bio207: lecture 1, observing microbes

detection

object able to be observed

resolution

the smallest distance where you are able to distinguish two objects as separate

magnification

allows us to see objects smaller than the resolution of our eyes; an increase in apparent size of an image

what is the resolution of the human eye?

around 100 to 200 um; this means that two objects can be at about this distance apart for us to still see them with our naked eye

a 1000x magnification would increase our resolution from (?) to (?)

~200 um; 200 um / 1000 = 200 nm

this means that we would be able to see objects up to 200 nm small through a lens that is magnified 1000x

microbes can be (detected/resolved) in a culture or environment, but only magnification (detects/resolves) single cells

detected; resolves

the resolution of the human eye is limited and determined by

the distance between photoreceptor cells in the retina

an image is magnified by

refraction of light waves by a curved lens

magnification is limited by the wavelength of electromagnetic radiation used because

details that are smaller than the wavelength of light will not be detected

this creates the rule of thumb that the distance between points that can be resolved is half the wavelength of the light used (ex. visible light at 750 nm, highest, can resolve objects with sizes down to 325 nm; at 400 nm can resolve down to 200 nm, lowest)

how should you choose what microscopy method to use?

• what size is the object (and what wavelength or electromagnetic radiation is needed?)

• is the goal to detect or to resolve?

• do I need to use stains, fluorescence, light scattering?

• do the cells need to be fixed/dead to visualize? does the microscopy method require fixation or staining/hybridization?

microscopy rundown on eukaryotic microbes

• include protozoa, algae, fungi

• 10 to 100 um

• structures can be seen under a light microscope

microscopy rundown on prokaryotic microbes

• includes bacteria, archaea

• 0.4 - 10 um - usually smaller than eukaryotes

• structures typically cannot be resolved under light microscope

microscopy rundown on phage and viruses

• mostly 5 - 200 nm (the smallest!!)

• cannot be resolved with light microscopy at all

light microscopy can only resolve an object if

• contrast exists between an object and its medium

• the wavelength of light follows the “rule of thumb:” the object is larger than half of 400 nm, or 200 nm (aka the wavelength is at most 2x the size of the object)

• magnification is able to resolve the object within 1000x magnification

light can interact with matter in four ways

absorption, reflection, refraction, and scattering

focal point

the point at which all light rays meet after refraction

why do we use immersion oil at high magnification?

maximizes the amount of light obtained by the objective lens by further refracting light inwards; immersion oil has a refractive index similar to the glass of the lens

different kinds of stains include

• simple stain: adds dark color specifically to cells, but not to the external medium

  • methylene blue

• differential stain: stains one type of cell only

  • gram stain (crystal violet + counterstain safranin)

i will review the microscopy chart in my notes

i promise