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 and immersion oil
• Refractive index of bacteria ~ water (so invisible)
Microscopy cont.
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.
Microscopy cont.
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?
Morphology cont.
• 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)
Fine Structure, Composition and Function
Bilayer
Sterols vs hopanoids
Bacterial, eukaryal and archaeal
membrane lipids
Ester link
Ether link
Side chains
are fatty
acids
Isoprene
side chain
= Archaea
Structure of Archaeal membranes
Note, monolayer
Structure and function-membrane
transport proteins
• 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 Nacetylglucosamine (like
Bacteria) and Nacetytalosaminuronic 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
Fine Structure, Composition and Function
• DNA , 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
Genome size
Flagella
• Filament
– Flagellin
– Hollow; self assembly
– wavelength
• Powered by PMF
• 1000 protons/rotation!
• FAST
Motility
Polar and peritrichous flagella
Taxis
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
Storage materials and inclusions
• Carbon-storage polymers
– Poly--OH-alcanoate
(PHA)
– Poly--OH-butyrate
(PHB)- lipid
– Glycogen, Starch (alfa 14 glucose linkages)
• Polyphosphate
• Sulfur
• Cyanophycin , (nitrogen
polymer in cyanobacteria)
• Magnetite (Fe3O4 )
• Gas vesicles (membrane
structures found in a wide diversity
of microbes, many aquatic
microbes for buoyancy)
(Antartica, Jim Staley, U
Washington)
Gas diffuses freely across the
membrane
Endospores
• Resistant to heat,
drying, etc.
• Survival, not
procreation• Spores in amber (25-40
My)
• Bacillus and
Clostridium
Early stages of endospore
formation
Middle stages of endospore
formation
Completion of endospore
formation
See table 3.2
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