MCB 2010C - Lab Practical #2
pyrimidine dimers
*cytosine - cytosine, cytosine - thymine, thymine - thymine* distort DNA molecule and interfere with DNA replication and transcription *occurs when DNA absorbs UV radiation at 254 nm*
e.coli uv repair methods
1. DNA photolyase (visible light @ 340 - 400 nm) 2. Excision Repair / Dark Repair a. Endonuclease (UvrABC) : breaks bonds 8 in 5', 4 in 3' b. Helicase (UvrD) : removes 13 nucleotide fragment (single stranded DNA) c. DNA polymerase 1 ( appropriate comp. nucleotides d. DNA ligase (repairs gap) *long, intense exposures can be lethal; cell can no longer repair*
yellow slant & butt in TSIA (triple sugar iron agar)...
FERMENTATION glucose, lactose and/or sucrose ACID PRODUCTION slant, butt
red slant & red butt in TSIA or KIA...
FERMENTATION none *proteins catabolized aerobically AND anaerobically with alkaline products* *not from enterobacteriaceae*
red slant & yellow butt in TSIA or KIA...
FERMENTATION glucose ACID PRODUCTION yes *proteins catabolized aerobically in slant with alkaline products*
yellow slant & butt in KIA (kliger - iron agar)...
FERMENTATION glucose, lactose ACID PRODUCTION slant, butt
red slant & no change in butt in TSIA or KIA...
FERMENTATION none *proteins catabolized aerobically with alkaline products* *not from enterobacteriaceae*
IMViC chart
Indole, Methyl Red, Voges-Proskauer, Citrate tests; chart used to differentiate enterics from various species
what is the control in pGLO experiment?
LB (Luria-Bertani) agar
part 2 in SIM medium lab
addition of Kovacs' reagent; depth of 2-3mm after 7 min observe upper formation of red color
part 2 in phenylalanine deaminase lab
addition of ferric chloride (reagent containing ferric chloride) *green color indicates presence of phenylpruvic acid -> phenylalanine deaminase* *yellow is negative*
part 2 in nitrate reduction lab
addition of reagent A & B (if no bubble in Durham tube) zinc - after addition of reagent A & B, and organism is not red
bound coagulase
aka clumping factor attached to bacterial cell wall; reacts directly with fibrinogen in plasma fibrinogen precipitates, causing cells to clump together
what is the sole nitrogen source in the citrate utilization test?
ammonium dihydrogen phosphate
nitrate reductase
an enzyme; performs a single step reaction of nitrate to nitrite (NO3 -> NO2)
DNA binding protein
araC; regulatory molecule for arabinose promoter *w/ arabinose:* araC has a shape that allows RNA polymerase to bind to promoter *w/out arabinose:* araC's shape prevents RNA polymerase from binding to promoter
"the pGLO plasmid used has been genetically engineered to contain..."
arabinose promoter (pBAD), gene for araC (DNA binding protein), antibiotic resitance gene (bla), green flourescent protein gene (GFP), and replication origin (ori).
staphylococcus aureus is resistant...
bc of the coagulase enzyme production
antibiotic resistance gene
bla; produces beta-lactamase (enzyme that hydrolyses certain antibiotics, including ampicillin) *provides a mean of differentiating cells that were transformed and those that weren't*
ferric sulfide (Fes)
black precipitate; when either cysteine desulfurase or thiosulfate reductase reacts with SIM Medium, H2S (hydrogen sulfide) COMBINES with iron (ferrous ammonium sulfate)
organisms that produce phenylalanie deaminase...
can be identified by their ability to remove the amine group (NH2) from amino acid phenylalanine *requires oxygen; produces ammonia and phenylpyruvic acid*
decarboxylation test
can include several amino acids; are used to differentiate Enterobacteriacae organisms and to distinguish them from other gram-negative rods
moller's decarboxylase base medium
contains peptone, glucose, pH indicator bromescrol purple, and coenzyme pyroxidal phosphate
denitrifiication
converting nitrate to molecular nitrogen (NO3 -> N2)
how does the broth in phenol red turn pink?
deamination of peptone amino acids produces ammonia (NH3)
pink broth & no bubble in tube in PR broth...
degradation of peptone; alkaline end products
phenol red broth
differential test medium prepared as a base to which a carbohydrate is added *includes peptone and pH indicator phenol red*
urea hydrolysis test
differentiates organisms based on their ability to hydrolyze urea with urease )(enzyme)
organisms used in UV lab
e. coli , s. marcensens , b. cereus, a. niger
cysteine desulfurase
enzyme catalyses putrefaction of amino acid to pyruvate
thiosulfate reductase
enzyme cataylses reduction of sulfur (in the form of sulfate) at the end of the anaerobic respiratory electron transport chain
citrate-permease
enzyme needed to metabolize citrate
what microorganism was used in the pGLO lab?
escherichia coli / e.coli
free coagulase
extracellular enzyme (released from cell) that reacts with a plasma component (coagulase- reacting factor , CRF)
what is the hydrogen sulfide indicator in TSIA (triple sugar iron agar)?
ferrous sulfate
coagulase enzymes occur in two forms...
free & bound coagulase
cracks or lifting in TSIA or KIA...
gas production
if gelatin is liquid
gelatinase is present
catalase test
identifies organisms that produce catalase; most commonly used to differentiate members of catalase positive (micrococcae) from catalase negative (streptococcae) can be performed in a tube or slide
no color change in phenylalanine test...
indicates a negative reaction - phenyalanine deaminase is absent
liquid medium in coagulase tube test...
indicates a negative reaction - plasma hasn't been coagulated
no clumping of cells in coagulase slide test...
indicates a negative reaction - plasma hasn't been coagulated
clumping of cells in the coagulase slide test...
indicates a positive reaction - indicates plasma has been coagulated
green color in phenylalanine test...
indicates a positive reaction - phenylalanine deaminase is present
solid medium in coagulase tube test...
indicates a positive reaction - plasma has been coagulated
yellow broth & no bubble in tube in PR broth...
indicates fermentation with acid end products *no gas produced*
yellow broth & bubble in tube in PR broth...
indicates fermentation with gas end products
red broth & no bubble in tube in PR broth...
indicates no fermentation
enterobacter aerogenes
indole negative methyl red negative voges proskauer positive / negative citrate positive / negative
proteus mirabalis
indole negative methyl red positive voges proskauer positive citrate positive / negative
citrobacter freundii
indole negative, methyl red positive , voges-proskauer negative, citrate positive / negative
escherichia coli
indole positive methyl red positive voges proskauer negative citrate positive / negative
morganella morganii
indole positive methyl red positive voges proskauer negative citrate positive / negative
no color change or growth in citrate utilization test
is a negative reaction - indicates citrate is not utilized
red color (after addition of zinc dust) in nitrate reduction lab
is a negative reaction - indicates no nitrate reduction
orange / yellow color in urea hydrolysis test
is a negative reaction - no urea hydrolysis; organism doesn't produce urease or can't live in both
no radiating growth from stab line in SIM medium...
is a negative reaction - nonmotile
no black in SIM medium...
is a negative reaction - sulfur is not produced
reagent color is unchanged in SIM medium..
is a negative reaction - tryphotan is NOT broken down into indole, pryruvate
yellow color in decarboxylation medium...
is a negative result - indicates fermentation, no decarboxylation
no color change in decarboxylation medium...
is a negative result - no decarboxylation
blue color (even a small amount) on citrate utilization test
is a positive reaction - indicates citrate is utilized
no color change; growth on citrate utilization test
is a positive reaction - indicates citrate is utilized
gas (nonfermenter) in nitrate reduction lab
is a positive reaction - indicates denitrification (production of nitrogen gas / NO3 -> NO2 -> N2)
growth radiating outward from stab line in SIM medium...
is a positive reaction - indicates motility
red color (after reagents a & b) in nitrate reduction lab
is a positive reaction - indicates nitrate reduction to nitrite (NO3 -> NO2)
no color change (after the addition of zinc) in nitrate reduction lab
is a positive reaction - indicates nitrate reduction to nongaseous nitrogenous compounds (NO3 -> NO2 -> nongaseous nitrogenous products)
pink color in urea hydrolysis test
is a positive reaction - indicates rapid urea hydrolysis; strong urease production
black in SIM medium...
is a positive reaction - sulfur reduction (H2S, hydrogen sulfide)
red in alcohol layer of Kovac's reagnent in SIM medium...
is a positive reaction - trytophan is broken down into indole and pyruvate
purple (even slightly) color in decarboxylation medium...
is a positive result - indicates decarboxylation *organism produces specific decarboxylase enzyme*
no color (after the addition of reagents) in nitrate reduction lab
is an unknown reaction - incomplete test; requires the addition of zinc dust
gas (fermenter, unknown status) in nitrate reduction lab
is an unknown reaction - source of gas is unknown *would require additional reagents*
pGLO lab
kit created by Bio Rad Laboratories that efficiently illustrates the principles of microbial genetics: 1. bacterial transformation 2. use of antibiotic selective medium to identify transformed cells 3. operon as a mechanism of microbial genetic regulation
crateriform liquefaction
may also be of diagnostic use since not all gelatinase-positive microbes liquify gelatin completely
triple sugar iron agar (TSIA)
medium designed to differentiate bacteria on the basis of: 1. glucose fermentation 2. lactose fermentation 3. sucrose fermentation 4. sulfur reduction includes animal proteins as carbon and nitrogen sources and oxidized sulfur (ferrous sulfate, sodium thiosulfate)
what is a disadvantage of nutrient gelatin?
melts at 28C (82F) ; inoculated stabs are incubated at 25C along with a control to verify liquefaction isn't temperature related
if gelatin is solid
no gelatinase is present
reversion
occurs in TSIA when inoculated with glucose - only fermenter . organisms in aerobic region will begin to break down amino acids, producing NH3 and raising pH *takes 18 - 24 hrs to complete; limited to slant bc of anaerobic conditions in butt*
no change in slant or butt in TSIA or KIA...
organism is growing slowly or not at all *not from enterobacteriaceae*
replication origin
ori; necessary for DNA replication; allows replication for pGLO plasmid *result of the replication: allows copies of plasmid to be distributed to descendants of original, transformed E.coli cell*
arabinose promoter
pBAD; attachment site for RNA polymerase during transcription of GFP gene on pGLO plasmid *is normally attachment site for RNA polymerase during transcription of araB, araA, and araD genes*
plasmid
pGLO; used as a vector to introduce the GFP gene into recipient e.coli cells
bromthymol blue dye
pH indicator -green @ pH 6.9 -blue @ pH 7.6
phenol red
pH indicator yellow below pH 6.8 pink - magenta above pH 7.4 red in between *in lab pH is adjusted to 7.3 to appear red*
bromescrol purple
pH indicator in moller's decarboxylation medium; purple at pH 6.8 and above yellow at pH below 5.2
what is the pH indicator in TSIA (triple sugar iron agar)?
phenol red
what two forms of the coagulase test can be done?
slide & tube
black precipitate in TSIA or KIA...
sulfur reduction *from fermentation of glucose or lactose exists in butt even if yellow is obscured by black precipitate*
what is the purpose of a durham tube in phenol red broth?
to indicate gas producer; durham tube is filled with liquid initially *gas bubble indicates gas production*
mineral oil is added to decarboxylation medium...
to seal medium from external oxygen and promote fermentation *lysine / orinthinine were the mineral oils used in this lab*
how can sulfur reduction to H2S (hydrogen sulfide) be done by bacteria?
two ways: 1. enzyme cysteine desulferase 2. enzyme thiosulfate reductase
how do you make the e.coli cells competent?
use a CaCl2 (calcium chloride) transforming solution and heat shock
sim medium
used for determination of 3 bacterial activities: sulfur reduction, indole production from tryphtophan and motility
GFP (green fluorescent protein)
used in pGLO experiment for bio-luminescence; creates luminescence in jellyfish aequorea victoria *visual evidence of successful transformation and subsequent gene expression*
gelatin hydrolysis lab
used to determine the ability of a microbe to produce gelatinases staphylococcus aureus vs. s. epidermis enterobacteriae: serratia & proteus species (+) bacillus anthracis, b. cereus, clostridium tetani, c. perfringens are (+) including several other members
citrate utlization test
used to determine the ability of an organism to use citrate as its sole source of carbon differentiates members of enterobacteriacae which are facultative anaerobes
phenylalaine deaminase test
used to differentiate genera morganella, proteus, and providencia
coagulase test
used to differentiate staphylococcus aureus from other gram-positive cocci
coagulase
works with normal plasma components to form protective fibrin barriers around individual cells or groups of cells *shields cells from phagocytosis and other forms of attack*
