Lecture 2: Cell Structure and Function Flashcards
Cell Structure and Function
four types of light microscopy:
bright-field
phase contrast
dark-field
fluorescence
________ scope
Specimens are visualized because of differences in contrast between specimen
and surroundings
Two sets of lenses form the image
* Objective lens (usually 10x -100x mag.) & ocular lens (usually 10x – 20x mag.)
Bright-field
how to calculate total magnification?
Total magnification = objective
magnification ✕ ocular magnification
- Maximum magnification is ~2,000X
_______: the ability to make an object larger
Magnification
_______: the ability to distinguish two adjacent objects as separate and distinct
Resolution
what is the limit of resolution for a light microscope?
0.2 μm (wavelength of light source can’t fit through anything smaller)
every light source produces a ____
wavelength
low wavelength = high energy
high wavelength = low energy
as wavelength ____, resolution improves
decreases
improving contrast results in a better final image, how do we improve contrast?
staining! we use organic dyes (contain Carbon) that bind to specific cellular materials, common stains are methylene blue, safranin, and crystal
violet
________ – One dye used to color specimen
Simple staining, sticks to everything possible! (non-specific)
________: coloured portion of a dye
Chromophore
what are the two types of simple stains?
Basic dye – positively charged chromophore
* Binds to negatively charged molecules on cell
surface
Crystal violet – basic
Acidic dye – negatively charged chromophore
* Repelled by cell surface
* Used to stain background
* Negative stain
what charge does the cell surface have?
negative charge
what are the three types of differential stains?
gram stain, acid fast stain, endospore stain
______: Separates bacteria into 2 groups based on cell wall
structure
The gram stain
_______ – cells that
retain a primary stain, crystal violet
* Purple
Gram positive
_______ – cells that
lose the primary stain
* Take color of counterstain, safranin
* Red or pink
Gram negative
gram negative cells have how many membranes?
2
gram positive cells have how many membranes?
1
what are the four steps of a gram stain?
Step 1: Flood a heat-fixed smear with crystal violet for 1 minute (all cells are stained purple)
Step 2: Add iodine solution for 1 min (helps crystal violet stick)
Step 3: decolourize with alcohol briefly, washes of crystal violet that is on outer membrane of gram negative cells to make them colourless, gram positive cells remain purple
Step 4: Counterstain with safranin for 1-2 minutes, all cells will be dyed pink but Purple overshadows Pink…
this results in gram positive cells being purple, and gram negative cells being pink-red
_________:
* Detects mycolic acid in the cell wall of the genus Mycobacterium
* Mycobacterium– retains primary stain (Fuchsia (pink))
* Anything else on slide – colour of counterstain
* Blue
Acid fast stain
what does an acid fast stain detect in cell wall of Mycobacterium?
mycolic acid, dyes it pink!
_________:
* Endospores retain primary stain (Green)
* Cells counterstained (Pink)
Endospore stain
_______:
* Phase ring amplifies differences in the refractive index of cell and
surroundings
- Improves the contrast of a sample without the use of a stain
- Allows for the visualization of live samples
- Resulting image is dark cells on a light background
Phase-contrast microscopy