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
Isoprene side chain = Archaea
Side chains are fatty acids
Ester linkEther link
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 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
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
MotilityPolar 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 1-4 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