chapter 4a: microscopy & microbial classification · microbial classification 1. ......
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Chapter 4A:Microscopy &
Microbial Classification
1. Types of Microscopy
2. Staining
3. Classification & Nomenclature
Visible light
10-8m
700 nm400 nm
Increasing resolving power
Increasing wavelength
Trough
100m10-4m10-12m
Crest One wavelength
103m
UVlight
Infra-red
Micro-wave
Xrays
Radio waves andTelevisionGamma rays
The Electromagnetic Spectrum
Diameterof DNA Ribosomes
Mitochondrion
Typical bacteriaand archaea
VirusesProteins
Aminoacids
Atoms
Largeprotozoan (Euglena)
Human redblood cell
Chloroplasts
Flea
Chickenegg
Pig
Unaided human eye200 µm–
Transmission electron microscope (TEM)10 nm–100µm
Scanningtunneling
microscope(STM)
0.5 nm–10 nm
1 nm 10 nm 100 nm0.1 nm 1 µm 10 µm 100 µm
Scanning electron microscope (SEM)10 nm–1 mm
1 mm 10 mm 100 mm 1 m 10 m
Atomicforce
microscope(AFM)
1 nm–10 nm
Compound light microscope (LM)200 nm–10 mm
Scale of Magnification
Microscopeobjective
Refractedlight rays
lost to lens
Light sourceSpecimen
Slide
Glass cover slip
LensesMicroscope
objective
Light sourceSpecimen
Slide
Glass cover slip
Without immersion oil With immersion oil
Immersion oil
Unrefractedlight rays enter lens
Oil Immersion & Light Refraction
Different media (air, water, glass, oil…) bend lightto different degrees. • i.e., have different refractive indexes• immersion oil has refraction index similar to glass,allows more light to enter the lens
Bright vs Dark Field Microscopy
Light refractedby specimen
ObjectiveLight unrefracted
by specimen
Specimen
Condenser
Dark-field stop
Dark-field stop
only light refractedby the specimen
will enter theobjective lens
(“normal” light microscopy)
Nucleus
Phase contrast
Rays in phase
Ray deviated byspecimen is 1/4 wavelength out
of phase.
Phase plate
Rays out of phase
Bacterium Deviated rayis now 1/2
wavelength out of phase.
Phase Contrast Microscopy
• reveals internal detailwithout staining
• useful for live specimens
Enhances misalignment of light waves to create contrast
Bacteria
Nomarski
Differential Interference Contrast (DIC) Microscopy
A variation on phase-contrast microscopy involving a more complex combination of filters and prisms.
• also referred to as “Normarski Microscopy”
• creates an image with evengreater detail and contrast
• image has a 3-dimensional appearance as if it was illuminated from the side
Fluorescent Microscopy Fluorescent dyes or antibodies with a fluorescent tag stick to specific targets.
Under UV light, dye fluoresces, onlylabeled cells or structures are seen.
confocalstandard
Confocal Fluorescence Microscopy
Only light from a given depth or plane is transmitted, “out of focus” light is excluded
Electron Microscopy Electromagnetic lenses focus electron beam onto metal-stained specimen.
• electron beams have veryshort wavelengths• allows far greater resolution
than with light microscopy
Transmission EM (TEM)• thin sections of specimen,
highest resolution
Scanning EM (SEM)• reveals surface features
Other types of Microscopy Scanning-Tunneling & Atomic Force microscopy use special fine-tipped probes to produce highest resolution.
SCANNING-TUNNELING• distance between probe and specimen determined by electron flow between them
scanning-tunneling (STM)
atomic force (AFM)
• deflection of laser aimed at probe tip produces image
ATOMIC FORCE
DNAdoublehelix
PlasmidDNA
Why the Need for Stains?Because, no matter how high the magnification or resolution, you need contrast to be able to see anything.
If contrast is not sufficient in the sample orthe microscopic method used, staining canprovide the necessary contrast:
• stains used for viewing bacteria via light microscopy aretypically positively charged chromophores (basic dyes)
• chromophore = “color-bearing” ion of a salt
• bacteria have a net negative charge (i.e., bind positive ions)
General Types of StainsSimple stain• dye that non-specifically stains all organisms, features
Differential stain• dye that binds various structures or organisms differently
Negative stain• dye that stains background, not specimen
Special stain• dye that specifically stains certain subcellular structures
**a mordant is any chemical added to enhance a stain**
Counter stain• a 2nd dye added that is a different color than original dye
Spread culture inthin film over slide
Air dry
Pass slide throughflame to fix it
Fixing the Specimen on a SlideSpecimens must first be “fixed” to the slidesurface before they can be stained.
• generally done by smearing a sample on the slide, airdrying, and passing briefly through flame to “heat fix”• specimens can also be fixed to the slide surface bychemical means
Gram Staining A very common stain to distinguish 2 bacterial types:
Process:1) 1o stain
(crystal violet)
2) mordant (iodine)
3) decolorize* (alcohol)
4) counter stain (safranin)
• retains primarystain (due to thickpeptidoglycan layerand teichoic acids)
Gram positive
• does NOT retainprimary stain, onlycounter stain
Gram negative
1 2 3* 4
* key step
Acid-Fast Staining Most bacteria are not “acid-fast” (i.e., don’t retain
primary stain afteracid wash).
• “acid fast” bacteriadetected by this stain include those in the genera:
• “non-acid-fast”cells revealed by counter stain
MycobacteriumNocardia
acid fast non-acid fast
Species Genus Family Order Class Phylum Kingdom Domain
Ursus americanus(American black bear)
Ursus
Ursidae
Carnivora
Mammalia
Chordata
Animalia
Eukarya
Taxonomic Hierarchy
species can also be subdivided
different strains
Scientific Nomenclature To avoid confusion, every type of organism must be referred to in a consistent way.The current system of nomenclature (naming) has been in use since the 18th century:• every type of organism is referred by its genus name
followed by its specific epithet (i.e., species name)
Homo sapiens (H. sapiens) Escherischia coli (E. coli)
• names are Latin (or “Latinized” Greek) with the genusbeing a noun and the specific epithet an adjective
• name should be in italics and only the genus is capitalizedwhich can also be abbreviated
**strain info can be listed after the specific epithet (e.g., E. coli O157:H7)**
Key Terms for Chapter 4A
• microscopy: bright & dark field, phase contrast, DIC, fluorescent, confocal, transmission & scanning EM,scanneling-tunneling, atomic force microscopy
• resolution, refraction & oil immersion
• simple, differential, counter, negative stains• mordant, chromophore
Relevant Chapter Questions MC: 1-10 FB: 1-5 Labeling SA: 1, 3-6
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