The Size of It All
Types of Microscopes
The Size of It All
Remember that 1 inch = 2.54 cm and that
1 meter contains 1000000 micrometers (µm) or
1000000000 nanometers (nm)
Microorganisms are measured in µm or nm.
Question??
If a typical E.coli is 1 µm in length, how many
inches would that be?
1 µm x
1m
x 100 cm x 1 in. = ?
1000000µm
1m
2.54cm
0.00003937 inches!!
Types of Microscopes
Light Microscopes:
1. Compound Light Microscope (LM)
2. Dissection or Stereoscope
3. Darkfield Microscope
4. Phase-Contrast Microscope
5. Differential Interference Contrast (DIC)
6. Fluorescence Microscopy
7. Confocal Microscopy
Types of Microscopes
Electron Microscopes:
1. Transmission Electron Microscope (TEM)
2. Scanning Electron Microscope (SEM)
Scanned Probe Microscopes:
1. Scanning Tunneling Microscope (STM)
2. Atomic Force Microscope (AFM)
Compound Light Microscope
• Uses visible light
• Magnifies up to 2000x, but generally only
1000x
• Good for:
magnification
resolution
refractive index
bright field illumination
can examine live organisms
Darkfield Microscope
• Good for examining live organisms
Phase-Contrast Microscope
• Can be used to visualize internal organelles of
a cell
• Uses one beam of light
Differential Interference Contrast
• Similar to phase-contrast except it uses two
beams of light
Fluorescence Microscope
• Used to rapidly detect and identify microbes
in tissues or clinical specimens
Technique: fluorescent antibody (FA) or
immunofluorescence. Specific antibodies are
treated so that they are fluorescent. The
antibodies are then applied to the specimen, if
the antigens are present the specimen will
appear to fluoresce.
Confocal Microscopes
• Similar to fluorescent microscopy except it uses laser
light
Transmission Electron Microscope
• An ultra thin section of specimen is placed on a copper
mesh grid where a beam of electrons pass through to
an electromagnetic objective lens, which magnifies the
image. The final image is seen as light and dark areas,
referred to as a transmission electron micrograph.
• Disadvantages:
1. requires a very thin slice of the specimen
2. no 3-D aspect
3. specimens must be fixed, stained and placed under a
vaccum which can cause distortion and shrinkage.
Scanning Electron Microscope
• Provides a 3-D image, the stream of electrons
are provided by an electron “gun”
• The image is referred to as a scanning electron
micrograph
• Advantages: intact cells and viruses can be
seen
• Disadvantages: surface structure only
Scanned Probe Microscopy
• Uses a probe to map atomic and molecular
shapes (surface).
• Can be used to determine magnetic, chemical
and temperature characteristics of the cell.
Making Things Visible - Stains
Staining: A technique used in which color (dye)
is added to a specimen to aid in visualizing
structures.
Stains (Dyes):
Basic dyes adhere to anions.
Acidic dyes adhere to cations.
*Bacteria have a slightly negative charge at
neutral pH so they require basic dyes.
Simple Stains:
A single stain is used to high light basic cellular
shape and structure. (Mordants maybe required to
intensify the stain.)
Differential Stains:
A series of dyes are used to distinguish between
various organisms and structures. Generally a
primary stain is applied followed by a rinse or
decolorizer and then finished with a counterstain to
color structures that did not retain the primary
stain.
Making A Slide
• Label the slide
• Apply the specimen to the slide
• Fixing the specimen to the slide
– Heat fixing
– Spray fixative (generally alcohol based)
– Wax setting
– Frozen
The “How” of the Gram Stain
1. Crystal Violet (primary stain) enters the cytoplasm of all
bacteria.
2. Gram’s Iodine (mordant) forms a large chemical complex
with the crystal violet.
3. Decolorizer dehydrates the peptidoglycan layer of the
gram positive cells making it harder for the crystal violetiodine complex to leave the cell. This will cause the gram
positive bacteria to be seen as bluish-purple.
In gram negative cells the decolorizer dissolves
the outer membrane leaving small holes that
the crystal violet-iodine complex to wash out –
leaving the cell “colorless”.
4. Safranin (counterstain) enters the cytoplasm of the bacteria
allowing the gram negative bacteria to be seen as reddishpink.
It’s About the Cell Wall
• Gram Positive Cells: the cell walls have a thick
layer of peptidoglycan and lower lipid content.
The cell walls will dehydrate when exposed to
the decolorizer retaining the crystal violetiodine complex.
• Gram Negative Cells: the cell walls have a
thinner peptidoglycan layer and a layer of
lipopolysaccharides. The outer layer will
dissolve when exposed to decolorizer leaving
small holes that will allow the crystal violetiodine complex to diffuse out of the cell.
Types of Differential Stains
• Gram’s Stain: (1884) Hans Christian Gram
1. Crystal Violet (primary stain) – up to 1 minute
2. Gram’s Iodine (mordant) – 30 seconds
3. Decolorizer – 50/50 alcohol acetone – 10
seconds
4. Safranin (counterstain) – 30 seconds
**Remember which stain the cell retains
depends on the cell wall structure.
• Gram Positive Cocci
Gram Positive Rods
Gram Negative Cocci
Gram Negative Rods
ACID FAST STAIN
• Used for bacteria with a waxy coat, primarily
used for mycobacterium and nocardia.
Technique:
1. Carbol fuchsin (primary stain)
2. Decolorizer
3. Methylene Blue (counter stain)
Wright’s Stain
• Also called Wright’s Giemsa Stain
• Used for differential staining of blood cells.
Technique:
Methylene Blue (primary stain) - basic
Phosphate buffer rinse
Eosin (counter stain) - acidic
Special Stains
Special stains are used to visualize specific parts
of the cell.
1. Capsule stain – generally done as a negative
staining technique where the background is
colored not the specimen.
India Ink is used to visualize the
Capsules around the yeast.
2. Endospore stain: stains the spore portion of
the bacteria. Fulton Endospore Stain is the most
commonly used.
Technique:
Malachite Green (primary)
Water Rinse
Safranin (counter stain)
3. Flagella stain: staining technique developed to
visualize the flagellum. It is a combination of
carbol fuschin and a mordant.
4. Trichrome Stain: often used for the
identification of parasites. It is a combination of
acid fuschin and picric acid.