Lab Exam 1 – Flashcards

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Supplemented thioglycollate
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used to assess aerotolerance but has added nutrients for growing bacterial cultures
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Kirby Bauer Test
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tests antibiotic sensitivity
-uses standardized conditions of 1/4 inch thick Mueller-Hinton agar plates; pH between 7.2-7.4; lawn made from .5 McFarland turbidity standard; antibiotic discs have a standard amount of antibiotic in them
-procedure: Label plate; innoculate plate to create lawn; apply antibiotic discs without overlapping zones of inhibition; press discs firmly into agar so they stick; invert plates and incubate.
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broad spectrum antibiotic
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effective against both gram positive and gram negative cells
-used when specific bacteria causing infection is unknown
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narrow spectrum antibiotic
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effective against either NOT BOTH gram positive and gram negative cells
-used when cause of infection is known - prevents killing off good bacteria in the body
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S10
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Streptomyocin
-gram negative narrow
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C30
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Chloramphenicol
-broad spectrum
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CF30
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Cephalothin
-broad spectrum
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TE30
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Tetracycline
-broad spectrum
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P10
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penicillin
-not effective
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NB 30
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Novobiocin
-gram positive narrow
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S10
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Streptomyocin
-gram negative narrow
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E15
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Erithromyocin
-gram positive narrow
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VA30
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Vancomyocin
-gram positive narrow
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E. coli gram ...
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negative
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S. aureus gram ...
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positive
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Antiseptic
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used on living tissue
-longer contact time (time it continues to work)
-iodine
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Disinfectant
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-used on inanimate objects
-decreases number of bacteria, but not sterilizing
-bleach
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confluent growth
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continuous bacterial growth without discrete colonies
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lawn on agar plate
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covering the entire surface of a plate with bacteria to grow
-start in middle and do one half, back and forth; then start in middle and do other half; rotate 90 degrees; start in middle and do one half; then start in middle and do other half
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gram negative bacteria are more resistant to antibiotics because
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of their outer membrane
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Gram positive cells are more or less susceptible to antibiotics than gram negative cells?
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Gram positive cells are more susceptible
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Zone of inhibition
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-area of no bacterial growth around an antibiotic disc
-measured in mm
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resistance OR susceptibility to antibiotic shown through zone of inhibition
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must compare measurement to standardized chart
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aseptic technique
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-used to prevent contamination/keep cultures pure
-procedure: use inner blue cone on bunsen burner; sterilize loop; remove cap of tube with little finger; flame tube; cool loop on tube; flame tube; close tube; innoculate culture with loop; sterilize loop
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streak plate technique
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-purpose: to isolate a single colony
-procedure: streak loop across plate in 1/4 of plate; sterilize loop; rotate plate; cool loop on agar; drag through previous section 2-3 times and then streak another 1/4 of plate; sterilize loop; rotate plate; cool loop on agar; drag through previous section 2-3 times and then streak another 1/4 of plate; sterilize loop; rotate plate; cool loop on agar; drag through previous section 2-3 times and then streak remainder of plate; sterilize loop
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errors in streak plate technique
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-digging loop into agar
-too many streaks through last quadrant before moving on to next quadrant
-overlapping streak sections creating confluent growth
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BSL
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Bio Safety Level
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precautions used for BSL 1 and 2
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-goggles
-gloves
-lab coats
-close toed shoes
-long pants
-hair pulled back
-disinfectant for tables
-sterilization of tools
-autoclave contaminated materials
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biohazard disposal
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-double bag anything that goes in biohazard bin
-biohazard glass or sharps go in red biohazard sharps container
-gloves inside out in biohazard bag
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Broth is used for:
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??
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slant is used for:
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-using less media than a plate (cost)
??
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plate is used for:
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-identifying growth patterns (shape, margin)
-isolating pure colonies
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aseptic
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pure (not sterile)
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sterile
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no living organisms
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Thioglycollate
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prepared using autoclave to remove all O2
-as it cools, the O2 reenters, creating an O2 gradient from aerobic to completely anaerobic in bottom of tube
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Obligate aerobe
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needs O2 to survive
-only on top of tube
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Obligate anaerobe
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needs no O2 to survive
-only on bottom of tube
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Facultative anaerobe
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prefers O2, but can adapt to grow without it
-grows anywhere, better on top
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microaerophile
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loves a LITTLE O2
-grows in a band about 1/3 way down the tube
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Aerotolerant anaerobes
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prefers no O2, but can tolerate growth with O2
-grows anywhere, but better on bottom
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maximum temperature
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highest temperature for cells to be able to grow
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minimum temperature
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lowest temperature for cells to be able to grow
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optimum temperature
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best temperature for cells to be able to grow - highest growth rate
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cardinal temperatures
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range defined by the three temperatures in the range:
-optimum
-maximum
-minimum
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temperature range
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the temps between the highest and lowest that an organism will tolerate to grow
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psychrophile cardinal temperatures
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Range: -5 to 15C
Optimal: 10C
icecap
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psychrotroph cardinal temperatures
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Range: -3 to 35C
Optimal: 25C
Santa Cruz
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mesophile cardinal temperatures
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Range: 15 to 45C
Optimal: 40C
rainforest; human pathogens
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thermophile cardinal temperatures
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Range: 40 to 80C
Optimal: 70C
hot spring
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extreme thermophile cardinal temperatures
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Range: 65 to 110C
Optimal: 95C
hot spring
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procedure for determining temperature category of microorganism
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-inoculate multiple specimens and grow at several different temperatures
-compare growth on all plates to rule out temperature ranges where there was no growth
-choose temperature category that includes all temperatures where there was some growth in medium
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hypotonic
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-fewer ions outside cell
-low osmotic pressure
-water moves into cell
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hypertonic
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-more ions outside cell
-high osmotic pressure
-water drawn out of cell, so plasma membrane shrinks away from cell wall (plasmolysis)
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isotonic
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-same amount of ions (osmotic pressure) inside and outside cell
-fluid moves at same rate into and out of cell
-0.85% osmotic pressure
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plasmolysis
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plasma membrane shrinks away from cell wall
due to cell being in hypertonic solution
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halophile
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loves hypertonicity
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facultative halophile
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range: 0.85% to 15%
-can grow with hypertonicity)
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obligate halophile
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range: must have over 3%
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extreme halophile
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range: must be over 15% up to 20%
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halotolerant
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can grow up to 5% but not well
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non-halophile
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grows most abundantly at 0.85%
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osmotic pressure order of organism tolerance
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non-halophile
halotolerant
facultative halophile
obligate halophile
extreme halophile
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order of organism tolerance (temperatiures)
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psychrophile
psychrotroph
mesophile
thermophile
extreme thermophile
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which osmotic pressure is closest to the average biological cell?
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0.85%
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purpose of including water as a sample in disinfectant experiment
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control
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procedure for preparing bacterial smear slide from broth culture
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-label slide
-use transfer technique to apply bacteria to slide (3-4 loops)
-heat fix bacteria using hot plate and/or flaming slide
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procedure for preparing bacterial smear slide from plate/slant culture
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-label slide
-add a drop of DI water to slide
-use transfer technique to apply small amount of bacteria to slide
-heat fix bacteria using hot plate and/or flaming slide
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what does heating a slide after applying bacteria do?
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-fixes the bacteria to the slide so they don't wash away
-kills the bacteria
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simple stain
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single stain applied to cells which makes them visible under the microscope
-allows determination of cell size, shape and arrangement
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procedure to prepare a simple stain
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-begin with prepared slide
-drop stain onto smear and let sit for 1 minute
-rinse with DI water
-blot w bibulous paper
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gram staining tell us what?
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-differential stain gives you information about whether a cell is gram positive or gram negative (has thick peptidoglycan VS outer membrane)
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differential stain
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allows for detection of differences between organisms or structures within one organism
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stains adhere through
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ionic bonding
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gram staining steps
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-primary stain - crystal violet
-mordant - iodine
-decolorizer - alcohol
-secondary stain (counterstain) - safranin
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general differential staining steps
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-primary stain
-mordant
-decolorizer
-counterstain/secondary stain
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gram stain pink results
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gram negative (outer membrane)
-E coli - short bacillus
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gram stain purple/black results
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gram positive (thick peptidoglycan)
-S aureus - coccus
-B subtilis - long bacillus
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Microscope parts
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-on/off switch
-rheostat (dimmer switch)
-light source (bulb)
-condenser lens
-condenser lens adjustment knob
-iris diaphragm adjustment lever
-specimen stage
-specimen stage positioning knobs
-coarse and fine focus knobs
-objective lens
-occular lenses
-diopter adjuster in left ocular lens
-interpupillary distance adjuster
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focal plane
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plane in space at which the lens is in focus (parfocal lenses have the same focal plane)
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how do you calculate magnification of an objective lens?
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multiply number on lens by 10 (for ocular lens magnification)
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parfocal objectives
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have the same focal plane; when they are exchanged they are very close to being in focus
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oil immersion improves resolution because
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it minimizes light refraction by removing air from the light path
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resolution
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sharpness of image
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magnification
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size of image
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Colony Morphology Shape (3)
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-circular

-irregular

-punctiform (pin-point)

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Colony Morphology Margin (4)
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-entire

-undulate 

-lobate 

-filamentous

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entire
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smooth edge

colony morphology margin

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undulate
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wavy edge

colony morphology margin

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lobate
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bumpy edge

colony morphology margin

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filamentous
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hairy edge

colony morphology margin

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colony or slant morphology texture (3)
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-moist

-mucoid

-dry

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colony or slant morphology optical properties (2 pair)
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-opaque/translucent

-dull/shiny

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slant morphology growth pattern (3)
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-filiform

-spreading

-friable

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friable
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crusty
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filiform
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-growth restricted to inoculation site

-edge is clear and defined
-appearance is creamy 

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Broth cultures morphology (4)
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-uniform fine turbidity

-pellicle

-sediment

-flocculent

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Colony or slant Morphology Pigment
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Any
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Uniform fine turbidity
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uniform cloudiness
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Pellicle
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raft floating on broth
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sediment
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accumulation on bottom
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Plate morphology categories (5)
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-shape

-margin

-texture

-optical properties

-pigment

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Slant morphology categories (4)
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-texture

-optical properties

-pigment

-growth

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procedure for loop transfer of culture (10 steps)
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-sterilize loop

-remove tube cap

-flame tube mouth

-cool loop on tube

-dip loop

-remove loop w/o flicking

-flame tube mouth

-close tube cap

-innoculate plate, slant or broth

-flame loop

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how to get single colonies on a streak plate (3 steps)
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-4 quadrant

-flame loop between each quadrant

-drag cool loop through previous quadrant before moving into next

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