Chapter 2 Observing the Microbial Cell Observing Microbes

Chapter 2 Observing the Microbial Cell

Observing Microbes n Human eyes have limited resolution ¨ Resolution ¨ 150 n = mm (1/7 mm, 1/200 inch) Microscope needed to see smaller objects ¨ Eukaryotic n n microbes Protozoa, algae, fungi 10– 100 mm ¨ Prokaryotes n n Bacteria, Archaea 0. 4– 10 mm

Relative sizes of different cells

Bacterial cell morphologies n cocci n rods, bacilli n n spirilla vibrios n spirochetes n irregula shapes

Optics and Properties of Light n Visible light has wavelengths 400– 750 nm ¨ Maximum n resolution is 1 wavelength Magnification spreads light rays out ¨ To n 150 mm, resolution of our eyes Distance between photoreceptor cells

Optics and Properties of Light n Refraction ¨ Passage through lens material bends light ¨ Parabolic lens brings rays to a focus point ¨ Lens with 2 differently shaped convex sides magnifies image

Bright-Field Microscopy n Increasing resolution ¨ Use n shorter wavelength light UV, X-rays ¨ But images aren’t visible to human eye ¨ Optimize n contrast Differentiate between objects ¨ Lens quality Collects more light from specimen n Wider lens closer to specimen n Higher numerical aperture (NA) n Use immersion oil n NA = n sin

Bright-Field Microscopy n Increasing resolution ¨ Multiple lenses Correct each other’s aberrations n Compound microscope n Need to focus two lenses n Objective ¨ Condenser ¨

Staining Fix cells to hold in position n Stain with dye n ¨ Reacts n with chemical structure of organism Gram stain reacts with thick cell wall ¨ Increases n absorbance Easier to find in low-contrast conditions Gram-negative cells Gram-positive cells

Dark-Field Microscopy n Light shines at oblique angle ¨ Only light scattered by sample reaches objective ¨ With enough light, some bounces off object n Even objects smaller than wavelength of light ¨ Makes n visible objects below resolution limit Flagella, very thin bacteria Helical bundle of flagella

Phase-Contrast Microscopy Light passes through and around sample n Light through sample is refracted n ¨ Changes phase of light ¨ Light waves out of phase cancel n Sample appears dark against light background ¨ Shows internal organelles of eukaryotes

Differential Interference Contrast (DIC) Microscopy n Polarized light passes through specimen ¨ Sample boundaries bend light ¨ Second polarized lens blocks light ¨ Bent light affects brighter or darker than Cell nuclei background Head of microscopic worm (C. elegans) Bacterium Pharynx (mouth) 10 mm

Fluorescence Microscopy n Fluorophores absorb high-energy light ¨ n Emit lower-energy light ¨ n UV blue green red Label molecules of interest in cell ¨ Marker for position of molecules within cell

Fluorescently Labeling Molecules n Attach directly to some molecules ¨ DAPI n binds DNA Attach labeled antibody to molecules ¨ Antibody n binds specific molecules Fluor covalently bound to antibody

Fluorescently Labeling Molecules n Attach labeled nucleotides to DNA ¨ Nucleotide n n probe base-pairs to DNA Fluor covalently bound to probe Gene fusion ¨ Protein n of interest fused to fluorescent protein GFP from jellyfish

Electron Microscopy n Electrons behave like light waves ¨ Very high frequency ¨ Allows very great resolution n n A few nanometers Sample must absorb electrons ¨ Coated with heavy metal ¨ Electron beam and sample are in a vacuum ¨ Lenses are magnetic fields

Transmission EM n Sample is fixed to prevent protein movement ¨ Aldehydes to fix proteins ¨ Flash-freezing ¨ High-intensity microwaves n Fixed sample is sliced very thin ¨ Microtome n Sample is stained with metal ¨ Uranium ¨ Osmium

Transmission EM n High resolution ¨ Can detect molecular complexes Ribosomes n Flagellar base n Strands of DNA n n Need many slices to determine 3 D structure

Scanning EM n Sample is coated with heavy metal ¨ Not sliced ¨ Retains 3 D structure ¨ Gives 3 D image n Only examines surface of sample

Atomic Force Microscopy

Visualizing Molecules n X-ray crystallography ¨ Locates all atoms in a large molecular complex ¨ Sample must be crystallized n Nuclear magnetic resonance (NMR) ¨ Measures resonance between chemical bonds ¨ Can locate all atoms in a small protein ¨ Shows atomic movement of proteins in solution n Proteins embedded in membranes

X-Ray Crystallography n X-rays have tiny wavelength ¨ Resolution less than 1 Angstrom n n No lenses to focus X-rays ¨ Shoot X-rays at crystallized sample Many molecules in identical conformation n X-rays diffract according to position of atoms n Compute position of atoms from pattern of scattered X-rays n

X-Ray Crystallography n Can detect position of thousands of atoms in a complex of proteins “Ribbon” shows position of “backbone” of amino acids in proteins

Ranges of resolution