic identification <ul><li>antibody response to the infecting agent </li></ul><ul><li>several weeks after an infection has occurred </li></ul>

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