Small, smaller, smallest Science uses the metric system –Each unit differs by 1000x (10 3 ) –Example: length. (same prefixes for moles, etc.) Millimeter = 1 x meter Micrometer = 1 x meter Nanometer = 1 x meter {Angstrom = 1 x meter} Picometer = 1 x meter 1

Not all “sizes” are measured in lengths Molecules are too small to talk about length –Units are molecular weight: grams/mol i.e. how much do x molecules weigh? –Molecular weight units often = daltons Large molecules: kilodaltons (Kd) = 1000 x dalton Some large molecules & Particles measured in Svedbergs –Svedberg: based on migration rate in a centrifuge –Both mass and shape affect size of units –RNA molecules, ribosomes, viruses 2

3 Something can’t be smaller than the parts it is made of! Sand is used to make bricks, and bricks are assembled to make a house. A house can’t be smaller than a brick; a brick can’t be smaller than grains of sand. Likewise, small molecules are combined to make polymers and polymers are used to make cells. cic.nist.gov/lipman/sciviz/scan/jun24_ptC1a.jpg mages/BrickLayer.gif

In the world of small, what’s big? Cells of eukaryotic organisms are big –Nerve cells can be quite long –White blood cells are about 10 µm in diameter –An amoeba may be around 20 µm Prokaryotes and cell organelles are smaller –E. coli is about 1 µm long –A mitochondrion is about the same size Particles are smaller –Viruses range from 20 to 200 nm (0.02 – 0.2 µm) –Ribosomes, found inside cells, are about 20 nm 4

In the world of small, what’s smaller? Ribosomes, viruses, cell walls are made of polymers –Ribosomes and viruses are combinations of proteins and nucleic acids –Cell walls are made of large molecules like peptidoglycan and lipopolysaccharide Polymers are larger than the monomers they are made of –Proteins range from 10,000 to 500,000 MW Most enzymes are proteins (if it ends in “-ase”…) –Bacterial DNA is over 1 mm long! (but very skinny) –Polysaccharides can be > 100,000 MW (grams/mol) 5

In the world of small, what’s smallest? These are all small molecules ranging from 18 g/mol to 1,000 g/mol –Water, oxygen gas, nitrogen gas –Sugars (glucose, sucrose, etc.) –Amino acids –Nucleotides –Fatty acids, cholesterol, (even phospholipids aren’t big) –Organic acids found in metabolism –Vitamins –Antibiotics and most other drugs 6

7 The study of chemistry and biochemistry is necessary to understand Microbiology Habitat Diet

8 Why Chemistry? Conflict between microbe and host. Vibrio cholera toxin inserting into intestinal cells. Review Chemistry

Microscopy 3 Basic terms –Magnification: to make things appear larger –Resolution (resolving power): the ability to distinguish two points as being separate –Contrast: differences in light intensity between an object and the background. Empty magnification –An increase in magnification without an accompanied increase in resolution (points larger, but conitnue to overlap) 9

Resolution 10 On the left: low resolution, multiple objects appear continuous. On the right: improved resolution; individual points are now visible. Note that in both, contrast is good. The white bar is 200 nm, so magnification is also quite good. The limit of resolution of the human eye is about 0.1 mm. Since microbes are typically – mm, magnification is required to seem them individually.

Optical laws Light travels in waves –λ = wavelength of light –visible light: 400 – 700 nm Numerical aperture –a characteristic of the quality of the lens –a measure of the light gathering capacity of the lens Resolution is affected by both – Resolution (r) = 0.61 λ /NA –where r is the smallest distance at which two points can be distinguished as being separate. 11

Types of Light microscopy Brightfield –specimen appears grey/stained; background is bright Darkfield –specimen appears bright; background is dark Phase contrast –light is slowed down after hitting specimen, becomes out of phase w/ background light (destructive interference) –specimen appears very dark (or bright); light background Differential interference Contrast (DIC) –“Nomarski optics”, prism changes speeds of light –shadowed effect, lights, darks, 3D appearance 12