Transcript Microscopy and Immunoassays

Sampling Criteria
• Sampling plans will depend on the
question that they are designed to answer
• Basic criteria
– Replication
– Representative
– Random
– Controls
– Method Validation
Detection of Pathogens in the
Environment
Detection of Pathogenic Microbes in Environmental
Media
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Three main steps:
(1) recovery, extraction and concentration,
(2) purification and separation, and
(3) assay and characterization.
Assay Methods for Pathogens
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culture or infectivity
viability or activity measurements
immunoassays
nucleic acid assays
Protein or other macromolecular/biochemical
assays
• microscopic examinations
Microscopic Methods
History of Microscopy
• 1595 First Microscopes (tube with lens at
each end; 3X to 9X magnification)
– Hans and Zacharias Janssen, Dutch eyeglass makers
• Improvement on Compound Microcope
design and Publication of Micrographia
– Robert Hooke; 1665; coined term “cell”
• First description of Bacteria (tooth
scrapings) and Protozoa (pond water)
– Anton van Leeuwenhoek
Detecting Pathogens and
Indicators in the
Environment
Microscopy
• Goal of microscopy is to improve resolving
power
– Resolving power is ability to distinguish two
points as separate
– Function of light and aperture of objective
• Resolution=smallest visible distance
between two points
– Human eye can resolve about 150μm
between two points
– Most light Microscopes can resolve ~0.2 μm
Magnification
• The ability to enlarge the apparent size of
an image
– Function of resolving power of microscope
and the eye
• Limit of resolution of eye/limit of resolution
of microscope = magnification
– e.g. 0.15mm/0.0002mm = 750X
Microscopic and Imaging Detection of Pathogens
• Still widely used for parasites and bacteria
• Specific staining and advanced imaging to distinguish
target from non-target organisms
– Differential interference contrast microscopy
– Confocal laser microscopy
• Distinguish infectious from non-infectious organisms
– Combine with infectivity, viability or activity assays
• Overcome sample size limitation due to presence of nontarget particles
– Flow cytometry and other advanced imaging
techniques
– Advanced imaging methods require expensive
hardware
Types of Microscopes
• Light microscopes
– Compound
– Dissection/stereo
– Inverted
– Confocal
• Electron Microscopes
– Scanning
– Transmission
Types of Microscopes
• Light microscopes
– Compound
– Dissection/stereo
– Inverted
– Confocal
• Electron Microscopes
– Scanning
– Transmission
Types of Microscopes
• Light microscopes
– Compound
– Dissection/stereo
– Inverted
– Confocal
• Electron Microscopes
– Scanning
– Transmission
Types of Microscopes
• Light microscopes
– Compound
– Dissection/stereo
– Inverted
– Confocal
• Electron Microscopes
– Scanning
– Transmission
Types of Microscopes
• Light microscopes
– Compound
– Dissection/stereo
– Inverted
– Confocal
• Electron Microscopes
– Scanning
– Transmission
Types of Microscopes
• Light microscopes
– Compound
– Dissection/stereo
– Inverted
– Confocal
• Electron Microscopes
– Scanning
– Transmission
Light Microscopy
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Bright Field
Dark Field
Phase Contrast
Differential Interference Contrast
Epifluorescence
Confocal Scanning
Kohler Alignment
Bright field
• Most common of all light scopes
• Light is transmitted through specimen
• Specimen appears darker than
surrounding field
• Typical use: Gram Stains
Gram Stain
Microscopic Detection of Pathogens:
Still Widely Used in Clinical Diagnostic Microbiology
C. parvum
oocysts ~5 um
diam.
Acid fast stain of
fecal preparation
Dark Field
• Used to increase the contrast of a
transparent specimen
– Contrast = ability to distinguish an object from
surrounding medium
• Specimen appears as a bright image
against dark background
• Often used to observe live nonfixed/stained samples, e.g. observe
motility and growth
Darkfield Microscopy
Phase Contrast
• Used to observe fine internal detail
• Takes advantage of differences in density
of transparent internal cell components
• Uses a series of diaphragms to separate
and recombine direct versus diffracted
light rays
DIC
• Illuminating light beam is split such that
one beam passes through the specimen
creating a phase difference with the
second reference beam
• Beams are then combined so that they
interfere with eachother
• Allows detection of small changes in in
depth or elevation of the surface of the
specimen
– Thus gives 3d appearance
Cryptosporidium parvum
Differential Interference Contrast Microscopy
Image courtesy of O.D. “Chip” Simmons, III
Fluorescent
• Uses UV light source to illuminate
fluorescent dyes that then emit visible light
– e.g. FITC, Acridine Orange, Rhodamine
• Specimens appear as bright colored
objects in front of black background
• Often used with immunologic procedures
Cryptosporidium parvum:
Microscopic Analysis of NC field isolate
Immunofluorescence
Differential Interference
Contrast
DAPI stain
Images courtesy of O.D. “Chip” Simmons, III
Electron Microscopy
• SEM-Scanning Electron Microscopy
– Image is formed as electron probe scans the
surface of the specimen
– Produces 3d images
• TEM-Transmission Electron Microscopy
– Image formed as electrons pass through
specimen
– Specimens must be thin cut
– Used to view internal structure
Activity Assays/Vital Dyes
Detection of Pathogens by Viability or
Activity Assays
Assay bacteria for viability or activity by combining microscopic
examination with chemical treatments to detect activity or
"viability".
– measure enzymatic activities, such as dehydrogenase,
esterase, protease, lipase, amylase, etc.
• Example: tetrazolium dye (INT) reduction:
2-[p-iodophenyl]-3-[p-nitrophenyl]-5-phenyltetrazolium
Cl (measures dehydrogenase activity).
• Reduction of tetrazolium dye leads to precipitation of
reduced products in the bacterial cells that are seen
microscopically as dark crystals.
FISH:
DAPI-stained Bacteria Incubated with INT (Tetrazolium Salt)
Enhanced image with
artificial colors.
•Blue: DAPI stain
•Red: INT grains;
indicate respiratory
active bacteria.
Progress in Detection of Bacteria by
Viability or Activity Assays
• Combine activity measurement and immunochemical assay (for
specific bacteria).
– Combine fluorescent antibody (FA) (for detection of specific
bacterium or group) with enzymatic or other activity
measurement
• Use image analysis tools to improve detection and quantitation
– Flow cytometry
– Computer-aided laser scanning of cells or colonies on filters
Viability or Activity Assays for Protozoan Cysts and
Oocysts
• Example: Stain with DAPI (the fluorogenic stain
4',6-diamidino-2-phenylindole; taken up by live
oocysts and propidium iodide (PI; taken up by dead
oocysts).
– Viable Cryptosporidium oocysts are DAPI-positive and PInegative
– Non-viable oocysts are DAPI-negative and PI-positive
• Alternative stains may be more reliable
• Viability staining is often poorly associated with
infectivity
Detects cysts and oocysts inactivated by UV and chemical
disinfection
C. parvum oocysts
Dual stain : DAPI (blue) and propidium iodide (red)
Immunological Methods
Immunoglbulins
• 5 Classes
– IgA secretory
– IgD found in plasma but not serum
– IgE involved in allergic reactions
– IgG humeral response
– IgM humeral response
• IgG and IgM most commonly used in
immunoassays