PPT PowerPoint Presentation

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Culture and identification of infectious agents

Dr. Abdullatif Neamatallah

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Key Terms 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)



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Taxonomy

• Defines common traits among strains for a bacterial species

• Usually genetic

• Allows development of diagnostic kits

Species versus strains

- selecting discriminating features selecting discriminating features

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Classification Classification

Strain: one single isolate or lineStrain: one single isolate or line

Type: sub-set of speciesType: sub-set of species

Species: related strainsSpecies: related strains

Genus: related speciesGenus: related species

Family: related generaFamily: related genera

Identification of infectious agents Identification of infectious agents

in the diagnostic laboratory in 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 identification Steps 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

CDC/Dr. James Feeley

Sheep blood agar plate culture

Bacillus anthracis Bacillus cereus.

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Mixed colonies

Isolation and identification Isolation and identification

• Step 2: Colonies Gram stained Step 2: Colonies Gram stained

– cells observed microscopically cells observed microscopically

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Gram negative

Gram negative Gram positiveGram positive Heat/Dry

Heat/Dry

Crystal violet stain Crystal violet stain

Iodine

Iodine FixFix

Safranin stain Safranin stain Alcohol

Alcohol de-de stainstain

Gram stain morphology Gram stain morphology

• Shape_Shape

– 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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ةيقن ةحيرش

SLIDE

ةيقن ريغةحيرش

MIXED SLIDE

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Step 3:

Step 3:

Isolated bacteria are speciated Isolated bacteria are speciated

• Generally using physiological testsGenerally using physiological tests

Clinical Microbiology Clinical Microbiology

Laboratory Bench

Laboratory Bench

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Step 4:

Step 4:

Antibiotic susceptibility testing

Antibiotic susceptibility testing

No No

growth growth

Antibiotic susceptibility testing Antibiotic susceptibility testing

Susceptible

Susceptible Not susceptibleNot susceptible Bacterial

Bacterial lawnlawn

Growth Growth

Antibiotic disk Antibiotic disk

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Molecular Molecular

differentiation differentiation

• GenomicsGenomics

• Gene characterization Gene characterization – SequencingSequencing

– PCRPCR

– Restriction digestsRestriction digests

• HybridizationHybridization

• % guanine + cytosine% guanine + cytosine

16S rRNA Sequencing 16S 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 DNA ds DNA

DyeDye

DNA-DNA hybridization DNA-DNA hybridization

100% Homology ++

HeatHeat

0% Homology

Strain 1 Strain 1

Strain 2 Strain 2

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Profiles Profiles

• 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

Protein profiling Protein 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 culture Rapid diagnosis without culture

• WHEN AND WHY?

• grow poorly

• can not be cultured

Streptococcal Agglutination Test

Latex beads Latex beads

Streptococcal Streptococcal

antigenic extract antigenic extract Antibody

Antibody

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Bacterial DNA sequences amplified Bacterial DNA sequences amplified

directly from human body fluids 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.

Microscopy Microscopy

• spinal fluids (meningitis) spinal fluids (meningitis)

• sputum (tuberculosis)sputum (tuberculosis)

• sensitivity poorsensitivity poor

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Serologic identification Serologic identification

• antibody response to the infecting agent

• several weeks after an infection has occurred