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Culture and identification of infectious agents
Dr. Alvin Fox
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Key TermsKey TermsIsolation (culture)
Agar plate plate/coloniesLiquid media Identification & taxonomyFamilyGenusSpeciesTypeStrain
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)PCRAntigen detection Staining (e.g. Gram stain)Serology (antibody detection)
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Taxonomy
• Defines common traits among strains for a bacterial species
• Usually genetic
• Allows development of diagnostic kits
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Species versus strains- selecting discriminating featuresselecting discriminating features
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ClassificationClassificationStrain: one single isolate or lineStrain: one single isolate or lineType: sub-set of speciesType: sub-set of speciesSpecies: related strainsSpecies: related strainsGenus: related speciesGenus: related speciesFamily: related generaFamily: related genera
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Identification of infectious agentsIdentification of infectious agentsin the diagnostic laboratoryin the diagnostic laboratory
• Aids treatmentAids treatment• Helps antibiotic selectionHelps antibiotic selection• General hospital laboratoryGeneral hospital laboratory
– physiological testsphysiological tests• Reference laboratoriesReference laboratories
– Genetic (less commonly protein) testsGenetic (less commonly protein) tests
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Steps in isolation and identificationSteps in isolation and identification
• Step 1: Step 1: Streaking culture plates Streaking culture plates – colonies on incubation (e.g 24 hr)colonies on incubation (e.g 24 hr)– size, texture, color, hemolysis size, texture, color, hemolysis – oxygen requirement oxygen requirement
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CDC/Dr. James Feeley
Sheep blood agar plate culture
Bacillus anthracisBacillus cereus.
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Mixed colonies
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Isolation and identificationIsolation and identification
• Step 2: Colonies Gram stained Step 2: Colonies Gram stained – cells observed microscopicallycells observed microscopically
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Gram negativeGram negative Gram positiveGram positive
Heat/DryHeat/Dry
Crystal violet stainCrystal violet stain
IodineIodine FixFix
Safranin stainSafranin stain
AlcoholAlcohol dede-stainstain
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Gram stain morphologyGram stain morphology
• ShapeShape
– cocci (round)cocci (round)
– bacilli (rods)bacilli (rods)
– spiral or curved (e.g. spirochetes)spiral or curved (e.g. spirochetes)
• Single or multiple cellsSingle or multiple cells
– clusters (e.g. staphylococci)clusters (e.g. staphylococci)
– chains (e.g. streptococci) chains (e.g. streptococci)
• Gram positive or negativeGram positive or negative
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Step 3:Step 3:
Isolated bacteria are speciatedIsolated bacteria are speciated
• Generally using physiological testsGenerally using physiological tests
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Clinical Microbiology Clinical Microbiology Laboratory BenchLaboratory Bench
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Step 4:Step 4: Antibiotic susceptibility testing Antibiotic susceptibility testing
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No No growthgrowth
Antibiotic susceptibility testingAntibiotic susceptibility testing
SusceptibleSusceptible Not susceptibleNot susceptible
BacterialBacterial lawnlawn
GrowthGrowth
Antibiotic diskAntibiotic disk
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Molecular Molecular differentiationdifferentiation• GenomicsGenomics
• Gene characterization Gene characterization – SequencingSequencing– PCRPCR– Restriction digestsRestriction digests
• HybridizationHybridization
• % guanine + cytosine% guanine + cytosine
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16S rRNA Sequencing16S rRNA Sequencing
• Differentiates bacterial species Differentiates bacterial species
• Development of clinical tests based Development of clinical tests based on sequence (e.g. PCR)on sequence (e.g. PCR)
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Real-time PCR
Cycle one
Cycle two
Cycle 30
2 2 3030
ds DNAds DNA
DyeDye
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DNA-DNA hybridizationDNA-DNA hybridization
100% Homology
++
HeatHeat
0% Homology
Strain 1Strain 1
Strain 2Strain 2
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ProfilesProfiles• Long chain Long chain fatty acidsfatty acids
- structural (e.g. cell membrane)- structural (e.g. cell membrane)
• Short chainShort chain- metabolic- metabolic- volatiles - volatiles
- Fatty acids/alcohols - Fatty acids/alcohols
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Protein profilingProtein profiling
• M.W. of a few characteristic proteinsM.W. of a few characteristic proteins
not “proteomics” not “proteomics”
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Rapid diagnosis without cultureRapid diagnosis without culture
• WHEN AND WHY?• grow poorly• can not be cultured
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Streptococcal Agglutination Test
Latex beadsLatex beads
StreptococcalStreptococcalantigenic extract antigenic extract
AntibodyAntibody
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Bacterial DNA sequences amplified Bacterial DNA sequences amplified directly from human body fluidsdirectly 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.
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MicroscopyMicroscopy• spinal fluids (meningitis) spinal fluids (meningitis) • sputum (tuberculosis)sputum (tuberculosis)
• sensitivity poorsensitivity poor
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Serologic identificationSerologic identification
• antibody response to the infecting agent
• several weeks after an infection has occurred