- 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.
streak plate method
In the streak plate method of isolation, a bacterial
sample (always assumed to be a mixed culture) is streaked
over the surface of a plated agar medium. During streak-
ing, the cell density decreases, eventually leading to
individual cells being deposited separately on the agar
surface. Cells that have been sufficiently isolated will grow
into colonies consisting only of the original cell type.
Because some colonies form from individual cells and
others from pairs, chains, or clusters of cells, the term
colony-forming unit (CFU) is a more correct description
of the colony origin.
streak plate patterns
Several patterns are used in streaking an agar plate,
the choice of which depends on the source of inoculum
and microbiologist’s preference. Although streak patterns
range from simple to more complex, all are designed to
separate deposited cells (CFUs) on the agar surface so
individual cells (CFUs) grow into isolated colonies. A quadrant streak is generally used with samples suspected
of high cell density, whereas a simple zigzag pattern may
be used for samples containing lower cell densities.
path of light
Light from
the source is focused on the specimen by the condenser lens. It then
enters the objective lens, where it is used to produce a magnified real
image (real image is produced by refraction of light as it passes thru obj lens). The real image is magnified again by the ocular lens to produce
a virtual image that is seen by the eye.
condenser lens
h concentrates the light
and makes illumination of the specimen more uniform, condenses light before it passes thru the specimen
turning knobs
changes the dist btwn the objective lens n the specimen
body tube
contains mirrors n prisms that transmit the image frm the obj lens to the ocular lens
obj lens
primary lenses that magnify specimen
iris diaphragm
controls amnt of light entering the condenser
fermentation tubes
contain a small inverted durham tube to trap any gases produced by microbial metabolism; used to indicate the anearobic breakdown of specific sugars
inoculated plates
inverted to prevent the agar from drying out too rapidly and ot prevent ocndenstaion droplets disturbing the isolated colonies
- used to isolate microo in the form of colonies, to test bacterial sensitvity to antiobitics and chems, to count the number of microos contaminating foods and water
pipet
piece of glass/plastic tubing that has been calibrated n drawn out to form a capillary-like topl cotton plug placed w/in mouthpiece for protection; used to deliver measured quantities of broth cultures/soln’s
loops
transfer, loop dilution studies to enumerate bacteria
needles
pick up isolated cultures frm agar plates for transfer to pure culture media n to inoculate agar deeps by stabbing
swabs
most useful piece of collecting equipment. wooden applicator stick tipepd w/ dacron batting or long-fibred, medicinal cotton
- inoculation
dry heat
- glassware n inert objects
- high temps for long periods of time
- 160 180 C 1.5 - 3 h
moist heat
autoclave: stem under pressure. useful for things thatd be destroyed by dry heat
- 121C 15min sea level
filtration
remove bacteria by sieve-like action of the minute pores of the filter. used to sterilize heat-labile soln’s that are destroyed/decomposed by high temps n to determine bacterial concentrations in fluids
incubators n incubation
- aerobic n facultative anaerobic: 18h 37C
- strict anaerobes: 48h
- organisms incubated at room temp will grow in 48h
vibrios
slightly curved rods; 1/2 spiral turn
coccobacilli
short rods
spirillum vs spirochete
lum: movement rotationa lalong long axis
chete: mvmnt is a helical wave
cocci pairs
diplococcus
cocci chaisn
streptococcus
grapelike cocci
staphylococcus
8 cocci
dubelike - sarcina
side by side bacilli
palisading
V bacilli
snapping
immersion oil
reduce amnt of light refraction so that more light passes thru the microscope slide n int the very narrow diameter of a higher power objective lens. In microscopy, more light = clear and crisp images. By placing a substance such as immersion oil with a refractive index equal to that of the glass slide in the space filled with air, more light is directed through the objective and a clearer image is observed.
cleaning microsceop
lens paper: condenser n obj
To clean an ocular, moisten a cotton swab with
cleaning solution and gently wipe in a spiral motion
starting at the center of the lens and working outward.
Follow with a dry swab in the same pattern.
microscope step 1
Raise the substage condenser to a couple of milli -
meters below its maximum position nearly even with
the stage and open the iris diaphragm.
adjusting light rule
Adjust the iris diaphragm and condenser position to
produce optimum illumination, contrast, and image.
(As a rule, use the maximum light intensity combined
with the smallest aperture in the iris diaphragm that
produces optimum illumination. DONT close down IRIS DIAPHRAGM too much
parfocal
A parfocal lens is a lens that stays in focus when magnification/focal length is changed. There is inevitably some amount of focus error, but small enough to be considered insignificant. : For most microscopes, the
distance from the nosepiece opening to the focal
plane of each lens has been standardized at 45 mm.
This makes the lenses parfocal and gives the user an
idea of where to begin focusing.
