Clinical microbiology dr. d.p. rajani
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Transcript of Clinical microbiology dr. d.p. rajani
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Clinical Microbiology
Dr. Dhanji P. Rajani
Microbiologist
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Clinical Diagnostic
Microbiology
All aspects of infectionInitial isolation/diagnosisTreatmentInfection controlSurveillance
InfectionAntimicrobial
Clinical managementPublic health
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Microcare Laboratory
General Microbiology
– Routine Culture & Sensitivity
– Molecular Diagnostics
– Media Preparation
– Fungal Culture & Sensitivity
– Anaerobic Culture
National TB Ref Lab
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Specimen Investigation
Direct
– Microscopy
– Culture & AST
– PCR
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Direct methods
1. Macroscopic examination
2. Microscopic examination
1. Direct
2. Stain
3. Molecular methods
4. Specimen Culture
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Microscopy
Direct
– WCC
– Parasites
– Bacteria
Stain
– Simple
– Differential
– Structural
– Flourescent
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Staining
Increase contrast of microorganisms
– Identify organism
– Structural characteristics
Classified into types of stains
– Simple stain:
– Differential stain:
– Structural or special stains
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Gram stain
Most common stain
Valuable first step in identification
Differentiates into two groups
Physicochemical cell wall properties
– crystal violet to a heat-fixed smear
– Lugol’s iodine as a mordant
– rapid decolorization with alcohol /acetone
– counterstaining with safranin/carbol fuchsin
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Gram stain morphology
Shape
– cocci (round)
– diplococci
– bacilli (rods)
– spiral or curved (spirochetes)
Single or multiple cells
– clusters (staph)
– chains (strep)
Gram positive or negative
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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.
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Bacterial Culture
Media
– Solid
– Liquid
25 different types of solid & liquid media used routinely in Microcare Laboratory
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Culture Media
– Nutrient
BA
CA
– Selective
SS Agar
XLD Agar
– Differential
MacC
– Chromogenic
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Incubation
Temperature
– 37oC, 30oC,22oC,40oC
Time
– 18 hours
Atmospheric conditions
– Air
– CO2 / Microaerophilic
– Anaerobic
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Bacterial Identification
Fermentation
Nutritional requirements
Enzyme detection
Metabolic activity
Antigenic determinants
Genomic
– PCR
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IdentificationIsolation (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 (antibody detection)
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Automated BC
Continuous monitoring
Early detection
CO2 production
Closed system
Reduced risk of laboratory contamination
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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
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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)
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Disc Diffusion
Classic Microbiology Technique
Standardised suspension swabbed onto plate
Discs placed on the surface
Zones read and compared to standard
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Microbiology, St. James's Hospital
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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
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Dilution in liquid broth
increasing antibiotic concentrations
Standard concentration
Incubation for 18 hr at 37°C
(Control 0,25 0,50 1 2 4 8 mg/l
MICBacterial growth Inhibition
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E-test