- Flashcards
The various types of light microscopy include
bright-field, dark-field, fluorescence,
and phase con trast microscopy
bright-field
Student lab microscopes tend to be brightfield microscopes, meaning that visible light is passed through the sample and used to form an image directly, without any modifications.Bright-field microscopy produces an image made from
light that is transmitted through a specimen.
The specimen restricts light transmission and appears
“shadowy” against a bright background (where light
enters the microscope unimpeded).
price of staining
Because most bio -
logical specimens are transparent, the contrast between
the specimen and the background can be im proved with
the application of stains to the specimen. The “price” of the im proved
contrast is that the staining process usually kills cells.
This is especially true of bacterial-staining protocols.
compound microscopes
have multiple lenses. Because of the way these lenses are arranged, they can bend light to produce a much more magnified image than that of a magnifying glass.
In a compound microscope with two lenses, the arrangement of the lenses has an interesting consequence: the orientation of the image you see is flipped in relation to the actual object you’re examining. For example, if you were looking at a piece of newsprint with the letter “e” on it, the image you saw through the microscope would be “ə.”
light microscopes
In a light microscope, visible light passes through the specimen (the biological sample you are looking at) and is bent through the lens system, allowing the user to see a magnified image. A benefit of light microscopy is that it can often be performed on living cells
stains
Stains are solutions consisting of a solvent (usually water
or ethanol) and a colored molecule (often a benzene
derivative), the chromogen.
chromogen
The portion of the chromogen that gives it its color is the chromophore. A chromogen may have multiple chromophores, with each adding
intensity to the color.
auxochrome
The auxochrome is the charged
portion of a chromogen and allows it to act as a dye
through ionic or covalent bonds between the chromogen
and the cell.
basic stains
Basic stains (where the auxochrome becomes
positively charged as a result of picking up a hydrogen
ion or losing a hydroxide ion) are attracted to the negative
charges on the surface of most bacterial cells. Basic stains have a
positively charged chromogen,
which forms an ionic bond
with the negatively charged bacterial cell, thus colorizing the cell. Common basic stains
include methylene blue, crystal violet and safranin.
heat-fixed
Basic stains are applied to bacterial smears that have
been heat-fixed. Heat-fixing kills the bacteria, makes
them adhere to the slide, and coagulates cytoplasmic
proteins to make them more visible. It also distorts the
cells to some extent.
how to avoid producing aerosols
Do not spatter the smear as you mix it, do not
blow on or wave the slide to speed-up air-drying, and do not
overheat when heat-fixing.
undefind / compelx media
digests of plant material
(phytone) or animal material (peptone and others). Be-
cause the exact composition and amounts of carbon
and nitrogen in these ingredients are unknown, general
growth media are considered to be undefined.
nutrient broth
3g beef extract
5g peptone
1L distilled/deionized water
pH 6.6-7.0 25C
nutrient agar
3g beef extract 5g peptone 15g agar 1L distilled/deionized water pH 6.6-7.0 25C
agar deep
used to study gaseous requirements of microorganisms.
- oxygen relationships, hydrogen sulfide production
purpose of slant
- greater SA
- easier to remove samples
Agar slants generally are used for growing stock cultures
that can be refrigerated after incubation and
maintained for several weeks. - used to maintain pre-cultures for subculturing and for short term storage. the flat surface area permits the study of colony characteristics as well as certain biochemical reactions.
bunsen burner flame
Sterilization of inoculating instruments is done in the hottest part of the flame—the tip of the inner cone. Heat-fixing bacterial smears on slides and incinerating the mouths of open glassware items may be done in the outer cone.
flaming the loop
Incineration of an inoculating loop’s
wire is done by passing it through the tip of the flame’s inner
cone. Begin at the wire’s base and continue to the end, making
sure that all parts are heated to a uniform orange color. Flaming in this direction limits aerosol production
by allowing the tip to heat up more slowly than if
it were thrust into the flame immediately. Allow
the wire to cool before touching it or placing it on/in a culture.
The former will burn you; the latter will cause aerosols of
microorganisms.
handling the tube
- Hold a culture tube in your nondominant hand and
move it, not the loop, as you transfer. This will minimize
aerosol production from loop movement - Grasp the tube’s cap with your little finger and remove
it by pulling the tube away from the cap. Hold the
cap in your little finger during the transfer - Hold open tubes at an angle to minimize the chance
of airborne microbes getting into it - flamed to sterilize the
tube’s lip and the surrounding air. Notice that the tube’s cap
is held in the loop hand.
broth cultures
Broth cultures are often used to grow cultures for use
when fresh cultures or large numbers of cells are desired.
