Ch. 3 Observing Microorganisms through a Microscope Flashcards
Microorganisms are measured in
micrometers and nanometers
Types of light microscopy
– Compound light microscopy
– Darkfield microscopy
– Phase-contrast microscopy
– Differential interference contrast (DIC) microscopy
– Fluorescence microscopy
– Confocal microscopy
Compound microscope
image from the objective lens is magnified again by the ocular lens
Total magnification =
objective lens x ocular lens
Resolution
the ability of the lens to distinguish two points
refractive index
a measure of the light-bending ability of a medium
Brightfield illumination
– Dark objects are visible against a bright background
– Light reflected off the specimen does not enter the
objective lens
Darkfield Microscopy
- Light objects are visible against a dark background
- Opaque disk placed in condenser
- Only light reflected off the specimen enters the objective
lens
Phase-Contrast Microscopy
- Allows examination of living organisms and internal cell
structures - Brings together two sets of light rays, direct rays, and
diffracted rays to form an image
Differential Interference Contrast Microscopy
- Similar to phase-contrast
- Uses two light beams and prisms to split light beams,
giving more contrast and color to the specimen
Fluorescence Microscopy
- Uses UV (short wavelength) light
- Fluorescent substances absorb UV light and emit longer
wavelength (visible) light - Cells may be stained with fluorescent dyes
(fluorochromes) if they do not naturally fluoresce
Confocal Microscopy
- Cells are stained with fluorochrome dyes
- Short-wavelength (blue) light is used to excite a single
plane of a specimen - Each plane in a specimen is illuminated and a three-
dimensional image is constructed with a computer - Can examine layers of cells to a depth of 100 μm
Two-Photon Microscopy
- Cells are stained with fluorochrome dyes
- Two photons of long-wavelength (red) light are used to
excite the dyes - Can study living cells up to 1 millimeter deep
Super-Resolution Light Microscopy
- Uses two laser beams
– One wavelength stimulates fluorescent molecules to
glow
– Second wavelength cancels out all fluorescence
except for that in one nm - A computer scans the specimen nm by nm, then puts the
images together
Scanning Acoustic Microscopy
- Measures sound waves that are reflected back from a
specimen - Used to study cells attached to surfaces
- Resolution of 1 μm
Electron Microscopy
- Uses electrons instead of light
- The shorter wavelength of electrons gives greater
resolution - Used for images too small to be seen with light
microscopes, such as viruses
Transmission Electron Microscopy
- A beam of electrons passes through ultrathin sections of
a specimen, then through an electromagnetic lens, then
focused on a projector lens - Specimens may be stained with heavy-metal salts for
contrast - Magnifies objects 10,000 to 10,000,000x; resolution of 10
pm
Scanning Electron Microscopy
- An electron gun produces a beam of electrons that scans
the surface of an entire specimen - Secondary electrons emitted from the specimen produce
a three-dimensional image - Magnifies objects 1,000 to 500,000x; resolution of 10 nm
Scanning Tunneling Microscopy
- Uses a tungsten probe to scan a specimen and reveal
details of its surface - Resolution of 1/100 of an atom
Atomic Force Microscopy
- Uses a metal-and-diamond probe placed onto a
specimen; movements are recorded - Produces three-dimensional images at near atomic detail
Staining
coloring microorganisms with a dye that emphasizes certain structures
Smear
a thin film of a material containing microorganisms spread over a slide
Fixed
Microorganisms are attached to the slide, which kills the microorganisms
Chromophore
colored stain consisting of a positive and negative ion
basic dye
chromophore is a cation
acidic dye
chromophore is an anion
negative staining
staining the background instead of the cell
Simple stain
use of a single dye
simple stains highlight the entire microorganism to
visualize cell shapes and structures
Mordant
used to hold the stain or coat the specimen to enlarge it
Differential Stains are used to
distinguish between bacteria
-Gram stain
-Acid-fast stain
Gram-positive bacteria
have thick peptidoglycan cell walls
Gram-negative bacteria
have thin peptidoglycan cell walls and a layer of lipopolysaccharides
Acid-Fast Stain
binds only to bacteria that have a waxy material in their cell walls, which is not decolorized by acid-alcohol
Acid-Fast Stain used for the identification of
-Mycobacterium
-Nocardia
Special Stains used to distinguish parts of microorganisms
-capsule stain
-Endospore stain
-Flagella stain
Capsules
are a gelatinous covering that do not accept most dyes
Suspension of India ink or nigrosin
contrasts the background with the capsule, which appears as a halo around the stain
Endospores
resistant, dormant structures inside some cells that cannot be stained by ordinary methods
Primary stain
Malachite green, usually with heat
Decolorize cells
water
counterstain
safranin
spores appear
green within red or pink cells
Flagella
structures of locomotion
flagella uses a mordant and carbolfuchsin to
thicken appearance of flagella, making them visible under the light microscope
