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Microscopy, Staining and Classification - Chapter 4

Most of these sections will be covered in lab


-- Principles of Staining
-- Taxonomic and Identifying Characteristics

Relevant metric units


mm = 10-3 meter
m (micrometer)
nm (nanometer)
Resolution vs
Magnification

Microscopy and Classification

Microscopy
How does a lens work?
Optical properties
refraction
magnification
resolution
distortion

Limitations of a single lens


magnification
illumination
aberration

Wikipedia link
http://en.wikipedia.org/wiki/Lens_%28optics%29
Microscopy and Classification

Some principles of a
compound microscope
Ocular lens
Objective lens
Condenser lens

How is magnification calculated?

Limitations to a compound
microscope

Microscopy and Classification

What were the key advances


in light microscopy?
1) Oil immersion lens
2) Fused lenses
3) Advanced optics
How do oil immersion and
fused lenses improve resolution?
Refractive index =

Speed of light in vacuum


Speed of light in medium

Borosilicate glass = 1.52


Air = 1.00
Water = 1.33
Immersion oil = 1.52

Fused lenses combine different types of glass


-- compensate for aberrations
Microscopy and Classification

Advanced optics only improve contrast


Theoretical limit of resolution
= 0.2 uM

Dark field

Bright field

Dark field
manipulates the
path of light

DIC

Phase contrast
and DIC
manipulate
phase of light

Microscopy and Classification

Fluorescence Microscopy allows precise


staining of cells and cellular components

Standard fluorescence
manipulates the
path of light

Standard Fluorescence vs Confocal image of Tetrahymena

Confocal
-- puts everything in focus

Microscopy and Classification

How does electron


microscopy yield
higher resolution?
Resolution
- Eye ~ 0.2 mm
- LM ~ 0.2 uM
- EM ~ 0.2 nM (0.0002 uM)
Wavelengths of
of electrons ~ 0.005 nm
visible light ~ 550 nm

Microscopy and Classification

What are types of


electron
microscopy?
Scanning vs Transmission
STM is different

Driscoll et al. 1990.


Nature, 346, 6281

Microscopy and Classification

Biological classification

Domain: Bacteria
Kingdom: Eubacteria

Biological taxa

Phylum: Firmicutes
Class: Bacilli

-- Muddled in the middle

Order: Bacillales

-- Blurry at the bottom

Family: Staphylococcaceae
Genus: Staphylococcus

Binomial naming

Species: aureus

Writing conventions
Staphylococcus aureus
Staphylococcus aureus

S. aureus
Common names and strains
Pneumoncoccus
E. coli O157 H7
Microscopy and Classification

What are the 3 domains of life?


Eukarya Archaea Bacteria
What traits do Archaea
and Bacteria share?
-- prokaryotic cell structure
-- circular chromosome

What traits do Archaea


and Eukaria share?
-- Nucleosomes
-- Multiple RNA Polymerases
-- Ribosome structure

Some Archaea have unique properties


-- Genetically distinctive
-- Cell membrane
-- Cell wall structure

The Antiquity of Microbes


Adam
Had em
-- Strickland Gillilan

Microscopy and Classification

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What are the characteristics of


Eukaryotes vs Prokaryotes?
-- all cellular organisms

Basic similarities?
membrane
genetic information
biochemistry
Distinctive properties of eukaryotes
eu = true karyo = nucleus
size
organelles
mitochondria, chloroplasts, etc
chromosomes
vs prokaryotes pre-nucleus

Microscopy and Classification

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Archaea Extremophiles
-- some notable ones
Pyrolobus fumarii -- Fire lobe of the chimney
Hyper-thermophile -- 90OC 113OC (106OC)
hydrothermal vents
Strain 121:
http://www.genomenewsnetwork.org/articles/08_03/hottes
t.shtml

Picrophilus spp.
Extreme acidophiles -- pH <4 (opt 0.7)
Volcanic steam vents

Halobacterium spp.
Extreme halophiles -- 3 5 M salt
Dead sea; great salt lake;
Salted foods

Microscopy and Classification

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