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Chapter 10Prokaryote Identification
• Microscopy– Chapter 3
• Other Phenotypic Characteristics– Chapters 4, 17
• Genomic Identification
• Strain Differences
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Prokaryote Identification
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Microscopy
1670sAntony van Leeuwenhoek
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Microscopy
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Microscopy: Stains
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Gram Stain
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Gram Stain: Clinical Specimen
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Acid-Fast StainMycobacterium species
Cell wall composition prevents dye uptake
Red dye added heated over boiling water or concentrated dye added
Rinse
Decolorizing acid-alcohol wash
Blue counterstain
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Capsule Stain / Negative Stain
Cryptococcus neoformans
Capsule (glycocalyx)Gel-like layer for protection or attachment
Distinct and gelatinousStains poorly :
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Endospore Stain
Green dye added heated gently
Rinse
Red counterstain
Bacillus and Clostridium species
Endospores resistant to Gram stain
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Fluorescent Dyes and Tags
Living: greenDead: red
AuramineMycobacterium cell wall
(a) Metabolic activity alters fluorescent properties of a dye (b) Fluorescent dyes bind to pathogen-specific compound (c) Dye-conjugated antibodies: recognize pathogen-specific antigens
S. pyogenes protein
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Identification by Microscopy
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Prokaryote Identification
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Enrichment Culture
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Streak Plate
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Types of Growth Media
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Complex vs. Defined Media
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Selective & Differential Media
Selective MediaInhibits growth of all but target organism
Differential MediaTarget bacteria changes in a recognizable way
MacConkey AgarIsolate/identify Gram negative rods from the intestine
Selective: bile salts/crystal violet dye inhibit growth of all but Gram negative rodsDifferential: lactose fermenting bacteria produce pink colonies (pH indicator)
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Differential Media: Blood Agar
Differential media detects bacteria that lyse red blood cells (hemolysin production)Red blood cell lysis generates a zone of clearing (a) clear zone = beta hemolysis = S. pyogenes (b) greenish zone = alpha hemolysis = non-pathogenic Streptococcus
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Prokaryote Identification
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Biochemical Tests
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Biochemical Tests
Catalase testbubbles = catalase activity
Sugar fermentation testGas and acid production = fermentation activity
Urease testGas and ammonia (base) production = urease activity
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Dichotomous Key
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Biochemical Tests:Commercial Arrays
API strip testadd liquid culture to dehydrated media
EnterotubeInnoculation from rod drawn through liquid compartments
Other formats: 96 well plates, Miniaturized systems
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Prokaryote Identification
Serology:1. Recognition of host antibody produced in response to bacterial antigen2. Direct recognition of bacterial antigen
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SerologySeronegativeNo specific antibodies to a pathogenbecause no prior exposure
SeropositiveProduction of specific antibodiesto a pathogen
SeroconversionSwitch from seronegative to positive
SerologyIn vitro study of antibody-antigeninteractions
SerumFluid portion of the blood that remains after blood clots
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Serology: Indirect ELISAELISAEnzyme-Linked Immunosorbent Assay
Bacterial antigen affixed to substratePatient serum added if antibody vs. antigen present, it will stay in the wellPeroxidase-conjugated anti-human IgG antibody added color reaction = antibody in serum recognizes antigen
Testing for antibody production in response to antigen
Often used as HIV test (anti-gp120, anti-p24 antibodies)
“Indirect” because second antibody required for signal
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Western Blot Analysis
To confirm positive HIV ELISA result
HIV proteins run on SDS-PAGE gel protein separation based on sizeSeparated proteins transferred to membrane Test serum addedEnzyme-labeled secondary antibody added
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Direct ELISA
Known antibodies vs. antigen attached to substratePatient sample added if bacterial antigen is present, it will stay in the wellPeroxidase-conjugated antibody added
Direct testing for presence of antigen
“Direct” because antibody directly conjugated to peroxidase
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ELISA plates
96 well format for ELISA, other high-throughput protocols
Antibody titer (concentration): serial dilutions of serum testedreciprocal of last dilution with positive reaction = titer (1:256 dilution = 256 titer)
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Fatty Acid Analysis
Bacteria differ in the type and quantity of membrane fatty acids
Fatty acids removed and converted to methyl ester form (Fatty Acid Methyl Ester)Gas chromatography analysis
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Prokaryote Identification
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Nucleic Acid Hybridization
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Polymerase Chain Reaction
Nucleotide sequence must be known for primer design
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Polymerase Chain Reaction
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Polymerase Chain Reaction
30 cycles = billion-fold amplification
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Prokaryote Identification: PCR
Could also use DNA probe instead of gel
Requires knowledge of a specific nucleotide sequence from the target pathogen
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rRNA Sequencing
Ribosomes protein + rRNA components S = Svedberg unit measure of sedimentation
mRNA translation & protein synthesis important / conserved process
Sequence rRNA directlySequence DNA that codes for rRNA
Usually 16S rRNA sequenced
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Strain Differences
E. coli strainsK-12
EPECEHECSTECETECEIEC
EAggEC
Strains: related, but not identical, isolates of a species
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Detecting Strain Differences
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Biochemical Typing
• Some bacterial strains can be differentiated with biochemical tests
• Biovar/Biotype:strain with a characteristic biochemical pattern
• Vibrio cholerae:– Pandemics 1-6 = Classical Biotype– Pandemic 7 = El Tor Biotype
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Serological Typing
• Strain differentiation based upon antigenic (protein/carbohydrate) differences
• Serovar/serotype: strain with characteristic antigen pattern
• V. cholerae O1 and O139 (LPS O region)
• E. coli O157:H7– 1st antigen = carbohydrate (LPS O region)– 2nd antigen = protein (flagella)
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Genomic Typing
RFLP: Restriction Fragment Length Polymorphism
Strain differences based on subtle differences in DNA sequence
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Genomic Typing
PulseNet: national database of RFLPs from foodborne pathogens
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Phage Typing
Strain differences based on bacteriophage susceptibility patterns
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Antibiograms
Strain differences based on antibiotic susceptibility patterns
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SummaryProkaryote Identification / Classification
• Microscopy– Chapter 3
• Other Phenotypic Characteristics– Chapters 4, 17
• Genomic Identification
• Strain Differences