Microscopy and Classification 1

Microscopy, Staining and Classification - Chapter 4

Most of these sections will be covered in lab-- Principles of Staining -- Taxonomic and Identifying Characteristics

Relevant metric unitsmm = 10-3 meter

μm (micrometer)

nm (nanometer)

Resolution vs Magnification

Microscopy and Classification 2

Microscopy

How does a lens work?

Optical propertiesrefraction

magnification

resolution

distortion

Limitations of a single lens

magnification

illumination

aberration Wikipedia linkhttp://en.wikipedia.org/wiki/Lens_%28optics%29

Microscopy and Classification 3

Some principles of a compound microscope

Ocular lens

Objective lens

Condenser lens

How is magnification calculated?

Limitations to a compoundmicroscope

Microscopy and Classification 4

Speed of light in vacuumSpeed of light in medium

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 =

Borosilicate glass = 1.52Air = 1.00Water = 1.33Immersion oil = 1.52

Fused lenses combine different types of glass-- compensate for aberrations

Microscopy and Classification 5

Advanced optics only improve contrast

Theoretical limit of resolution = 0.2 uM

Dark fieldmanipulates the path of light

Phase contrast and DIC manipulate phase of light

Dark field

Bright field

DIC

Microscopy and Classification 6

Fluorescence Microscopy allows precise staining of cells and cellular components

Standard fluorescencemanipulates the path of light

Confocal -- puts everything in focus

Standard Fluorescence vs Confocal image of Tetrahymena

Microscopy and Classification 7

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 8

What are types of electron microscopy?

Scanning vs Transmission

STM is different

Driscoll et al. 1990. Nature, 346, 6281

Microscopy and Classification 9

Biological classification

Biological taxa

-- Muddled in the middle

-- Blurry at the bottom

Binomial naming

Writing conventionsStaphylococcus aureusStaphylococcus aureusS. aureus

Common names and strainsPneumoncoccusE. coli O157 H7

Domain: Bacteria

Kingdom: Eubacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Staphylococcaceae

Genus: Staphylococcus

Species: aureus

Microscopy and Classification 10

The Antiquity of MicrobesAdamHad ‘em

-- Strickland Gillilan

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

Microscopy and Classification 11

What are the characteristics of Eukaryotes vs Prokaryotes?

-- all “cellular organisms”

Basic similarities?membranegenetic informationbiochemistry

Distinctive properties of eukaryotes• ‘ eu’ = true ‘karyo’ = nucleus• size• organelles

mitochondria, chloroplasts, etc• chromosomes

…vs prokaryotes ‘pre-nucleus’

Microscopy and Classification 12

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/hottest.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