Microscopy

Compound Light Microscope
Objective lens = real image
Eye piece = virtual image
Magnification
Condenser lens and iris diaphragm
Other terms
Resolving power (resolution)
Refractive index of bacteria ~ water (so invisible)

Bright Field
Stains, Gram, spore, flagella, Sudan black etc.
Cell surface is negative therefore stains positive
Simple stains
Differential stains

Phase Contrast and Dark Field
Phase: amplifies the slight difference in the refractive index and converts the difference into contrast.
Dark:special condenser only light from specimen enters objective

Fluorescence
Uv or halogen light source
Illuminate from above
Use different filters to select for different wavelengths
Cyanobacteria glow red =chlorophyll and other pigments =autofluorescence
Stains, ribosomal RNA probes, DAPI etc.

3-D imaging
Differential Interference Contrast (plane polarized)
Atomic Force (repulsive atomic forces)
Confocal Scanning Laser Microscopy (laser light)

Electron Microscopy
TEM
Electron gun = e- which is the illumination
Get REALLY short wavelengths = greater resolution
Need to make thin sections
SEM


Morphology
Cell size, why be small?
Unicellular
Rods or bacillus, vibrio spirullum
Cocci (chains, diplococci, grapes)
Filaments, sometimes filaments can be deceptive. To the naked eye you think they are filaments but under the microscope short rods stick together as filaments (PIC from Yellowstone)
Multicellular, like actinomycetes, mycelium (PIC deep-sea Actinomycetes)
Oscillatoria makes a trichome (PIC)
Shealthed and filamentous

Division
Binary, septum produced along transverse axis
DNA replication occurs before septum formation
Budding, less common in prokaryotes (some Archaea like Sulfurococcus)
Fragmentation, actinomycetes do this, filament fragments to form unicellular
rods


Fine Structure, Composition and Function
Cell membranes
Cell membranes, the ultimate barrier between cytoplasm and external environment, gases & water (small uncharged molecules pass through) diffuse readily, ions do not
Bacteria, ester linked
Archaea, glycerol linked ethers (thermophilic microbes >>> tetraethers)

Bacterial, eukaryal and archaeal membrane lipids

Structure of Archaeal membranes

Structure and function-membrane transport proteins

Group translocation
Substrate chemically altered

Cell walls
Bacteria, almost all have peptidoglycan (murein), over 100 different peptidoglycan structures , differences are based on the amino acids and how they cross link
N-acetylglucosamine
N-acetylmuramic acid
Lysozyme sensitive

Archael cell walls
Archaea have pseudopeptidoglycan contains N-acetylglucosamine (like Bacteria) and N-acetytalosaminuronic acid (unlike Bacteria which have of N-acetylmuramic acid)

Bacterial cell walls
BACTERIA
Gram +
40-80% is peptidoglucan
teichoic acids, polyol phospate polymers
Gram -
No teichoic acids
Only one layer of peptidoglycan, (5% of cell wall weight)
Outer membrane similar to cytoplasmic membrane, lipids, proteins but also polysachharides
LPS or lipopolysaccharide layer (lipid A= endotoxin, bubonic plague, typhoid fever etc.)
Proteins like porins Omp C and Omp F

Antibiotics and cell walls
Antibiotics and bacterial cell walls
Function to inhibit production of enzymes that make peptidoglycan (eg Penicillin)
Why are Gram + more sensitive than Gram -?

LPS-Gram ive Bacteria
LPS or lipopolysaccharide layer (lipid A= endotoxin, bubonic plague, typhoid fever etc.)
Proteins like porins Omp C and Omp F

Capsules
Protective outer layer made up of polysaccharides, some polypeptides
Often house the virulence factors eg. Steptococcus pneumoniae

Protein layers, -S-layer, sheaths
S-layer, perhaps involved in mineral precipitation?
Very fragile
Sheaths-complex composition, important to many iron oxidizing bacteria

concentrates in an area in cytoplasm= nucleoid
In general: 4 X 106 Mbp
Plasmids (carry important functions like metal resistance, naphthalene degradation, antibiotic resistance; also may be important for lateral gene transfer)
Transcription
Transduction, tranformation, conjucation

Ribosomes, 30S + 50S = 70S (cause Svedberg unit not directly related to molecular mass, rather density!)
Antibiotic sensitivity
translation

Flagella
Filament
Flagellin
Hollow; self assembly
wavelength
Powered by PMF
1000 protons/rotation!
FAST

Motility and peritrichous flagella

Cell surface structures
Fimbria (-ae)
Structurally like flagella, but shorter
Various functions including adhesion
Pilus (-i)
Longer than fimbriae
Functions include conjugation
S-layer
Capsule or slime layer
Collectively called glycocalyx
Avoidance of phagocytosis, dessication

Endospores
Resistant to heat, drying, etc.
Survival, not procreation-
Spores in amber (25-40 My)
Bacillus and Clostridium

Eukaryotic cells
DNA in nucleus
DNA arranged in chromosomes
Ribosomes: 80S
Organelles
Mitochondrion (-ia)
Chloroplast

Mitochondria and chloroplasts are prokaryotes
Contain DNA
Closed, circular
Prokaryotic ribosomes (70S)
Antibiotic sensitivity
Ribosomal phylogeny
Mitochondria are related to proteobacteria
Chloroplasts are related to cyanobacteria