Specimen Processing
Introduction to
Medical Microbiology Part III
• Some specimens require special
preparation before culturing
• Techniques
– Concentration
• Centrifugation (Liquid specimens >2 ml
centrifuge first and plate the sediment)
• Filtration
Student Lab
Division of Medical Technology
Carol A. Larson MSEd, MT(ASCP)
Specimen Processing
– Homogenization – tissue grinding
– Decontamination
Specimen Processing
Selection of Culture Media
• Purpose: promote growth of all possible
pathogens in specimen
• Media requirements
– Proper pH
– Essential nutrients
– Moisture
Specimen Processing
General Purpose Media
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Blood agar
Chocolate agar
MacConkey agar
CNA agar
Media Types
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Nutrient
Enriched
Selective
Differential
Selective & Differential
Enrichment / Broth
Transport
Specimen Processing
Culture Setup
• Specimen plated to media right away
• Use biological safety hood
• Inoculate media using loop, swab or
other devise according to specimen
• Strive for ISOLATED colonies
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Specimen Processing
Specimen Processing
Inoculation of Media
1. Label and date all media
Inoculation of Media
3. Flame loop and make second streak
2. Inoculate plate with
sterile loop / swab
(making first streak)
Specimen Processing
Specimen Processing
Inoculation of Media
4. Flame loop and make third streak
5. Flame loop
Specimen Processing
Specimen Processing
Incubation of Media
• Provide proper environment for optimal
recovery of all possible pathogens
• Components
– Atmosphere
– Temperature
– pH
– Moisture
Atmosphere
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Obligate aerobe
Microaerophile
Obligate anaerobe
Facultative anaerobe
Capnophilic
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Specimen Processing
Specimen Processing
Temperature
pH and Moisture
• Psychrophiles
• Mesophiles
• Thermophiles
• pH
• Most organisms grow at 35°C
• Moisture
– Range = 6.5-7.5
– Buffered media
– Evaporation concerns
– Need 70-80% humidity
Specimen Processing
Incubators
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Ambient air incubator
CO2 incubator
Anaerobic incubator
Biobags providing
specialty atmosphere
In Summary …
• Specimen collection & transport
• Specimen processing
– Evaluate specimen
– Direct examination – gram stain
– Specimen preparation
– Culture setup
• Media selection and inoculation
• Media incubation
Interpretation of Cultures
Interpretation of Cultures
• Evaluate growth
– Colony morphology
– Media type
– Specimen source
– Identification of organisms
– Antimicrobial susceptibility testing
– Correlation of results
Colony Morphology
• Appearance
– Colony size
– Color (pigment)
– Opacity
– Elevation
– Shape
– Consistency
– Hemolytic reaction
– Odor
• Number of colony types
• Quantitation of colonies
• Reporting results
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Interpretation of Cultures
Interpretation of Cultures
Type of Media Supporting Growth
• Blood agar
– Majority of organisms
• Chocolate agar
Source of specimen
• Determine if organism is pathogen or
normal flora / contaminant
• Patient history valuable also
– Same as BAP plus fastidious organisms
• MacConkey agar
– Most GNR’s
• CNA agar
– GPC
Interpretation of Cultures
Interpretation of Cultures
Identification of Organism
• Must ID potential pathogens
• Process
– Gram stain and other staining techniques
– Rapid biochemical tests
– Conventional biochemical tests
– Immunochemical and serological tests
– Molecular tests
Interpretation of Cultures
Antimicrobial Testing
• Performed on clinically significant
organisms
• Standards must be followed
• Aid MD in
determining
appropriate
treatment
Interpretation of Cultures
Correlation
• All findings must correlate
– Growth characteristics
– Specimen and patient history
– Specimen gram stain
– Organism identification
– Organism antimicrobial pattern
Reporting Results
• Report types
– Preliminary
– Interim
– Final
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Specimen gram stain
Pathogens, quantitation
Note n. flora/contaminants, quantitation
Antimicrobial susceptibility
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In Summary …
• Interpretation of culture
– Organism identification & significance
– Correlation with specimen type and patient
history
• Antimicrobial susceptibility testing
• Reporting results
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