Microbiology - microscopy

light path

light source —> condenser lens —> objective lens —> ocular lens —> image to eye

magnification definition

the extent to which the image of an object is larger than itself

• the product of the objective and ocular lenses

  • ex: 10X and 20X give 200x magnification

resolution (resolving power) definition

the degree not which details are retained in a magnified image; ability to distinguish between two points

• R = 0.5λ / NA

• use of immersion oil increases NA of lens

bright field microscopy

light is transmitted through the specimen

• contrast between background and cell

• staining is generally required

simple stains

charged molecules

• positive charge: basic dyes, cationic, bind to negative charges

  • ex: carboxyls, phosphates

• negative charge: acidic dyes, an ionic, binds to positive charges

  • ex: amino groups

what stain is most commonly used?

a basic simple stain, since bacterial cell surfaces carry negative charges

differential stains

allows classification of a variety of bacterial species into 2 classes: positive or negative

• ex: gram stain —> gram pos or gram neg

gram stain

1) flood heated smear with violet (all purple)

2) add iodine (all purple)

3) decolonize with alcohol (some purple, some colorless)

4) counter stain with safranin (+ are purple, - are pink/red)

why do gram stains work?

the removal of the stain shows which sample is negative or positive

gram pos: thick cell wall, still purple color

gram neg: thin wall, can easily remove stain, pink/erd color

dark field microscopy

light enters through the side of the specimen

• cells bright against dark background

• no stains

• for smaller cells/structures b/c better resolution

phase contrast microscopy

special condenser places all light in same phase, objective lens blocks out-of-phase light

• no stains

fluorescence microscopy

light source causes specimen to fluoresce at a different wavelength

• uses stains in specific structures

• proteins can be localized in cells

how it works:

• UV and visible light —> filter to choose one wavelength —> apply light to specimen —> filter —> eye

transmission electron microscopy

electrong passing through sample are collected to make the image

• resolution better than light

• stain with heavy metal

scanning electron microscope

electrons that scatter off the sample, collect to produce image

• stain sample with heavy metal

atomic force microscope

tiny stylus is scanned across & just above the surface of the sample

• produces surface image, extremely fine detail

• caused by weak repulsive forced between stylus and sample —> stylus deflects —> deflection records on surface

• challenging with live samples