Download Gwy-Am Shin

Sampling and detection
of microorganisms
in the environment
Gwy-Am Shin
Department of Environmental and
Occupational Health Sciences
Sampling
The challenges
• Different microbe types
• Different media types
• Low numbers of pathogens in the
environment
Infectious diseases
• 1415 human pathogens (2001)
– 217 viruses and prions
– 538 bacteria and rickettsiae
– 307 fungi
– 66 protozoans
– 287 helminths
Transmission of infectious disease
• Person-to-person
– Direct: person-to-person or animal-to-person
– Indirect : droplet, fomites (toys), other vehicles
• Environment
– Airborne
– Waterborne
– Foodborne
– Vectorborne
Low numbers of pathogens in
the environment
Transmission of enteric pathogens
Low number of microbes in the
environment
• Need large volumes
• Need to separate microbes from other
materials
Steps in pathogen sampling in the
environment
• Concentration
• Purification/Reconcentration
• Analysis
Concentration in Individual
media
Sampling microbes in water
• Filtration is typically used for concentration
• Several formats utilized:
– Membrane, pleated capsule, cartridge, hollow
fiber
• Several types of media
– cellulose ester, fiberglass, nylon, polycarbonate,
diatomaceous earth, polypropylene, cotton, polysulfone,
polyacrylonitrile, polyether sulfone
Filters to Recover and Concentrate Microbes from Liquids
Sampling microbes in air
• Filters
– Not recommended due to low sampling efficiency
• Impingers
– AGI sampler
– Biosampler (SKC) sampler
• Impactors
– Anderson single and multistage sampler
– Slit sampler
– Rotary arm sampler
• Centrifugal samplers
– Cyclone sampler
– Centrifugal sampler
Impingers
Impactors (I)
Impactors (II)
Centrifugal samplers
Sampling microbes from surfaces
• Swabs
– cotton, dacron, calcium alginate, sponge
• Swipes/Wipes
– cotton, nitrocellulose membranes, polyester bonded
cloth, velvet or velveteen
• Vacuum Filtration
– Hepa bag vac, wet vac
• Contact Plates and Paddles (RODAC)
• New Methods
– Adhesive Strips and Paddles
– Scraping/Aspiration
Yamaguchi, et al. 2003; Cloud, et al. 2002; Lemmen, et al, 2001; Poletti, 1999;
Craythorn, et al. 1980; Osterblad, et al. 2003; Taku, et al. 2003
Purification/re-concentration
Purification/re-concentration
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PEG (polyethylene glycol)
Organic Flocculation
IMS (Immunomagnetic separation)
Ligand capture
BEaDs (Biodetection Enabling Device)
Capillary Electrophoresis
Microfluidics
Nucleic Acid Extraction
Spin Column Chromatography
Floatation
Sedimentation
Enrichment
Immunomagnetic Separation
Antibody
Y
Bead
Microbe
Immonomagnetic separation assay
Summary (Sampling)
• Sampling methods are lagging behind detection
methods
• Speed isn’t everything
• Negative results don’t necessarily mean target
not there
• There is a need to focus on the reliability and
sensitivity of concentration methods
• Difficulties with a single platform for any one
media because of wide range of organisms and
environmental conditions
Detection methods
Light microscope
Electron microscope
Sizes of microorganisms
Cultural methods (bacteria)
Traditional approach
• 1st step
– pre-enrich and/or enrich using non-selective and then selective broth
media
– grow colonies on membrane filters
• 2nd step
– Transfer to differential and selective agars
– Recover presumptive positive colonies
– Biochemical, metabolic and other physiological testing
– Serological or other immunochemical typing
– Other characterization: phage typing, nucleic acid analyses,
virulence tests
Enrichment Cultures
• Observe for growth by
turbidity, clearing, gas
production, color change,
etc.
• Score as presenceabsence (positive or
negative)
• (sometimes) Quantify
using replicate and
different volumes to
compute a Most Probable
Number
Left: negative
Right: positive (color
change)
Cultural methods (bacteria)
• Plating methods
– Spread plating technique
– Pour plating technique
• Most Probable Number (MPN) technique
Different bacterial colonies on general
media
Cultural methods (viruses and protozoa)
• Animal infectivity assays
– Mouse
– Gerbils
– Champagnes
– Human
• Cell-culture infectivity assays
– Primary cell lines
– Established cancer cell lines
Immunological methods
Antigen and antibody reaction
Immunological methods
• Immunoprecipitation assays
• Immunoblotting assays
• Enzyme-Linked Immunosorbent assays
(ELISA)
Nucleic acid-based methods
Structure of DNA
Nucleic-acid based methods
• Gene probing
– Southern and northern hybridization
– Microarray
• Polymerase Chain Reaction (PCR)
Gene probe detection
Real-Time PCR and Quantitative Fluorogenic Detection
• Molecular beacon. Several 5'
bases form base pairs with
several 3' bases; reporter and
quencher in close proximity.
– If reporter is excited by light,
its emission is absorbed by
quencher & no fluorescence is
detected.
• Detection of PCR product by
molecular beacon.
– Beacon binds to PCR product
and fluoresces when excited
by the appropriate light.
– [Fluorescence] proportional to
[PCR product amplified]