Lab Quiz 8: Water Quality Testing, Genetic Transformation, Lactose Operon Regulation, and Enterobacteriaceae Biochemistry (Bio 286 - Microbiology) Flashcards
indicator organisms
always present when feces are present (PRESENT IN FECES); should be absent when feces are absent (ABSENT WITHOUT FECES); organism should be very EASY TO GROW; organism should be EASY TO IDENTIFY (using differential media); organism should persist in the environment slightly longer than the pathogens (there is less problem with false positives than false negatives)
fecal coliforms
indicator organism used to detect fecal contamination of mammals (humans); lactose-fermenting gamma proteobacteria
fecal streptococci (enterococci)
indicator organism used to detect fecal contamination of animals (like ducks) but not humans; glucose-fermenting bacteria
ratio of coliforms to streptococci
indicates where fecal contamination originated
wastewater is not contaminated
< 400 FC/100 mL
surface water is not contaminated
<160 FC/100 mL
drinking water is not contaminated
< 1 FC/100 mL
coliforms : enterococci ≥ 4:1
source of fecal contamination is human
2:1 ≥ coliforms : enterococci < 4:1
source of fecal contamination is a mix between human and animal
0.7:1 ≥ coliforms : enterococci < 2:1
source of fecal contamination is unknown
coliforms : enterococci < 0.7
source of fecal contamination is animal
mFC plate
used to detect FECAL COLIFORMS… selective with BLUE ACIDS… differential with LACTOSE… colony color is BLUE… incubation temperature is 44.5 degrees Celsius
mEA plate
used to detect FECAL STREPTOCOCCI (Enterococcus)… selective with SODIUM AZIDE… differential with GLUCOSE… colony color is RED… incubation temperature is 35 degrees Celsius
dilution calculation (calculating number of fecal coliforms or streptococci per 100 mL)
[(cfu * SDF) / volume plated] x 100
ratio of coliforms to streptococci calculation
Blue Colonies (fecal coliforms) / Red Colonies (enterococci)
bacteria can horizontally transfer genes through
transformation, conjugation, and transduction
Frederic Griffith
first described the process of transformation using rough and smooth strains of Streptococcus pneumoniae… mixing the dead smooth strain with the live rough strain killed injected mice, but live smooth bacteria were isolated from these dead mice… indicating that something had transformed the rough strain into a smooth strain
rough strain
made small compact colonies; avirulent when injected into mice
smooth strain
made larger mucoid colonies; efficiently killed mice when injected into them and could be isolated from the mice postmortem (Koch’s postulates)
heat killing the smooth strain
made them nontoxic
mixing the dead smooth strain and the live rough strain
killed injected mice (despite non being virulent when injected individually); live smooth bacteria were isolated from the dead mice because something had transformed the rough strain into a smooths train
Oswald Avery
proved that the transforming material was DNA
competent
bacteria that are capable of transporting exogenous DNA into the cell and incorporating it into their genomes (ex: streptococcus pneumoniae)
on the nutrient agar plate without antibiotics
all strains of the acinetobacter calcoaceticus grow… but quadrant 3 (with only the heat killed smooth strain) does not grow anything as only the DNA genomes are present
on the nutrient agar plate with antibiotics (streptomycin)
only the smooth, resistant strain (quadrant 1) and the transformed rough -> smooth, resistant strain (quadrant 4) grows because the rough strain (quadrant 2) is susceptible and quadrant 3 only contained DNA
Griffith’s transformation experiment – quadrant 1
grow the rough, susceptible strain
Griffith’s transformation experiment – quadrant 2
grow the smooth, resistant strain
Griffith’s transformation experiment – quadrant 3
heat kill the smooth, resistant strain
Griffith’s transformation experiment – quadrant 4
mix the dead smooth strain with the rough, susceptible strain
proteins encoded by the lac operon
β-galactosidase (product of lacZ gene), lactose permease (product of lacY gene), and lactose transacetylase (product of lacA gene)
lac transcript is polycistronic
it contains more than one coding sequence
lacI
lac repressor; tetramer of four identical subunits… it binds to DNA near lac promoter and prevents transcription of the structural genes and it binds to the INDUCER (allolactose)– this is cooperative [binding of one molecule of inducer makes binding of the next one more favorable… repressor binds inducer in all or none fashion]… or it binds to the OPERATOR (lacO) which partially overlaps promoter [allows RNA polymerase to bind to promoter and form an open complex but not to elongate transcription]
lacI^s
REPRESSED EXPRESSION; proteins never leave operator
lacO^c
loss of repressor binding; CONSTITUTIVE EXPRESSION
merozygote
two copies of the relevant control genes exist
Jacob and Monod
used fertility plasmid (F) from E.coli to study regulation of lactose operon; used a variant (F’) that contained a normal functional copy of the lactose operon genes (normal LacI and LacZ proteins)
ONPG
a colorless chemical analog of lactose used to measure enzyme activity of LacZ… when cleaved by LacZ it become yellow ortho-nitrophenol
two grey tubes -> one grey and one pale yellow
lacZ
one grey and one pale yellow -> one grey and one bright yellow
WT (wild type)
two pale yellow tubes -> one grey and one pale yellow
lacI-
two pale yellow tubes -> one pale yellow and one bright yellow
lacO^c
two grey tubes -> two grey tubes
lacI^s
enterobacteriaceae
family of gamma proteobacteria that includes Escherichia, Salmonella, Yersinia… BACILLI that are typically 1-5 micrometers in length… stain GRAM NEGATIVE and are FACULTATIVE ANAEROBES… can ferment sugars (all can ferment glucose and some can ferment lactose) and reduce nitrate to nitrite via ANAEROBIC RESPIRATION… generally LACK CYTOCHROME C OXIDASE… most have FLAGELLA… NON-SPORE FORMING… PRODUCE CATALASE
coliforms
Members of the family enterobacteriaceae that ferment lactose
MacConkey agar
if the microbes ferment lactose then the acids they produce turn the indicator dyes red… a positive result is red and a negative result is no change
indole
if microbes breakdown amino acid tryptophan to indole, the indole reacts with Kovac’s reagent to turn bright red… a positive result is red and a negative result is yellow
triple sugar iron agar (slant)
if ammonia is produced then the pH of the media becomes neutral – red and if lactose/sucrose is fermented then the tube will remain yellow… a positive result is yellow (A) and a negative result is red (K)
triple sugar iron agar (butt)
the butt of the TSI is always yellow – a positive result is yellow (A)
triple sugar iron agar (gas)
gases produced during fermentation will appear as bubbles or cracks… a positive result is cracking and a negative result is no change/no cracking
triple sugar iron agar (H2S)
hydrogen sulfide produced from breakdown of amino acid cysteine reacts with the iron to form ferric sulfide which is black… a positive result is black and a negative result is no change
MRVP (methyl red)
turns red in the presence of organic acids… a positive result is red and a negative result is yellow
MRVP (voges proskauer)
detects acetoin to turn red in its presence… a positive result is red and a negative result is coppery brown/no change
citrate
at acidic pH (citrate present) the media is green and if the citrate is metabolized the pH will turn blue… a positive result is blue and a negative result is green
urea
under alkaline conditions the indicator will turn pink… a positive result is bright pink and a negative result is yellow/no change
indole negative result
yellow
indole positive result
RED
methyl red negative result
yellow
methyl red positive result
RED
voges proskauer negative result
no change (yellow brown)
voges proskauer positive result
RED
citrate negative result
green
citrate positive result
BLUE
MacConkey Agar (MAC) negative result
no change (not red– brown or tan)
MacConkey Agar (MAC) positive result
RED
urea negative result
no change (yellow)
urea positive result
PINK
TSI (slant) negative result
K - red
TSI (slant) positive result
A - YELLOW
TSI (Butt) negative result
K - red
TSI (Butt) positive result
A - YELLOW
TSI (gas) negative result
no change/no cracks
TSI (gas) positive result
CRACKS
TSI (H2S) negative result
no change/yellow
TSI (H2S) positive result
BLACK
