Microbio Module 3

Micrometer and Nanometer measurements

micrometer: 10^-6m

nanometer: 10^-9m

how tiny can human eyes vs light microscopes vs electron microscopes see

human: >200 µm

light: >0.2 µm

electron: <1nm

Resolution

distinguish 2 things as separate (how close can 2 objects be before they look like 1?)

higher resolution = more detail

Contrast

difference in light between object and background

higher contrast = easier to see

Brightfield Microscope

most common

light passes THROUGH specimen

light source —> condenser —> speciment —> objective lens —> ocular lens/eyepiece

disadvantages of Brightfield

unstained cells = basically invisible

requires staining

staining kills/fixes cells

magnification formula

objective x ocular

illuminator

light source

Iris diaphragm

controls amount of light entering condenser

condenser

focuses light through specimen

objective lens

primary lens that magnifies specimen

• closest to specimen

• provides most magnification

body

transmits image from objective to ocular lens using prisms

ocular lens (eyepiece)

remagnifies image formed by objective lens

usually 10x

Phase Contrast Microscope

Enhances tiny differences b/w cell and its background

• best for LIVE cells (no staining needed)

• can look at motility + internal structures

• more specialized than brightfield

Dark Field Microscope

light reflects OFF speciment at an angle

(GREAT contrast) black background, bright/glowing specimen

• best for THIN organisms

• can’t see internal structures

Fluorescence Microscope

uses fluorophores (flourescent dyes/proteins)

uses UV light —> excites fluorophores at diff wavelengths —> glow diff colors (wide variety)

dark background, bright colored objects

• ex: GFP, RFP, YFP

• used to locate specific proteins, detect antibodies

• can’t visualize intracellular structures

how are fluorescent proteins expressed in a cell alone

1. nonspecifically illuminating cell as whole

2. linked (coupled) to normal protein of interest

3. tags on molecules/antibodies —> designate presence/absence of specific protein target

fluorescence detected: protein present

no fluorescence: protein absent

Confocal (Laser Scanning) Microscope

fluorescence microscope + 3D imaging

high-resolution, 3D, multiple cell layers, light = laser light

layer scans 1 thin layer at a time —> computer stacks images together —> 3D image

• GREAT resolution + contrast

• 3D

  • individual organelles visible

Why use Electron Microscopes

light microscopes stop at 0.2 µm

viruses < 0.2 µm

electron microscopes can see viruses (resolution <1nm) = 200x better than light microscopes

uses beams of e-

• e- wavelength < light wavelength

• shorter e- wavelength —> higher resolution

Disadvantages of electron microscopes

expensive, complex, labor intensive

sample must be DEAD

process may alter cell structure

TEM

electron passes THROUGH thin slices of specimen (transmission = through)

coated in preservatives, heavily treated

sample in b/w e- beam source and detector

• 2D

• subcellular structures (organelles), viruses

SEM

electrons bounce OFF the surface (scanning = surface)

specimen coated w/ gold or palladium (e- coated)

• 3D appearance

• surface details, external shape

STEHM

HIGHEST resolution (35 pm)

• protein surfaces

• subatomic structures

stains used to examine

b/c most cells = transparent

chemical dye = makes microorgs easier to see

• tissues

• specific cell types

• organelles w/in a cell

differential stain

staining technique that separates specimens into subgroups

• usually uses at least 2 dyes

1. gram

2. acid-fast

3. giemsa

simple stain

uses 1 positive charged dye

examine: size, shape, arrangement

gram stain

developed by Hans Christian Gram (1884)

distinguishes thickness of peptidoglycan cell wall (thick/thin)

Gram Positive

very thick peptidoglycan cell wall (overlapping layers)

• permeable

• no outer membrane

+crystal violet —> +iodine —> stable complex —> thick peptidoglycan traps dye

—> cell stays PURPLE

Gram negative

thin peptidoglycan cell wall

• outer membrane = made of LPS (lipopolysaccharides)

+crystal violet —> +iodine —> thin wall can’t trap dye —> alcohol wash removes dye —> LPS loses color —> +safranin —> cell becomes PINK

Gram Stain Steps

Color It And Save (CIAS)

1. crystal violet — primary stain

2. iodine — forms stable complex

3. alcohol — decolorization wash

4. safranin — counterstain

why does alcohol matter

removes crystal violet from gram negative

BUT NOT

gram positive (b/c it has thick peptidoglycan)

heat fixation

cells must stick to slide before staining —> prevents cells from washing away BUT kills microorg (can’t observe motility)

1. air dry sample

2. pass through flame (cell attaches)

3. can stain

Chemical fixation

instead of heat, chemicals can attach cells (still kills cells)

ex:

• formaldehyde

• ethanol

• methanol

wet mount

opposite of Gram stain

wet = alive: no fixing, no heating, no killing

can observe movement, behavior, motility

1. drop of liquid on glass slide

2. cover slip

Simple stain

1 positively charged dye to stain organism

quickly determine: shape, size, arrangement

ex:

• methylene blue

• crystal violet

• safranin

• fuchsin

why positive dye?

bacterial membrane = neg charge

opposites attract, positive dye sticks

Negative stain

opposite of simple stain

stain everything BUT organism

dye = nigrosin (India Ink) = neg charge

dye + membrane = both neg charge, like repels, dye can’t attach

positive vs negative dye

negative = background gets color

positive = cell gets color

acid-fast stain

some bacteria do not Gram stain well

• esp Mycobacterium TB b/c very thick, lipid-rich protective membrane

1. primary stain = Carbolfuchsin (red)

2. alochol wash (acid-fast stay red)

3. counter stain = methylene blue

4. non-acid fast-become blue

TB (acid-fast)= red

healthy background = blue

Giemsa Stain

combined with Wright’s stain

used for blood smears

• Malaria

• blood parasites

human blood cells: purple

bacteria: pink

structural stains

features of bacterial cells

1. endospore

2. capsule

3. flagella