Microscopy

How do compound light microscopes work

they use visible light to illumnate specimens

Types of light microscopy

• brightfeild (what we use)

• dark feild

• phase-contrast

• fluorescene

Lenses

• light is refracted when passing from one medium to aother

• F = focal point

• f = focal length

• shorter focal length = greater magnifcation

Brightfeild microscope

• specimens are vidualized because of diffrence in contrast (denstity) between the speciment and its surondings

• total magnification = mag. of ocular lens * mag. objectve lens

• upper limit of magnification = 1000x

• upper limit of resolution = 0.2um (with blue filter)

Microscope resolution

• resolution (d) ability to distinguish two objects as distinct and seperate when viewed under a microscope clarity

• wavelength of light (shorter wavelength) leads to better resolution (or smaller d)

Why do we use blue light

decreases light

leads to better reoslution

Numerical aperature (NA)

measure of light gathering ability

higher NA —> better resolution

Increase refraction index —> icnrease NA

Formula for resolution (D)

1st step for improving contrast in light microscopy

Fixation

• preserves specimens and fixes them own position

• organisms usually killed and firmly attached to microscope slide

  • heat fixation; used for bacteria and archaeons

  • chemical fixation; used with larger more delicate organisms

2nd step for improving contrast in light microscopy

staining

• make internal and eternal structure of cell more visible by increasing (contrast) with backround

two common features of cells

chromophore groups

• chemical groups with conjugated double bonds

• give stain its color

ability to bind cells

2 types of stains

Basic stains

• dyes with postive charges

• bind to negativly charged structures

Acidic stains

• dyes with negative charges

• bind to postiviely charged structures

Define simple stain

a single staining agent is used

Differential staining

use more than one dye to preferentially stain features

• used to detect presense or absence of structures

• divides microorganisms into groups based on their staining properties

  • gram stain

  • acid-fast stain

  • endospore stain

Gram staining

• most widely used differential staining procedure

• divides bacteria into two groups based on diffrences in cell wall structure

  • gram postive, gram negative

Gram staining steps

1. flood the heat-fixed smear with crystal violet for 1 min (all cells purple)

2. add iodine solution for 1 min (all cells purple)

3. decolorize with alcohol briefy for 20 seconds (Gram postiive cells are purple, Gram negative cells are colorless

4. Counterstain with safranin for 1-2 mins (Gram positive are purple, gram negative are pink to red)

Acid fast staining

• helpful for staining members of the genus mycobacterium

• high lipid myotic acid contect in cell walls is responsible for thier staining characterists

Endospore staining

• heated, double staining technique

• bacterial endospore is one color and vegatative cell is a diffrent color

Capsule staining

negative stain - capsules are colorless against a stained background

Flagella staining

mordant applied to increase thickness of flagella