BIO TEST #5
Terms in this set (116)
Mannitol Salt Agar (MSA)
What is the Selective medium for Gm+ Staph?
high salt concentration
MSA: Selective medium for Gm+ Staph; _______ _____
______________ inhibit Gm- bacteria
red to yellow
Mannitol Salt Agar (MSA): Medium contains a color pH indicator that turns from ____ to __________
MSA: If bacteria can ferment mannitol, _____ are
If bacteria can ferment mannitol, acids are
What is the Typical hallmark of pathogenic Staph?
pH turns acidic (lower pH)
Mannitol Salt Agar (MSA): If acids are produced, then what happens?
we see a color
MSA: When pH changes to acidic, what happens?
Staphylococcus Medium 110
what is the Selective medium for Gm+ Staph; high salt concentration?
Differential medium -
What Differentiates species
of Staphylococcus on the basis of pigment
production, i.e., different species will
produce different pigments?
Staphylococcus from the M-Staph broth from last lab by streaking on MSA and SM110 plates
Last time...We inoculated M-Staphylococci broth with the nose swab and the fomite swab
NEXT lab... we will isolate what by doing what ?
expected results: MSA plate:
S. epidermidis -> non-pathogenic, cannot
ferment mannitol -> medium = what color?
expected results: MSA plate:
S. aureus -> ferment mannitol-> medium = what color?
no pigment production
expected results: SM110:
S. epidermidis -> ?
expected results: SM110:
S. aureus -> ?
hemolysis identification: (none)
hemolysis identification: (complete)
hemolysis identification: (partial)
What is a chemical that is purple when
oxidized; colorless when reduced?
Actively growing _______ consume oxygen in
milk, which reduces the milk.
___________ ________: gain of electrons/loss of
oxygen (the opposite of oxidation)
Measuring __________ is a measure of the
number of bacteria in a milk sample
-Resazurin will be added to milk; as milk is
reduced by bacteria, resazurin will turn from
blue/purple to pink to colorless over time
-I will add 1.0 mL resazurin to 10 mL high- and
what is the ONLY test that is done on the farm (easily done)?
Incubate in 37°C water bath; check every 30
minutes to record time it takes for the color
to change and disappear
What Method for preserving milk and other dairy
heating milk to a temperature below
the boiling point (usually 60°C) for 20
minutes; brought down to 10°C
Describe the process of pasteurization.
_______ ____________: 72°C for 15 seconds;
rapid cooling to 10°C
it reduces their numbers
Pasteurization does not kill all microbes - but it does what?
standard plate count
Method used to determine
the quality of milk
High standard plate counts may indicate what 3 things?
"high-quality" milk (Pasteurized whole milk)
and "low-quality" milk (raw milk)
What are the different types of milk we used in our experiment? (2)
(tryptone glucose extract agar)
What does TGEA stand for?
-Make your dilutions first! Keep TGEA in water
bath until you are ready to use it
-Mix milk (or diluted milk) with the liquid
TGEA, then pour into a sterile Petri plate
-Let agar solidify, then we will incubate at
What are the 3 proper steps when dealing with milk and TGEA plates?
DO THIS FIRST:
Add 1 mL of highquality milk!
milk microbiology exercise: what did we do very first?
Standard Plate Count?
What is this?
half-plate T streak
Divide in half, then
make 2 T's; streak
as usual, but only
taking up half the
plate with each
half-plate T streak
what is this?
Plate 1 of both MSA and simulated SM110 = Halfplate T-streak with S. epidermidis and S. aureus
Ex. 18 inoculations: plate 1 gets?
Plate 2 of both = full T-streak with nose sample
Ex. 18 inoculations: plate 2 gets?
Plate 3 of both = full T-streak with fomite sample
! Incubate at 35°C
Ex. 18 inoculations: plate 3 gets?
Ex. 19 once you have found a hemolytic colony, Perform the catalase test on controls & ONE
colony from throat plate (do not use the entire
colony!) Look for __________ result!
TH (Todd Hewett) tube (tighten cap and NO candle jar)
Ex. 19 Inoculate the catalase negative colony into a _________.
1. Add 1-2 drops of H2O2 onto slide
2. Use sterile toothpick to pick single, isolated colony
3. Careful not to pick agar!!
4. Swirl colony into drop & look for immediate bubbles (May need slight magnification to see weak positive)
Catalase Test: Slide Method: what are the 4 steps?
Catalase Test: Slide Method: Water + O2 = ?
no bubbles (-)
Catalase Test: Slide Method: No O2 = ?
Streptococcus is catalase (-/+)
Ex. 19 Choose negative colony for ___ broth inoculation (TIGHTEN CAP THIS TIME! No candle jar)
# CFUs (count)/
Volume plated (we know)
units = cfu/mL
final conc = ?/?
a Differential medium
Blood Agar Plates is considered what?
Used to differentiate bacteria on the basis of
their hemolytic properties
what are blood agar plates/ differential medium used for?
What uses BAPs along with other characteristics
(morphology/arrangement, catalase test) to
identify Gm+ UTI-causing organisms
-metallic green sheen = lactose +
-purple (middle) = lactose +
-light pink (left end) = lactose -
Which is lactose pos? neg?
Eosin-Methylene Blue (EMB) Agar
What has Selective and differential
EMB: _______ media grows Gm-bacteria; most Gm+ bacteria will not grow
EMB: _______ media in that different
Gm- bacteria will have
characteristics on EMB,
allowing for putative
Gram-negative rods and Gram-positive cocci
Most bacteria that cause UTIs will fall under
one of two groups: what two groups?
Where does the following belong? (e.g. E. coli, Proteus spp., Enterobacter spp., and Klebsiella spp.)
Where does the following belong? (e.g. Streptococcus spp.,
Staphylococcus spp., and Enterococcus spp.)
evidence of UTI
In clean-catch urine: >100,000 bacteria/mL = ?
(patient must exhibit other symptoms)
In clean-catch urine: 10,000-100,000 bacteria/mL = ?
In clean-catch urine: <10,000 bacteria/mL = ?
Name the process in which milk is heated to
72°C for 15 seconds and then rapidly cooled
Sick cows, Unsanitary handling, Inadequate
Name one of the three ways we discussed
milk going bad. (too many microbes in it)
To cut open or break open red blood cells
Succinctly, define hemolysis.
2 H2O2 -> 2 H2O + O2
What chemical reaction is catalase
In BSL 2, up to what portion of the stick is
allowed to go into culture? (in other words,
no more than what fraction of the stick?)
1. Coagulase test
2. DNase test
3. Hemolysis test
After selecting nose and fomite colonies... what are the 3 tests you will perform?
What solidifies blood plasma, making a
tough layer around Staph cells to shield them
from the host immune system?
Fibrinogen (soluble) -> fibrin (tough)
Fibrinogen (?) -> fibrin (?)
What an enzyme
produced by S. aureus
that can break down
DNA (+) ?
Name the test: Evasion of immune
extracellular traps, or
Blue = DNase negative
Pink = DNase positive
Blue = ?
Pink = ?
NETs are made of _____, and will
bind and kill pathogens.
_______ is a
virulence factor that can break
down NETs, inhibiting microbial
killing by this immune mechanism.
What breakdown of
red blood cells ?
Staph species may
produce ________, an
enzyme that forms a sort
of pore that inserts into
the RBC membrane.
sheep blood agar
What agar do you use to
test for hemolysis?
what is complete
complete clearing of
RBCs in plate around
________+ = no hemolysis
A strep (S. pyogenes)
Bacitracin (BAC disc) Susceptibility Test: Only Group ________ (______) is sensitive
Bacitracin is an ___________
Bacitracin is isolated from what
what is Toxic and difficult-to-use antibiotic; not a good
What is described:
! Very effective antibiotic
! Affects Gm+ cell wall synthesis
! Differentiates group A from other Strep
Optochin (P disc) Susceptibility Test: Only ________ is sensitive to optochin
________ is used for the presumptive
identification of S. pneumoniae and
differentiation from other α-hemolytic
Bile Esculin Hydrolysis
What distinguishes group D streptococci (E. faecalis) and
other α-hemolytic strep?
Bile esculin slant contains _% bile and esculin
Only Group __ can grow on 4% bile, which inhibits
esculin to esculetin.
Group D can hydrolyze ________ to _________.
Group __ can grow in high salt concentration
________ reacts with ferric citrate in agar to form
Perform catalase test
(If catalase (+) -these are the selected nose
and selected fomite colonies)
Note the color of colonies on MSA and SM110
plates for S. epidermidis and S. aureus...
Choose two colonies on fomite and nose
plates that look like both S. epi and S. aureus... perform what?
Inoculate each quadrant with a dime-sized, relatively thick inoculum of each of your samples (Positive Control, Negative Control, selected fomite, selected
nose) Which test?
Each table gets two blood agar plates (BAP) (T-streak each plate with: Selected nose and Selected fomite)
BAC disk and SXT disk
Inoculation of disc:
β-hemolytic strep -> Use _____ disk and _____ disk
(middle of two halfs)
Inoculation of disc:
α- or γ-hemolytic strep -> use __ disc (middle)
Bring up to the candle jar for incubation
After using forceps and inoculating the disk, then what?
Bring to beaker in front incubate with tight
Bile Esculin Hydrolysis and Growth in 6.5% NaCl: what do you do with cap?
Bile Esculin Hydrolysis helps distinguish what
group of Streptococci?
True or False: Bacitracin is an antibiotic that is
True or False: Alpha (α) hemolysis is a definitive
sign of a pathogen.
Evasion of the Immune System
Some Staphylococci produce DNase. How does
this enzyme help them? (think big picture)
Cell Arrangement (Streptococci - Chains
Staphylococci - Clusters)
What might be one feature we learned about
that could differentiate Staph and Strep
Media solidified = fibrinogen -> fibrin =
Media still liquid = no fibrin = Coagulase(+/-)
Coagulase(+) = S. aureus or S. epidermidis
Coagulase(-) = S. aureus or S. epidermidis
Blue -> pink/purple = DNA broken down by
DNase = DNase(+/-)
Still blue (or clear) = no DNase activity =
DNase(+) = S. aureus or S. epidermidis
DNase(-) = S. aureus or S. epidermidis
S. aureus (complete hemolysis)
beta = S. aureus or S. epidermidis
S. epidermidis (no hemolysis)
gamma = S. aureus or S. epidermidis
Final concentration/ (from last step)
Final Dilution (fraction) (we know this)
original total conc?
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