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 link Ether 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 % 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 10 6 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 1- 4 glucose linkages) Polyphosphate Sulfur Cyanophycin, (nitrogen polymer in cyanobacteria) Magnetite (Fe 3 O 4 ) 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 ( 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