Lab Quiz 3 Flashcards
Molecular cloning
Recombinant DNA molecules are assembled and expressed in a host organism
Enable the alteration of genetic information for research and industrial purposes
Cutting, pasting, and rearranging of pieces of DNA
Often involves the insertion of a gene-of-interest into a plasmid
restriction enzymes
Perform the cutting
Bind and cleave (digest) a restriction site
Different restriction enzymes have different
restriction sites
Cleave both strands of DNA
Restriction site
A specific DNA sequence where the restriction enzyme(s) cleave
Are palindromic – the 5’ –> 3’ sequence is identical on
both strands
Plasmid
A circular DNA molecule that replicates independent of the chromosome
Used as vectors to facilitate the transfer of a gene into an organism
During cloning, the plasmid is digested by a restriction enzyme to allow the insertion of the gene-of-interest
Plasmid cutting
A plasmid that has been cut by a restriction
enzyme in one location will become a single linear piece of DNA
A plasmid cut in two locations will yield two linear pieces of DNA, whose combined size is equivalent to the full-size plasmid
Gel electrophoresis
Used to visualize the size and quantity of DNA in a sample
An electrical current is passed through a gel. Since DNA has a intrinsic negative charge (due to its phosphate backbone), DNA moves away from the negative
electrode and toward the positive electrode
The gel consists of an agarose matrix that enables
DNA to be separated by size: smaller DNA fragments weave through the pores of the agarose
matrix easier than larger DNA fragments.
A DNA marker (or ladder) is included on a gel as a
reference to indicate fragment size
Do larger or smaller pieces go to the negative end (up top) on gel electrophoresis?
Larger
Within a bacterial cell, a plasmid naturally exists in a _________ conformation
supercoiled
What converts a plasmid to a relaxed, circular conformation?
When a plasmid is isolated from bacterial cells, the isolation process can damage the DNA backbone (nick in one strand)
When isolating plasmid from a bacterial cultures, many
copies of the plasmid are harvested, some in a supercoiled confirmation and some in the relaxed,
circular conformation - which move faster?
Supercoiled plasmids move slightly faster through the gel matrix than expected (compared to linear DNA) while circular plasmids migrate a little slower
All microbial species are unique, often due to the ______ they produce.
enzymes
Microbial enzymes catalyze the _________ of biological molecules, thereby enabling microbes to grow on particular substrates
metabolism
Microbiologists take advantage of differences in biochemical activities to distinguish between
microbial ______.
species
Enteric bacteria
Gram negative rods that ferment glucose
Can be identified using a group the IMViC tests
IMViC: Indole (I), Methyl Red (M), Voges-Proskauer (V), Citrate (C)
Indole Test
Determines if the bacteria can metabolize the amino acid tryptophan to produce indole using the
enzyme tryptophanase.
Media containing tryptophan (ex.SIM stab) is inoculated and incubated.
After growth occurs, Kovac’s reagent is added and will react with indole if present.
+ : red color in the layer atop the media.
- : yellow color in the layer atop the media
Methyl Red Test
Determines if mixed acids are produced during fermentation of glucose
Mixed acid fermentation produces three acids (acetic, lactic, succinic).
Glucose-containing MR-VP broth is inoculated. Then methyl red (pH indicator) is added after growth in MR-VP broth.
+ : carbohydrate utilization test: red color in the medium
- : carbohydrate utilization test: yellow color in the medium
Voges Proskauer (VP) Test
Determines if bacteria produce acetoin during fermentation of glucose. Acetoin is an organic molecule used by some bacteria to store energy.
Glucose-containing MR-VP broth is inoculated. After growth, Barritt’s reagent A (alpha-naphthol) then Barritt’s reagent B (potassium hydroxide) are added.
+ : development of a red color 15min after the addition of Barritt’s reagents indicating the presence of diacetyl, the oxidation product of acetoin.
- : absence of a red color 15min or more after the addition of Barritt’s reagents.
Citrate Test
Determines if the bacteria can utilize citrate as a sole
carbon source; if a microbe can use citrate as its sole source of carbon and energy for growth
Media contains the pH indicator bromothymol blue to detect the production of alkaline products that increase the pH of the media.
+ : media turning blue or the appearance of bacterial growth on the slant. Sometime the blue is faint.
- : media remaining green.
SIM Stab
A single medium that tests for three characteristics: sulfur reduction, indole production, and motility.
SIM medium is inoculated by stabbing through the agar with an inoculation needle.
SIM stab contains 2 sources of sulfur: sodium thiosulfate and cysteine (an amino acid).
Hydrogen sulfide gas is produced if the bacteria possess enzymes that metabolize either source of sulfur (either thiosulfate reductase cysteine desulfurase).
In the media is the H2S indicator ferrous ammonium sulfate, which reacts with hydrogen sulfide gas to produce an insoluble, black precipitate.
SIM Stab: Sulfur Reduction
Determined based on the ability of bacteria to produce hydrogen sulfide gas (H2 S).
+ : presence of black precipitate; precipitate may be localized to the stab line or dispersed throughout the
tube, depending on the motility of the bacteria.
- : absence of black precipitate
SIM Stab: Indole
+ : red color in the layer atop the media.
- : yellow color in the layer atop the media
SIM Stab: Motility
Motility is determined based on turbidity within the medium.
o SIM medium is semi-solid due to low agar concentration so that motile bacteria can
freely migrate within the tube.
o Motility is indicated by turbidity away from the stab line (often throughout the tube).
o Non-motility is indicated by turbidity localized to the stab line
Urease Test
Determines if the bacteria can break down urea using the enzyme urease
Microbes that utilize urea have urease which breaks it down to ammonia, CO2 , and water.
If ammonia is produced, the media will become alkaline resulting a colorimetric change in the pH indicator phenol red.
+ : pink color.
- : absence of pink/red
Carbohydrate Fermentation Test
Determines if a microbe can ferment different carbohydrates such as glucose and sucrose.
Carbon dioxide, ethanol, acids, and hydrogen gas are
typical products of carbohydrate fermentation.
A pH indicator (phenol red) detects acids produced during a fermentation reaction.
A Durham tube is present to capture gas production.
+ : either the development of a yellow color in the medium or the presence of bubble in the Durham tube.
- : media remains red
Oxidase Test
Determines if oxygen is utilized as the terminal [final] electron acceptor by the enzyme cytochrome oxidase.
The DrySlide Oxidase test (window thing) consists of a slide coated with a chromogenic reducing agent that changes color if oxidized.
+ : production of a blue/purple color within 20sec
- : no color change or blue/purple after 20sec
Catalase Test
Determines if hydrogen peroxide can be broken down to O2 and H2O by the enzyme catalase.
Catalase protects the cell from oxidative damage caused by reactive oxygen species such as hydrogen peroxide.
Done on a microscope slide
+ : bubbling (indicates the production of oxygen gas)
- : the absence of bubbling (no reaction)
Nitrate Reductase Test
Tests for the ability to dissimilate nitrate to nitrite (incomplete) or nitrogen gas (complete).
* Broth contains nitrate and a Durham tube to capture gas production.
* Nitrate reagents A (sulfanilic acid) and B (N,N-dimethyl-1- naphthylamine) are added after growth to detect the presence of nitrite.
* Nitrate reagent C (zinc) is added after A and B to detect if nitrate was reduced beyond nitrite (e.g. ammonia or nitrogen gas).
* A bubble in the Durham tube indicates the reduction of nitrate to nitrogen gas, while no bubble indicates no nitrogen gas production.
* After addition of Nitrate reagents A and B, a red color indicates the reduction of nitrate to nitrite, while no color change indicates no nitrite production.
- After addition of Nitrate reagent C, a red color indicates that nitrate was not reduced, while no color change indicates that nitrate was reduced beyond nitrite
Starch Hydrolysis Test
Tests for the presence of alpha-amylase (hydrolase) that
breaks down the amylose and amylopectin components
of starch; can starch be broken down and used as an energy source
Starch plate, opaque/white in color
Split plate in half, innoculate a line on each side
Put gram’s iodine on plate; stain creates a halo
Agar plates containing starch are inoculated. After
growth, iodine is added to the top of the agar. Iodine
reacts with intact starch but not hydrolyzed starch.
+ : halo surrounding the bacterial growth
- : absence of a halo surrounding the bacterial growth
microbiota [or flora]
The collection of microbes associated with the human body
Most of these microbes are harmless
While opportunistic pathogens may be present, they are
held in-check by other members of the microbial community
As symbionts, a healthy microbiota confers many benefits to humans: these microbes help to digest food, produce vitamins, and provide a physical and chemical barrier to pathogen invasion.
dysbiosis
an imbalance in the microbiota – leading to disease
Perturbations
Antimicrobial drugs or abnormal diets
Cause dysbiosis
It has been estimated that the human body contains more microbial cells than _____ cells (and
severalfold more microbial genes than human genes).
human
Microbes are found throughout the human body including the nose, mouth, throat, skin, large intestine, urethra, and vagina.
The conditions (temperature, pH, moisture) at each site influence the type of microbes that reside there
The nose and throat are inhabited by many bacterial species including those in the genera…
Staphylococcus, Streptococcus, and Corynebacterium
The intestines are colonized by various Gram negative bacteria, called enterics, such as…
Escherichia coli, Klebsiella spp, and Salmonella spp
Selective media
contains components that inhibit particular types of bacteria
Differential media
contains components that change the appearance of media or growth depending on the type of bacteria
MacConkey agar
Both selective and differential
Commonly used for the isolation of enteric bacteria.
Crystal violet and bile salts inhibit the growth of Gram positive bacteria.
The media contains lactose and neutral red (pH indicator) that enables differentiation of bacteria based on lactose fermentation.
Lactose fermenters (such as coliforms) break down lactose to acidic products that react with the pH Indicator turning the colonies pink-red
Mannitol Salt Agar (MSA)
Both selective and differential.
A high concentration of salt (NaCl) selects for Gram positive bacteria.
The presence of mannitol and a pH indicator enables differentiation between S. aureus and other Staphylococcus species.
S. aureus, but not other Staphylococcus species, ferments mannitol producing acidic products that react with a pH indicator turning the agar yellow
Bile Esculin Agar (BEA)
Both selective and differential.
Bile esculin agar is used to differentiate between group D streptococci and other streptococci.
Bile salts inhibit the growth of most Gram positive bacteria.
Group D streptococci (such as Enterococcus
spp) can hydrolyze esculin (a glucoside produced by several plants) in the presence of bile salts.
The products of esculin hydrolysis react with ferric ions in the media to produce a black color
Blood agar
Used to differentiate between species of Streptococcus based on pattern of hemolysis.
For alpha hemolysis, the agar turns green/dark due to hemoglobin reacting with hydrogen peroxide produced by the bacteria.
For beta-hemolysis, the agar surrounding the growth becomes clear due to the lysis of red blood cells by the enzyme streptolysin.
For gamma hemolysis, there is no change in the appearance in the agar surrounding the growth – indicating no lysis occurred
Antibiotics are commonly classified based on three characteristics:
their effect, their spectrum, and their mechanism of action
Antibiotic Effect
Two categories:
They can kill the bacteria directly –> bactericidal
They can inhibit growth rather than killing the already existing cells –> bacteriostatic
Antibiotic Spectrum
The range of bacteria against which the antibiotic is effective.
Broad spectrum antibiotics: are effective against many bacterial species, often both Gram negative and Gram positive bacteria.
Narrow spectrum antibiotics: are only effective against very specific bacteria.
Ex. ethambutol; only acts on Mycobacteria species (e.g. tuberculosis) as it inhibits integration of mycolic acid into the cell wall (mycolic acid is only a component of the cell wall in Mycobacteria).
Antibiotic Mechanism of Action
The molecular explanation for its effect.
Some antibiotics inhibit cell wall synthesis. Others inhibit translation, preventing protein synthesis. Others inhibit DNA replication. Some inhibit transcription, interrupting RNA synthesis.
Kirby-Bauer test
Uses paper disks containing different antibiotics to test antibiotic susceptibility. Sensitivity to the antibiotic is
determined by measuring the ZOI around the disk where bacteria did not grow after an overnight incubation.
Mueller-Hinton agar is used for this because its low thymine-thymidine concentration prevents inhibition of some antibiotics, and it contains starch to absorb
toxins released by bacteria that inhibit some antibiotics.
Large ZOI
The antibiotic is more effective against bacteria
Bacteria is sensitive to the antibiotic
Small ZOI
The antibiotic is not effect against bacteria
Bacteria is resistant to the antibiotic
