Phenotypic
Methodology
Helen Barry
Microbiology Department
St. James’s Hospital
Clinical Diagnostic
Microbiology
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All aspects of infection
Initial isolation/diagnosis
Treatment
Infection control
Surveillance
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Infection
Antimicrobial
– Clinical management
– Public health
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National
European
Microbiology, St. James's Hospital
SJH Microbiology
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LabMed Directorate
General Microbiology
– Virology serology
– Molecular Diagnostics
– Media Production
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National MRSA Ref Lab
Irish Mycobacteria Reference Lab
Microbiology, St. James's Hospital
Specimen Investigation
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Direct
– Microscopy
– Culture
– PCR
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Indirect
– Antibody
– T cell response
– Interferon
Microbiology, St. James's Hospital
Direct methods
1.
2.
Macroscopic examination
Microscopic examination
1. Direct
2. Stain
3. EM
3.
4.
5.
Rapid tests
Molecular methods
Specimen Culture
Microbiology, St. James's Hospital
Microscopy

Direct
– WCC
– Parasites
– Bacteria
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Stain
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Simple
Differential
Structural
Flourescent
Microbiology, St. James's Hospital
Microscopes
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Use of light
– Visible
– UV
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Magnifying lens
Microbiology, St. James's Hospital
Microscopy
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Light Microscope
– Monocular
– Binocular
– Stereo/dissecting
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Phase contrast
Dark ground
Flourescent
Electron Microscopy
Microbiology, St. James's Hospital
Light Path
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Passes through opening in stage to
slide
Light enters objective lens
Image magnified by ocular lens
Microbiology, St. James's Hospital
Staining
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Increase contrast of microorganisms
– Identify organism
– Structural characteristics

Classified into types of stains
– Simple stain:
– Differential stain:
– Structural or special stains
Microbiology, St. James's Hospital
Gram stain
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Most common stain
Valuable first step in identification
Differentiates into two groups
Physicochemical cell wall properties
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crystal violet to a heat-fixed smear
Lugol’s iodine as a mordant
rapid decolorization with alcohol /acetone
counterstaining with safranin/carbol fuchsin
Microbiology, St. James's Hospital
Gram stain morphology

Shape
– cocci (round)
– diplococci
– bacilli (rods)
– spiral or curved (spirochetes)
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Single or multiple cells
– clusters (staph)
– chains (strep)
Gram positive or negative
Microbiology, St. James's Hospital

Bacterial
isolation and identification
Samples
streaked on culture plates
isolated colonies of bacteria appear after incubation.
Key step in identification – colonial morphology
size,
texture,
colour,
haemolysis ,
smell.
Incubation temperature, time and atmospheric conditions
important characteristic.
Microbiology, St. James's Hospital
Bacterial Culture
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Media
– Solid
– Liquid
30 different types of solid media used
routinely in SJH
Microbiology, St. James's Hospital
Culture Media
– Nutrient
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BA
CA
– Selective
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Neomycin = anaerobes
CCDA=Camp
Brazier=Cdiff
– Differential
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MacC
– Chromogenic
Microbiology, St. James's Hospital
Incubation
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Temperature
– 37oC, 30oC,22oC,40oC
Time
– 18 hours
Atmospheric conditions
– Air
– CO2 / Microaerophilic
– Anaerobic
Microbiology, St. James's Hospital
Key function

Bacterial Identification
– Investigator experience

limited range of tests to confirm
– Commercial ID systems
– Additional biochemical tests
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Systematic approach
Antimicrobial susceptibility
– Resistance determinants
Microbiology, St. James's Hospital
Bacterial Identification
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Fermentation
Nutritional requirements
Enzyme detection
Metabolic activity
Antigenic determinants
Proteomic
– Mass spec

Genomic
Microbiology, St. James's Hospital
Metabolic activity
Measure various metabolic activities
– acidification,
– alkalinisation,
– enzyme hydrolysis

growth in the presence of inhibitory
substances.
Microbiology, St. James's Hospital
Mass spectrometry
MALDI TOF
Matrix-assisted laser
desorption/ionisation – time of flight
 Ionisation
 Profile spectra in linear positive mode
of constantly expressed high abundant
proteins e.g. Ribosomal

Microbiology, St. James's Hospital
Chemical analysis
Characterization of secreted metabolic products
volatile alcohols
short chain fatty acids
Microbiology, St. James's Hospital
Identification
Isolation (culture)
Agar
plate/colonies
Liquid media
test tube - bulk
Identification & taxonomy
Family
Genus
Species
Type
Strain
Biochemical (physiological) tests
Fermentation
Metabolic characteristics
Molecular tests
DNA-DNA homology
16S rRNA sequencing
Chemical profiling
Mass Spectrometry
Non culture based detection
Polymerase chain reaction- (PCR)
Agglutination (antigen detection)
Stain
Serology
Microbiology,
St. James's Hospital (antibody detection)
Rapid Detection

Continuous monitoring
– Blood or body fluid culture
– Small numbers of organisms
– Critical specimen
– Rapid results
Microbiology, St. James's Hospital
Automated BC
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Continuous monitoring
Early detection
CO2 production
Closed system
Reduced risk of laboratory
contamination
Microbiology, St. James's Hospital
Antimicrobial susceptibility test
Minimum inhibitory concentration [MIC]
– The smallest concentration of antibiotic that inhibits
the growth of organism
Liquid media (dilution) allows MIC estimation
Solid media (diffusion)
– Disk diffusion CLSI/EUCAST
– E-tests
– Allows MIC estimation
Identification of resistance determinants
Natural & acquired
resistance
Natural resistance
– Affect almost all species strains
– Existed before antibiotic use (Enterobacter sp. amoxicillin)
Acquired resistance (mutation)
– Chromosomic, plasmidic
– Affects a fraction of strains
– Increased with antibiotic use
(extended spectrum beta-lactamase producing E. coli)
Disc Diffusion
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Classic Microbiology Technique
Standardised suspension swabbed
onto plate
Discs placed on the surface
Zones read and compared to standard
Microbiology, St. James's Hospital
Microbiology, St. James's Hospital
Common problems
Problem with the size of the
inoculum
Depth of medium
Type of medium
Moisture content
Solution:
 Use McFarland 0.5 photometer
 Scale -> same tubes
Dilution in liquid broth


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increasing antibiotic concentrations
Standard concentration
Incubation for 18 hr at 37°C
MIC
Bacterial growth
(Control
0,25
0,50
1
2
Inhibition
4
8
mg/l
E-test

AST automated systems
Allow analysis of a large range of
breakpoints
Computerised data management and
analysis
Advanced expert rules
Microbiology, St. James's Hospital
Pre and post analytical
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Automated inoculators
Smart incubators
Waste management
Biosecurity
Microbiology, St. James's Hospital