chapter 3 Flashcards
magnification
ability to enlarge object
-work on microscope because of visible light waves that interact between the curvature of the lens
-image formed by objective lens
resolving power
ability to show detail
where is the real image projected on microscope?
on ocular where it is magnified again to from the image
total mag equation
objective power x ocular power
below 400 nm
ultraviolet light
above 700 nm
infarred light
average white light nm
550 nm
resolving power equation
wavelength of light nm divided by 2 (numerical aperture of objective lens)
is a smaller resolving power better?
-yes because you see smaller things better
-it is the ability to distinguish two adjacent objects
numerical aperture
property of the objective lens
what views smaller than .2 micron/nm
electron microscope
light microscope limit
200/ .2 micron
why is 200 the limit of microscope
because it is all the human eye can handle, visible light limit
numerical apeture
set value of each lens and defines the amount of light that is able to be captured
oil immersion does?
the oil captures the larger cone of light when using 100x
-oil has refractive properties as the glass, so more light is able to be captures
what happens without oil?
light refracts out of lens
bright field
specimen is darker than surrounding field
-live or preserved samples
-white light
dark field
dark surrounding, bright specimen
-live or unstained specimens
-there is a ring of light that interacts with the sample and bends the light/ illuminates the sample (coin on regular scope)
florescence scope
-with UV radiation source and filter
-uses dyes/ florescent molecules that emit visible light when bombarded with shorter UV rays
-can use to diagnose based on what colors light up
ex: antibodies with florescent properties bind to microbe
scanning confocal
3d like image
-laser beam of light to scan specimen surface
can the electron microscope samples be alive?
no
em types
-scanning (color)
-trans (BW)
em
-beam of electrons in wavelike patterns interact with sample
-waves are 100,000 times shorter than visible light
-5,000 to 1 mil x
-no objective lens it is a magnet that directs the electrons
trans em
through specimen
-darker areas are thicker/ denser parts
-specimen is in think slices
-shoot negative electrons through sample
-BW
why are there dark areas of trans em?
because they are thicker areas that are electron dense so they reflect the electrons being shot at them because electrons are neg and they repel each other
scanning em does what
detects the electrons that bounce off the specimen so it gets an image of the outside surface
-it is a metal coated specimen
-color
wet mounts
-live cells, examine size, motility, shape and arrangement
fixed mounts
-dry and heat specimen (heat fixing)
-smear is stained using dyes to permit visualization of cells or cell parts
positive staining
the microbes are negatively charged at the surface and attract basic dye
-opposites attract
what charge does basic dye have
positive
neg stain
microbes will repel the dye and the dye will stain the background (neg dyes/ anionic)
acidic dyes cant?
enter the microbe because they are neg and the bacteria is too
-like repel like
simple stain
one dye
-size, shape arangement
differential
primary stain and a counterstain to see cell types or parts
-gram stain, acid fast, endospore
endospore stain
-differentiates endospores from not endospores
structural stain
-reveal cell parts that you wouldn’t be able to see ex: capsular or flagellar stain
-ex: in capsule the sugar does not take the stain
can gram staining diagnose?
it helps eliminate 50% of possibilities either gram (+) or neg
-bacteria differentiated based on cell wall morphology
gram +
thick peptidoglycan
gram -
thin peptidoglycan and 2 membranes
gram + primary
crystal violet will clump/ mordant from iodine after rinse the color is there
-color is trapped in thick polyglycan layer
-crystal violet is positive and will enter the cell and get trapped when it is thick
-decolorizer removes it from the thin ones
saffronin
negative so it enters all the cells but only stains the ones that are gram negative because purple is the dom color
gram - is what color
-pink from safronin bc purple washed away bc cell wall too thick
how does endospore staining work?
the sample needs to be boiled for the stain to work bc they are heat resistant
-green first and it is dom over saffron
acid fast is for bacteria with?
mycolic acids (lipids)
-it creates the waxy cell wall
-TB bacteria is acid fast
-wax is drug resistant
-bright red
what makes TB drug resistant
mycolic acids
Kirby Bower assay
plate coated in bacteria
inoculation
introduce sample into a container of media to make a culture of growth
isolation
separate your colony from the growth (one species)
-streak
-pour plate
-spread plate
streak plate
what we did on agar, flaming loops
pour plate
3 liquids each with lower dilutions of bacteria poured into plates and as agar solidifies bacteria will grow
spread plate
diluted bacteria in a liquid sample spread on plate with hockey stick tool
single species growing
-pure culture
-mult. = mixed culture
other colors visible
-contaminant
16 s gene can?
identify microbes
-is the sequence gene
gene sequence on microbe
can show what they are resistant too
media properties
-physical state (liquid, semisolid, solid)
-chemical composittion (synth or complex)
-functional type (general purpose, transport, assay etc)
semisolid
has a solidifying solid agent in low amounts
-measures motility of microbes
-soft agar
solid
-surface for colony formation
-solidifying agent= agar
-powder heated in liquid then solidifies when cool
why do we use agar not jello?
jello is collagen based and comes form animals so bacteria would eat it and the agar would have holes
-agar is cellulose/ seaweed based so only marine bacteria might eat it
temp agros solids
42 celsius
temp agar liquid
powder melts at 100 celsius
synthetic media
-know exactly what in it/ formula
-pure organic and inorganic compounds
complex/ nonsynth
-not chemically definable
-brownie batter mix
-ex: we only know tsa and tsb is soy based
what enzyme digests protein in agar
tripsin
general purpose media
-typically non synth.
-grows lots of types of bacteria
enriched media
blood, serum, etc
-special growth factors for fastidious microbes
-often the microbes that live in people
blood agar
-strep throat or flesh eating disease (same bacteria)
-for microbes that destroy red blood cells
-clear areas shows circular zone clearing of cells
beta hemolysis
where bacteria have cleared red blood cells
chocolate agar
-bubonic plague
-killed blood cells that become brown when the hemoglobin/ iron is exposed to air
what media is used for isolation
selective because it has stuff that inhibits growth of some microbes ex: antibiotic in a plate when looking for antibiotic resistant microbes
-or salt
what makes microbes turn different color
differential media
-allows color differences
what does bile salt inhibit
-gram positive bacteria
what causes color change in plate
-microbe fermenting sugar producing acid and the H ions released cause a ph change which changes the color of the agar
selective and differential
mannitol salt agar that lets staph grow bc it likes salt and other microbes wont grow in salt
-as the salt is fermented the ph/ color changes
macconkey agar
-only gram negative will grow on this plate
-microbe has to eat lactose
-crystal violet and bile salt wont let gram positive grow
why does oxygen need to be in water?
oxy does not diffuse into water well so there would be less oxygen deeper in the water over time and life would die
what reduces oxygen in water?
boiling water
reducing medium
substance that absorbs oxygen so we can see how anaerobic baceria grow
-will grow in its optimal environment (tell based on area of water ex: at top the bacteria want the oxygen)
carbohydrate fermentation medium
-phenol red broth
-has sugar that can be fermented which changes the ph
-has upside down durum tube
