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Ch. 4.

Microscopy and Staining


Learning Objectives:
 Measurements
 Magnification
 Resolution
 Refraction
 Parts of the compound microscope
 Different microscopes
 Different Stains and Specimen Preparation
Chicken
egg
Human red
blood cell
Large
protozoan
(Euglena) Chloroplasts
Flea
Typical bacteria
and archaea
Diameter
of DNA
Viruses
Proteins
Ribosomes
Amino
acids
Atoms
Transmission electron microscope (TEM)

Compound light microscope (LM)

Mitochondrion
*textbook p.100
Two key characteristics of a microscope:

Magnification – ability to enlarge objects

Resolving power – ability to show detail

Ocular lens: the lens closest to the eye

Objective lens: the lens closest to the
specimen



Total power of magnification of the final image:
Power of objective X Power of Ocular = Total Magnification
10 x low power 10 x = 100 x
40 x high dry 10 x = 400 x
100 x oil immersion 10 x = 1,000 x
Refraction – bending or change in the angle of light as
it passes through a medium (e.g. a lens)
When using an oil immersion lens, need to use oil.
The oil has same optical qualities as glass and prevents
refractive loss and increases the numerical aperture.
Resolution – capacity of an optical system to distinguish or
separate two adjacent objects or points from one another.

Shorter visible wavelengths of light will provide better
resolution.

Blue filters may be placed over a light source to limit
longer wavelengths from entering the specimen.
Bright-field microscopy: image is
formed when light is transmitted
through the specimen
Phase-contrast microscopy: changes
in light waves passing through
specimen are transformed into
differences in light intensity.
https://www.youtube.com/watch?v=hC0jeNU
yQ9s
Dark-Field Microscopy: “stop” disc added to condenser
block all but peripheral light from entering objective
lens
• Reverse image
• Specimen appears
bright on a dark
background
Nomarski microscopy:

-uses two prisms and two beams
of light

-depends on differences in
refractive index

-provides a “3-D” image
Fluorescence
Microscopy:
Uses UV radiation source
and dyes that show
fluorescence.
Confocal Scanning Laser Microscopy:

-used to construct three-dimensional image of thicker
structures

-provides detailed sectional views of internal structures of an
intact organism

Confocal microscopy used to look at
Staphylococcus aureus biofilms
http://www.vcu.edu/micro/lab_web/jefferson/research/index.html
Electron Microscopy: Resolution is approx. 0.3 nm and
magnification 5,000 x to 1,000,000 x for biological specimens.

Uses a series of electromagnetic lenses, electrons, and fluorescent screen to
produce images

Transmission Electron Microscopy (TEM):
Used to observe fine detail inside the cell

Directs beam of electrons at specimen
• Electrons pass through or scatter at
surface

Specimen preparation through
• Thin sectioning

Scanning Electron
Microscopy (SEM):
Used to observe detail
on the surface of the
specimen

Specimen is coated with
metal (e.g. gold)

Beam of electrons scan
surface of specimen
Microscope Techniques, Dyes, and Staining
– Stains are made of organic salts

–Basic dyes bind to cell structures that
carry negative charge
» Commonly stain the cell

–Acidic dyes are repelled by cell structures
that carry negative charge
» Commonly stain the background
Spread culture in
thin film over slide
Pass slide through
flame to fix it
Air dry
Negative Staining:

– dye settles around specimen
e.g. capsule stain
Differential Staining:

Differential – primary dye and counterstain
e.g. Gram Stain
permits differentiation of major categories of
bacteria
Gram positive bacteria:
cells stain purple
Gram negative bacteria:
cells stain pink

Gram Stain Video
https://www.youtube.com/watch?v=tg5P6M
M7AAs
Ziehl-Neelsen acid-fast stain:
– Can be used for presumptive identification in diagnosis of
clinical specimens

- Primary dye
– Carbol fuchsin
» Colors acid-fast bacteria red

- Decolorizer
– Generally acid alcohol
» Removes stains from non acid-fast bacteria

- Counter stain
– Methylene blue
» Colors non acid-fast bacteria blue
» Color of acid-fast bacteria red
Schaeffer-Fulton endospore stain (spore stain):

- Dye is forced by heat into resistant bodies called spores

- Vegetative cells stain pink; spores stain green
Endospore stain video
https://www.youtube.com/watch?v=8tqNzsS
YQYA
Flagella stain:

-Staining increases
the diameter of the
flagella and
increases the
visibility of the
flagella