Microscopy and Specimen Preparation Flashcards
type of microscope that use visible light to illuminate specimens
compound light microscope
types of light microscopy (4):
- bright-field
- dark-field
- phase-contrast
- fluorescence
type of light microscopy; what we use; image is dark w/ bright background
bright-field
how many lenses does a compound microscope require?
2
type of lens: focuses the light
condenser lens
process of light being refracted (bent) when passing from one medium to another
refraction
why does light refract/bend when passing from one medium to another?
it slows the velocity of the light = bend (towards the normal)
F =
focal point
f =
focal length
where light rays converge on the other side of the lens
focal point (F)
the ______ the focal length, the stronger the lens
shorter
the shorter the focal length, the ______ the magnification
greater
magnification has to do solely with the _______
lenses
using the bright-field microscope, specimens are visualized because of differences in _______ between the specimen and its surroundings
contrast (density)
total magnification =
magnification of OCULAR lens x magnification of OBJECTIVE lens
upper limit of magnification =
2000X
upper limit of resolution =
0.2 micrometers
ability to distinguish two objects as distinct and separate when viewed under a microscope; clarity of an image
resolution (d)
T/F: resolution and magnification are the same thing
false
d =
resolution
you want d, or resolution, to be extremely ________
small
formula for d (resolution) =
0.5 * wavelength / NA
NA =
numerical aperture
measure of light gathering ability
NA (numerical aperture)
formula for d (resolution) can ALSO be:
0.5 * wavelength / n (sin θ)
n =
refractive index
ways to increase resolution (aka make it smaller - 4):
- decrease wavelength (ex: using blue light instead of white light)
- decrease working distance
- increase NA
- increase n (refractive index)
_____ working distance to increase resolution
decrease
higher numerical aperture = _____ resolution
better
you should shorten the working distance to make θ ______ AND to make NA ______
increase; increase
how much a light ray bends
refractive index (n)
if you decrease n (refractive index), you lose _____ _____ and make resolution worse
light rays
anything thicker than air will have a _____ n
hgiher
refractive index (n) of air =
1
refractive index (n) of oil =
1.25
increasing n (refractive index) results in an increase in _____ which results in a better resolution
NA
limit of resolution =
0.2 (~0.18) nanometers
improving contrast in light microscopy (2 main steps):
1) fixation
2) staining
preserves specimens and fixes them in position; organisms usually killed and firmly attached to microscope slide
fixation
type of fixation: routine used for bacteria and archaeons; what we usually do; kills them
heat fixation
type of fixation: use with larger, more delicate organisms
chemical fixation
make internal and external structures of cell more visible by increasing contrast with background
staining
2 common features of staining:
1) chromophore groups
2) ability to bind cells
chemical gropus with conjugated double bonds; gives stain its color
chromophore groups
stains ability to bind cells usually inovolves what types of bonds?
ionic (most are salts)
types of stains (2):
- basic stains (dyes)
- acidic stains (dyes)
type of stain: dyes with POSITIVE CHARGES; bind to negatively charged structures; when it ionizes, the chromophore group has a POSITIVE charge; attracted to surface of bacteria (mainly neg.)
basic stains
type of stain: dyes with NEGATIVE charges; bind to positively charged structures; chromophore group is neg. charged when released/ionized
acidic stains (dyes)
basic stains are colored _____ and acidic stains are colored ______
bases; acids
stain the background to stain; negative stain; REPELS bacteria
acidic stain
bacteria are mainly ____ charged
negatively
types of staining techniques (2):
1) simple staining
2) differential staining
type of staining technique: use of a single stain
simple staining
type of staining technique: use of two contrasting stains separated by a decolorizing agent
differential staining
purpose of simple staining =
for visualization of morphological shape + arrangement
purpose of differential staining =
identification + visualization of structure
examples of IDENTIFICATION differential staining (2):
- Gram stain
- Acid fast stain
examples of VISUALIZATION differential staining (2):
- spore stain
- capsule stain
a stimple stain looks at what 3 things of a cell?
1) size
2) shape
3) arrangement
differential staining use more than one dye to ______ stain features
preferentially
differential staining is used to detect the _____ or ______ of structures
presence or absence
differential stains that divides microorganisms into GROUPS based on their staining properties (3)
- Gram-stain
- acid-fast stain
- endospore stain
most widely used differential staining procedure; divides bacteria into two groups based on differences in cell wall structure
gram staining
when and by who was the Gram Stain discovered?
1884 by Christian Gram
what was Christian Gram trying to distinguish between when disovering the Gram Stain?
eukaryotic nuclei and bacteria
Procedure of Gram Staining (and color during steps):
1) flood the heat-fixed smear with crystal violet for 1 min (both purple)
2) add iodine solution for 1 min (both purple)
3) decolorize with alchohol briefly for 20 seconds (pos = purple; neg = colorless)
4) counterstain with safranin for 1-2 minutues (pos = purple; neg = pink)
both Gram-postive and Gram-negative bacteria are _____ charged on their SURFACE
negatively
useful for staining members of the genus Mycobacterium (ex: TB, leprisy); high lipid (mycolic acid) content in cell walls is repsonsible for their staining characteristicspi
acid-fast staining
acid-fast cells stain ____
pink
non-acid fast cells stain _____
blue
why are cells like Mycobacterium hard to stain?
bc they have a really high lipid content, known as mycolic acid, in their cell walls
“acid-fast if you hold on FAST to acidic conditions”
acid-fast uses _______ and ____ ____ as two of the staining materials
methylene blue and acid-alcohol
types of stains for SPECIFIC STRUCTURES (3):
- endospore staining
- capsule staining
- flagella staining
type of stain for a specific structure: heated, double staining technique; bacterial _____ is one color (GREEN) and vegetative cell is a dif. color (PINK)
endospore staining
in endospore staining, bacterial endospores are ______ and vegetative cells are _____
green; pink
type of stain for a specific structure: negative stain; _____ are colorless against a stained background
capsule staining
in capsule staining, capsules are ______ against a stained background
colorless
