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Information about vsv2

Published on December 5, 2007

Author: lebahiep

Source: slideshare.net

Culture and identification of infectious agents Dr. Alvin Fox

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) Serology (antibody detection)

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)

Serology (antibody detection)

Taxonomy Defines common traits among strains for a bacterial species Usually genetic Allows development of diagnostic kits

Defines common traits among strains for a bacterial species

Usually genetic

Allows development of diagnostic kits

Species versus strains - selecting discriminating features

Classification Strain: one single isolate or line Type: sub-set of species Species: related strains Genus: related species Family: related genera

Strain: one single isolate or line

Type: sub-set of species

Species: related strains

Genus: related species

Family: related genera

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

Aids treatment

Helps antibiotic selection

General hospital laboratory

physiological tests

Reference laboratories

Genetic (less commonly protein) tests

Steps in isolation and identification Step 1: Streaking culture plates colonies on incubation (e.g 24 hr) size, texture, color, hemolysis oxygen requirement

Step 1: Streaking culture plates

colonies on incubation (e.g 24 hr)

size, texture, color, hemolysis

oxygen requirement

CDC/Dr. James Feeley Sheep blood agar plate culture Bacillus anthracis Bacillus cereus .

Mixed colonies

Isolation and identification Step 2: Colonies Gram stained cells observed microscopically

Step 2: Colonies Gram stained

cells observed microscopically

Gram negative Gram positive Heat/Dry Crystal violet stain Iodine Fix Safranin stain Alcohol de - stain

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

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

 

 

Step 3: Isolated bacteria are speciated Generally using physiological tests

Generally using physiological tests

Clinical Microbiology Laboratory Bench

Step 4: Antibiotic susceptibility testing

Antibiotic susceptibility testing No growth Susceptible Not susceptible Bacterial lawn Growth Antibiotic disk

Molecular differentiation Genomics Gene characterization Sequencing PCR Restriction digests Hybridization % guanine + cytosine

Genomics

Gene characterization

Sequencing

PCR

Restriction digests

Hybridization

% guanine + cytosine

16S rRNA Sequencing Differentiates bacterial species Development of clinical tests based on sequence (e.g. PCR)

Differentiates bacterial species

Development of clinical tests based on sequence (e.g. PCR)

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 Long chain fatty acids - structural (e.g. cell membrane) Short chain - metabolic - volatiles - Fatty acids/alcohols

Long chain fatty acids

- structural (e.g. cell membrane)

Short chain

- metabolic

- volatiles

- Fatty acids/alcohols

Protein profiling M.W. of a few characteristic proteins not “proteomics”

M.W. of a few characteristic proteins

not “proteomics”

Rapid diagnosis without culture WHEN AND WHY? grow poorly can not be cultured

WHEN AND WHY?

grow poorly

can not be cultured

Streptococcal Agglutination Test Latex beads Streptococcal antigenic extract Antibody

Bacterial DNA sequences amplified directly from human body fluids Polymerase chain reaction (PCR) Great success in rapid diagnosis of tuberculosis.

Polymerase chain reaction (PCR)

Great success in rapid diagnosis

of tuberculosis.

Microscopy spinal fluids (meningitis) sputum (tuberculosis) sensitivity poor

spinal fluids (meningitis)

sputum (tuberculosis)

sensitivity poor

Serologic identification antibody response to the infecting agent several weeks after an infection has occurred

antibody response to the infecting agent

several weeks after an infection has occurred

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