Define microscopy

Microscopy

The usage of microscopes to view

microorganisms

Common units of
measurement

The Metric System

1m = 1m
1000mm = 1m
10^-6mm = 1 micron
10^-9mm = 1 nanometer

Define magnification

Magnification

allows microscopic specimens to become

visible to the naked eye

Define resolution

Resolution

the ability to resolve two objects as

separate, or distinguish objects that are
close together.

What is the resolution

distance dependent on?

Resolution
depends on the wavelength of
electromagnetic radtion, and the
numerical aperture of the lens (i.e. the
ability of a lens to gather light)

Define contrast

Contrast

the differences in intensity between the

specimen and its background

2 ways to enhance contrast

Contrast

1. staining techniques
2. in-phase wavelengths

What are the limits of light

microscopy?

The limits of light microscopy

1. max 2000x magnification

2. resolution is limited down to 0.2 microns;
anything less than that = no.

Explain (a) light refraction, and

(b) why we use an immersion oil
when using light microscopes

The refraction of light

Light passing through a lens refracts, or

bends, because light rays slow down as
they enter the glass. However, it doesn't
capture some light rays which is why we
use immersion oils.

Immersion oil
taking into account that light passes through
immersion oil at the same speed as it
does with glass, immersion oil is used as a
"bridge" between the slide so light travels
at a uniform speed

Bright-field microscope

Bright field microscope

illuminated background
stain cells to increase contrast
cheap, easy-to-use

Dark-field microscope

Dark-field microscope
light that is really reflected is magnified
no stain needed
specimen appears light against a dark
background
good for cells that move

Phase contrast microscope

Phase microscope
in-phase lightwaves = brighter image
out-of-phase lightwaves = darker image
bring both lightwaves together = phase-shift =
increased contrast
2 types: phase-contrast & DIC

Phase contrast microscope

Phase contrast
provide greater resolution of internal structures
simplest phase microscope

DIC microscope

DIC
differential interference contrast

use prisms to split light beams into their

component wavelengths = increase
contrast
3D

Electron Microscopes
(2 Types)

Electron microscopes
use electrons instead of light
can resolve down to 1nm
TEM & SEM

Electron Microscopes
(2 Types)

TEM
2d images w/ views of internal structures
works by "slicing" specimen
stain w/ uranium salts to increase contrast

SEM
3D images of external structures
paint surface of object w/ heavy metal
view the surface

Staining

Staining
increases contrast and resolution of a
specimen
dyes stick through charged
chromophores; positively-charged dyes
stick to the negatively charged surfaces
most cells have

Anionic and Cationic chromophores

Anionic and Cationic

chromophores
acidic dyes contain anionic chromophores
less commonly used
basic dyes contain cationic chromophores

Simple stains
(2 types)

Simple stains
direct and indirect

Direct stains

Direct stains
simple stains w/ basic dyes (such as CV
or methylene blue)
coating w/ positively-charged dye on
negatively-charged surface of the cell
stains the organism

Indirect stains

Indirect stains
negative stains (such as eosin)
slathering the slide w/ negatively-charged
dye
stains around the organism

Differential stains
(3 types)

Differential stains
Gram
Endospore
Acid-fast

Gram Stain

Gram Stain
used to distinguish thickness of PG layer
Gram positive = thick PG layer (purple)
Gram negative = thin PG layer (pink)
1. smear & heat fix
2. primary stain - basic dye (CV)
3. mordant - binds basic dye to cells (I)
4. decolorizer - thick PG layer harder to remove
(Acetone/Alcohol)
5. counterstain - stain decolorized cells; G- cells are now
pink (Safranin)

Endospore Stain

Endospore stain
stains resting body of dehydrated DNA w/ protective
coats
(endospores cannot be stained by normal staining
procedures)
1. smear and heat fix
2. primary stain (malachite green)
3. steam to push primary stain pass the coat and into the
endospore
4. decolorize w/ water - endospore remains green
5. counterstain (safranin) - cell body now pink

Acid-fast Stain

Acid-fast stain
used on G+ cells with thick PG layer and waxy lipid cell wall
1. smear and heatfix
2. primary stain (carbolfuschin)
3. heat (steam) to push dye into waxy cell wall (carbolfuschin
dissolves in wax)
4. decolorize w/ alcohol & HCl (if dye fastens to cells = acidfast = pink
if dye washes off easily = cell lacks waxes = clear)
5. counterstain (methylene blue) to stain bleached, non acid
fast cells

Negative (Capsule) Stain

Capsule layer
protein-sugar layer outside the cell wall
uncharged
use a mixture of a direct and an indirect stain
1. mix cells w/ congo red (negative, acidic stain)
2. let the smear air dry
3. flood cells w/ basic fuschin dye (sticks to PG layer)

5 main elements that make

up most of a cell's mass +
functions

PONCH
C - backbone off organic molecules
H - organic molecules and water; H+ released
by acids
O - ''; OH- released by bases
N - amino acids, proteins, nucelic acids
P - nucleic acids, atp, storage, transfer
membranes
(S) - proteins

Importance of minor and

trace elements

Minor & trace elements

elements that are required in small ("trace"; <1%)
amounts for proper growth
Minor elements: S, K, Mg, Ca, Na, Fe, Cl
Trace elements: Mn, Co, Zn, Cu, Mo

Covalent bonds

Covalent bonds
the sharing of a pair of electrons by two
atoms
includes H bonding
Polar covalent: charged due to unequal sharing
of electrons
Nonpolar covalent: atoms share electrons
equally = uncharged

Hydrogen bonds

H bonds

H bonded to a highly EN atom such as F, I,

Br, Cl, etc.
explains why water is cohesive
3d structure
weak; requires less energy to break
cells = 70% water = very important
allows signaling to take place

Ionic bonds

Ionic bonds
two atoms exhange electrons
usually between a metal and a nonmetal
charged
salts
dissolvable by water usually

Hydrophylic vs. Hydrophobic

Hydrophilic vs. Hydrophobic

Hydrophilic: water-loving; dissolvable by
water
Hydrophobic: water-fearing; NOT
dissolvable by water
Amphipathic: both properties

Acids vs. Bases

Acids vs. Bases

Acids: increases concentration of H+ in
solution when dissolved in water (H+
released)
HCL

-->

H+

Cl-

Bases: decreases concentration of H+

when dissolved (OH- released)
NaOH + H20 --> Na+ +

OH-

Cations vs. Anions

Cations vs. Anions

caTions
Anions

Cations vs. Anions

Cations vs. Anions

caTions
Anions

Organic vs Inorganic
compounds

Organic vs Inorganic
compounds
Inorganic:
compounds w/o C except CO2
water, O molecules, metal ions, acid
compse <2% of an organism's mass

Organic:
compounds w/ C made by cells except CO
and CO2
compose approx. 98% of cells

Hydrocarbons

Hydrocarbons
hydrophobic chains of C and H
dissolve and react through the addition of
functional groups

Functional groups

Functional groups
allow certain molecules (such as
hydrocarbons) to react with each other
*OH
*NH2
*COOH
SH
P

* adds polatiry due to their electronegativity

Polymers vs. Monomers

Polymers vs. Monomers

Monomers: basic building blocks
join together via dehydration synthesis (water is
released in the reaction, requires energy)

Polymers: chains of monomers

break down via hydrolysis (water is a reactant,
releases energy and monomers)

Make up carbohydrates, proteins, nucleic acids,

and lipids

Monomer: Monosaccharide