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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