CHAPTER-3

Microscopy
van Leewenhoek
Microscopy
relative
sizes of
things
Microscopy
light
 Microscopy
compound light microscope

be able to
name the
major parts
Microscopy
Light Path
 Microscopy
compound light microscope

MAGNIFICATION:

Objective lens: we use 4x, 10x, or 40x

Ocular lens: typically 10x (that’s what we use)

What is Total Magnification?

Magnification isn’t
everything

Why can’t you see a virus

with a light microscope?
Microscope Resolution
 ability of a lens to separate or
distinguish small objects that are
close together
 wavelength of light used is major
factor in resolution
shorter wavelength  greater resolution

9
 Resolution
RESOLUTION: the ability of the optical system
to distinguish between two points
at a certain distance

http://www.microscopyu.com/articles/formulas/formulasresolution.html

microscopyu.com
Lenses and the Bending of
Light
 light is refracted (bent) when passing
from one medium to another
 refractive index
 a measure of how greatly a

substance slows the velocity of

light

11
Refractive Index

REFRACTIVE INDEX (n):

how much a medium (like
air or glass or immersion oil)
bends light

n = 1.00 for air

n = 1.33 for water
n = 1.52 for oil

http://www.microscopyu.com/articles/formulas/formulasri.html
 Refractive Index and
Oil Immersion
Immersion oil keeps the refractive
index the same (as glass) from the
condensor to your eye

** No light is lost so you can get

better resolution at higher
magnification
 Other Light
Microscope Systems
Brightfield: what we use in the lab
-- usually need to stain specimen

Darkfield: uses a disc to block light from the

center of the condenser so only light reflected
from specimen gets to your eye
-- good for living specimens
-- good for specimens that cannot be
stained or that staining methods distort
-- often used for Treponema pallidum (syphilis)
Brightfield vs Darkfield
Phase-Contrast Microscopy

• uses two sets of light waves

-- one is directly from the light source
-- one has passed through the specimen

•
 Phase-Contrast Microscopy

-- good for observing living mammalian cells

www.microscopyu.com/
 Fluorescence Microscopy

• uses an ultraviolet light source (not visible)

• some organisms are naturally fluorescent
• stain non-fluorescent specimens

• used extensively in diagnostics with

organism-specific antibodies that fluoresce
Natural Fluorescence

Osamu Shimomura, MBL Woods Hole

Natural Fluorescence

www.upenn.edu/pennnews/article.php?id=704
 Fluorescence Microscopy

UV light source Visible light out

- higher energy fluorescent
- lower energy
- shorter wavelength molecule
- longer wavelength
 Fluorescence Microscopy

It’s E. coli !
UV light source
E. coli

visible light out

antibody against E. coli

fluorescent molecule on
antibody

www.giantmicrobes.com
 Electron Microscopes

How do you clearly see smaller organisms

or structures within larger organisms?

What limited the ability of the light

microscope? Resolution!

What’s another way around the

resolution issue?
 Electron Microscopes

Electrons travel in waves just like visible light,

but the wavelength is much shorter!
-- far better resolution
-- but, specimen preparation can be a pain
stuff.mit.edu/people/rsmith80/hiphopscience.htm
 Electron Microscopes

Two kinds of Electron Microscopes:

Transmission (TEM): allows very high resolution

(~2 nm vs 200 nm for a light microscope) but you
have to have ultra-thin section specimens and stain
them with heavy metals

Scanning (SEM): no sectioning of specimens, but you

can only see the surface
 TEM vs SEM
E. coli

TEM SEM
 TEM vs SEM
viruses

Ebola virus

HIV-1 budding
Herpesvirus from a cell
in a cell
SEM
Rotavirus
TEM thin
section
TEM
negative stain www.aecom.yu.edu
pathmicro.med.sc.edu
www.microbelibrary.org
 Staining

Most specimens are colorless (colorless)

Stains color a specimen by using dyes that bind

to specific structures in the organisms

Simple stain: colors an organism so you can see it

Differential stain: colors different organisms

differently to help in identification

Negative stains color the background, not the

organism, to increase contrast
 Staining

SMEAR: organisms in liquid spread in thin film on

a microscope slide

FIX: kills the organisms and fixes them to the

microscope slide
-- heat or alcohol typically used
 Staining

Most commonly used stains are positively

charged (cationic; basic)
-- bind to negatively charged
structures in and on cells
 Staining

Simple Stain: essentially colors entire organism in

a quick procedure
-- to determine basic cell shape and structure
-- methylene blue, crystal violet, safranin
 Negative Staining

Negative Stain:
Stain the background and see the bacteria
as light colored objects on a dark
background

Negative Staining of Staphylococci

(http://homepages.wmich.edu/~rossbach/bios312/LabProcedures)
 Staining

Differential Stain: stains organisms different

colors based on a difference in some physiological
characteristic

-- often first step in identification of an

unknown organism

-- examples: Gram stain and acid-fast stain

-- a mordant is a second chemical (or process)

that increases the intensity of the primary
staining dye
 Gram Staining

Named after Gram (Hans Christian)

Procedure:
1. Heat fix smear
2. Primary stain: crystal violet
3. Mordant: iodine
4. Decolorize: alcohol
5. Counterstain: safranin
Gram Staining
 Gram Staining

mixed laboratory culture

 Gram Staining

Gram positive cells are purple

Gram negative cells are pink

 Acid-Fast Staining

Procedure:
1. Heat fix smear
2. Primary stain: carbolfuchsin
3. Heat slide for dye penetration of cells
4. Decolorize: acid alcohol
5. Counterstain: methylene blue

Used mostly to stain Mycobacterium species

(tuberculosis and leprosy)
Acid-Fast Staining

Mycobacterium tuberculosis
in sputum sample
 Spore Staining

Endospore Stain:
1. Heat fix smear
2. Primary Stain: malachite green
3. Steam heat for dye penetration to spores
4. Rinse: water removes dye except from spores
5. Counterstain: safranin
Bacillus subtilis

Bacillus megaterium

spores