Chapter 3.2 Microscopy Flashcards
Dimensions of macroscopic organisms are given in terms of
meters (m) and centimeters (cm)
Dimensions of microscopic organisms are measured from
millimeters (mm) to micrometers (μm) to nanometers (nm)
Three properties on compound/light microscope
-Magnification
-Resolution
-Contrast
Magnification
-Glass sphere can make objects appear larger than they are
-Results from complex interaction between visible light waves and curvature of a lens
Magnifying power
Calculated by multiplying the power of the ocular lens by the power of the objective lens
Resolution
capacity of an optical system to distinguish or separate two adjacent objects or points from one another
Resolving power
-The limit up to which two small objects are still seen as separate entities
-Shorter wavelengths have increased power
Contrast
the difference in light intensity between the image and the adjacent background relative to the overall background intensity
Refractive index
The degree of contrast (the bending of light as it passes from one medium to another)
Four types of light microscopes
-Bright-field
-Dark-field
-Phase-contrast
-Interference
Bright-field microscope
white light transmitted through sample (simplest of microscopes)
Dark-field microscope
Used to enhance contrast in unstained samples causing them to appear bright against a dark background
Phase-contrast microscope
used to enhance contrast in clear or transparent samples
Inference microscope
Used to visualize surface features of sample
Fluorescence microscope
Uses ultraviolet radiation as the illuminating scource
Confocal microscope
Uses a laser beam as the illuminating source
Wet Mounts
-provide a true assessment of size,shape, arrangement, color and motility of cells
-Consists of a drop or two of culture placed on a slide overlaid with a cover slip
-Dry out quickly under microscope light/ for short term observation
Hanging drop Mounts
-provide a true assessment of size,shape, arrangement, color and motility of cells
-Prepared with concave slide, Vaseline and a cover slip
-Complex technique but allows for longer term observation and more reliable observation of motility.
Fixed smear technique
-Smear thin film made from a liquid suspension of cells on slide
-allow to air dry
-Heat or chemical fix kills the cells, secures specimen to slide, preserves cellular components in a natural state w/ minimal distortion
Staining fixed smears
Provides contrast with basic/cationic (positive) or Acidic/anionic (negetive) dyes
Basic (cationic)
Have positive charge, attracted to acidic, negatively charged components on bacterial cell walls
Acidic (anionic)
Have negative charge, repelled by acidic, negatively charged components on bacterial cell wall
Positive stain
-attracted to negatively charged cell walls
-Sticks to the cell and gives it color
Negative stain
-Repelled by negatively charged cell walls
-Produces dark background around cells
Types of stains
-Simple stains
-Differential stains
-Special stains
Simple stains
-crystal violet, safranin and methylene
-Used to determine bacterial species, morphology and arrangement (single, chains, clusters) but no additional info
Differential stains
-Gram stain
-Acid-fast stain
-Endospore stain
Gram Stain
-Universal diagnostic staining technique for bacteria
-Permits ready differentiation of major categories based on the color of cells
-Gram positive stains purple
-Gram neg stains pink
Acid-fastness
Physical property that gives bacterium ability to resist de-colorization from acids during staining procedures
Acid-fast stain
-Differentiates acid-fast bacteria from non acid-fast bacteria
-Acid fast bacteria stain red/pink
-Non acid-fast stain blue
-Detects agents of tuberculosis and leprosy
Endospore stain
-Distinguishes vegetative (active) cells from dormant endospores
-Endospores are green
-Vegetative cells are red
Special stains
-Capsule stain
-Flagellar stain
Capsule stain
-Special stain
-For viewing capsule
-negative staining with India ink or special positive stains
Flagellar stain
-Reveals flagella
-deposits coating on outside of filament and then stained
