Module 2 Flashcards
1 micrometer (μm) =
10^-6 m
1 nanometer (nm) =
10^-9 m
What makes a good microscope
adequate magnifying power
high resolving power
serves your purpose
provides good contrast
two main types of microscopes
light and electron
microscope that use light waves and mirrors
light microscopes
types of light microscopes
simple, compound/complex, bright-field, dark-field, phase contrast, differential interference contrast, fluorescence, confocal, two-photon
- use electron beams as energy source
- has higher magnification and resolving power
- for objects smaller than 0.2 mm in diameter
- in vacuum
- should have mo water when used
electron microscope
- objects under study are darker
- microscopic field is brightly lit
- gross morphology
- can be used stained or unstained
BRIGHT FIELD
- microscopic field is dark
- objects under study are luminous
- for specimens that are:
- invisible in the ordinary light microscope
- cannot be stained by standard methods
- distorted by staining
- has opaque disc that blocks light
DARK FIELD
- principle is based on variations in the refractive indices
(measure of bending or refracting of a beam of light on
entering a denser medium) - not necessary to fix or stain cells
- detailed examination of internal structure
PHASE CONTRAST
- principle is based on variations in the refractive indices
- advantage: no diffraction halo associated with phase contrast
- disadvantage: the three-dimensional appearance may not represent reality
DIFFERENTIAL INTERFERENCE CONTRAST
- makes use of fluorochromes
- visualizes specimens that fluoresce
- detection of immunological reactions
- can be direct (primary antibody fluorochromes) and indirect (secondary antibody is attached to primary antibody)
FLUORESCENCE
- useful for thick specimens like biofilms
- used to visualize structures
CONFOCAL
- useful for examining living cells within intact tissues
- currently limited to advanced clinical and research laboratories
TWO-PHOTON
types of electron microscopes
TRANSMISSION ELECTRON MICROSCOPE (TEM),
SCANNING ELECTRON MICROSCOPE (SEM),
SCANNING TUNNELING MICROSCOPE (STM),
ATOMIC FORCE MICROSCOPE (AFM)
- ultrastructure in thin section of the cells
- can project images in a much higher resolution—up to the
atomic level of thinner objects - examine viruses
- cells should be killed
- 100,000 X and above magnification
TRANSMISSION ELECTRON MICROSCOPE (TEM)
- surface features of viruses and cells
- reveals a 3-D image
SCANNING ELECTRON MICROSCOPE (SEM)
for observing materials like Pure Gold Surface
SCANNING TUNNELING MICROSCOPE (STM)
for observing Nanocellulose
ATOMIC FORCE MICROSCOPE (AFM)
advantages of examining organisms in living or natural state
- observation of unaltered/undistorted characteristics
- cellular processes can be studied
- motility can be observed
- simple to prepare
disadvantages of examining organisms in living or natural state
- refractive index of cells almost similar to that of water
advantages of examining organisms in stained organisms
- provides contrast
- slides can be preserved
- specimens are killed
disadvantages of examining organisms in stained organisms
- more complicated and tedious to prepare
- expensive
Three basic steps in staining microorganisms:
- Smear Preparation
- Fixation
- Staining
a thin, dry film of microorganisms
Smear
STEP 1: Smear Preparation
spread culture in thin film over slide -> dry in air
purpose of fixation
- kills the cells
- makes the cells sticky
- increases apparent diameter of cells
types of fixation
- Heat Fixation
- Chemical Fixation
- fixation through direct flame or steam
Heat Fixation
- fixation using alcohols
Chemical Fixation
- application of biological dyes
Staining
- organic compounds carrying chromophoric ions
- make cell’s internal and external structures more visible with the increased contrast with the background
Dyes (Stains)
Types of Stains:
- Basic or Positively-Charged Dye
- Acidic or Negatively-Charged Dye
- Neutral
staining which only uses one dye
SIMPLE STAINING
types of simple staining
Positive / Direct
Negative / Indirect
- cells are the same color as the dye
- crystal violet, malachite green, methylene blue, safranin
Positive / Direct
- cells are colorless or luminous
- acid fuchsin, eosin, rose bengal, india ink, nigrosin
Negative / Indirect
- two or more dyes and/or reagents are used
DIFFERENTIAL STAINING
examples of DIFFERENTIAL STAINING
Gram Staining
Acid-Fast Staining
- used in finding gram-positive / gram-negative
Gram Staining
- used in diagnosis of tuberculosis
Acid-Fast Staining
Gram staining process
1: crystal violet (primary stain) -> stain cells turn purple or blue
2: iodine (mordant makes dye less soluble so it adheres to cell walls) -> cells remain blue or purple
3: alcohol (decolorizer washes away stain from gram-negative cell wall) -> gram positive remains the same color, gram negative turns colorless
4: safranin (counterstain allows dye adherence to gram-negative cells) -> gram positive remains the same color, gram negative turns pink/ red
methods used in Acid-Fast Staining
- Ziehl-Neelsen Technique
- Kinyoun Technique
types of staining
simple staining
differential staining
structural staining
- two or more dyes and/or reagents to emphasize structures
STRUCTURAL STAINING
structural staining is used for:
- Capsules
- Endospores
- Flagella
- Storage Granules
