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Selecting a single colony from a streak plate for further growth in a broth or plate
Add DNA to competent cells on ice, flick to mix DNA and bacteria
Ice bath for 30 minutes
Place E. coli w/ DNA into 42 degree water bath for 30 seconds
Ice bath for additional 2 minutes
Add warm LB broth to competent bacteria/DNA soln
Transfer x-gal to agar, spread with glass rod, let dry
2 drops, 10 drops, control (contains no ampicillin - 10 drops)
Let plates briefly air dry and then incubate at 35 C overnight
Testing bacterial susceptibility to antibiotics/antiseptics/disinfectants
You have a streak plate of a mixed culture. What are some features of the bacteria you might use to tell the two colonies apart?
Elevation (flat, convex, umbonate), Shape (circular, irregular, rhizoid), smooth/granular, shiny/dull, adheres to media, sticky/viscous, opaque/translucent, color
What is a bacteriophage? How does a bacteriophage work?
Bacteriophage is a virus that infects bacteria – consists of a nucleocapsid containing genetic material. Bacteriophages are obligate intracellular parasites and use host machinery to reproduce, make viral proteins and genetic material within the cell.
Review the procedure for the bacteriophage lab- why do you start off with a lawn of bacteria?
Originally grown as a lawn so the plaques can be clearly seen. The introduced phage particles cause the host cell to lyse and the phage particles released after lysis infect neighboring cells. These cells are then lysed and the process continues.
How will you quantify the amount of phage present in an unknown stock?
(#PFU/vol of plate) x (1/dilution of tube) = PFU/mL Note: vol of plate is usually 1 mL
Phages are described in terms of plaque forming units per milliliter. What does this unit mean? (i.e. if I hand you a vial of phage and tell you that the concentration is 4.6x107 plaque forming units (PFU) per mL, what does that MEAN?)
Under the assumption that 1 phage creates a single plaque and that a plaque is created by a single phage. Therefore, it is synonymous to the number of phage/mL.
What is serial dilution and what is the benefit of the technique?
Series of stepwise dilutions. A serial dilution is constant so it progresses in a geometric fashion, allowing concentrations to be predicted/extrapolated off of graphs
You want to figure out a bacteriophage’s plaque forming units, and so you look at the plates you prepared. The 10-1 plate has 438 colonies, the 10-2 plate has 46 colonies, and the 10-3 plate has 3 colonies. Which plate would you choose to count for your calculation and why?
46 – use the plate that has the highest number of countable plaques
What is the tool called that you use to dispense small quantities of liquid?
What is horizontal gene transfer? How does it differ from vertical gene transfer? Is conjugation horizontal or vertical? Is transformation horizontal or vertical?
In horizontal gene transfer, cell acquires DNA from a different source (plasmid, naked DNA). In vertical gene transfer, a mutation arises in a single cell and the new variation is passed to offspring. Both conjugation and transformation are horizontal.
What is transformation?
The uptake of single stranded “naked” DNA by competent bacteria via a receptor on their cell surface
What is “naked” DNA? Does transformation involve uptake of naked DNA? Does conjugation involve uptake of naked DNA?
What does the term “episomal plasmid” mean?
The plasmid remains in the cytoplasm and is not homologously incorporated into the bacteria’s DNA. Still results in a recombinant organism
DNA taken up by bacteria during transformation can exist in what locations?
Cytoplasm or it can incorporate itself the bacterial chromosome
New genetic material is incorporated into the bacterial chromosome, can be homologous or nonhomologous
What is a competent cell?
Bacteria that are naturally able to take up and incorporate foreign DNA via transformation (most E. coli are not naturally competent), can be made chemically competent through chemical or physical treatment
Why do we subject our competent cells to drastic changes in temperature during transformation?
Creates a temperature gradient and allows the exogenous DNA to be transported through the cell envelope
The plasmid we used in lab for transformation has four critical components. What are they?
Origin of replication (pUC ori), multicloning site (MCS), selectable marker (Ampr – encodes for enzyme that inactivates penicillin), second marker (lacZ gene)
What is an origin of replication on a plasmid? Why does a plasmid need an origin of replication?
Site where replication begins – plasmids must be able to replicate themselves independent of chromosomes (pUC ori)
What is a multi-cloning site on a plasmid? Why is this important?
Contains recognition sequences of many different restriction enzymes. This allows the plasmid to be cut by several different restriction enzymes depending on where they need to be inserted into the bacterial DNA – contained within the genetic sequence of lacZ gene. When cut, it cannot make a functional lacZ protein – cannot generate blue color
What is a selectable marker on a plasmid? Why is it important to include a selectable marker?
Gene whose product allows cells to grow in conditions that would otherwise be inhibitory/lethal. (Amp^r – encodes for enzymes that inactivate penicillin). Can differentiate which cells are able to take up vector.
What is a “second marker” on a plasmid? Why is it beneficial to include a second marker?
LacZ gene - Used to distinguish cells that contain recombinant plasmids from those containing an intact vector (insertional activation). If no gene is inserted, a functional lacZ protein will be made that cleaves X-gal to turn X-gal blue. If a gene is inserted, a functional lacZ protein cannot be made and it cannot cleave X-gal, so the colonies are white.
pBluescript SK+ (no gene), second vector with gene inserted into MCS
Why do we let the plates air dry before flipping them over for incubation?
A (relatively) large volume of bacteria was put on the plates. It is important to ensure that all bacteria/broth is absorbed by the LBA or LBA-Amp plates so that maximum amount of bacteria can grow. If there's a lot of liquid left on top, it will just drip onto the lid once we flip the plate over for incubation. (also the reason why plates are flipped over: so that condensation from bacterial respiration does not drop down on the colonies and drown them)
Why is the plasmid designed so that the multi-cloning site is within the lacZ gene?
This way, the gene of interest will be inserted in the middle of the lacZ gene. If the lacZ gene is cut, it cannot produce functional proteins, which can cleave x-gal and change the color of the colony to blue
What is X-gal? Why do we use it in transformation?
Colorless chemical compound, turns blue when cleaved by lacZ. It is useful in transformation because it can differentiate between the cells that have taken up the gene of interest and those that haven’t.
If you transform bacteria using a plasmid with the LacZ second marker included and put Xgal on your plate, what does it mean if the colony that grows turns white? What does it mean if the colony turns blue?
White: contains gene of interest
Blue: intact vector, lacZ protein was formed which cleaves x-gal, turning it blue
You forgot to put Xgal on your transformation plates. What color do you expect the colonies to be?
Xgal helps colonies that have insertional inactivation of the lacZ gene look white, and those that have an intact lacZ look blue. If you did not use xgal on your plate, then, all colonies (lacZ intact and disrupted) will look white, so you will be unable to perform the blue-white screen.
What does insertional inactivation mean in the context of transformation?
When a gene is inserted into the secondary marker, the formation of the lacZ protein is inactivated
Why do we incubate the transformation plates overnight at 35° C?
Body temperature? Mesophils? Optimal growth temperature
You overhear your lab partner say that he has no bacteria on his skin because he just washed his hands. Do you agree with that statement?
No – we have normal, even beneficial bacterial flora on our skin
Benefits of normal flora: protective functions, metabolic functions, structural functions
Why did we use use both Sabouraud and blood agar plates for the handwashing experiment?
Sabouraud plates grow fungi, SBA plates grow bacteria. The Sabouraud plates help us see fungi that may have colonized/come for a ride on our hands.
What is the benefit of using differential/selective media?
Improves the likelihood of isolating the suspect organism from the “background clutter” of normal flora
Why do we use the mannitol salt plate agar plate in the nasal swab experiment?
Differential (pH effects – see below) and selective (high salt concentrations – 7.5% NaCl, only halotolerant bacteria can grow), used to select for Staphylococcus, plate contains phenol red
Why do some colonies of bacteria cause the media of a mannitol salt plate to turn yellow while others don’t?
Staph strains ferment mannitol, lowering the pH, which turns the phenol red indicator yellow
In one sentence, what is conjugation?
Genetic material from a donor bacterial cell is transferred to a recipient cell via a sex pilus, increases the diversity in a population which becomes the basis for selection
Strain I = resistance to streptomycin (on chromosome), Strain II = resistance to ampicillin (on plasmid)
What is the fertility factor gene?
“F-factor” – codes for the production of a sex pilus, transferred during conjugation
Where can the F factor exist in a bacterial cell?
Can exist in a plasmid outside of the chromosome or it may be integrated into the chromosome itself (HFr cell – high frequency recombination). HFr cells can only transfer part of chromosome because the origin of transfer is in the middle of the F gene
What is a pilus? What determines if a bacterium can form a pilus?
A pilus is a protein tube required during conjugation that is the mechanism of transfer of genetic information between donor and recipient cells. A pilus is coded for by the F factor gene (F+ or F-)
Can conjugation occur between two different species of bacteria?
What are the proximity requirements for conjugation to occur between two bacterial cells? Why is this?
Direct cell to cell contact – via a bridge like protein appendange
How can you make use of selective pressure to identify bacteria that have undergone conjugation?
Grow on mediums containing different antibiotics to see if bacterial growth has occurred, indicating the transfer of the plasmid that conferred resistance to the given antibiotic
Discuss the issues that conjugation presents with regards to antibiotic resistance and people not taking their full course of prescribed antibiotics.
If people don’t take their full course of antibiotics, the bacteria that are resistant will be selected for and they can spread the resistance gene to other bacteria through conjugation
Describe the cellular events that occur during conjugation
During conjugation, does the donor cell retain a copy of the F factor?
During conjugation, does the recipient cell receive a copy of the F factor? What might influence whether or not a recipient cell receives a copy of the F factor?
The recipient cell receives a copy of the F factor if a plasmid is transferred, not if it is an HFr cell because the entire chromosome is not transferred
Describe how conjugation might be used for chromosome mapping.
Since the chromosomal genes closest to the origin of transfer are transferred first, the location of genes on the chromosome may be mapped, based on the timed appearance of specific genetic components
What is a high frequency recombinant?
High frequency recombinant – the F plasmid is integrated into the bacterial chromosome – rarely transfers all of F factor
In the conjugation we performed in class, where would we see evidence of conjugation? Why do we not see conjugation anywhere but here?
Conjugation was seen on the amp/strep plate in the quadrant containing both strain 1 and strain 2. They transferred their individual antibiotic resistance genes to each other, allowing each type to grow. This is the only quadrant in which the bacteria were cultured together.
What is the difference between a disinfectant and an antiseptic? Which would you rather use on your hands?
A disinfectant is used on inanimate surfaces, an antiseptic is used for tissues. Use antiseptics on hands.
What is the difference between a bacteriostatic agent and a bacteriocidal agent?
A bacteriostatic agent inhibits the growth of microorganisms but do not kill them. When a bacteriostatic agent is removed, the microorganism can resume its growth. Bacteriocidal agents kill bacteria. Often, can be either type depending on concentration.
What factors influence the effectiveness of a chemical agent against microorganisms?
Type of microbial population (log phase most susceptible, presence of capsid – resistance factors, spores are very resistant), Environmental conditions (increasing temp increases rate of chem reactions of disinfectants, extreme pH is harmful to bacteria, blood/pus/tissue may hamper ability of bacteria to react with bacteria – cleaning increases effectiveness)
Are all microorganisms equally sensitive to all antibiotics?
NO (resistant, sensitive, intermediately sensitive)
What is an example of an antiseptic? What is an example of a disinfectant?
Antiseptic: Purell, Disinfectant: Lysol
Why don’t we place the paper discs in the bacterial susceptibility experiment right against the edge of the plate?
It could limit the size of the zone of inhibition, which we use to calculate the degree of susceptibility of the organism
Why might it be beneficial for a microorganism to produce an antibiotic?
Inhibits the growth of other microorganisms
What is the Kirby-Bauer technique used for?
The technique measures the sensitivity of a test microorganism to a wide range of antibiotics. It is useful to physicians so they choose the right antibiotic for the treatment of a patient from whom the specimen was tested, allows results from different hospitals to be compared
What clinical decisions can you reach after performing a Kirby-Bauer test with an organism isolated from a patient? How would you reach this decision?
Physicians can choose the right antibiotic treatment of a patient from whom the specimen was tested, based on the degree of sensitivity of the isolated bacteria to various antibiotics
Mueller-Hinton agar is the universal standard for performing clinical Kirby-Bauer tests. Why is it useful for everyone to use Mueller-Hinton agar instead of whatever plates are lying around?
Standardized, allows results to be compared across different labs/hospitals
What does the term “zone of inhibition” refer to?
Zones free of bacterial growth, measured in mm across the diameter and compared to a standardized table.
What does the size of the zone of inhibition tell you for a particular organism and antibiotic?
The size of the zone of inhibition is directly related to the degree of susceptibility of the organism.
How do you evaluate an organism’s susceptibility to an antibiotic? (Name of test is not sufficient, please describe in more detail.)
Kirby-Bauer technique, which measures the sensitivity of a test microorganism to a wide range of antibiotics. Filter paper discs impregnated with antibiotics are placed on the surface of a Mueller-Hinton agar plate that has been swabbed with the test organism
What are two factors influence the size of the zone of inhibition in a Kirby Bauer test?
Other factors also influence the degree of susceptibility: size of the inoculum, incubation time, temperature, diffusion rate of antibiotic, growth rate of organism
Antibiotic Y – it is more selective and will cause less harm to beneficial bacteria. Additionally, it won’t promote broad antibiotic resistance
An agglutination reaction is the creation of antigen-antibody complexes as a result of the invasion of particulate antigens and the body’s response by creating antibodies to counter the antigens
What is a selective medium? What are the selective media used in class?
What is a differential medium? What are the differential media used in class?
What is a pH indicator?
What are some examples of pH indicators that we have used in lab?
What color changes do pH indicators display? Under what conditions?
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