Culture and identification of infectious agents Dr Abdullatif
Culture and identification of infectious agents Dr. Abdullatif Neamatallah 1
Key Terms ØIsolation (culture) ØAgar plate/colonies ØLiquid media ØIdentification & taxonomy ØFamily ØGenus ØSpecies ØType ØStrain After culture q Biochemical (physiological) tests q Genetic tests q. Sequencing, q. Polymerase chain reaction (PCR) q. DNA-DNA homology q Restriction enzymes (digests) q Chemical - fatty acid/protein profiling q Immunological q. Direct detection (i. e. without culture) q. PCR q. Antigen detection q. Staining (e. g. Gram stain) 2 q. Serology (antibody detection)
Taxonomy • Defines common traits among strains for a bacterial species • Usually genetic • Allows development of diagnostic kits 3
Species versus strains - selecting discriminating features 4
Classification ü Strain: one single isolate or line ü Type: sub-set of species ü Species: related strains ü Genus: related species ü Family: related genera 5
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 6
Steps in isolation and identification • Step 1: Streaking culture plates – colonies on incubation (e. g 24 hr) – size, texture, color, hemolysis – oxygen requirement 7
Sheep blood agar plate culture Bacillus cereus. Bacillus anthracis CDC/Dr. James Feeley 8
Mixed colonies 9
Isolation and identification • Step 2: Colonies Gram stained – cells observed microscopically 10
Gram negative Gram positive Heat/Dry Crystal violet stain Iodine Fix Alcohol dede stain Safranin stain 11
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 12
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Step 3: Isolated bacteria are speciated • Generally using physiological tests 17
Clinical Microbiology Laboratory Bench 18
Step 4: Antibiotic susceptibility testing 19
Antibiotic susceptibility testing Susceptible Not susceptible Bacterial lawn No growth Growth Antibiotic disk 20
Molecular differentiation • Genomics • Gene characterization – Sequencing – PCR – Restriction digests • Hybridization • % guanine + cytosine 21
16 S r. RNA Sequencing • Differentiates bacterial species • Development of clinical tests based on sequence (e. g. PCR) 22
Real-time PCR ds DNA Cycle one Dye Cycle two Cycle 30 23
DNA-DNA hybridization Strain 1 Heat + 0% Homology Strain 2 100% Homology 24
Profiles • Long chain fatty acids - structural (e. g. cell membrane) • Short chain - metabolic - volatiles - Fatty acids/alcohols 25
Protein profiling • M. W. of a few characteristic proteins not “proteomics” 26
Rapid diagnosis without culture • WHEN AND WHY? • grow poorly • can not be cultured 27
Streptococcal Agglutination Test Streptococcal antigenic extract Antibody Latex beads 28
Bacterial DNA sequences amplified directly from human body fluids • Polymerase chain reaction (PCR) • Great success in rapid diagnosis of tuberculosis. 29
Microscopy • spinal fluids (meningitis) • sputum (tuberculosis) • sensitivity poor 30
Serologic identification • antibody response to the infecting agent • several weeks after an infection has occurred 31
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