(exam 1) ch 3 microscopy staining Flashcards
what is light microscopy?
microscopes that use visible light to observe specimens
what is a compound light microscope?
series of lenses for magnification which uses visible light; (no oil) 4X, 10X, 40X (oil) 100X
in a compound microscope the image from the _______ lens is magnified again by the _______ lens.
objective lens; ocular lens
total magnification =
object lens x ocular lens “add zero on”
what is resolution (or resolving power) ?
ability to distinguish between two points; greater resolution allows finer detail and structure between two points
what is refractive index?
measure of light bending ability of a medium; immersion oil has raw sane refractive index as glass and keeps light from refracting (changing direction) which increases the resolution
what is staining?
coloring microorganisms with a dye that emphasizes certain structures
what must happen before staining microbes?
microbes must be smeared (creating a thin film containing microbes) and fixed (attached with heat) to the glass slide
why is fixing microbes important?
1) causes them to stick to the slide so they do not wash off 2) kills them 3) preserves various structures for observation
what are three common staining techniques?
1) simple stain 2) differential stain 3) special stain
what is the primary stain?
first dye used to color the microbe
what is the counter stain?
second dye used after a wash step
what is mordant?
substance used to set / bind dye and acts to intensify staining (iodine)
what is a simple stain technique?
uses a single dye with a positive charge; *reveals the cells morphology* size, shape, and arrangement of bacterial cells
what is differential stains?
*Used to distinguish between different bacterial species* and the differences are based on the difference in bacterial structure; usually require more than one stain
what is the most common and most important type of differential stain in medical microbiology?
GRAM STAIN
what are the two large groups that a gram stain (differential stain) places bacteria into?
1) gram positive 2) gram negative
what is the structure of gram positive bacteria?
bacteria have a thick peptidoglycan cell wall
what is the structure of gram negative bacteria?
bacteria have a thin peptidoglycan cell wall and an outer membrane composed of lipopolysaccharides
what kind of information does a gram stain provide?
provides valuable information for the treatment of disease; allows for the proper selection of antibiotics; antiobiotics are typically effective against either gram positive or gram negative bacteria BUT NOT BOTH
what is the four step procedure of a gram stain?
1) application of crystal violet (purple dye) *primary stain* 2) application of iodine (mordant) *seal- sets crystal violet into gram positive stain* 3) alcohol wash (decolorization) * washes purple off anything gram negative* 4) application of safranin (counter stain) *turns anything gram negative pink*
what are the results of a gram stain (differential stain) ?
gram positive bacteria appear PURPLE; they retain crystal violet from the 1st stain
gram negative bacteria appear PINK; alcohol wash removes the crystal violet and bacteria appear pink after the second stain with safranin
what is another type of differential stain that was discussed?
acid-fast stain
what happens in an acid fast stain?
dye binds to bacteria that have a waxy material in thier cell walls - dye is not decolorized by acid acohol
what two bacteria genera (plural of genus) would you use an acid fast stain to identify them?
1) Mycobacterium
2) Nocardia
what are special stains used to distinguish and what are the three types?
used to distinguish specific structures of microorganisms
three types 1) capsule stain 2) endospore stain 3) flagella stain
what are bacterial capsules?
gelatinious outer covering; not found on all bacteria; they contribute to the ability of bacteria to cause disease (virulence)
how do you stain bacteria capsules (special stain)?
bacteria capsules are difficult to stain because they are water souble and dye washes away easily; so in order to stain them we use negative staining = the background is stained instead of the cell and the dark background contrasts w capsules so they appear as a halo around the cell
what are endospores?
resistant, dormant structures located inside some cells (protect bacteria from adverse environmental condition)
how do you stain endospores (special stain)?
endospores cannot be stained with ordinary methods because dye cannot penetrate endospore wall; procedure is to use a 1) primary stain of malachite green w/ heat 2) decolorize cells = water and 3) counterstain = safranin
result = spores appear green within red/pink cells
what are flagella?
structures used for locomotion and are too small to be seen with light microscopy
how do you stain flagella (special stain)?
use a mordant and carbolfuchsin dye to thicken the appearance of flagella which makes them visible under the light of a microscope
