BIO205 LAB
Following the transformation experiment, you spread cells onto brain-heart infusion agar with streptomycin added. After incubating the "D" plate and the "C" plate, what could it mean if colonies grew on your C (control) plate? Select all choices that are plausible.
- A. calcoaceticus StrepR cells, or their DNA, were accidentally added to the A calcoaceticus StrepS cells in the control tube. - The A. calcoaceticus StrepS cells in the control tube were transformed
What flagella arrangement is characterized as multiple flagella extending from random points all over the cell?
- Peritrichous - having or being flagella which are uniformly distributed over the body Lophotrichous - having a tuft of flagella at one end. Amphitrichous - Having a single flagellum on each of two opposite ends. (forward and reverse) Monotrichous - having a single flagellum at one pole.
Place the steps of this exercise into the correct order.
1 - Prepare the PCR reaction mixture and add to the PCR tubes. 2 - Add the water to the PCR reaction. 3 - Mix cells from your EI colony into sterile water. 4 - Run the PCR reaction in the thermal cycler.
Place the main steps for the lab in the correct order.
1- Extract RNA from "saliva sample" 2- Use reverse transcription to convert viral RNA to complementary DNA. 3- Use PCR with primers specific to viral coat protein gene to amplify viral genetic material. 4- Determine presence of band of amplified DNA using agarose gel electrophoresis.
RNA extraction ordering: Place the steps of RNA extraction from the sample in the correct order. Figure 1: Left - Silica column in collection tube with cap open. The collection tube is only for waste collection of flow through. Right - Silica column without collection tube. The white silica pad is visible at the bottom of the column chamber. Never place the silica column in the centrifuge by itself. It must always be inserted in a receptacle to catch flow-through liquid.
1- Mix sample with 500 μl RNA lysis and binding buffer, add combined volume to column chamber on top of silica pad. Centrifuge. 2- After discarding collection tube and waste liquid, place the blue column into a new collection tube. 3- Add 500 μl Wash Buffer 1 and centrifuge. Pour out waste flow through from colleciton tube. 4- Add 500 μl Wash Buffer 2 and centrifuge. Transfer blue column to new 1.5 ml microcentrifuge tube. 5- Add 40 μl RNAse-free water and centrifuge. Save the flow-though liquid in the 1.5 ml microcentrifuge tube on ice.
When preparing the pour plates, how much volume will be added from the 10-5 dilution to the empty 10-5 pour plate?
1.0 mL
Which of these is the correct way to express 125 x 10^4 in scientific notation?
1.25 x 10^6
Using the below figure, determine the burst size of this bacteriophage. Use the lines drawn on the chart for guidance.
60 bacteriphages
What is considered TNTC (too numerous to count)?
>300
After 48 hours of growth, what indicates a positive result for casein hydrolysis?
A clear area around the bacteria.
For preparing pour plates, which of these would be the last step performed?
Add the molten nutrient agar to each plate and gently swirl the plates.
Match each term with the best definition.
Adsoprtion - The attachment of the bacteriophage to the host cell. Replication - The host cell makes new bacteriophage DNA. Assembly - The host cell produces new bacteriophage. Lysis - The host cell bursts and releases mature bacteriophage.
What are the 4 steps of the lytic bacteriophage life cycle, in correct order?
Adsorption, Replication, Assembly, Lysis
In this exercise, which type of plate will you start with for the pour plate analysis?
An empty plate
What is the definition of an operon?
An operon is a unit of DNA that is transcribed to produce a single mRNA molecule.
Which type of bacterium would be able to obtain carbon from inorganic sources such as carbon dioxide? Choose the best option from the choices below.
Autotrophic bacterium
What will happen to the number of T4 phage particles present in a sample after all of the Escherichia coli cells have been lysed?
Bacteriophage cannot reproduce without host cells, so the number of T4 phage particles will remain the same
What is an example of an obligate intracellular parasite? (virus)
Bacteriophages
What is the units for titer?
CFU
In the enumerating bacteria exercise you used _____ units. In the bacteriophage exercise you used ____ units.
CFU/g ; PFU/ml
Which of the following is not used in a gram stain:
Carbol fuchsin
Match the following steps of PCR with the process that occurs during that step. Denaturation Annealing Extension
Denaturation- DNA unwinds and the two strands of DNA separate. Annealing- Primers attach to the template DNA. Extension- DNA Polymerase adds nucleotides to make a complimentary strand of DNA.
What is a primary purpose of the reagent guanidinium isothiocyanate in the "RNA Lysis and Binding Buffer"?
Disrupts or denatures proteins
Which bacteria would be expected to have the genes of the lac operon present?
Escherichia coli
Which type of bacteria must import a majority of the necessary components for larger molecules from their nutritional sources in a prefabricated state?
Fastidious bacteria
What is a direct method of counting cells?
Hemocytometer with a microscope
Match each technique with the category of bacterial enumeration that it falls under.
Indirect enumeration: - Measurements of dry (or wet) weight (or volume) - metabolic tests - spectrophotometer - viable plate count assay Direct enumeration: - coulter counter - hemocytometer
Why is it important that Taq DNA polymerase is heat-stable?
It allows the enzyme to withstand high temperatures in the PCR reaction.
What is the purpose of the reverse transcriptase enzyme?
It is used to convert the viral gene from RNA into a complementary DNA form.
What is the function of the lac permease protein, which is the product of the lacY gene?
It transports lactose into the cell.
When bacteriophages replicate, they undergo four main steps. In which step does the "rise period" occur?
Lysis
What are the three key factors in obtaining the best image while viewing organisms through the microscope?
Magnification, resolution, and contrast.
What are bacteria that require oxygen to survive and grow called?
Obligate aerobes
In which tube are the A. calcoaceticus StrepS cells and the lysed A calcoaceticus StrepR solution combined?
Only in the D tube
Determine the correct order of these steps in the Gram stain. You may omit rinsing steps, assume they are to be done after each stain.
Prepare dry mount of cells. Heat fix the slide. Apply crystal violet for 2 minutes. Apply Gram's iodine to the slide for 2 minutes. Apply 5 drops of 95% ethanol. Apply safranin to the slide for 5-7 minutes.
What are the main factors to obtain a good image used in microscopy? Select all correct answers.
Resolution Magnification Contrast
How many plates will be created to measure the growth cycle of the bacteriophage?
Seven
Which type of staining method uses only a single stain?
Simple stain
Label the four stages in the bacteriophage one-step growth curve below.
Stage 1 - Adsorption Stage 2 - latent period Stage 3 - rise Stage 4 - plateau
What virus will we be looking at ?
T4 Bacteriophage
Which of the tubes has the bacteriophage in it to begin with?
The ADS (adsorption) tube
In the bacteriophage exercise...
The T4 bacteriophage infects Escherichia coli strain B
What is the definition of competence as it relates to bacterial transformation?
The ability of a bacterial cell to pick up DNA from the environment
What is the purpose of a serial dilution in a viable plate assay? Select all correct answers.
The amount of bacteria in the original sample is unknown, and at least one plate needs to have a countable number of colonies The serial dilution allows us to calculate the amount of bacteria in the original sample
What is the definition of burst size?
The average number of phage particles released per infected bacterial cell
How did you know if the broth from your aseptic technique quiz was contaminated?
The broth was turbid
Why is the small subunit of ribosomal DNA (rDNA) a good gene to analyze for molecular identification of unknowns?
The gene is ubiquitous among organisms. It has conserved regions for primers to bind. It has variable regions that change over evolutionary time. All of the above
In a negative gelatinase test, what happens to the liquefied media when it is placed on ice?
The media will resolidify
Which component in the PCR reaction sets the specific starting point for DNA synthesis to occur?
The primers
Why is it important to wear gloves when preparing the PCR reaction?
To avoid introducing unwanted bacteria.
What is the purpose of re-streaking your environmental unknown?
To isolate a single species of bacteria and to keep it alive
Why perform serial dilutions?
To produce at least one plate that you are able to count
Match each acronym with its meaning.
Too Numerous To Count - TNTC Colony Forming Units - CFU Not Statistically Significant - NSS
What is conjugation?
Transfer of DNA between two cells that are in physical contact with each other
What needs to occur for a successful PCR? Select all correct answers.
annealing, extension, denaturation
A bacterial species that is not naturally competent cannot have its genome altered.
false
Escherichia coli is a naturally competent bacterial species.
false
For identifying an unknown bacterium, cell morphology, Gram reaction, and colony morphology are usually adequate measurements.
false
What are two attributes that can be revealed with a simple stain?
morphology and arrangement of cells.
Is E. coli naturally competent?
no
There are two broad classes of staining techniques: staining techniques use one dye, while staining techniques use two or more dyes.
simple; differential
To make a ____ plate, a diluted sample is added to the surface of a pre-made agar plate. Bacteria grow on the surface of the agar. To make a _____ plate, a diluted sample is mixed with molten agar in an empty Petri plate. Bacteria grow on the surface of the agar plate and grow within the agar.
spread; pour
At the 15 minute time stamp, in which tube do you mix the 0.1 mL from the row 2 tube and the E. coli B immediately before pouring on the agar plate?
the soft agar tube
A viable plate count assay is an example of an indirect counting method for enumerating bacteria.
true
Acinetobacter calcoaceticus is a naturally competent bacterium.
true
During the transformation lab, the DNA is extracted from the streptomycin resistant cells and the streptomycin sensitive cells are spread on the BHI-Strep agar plates.
true
Horizontal gene transfer is the exchange of genetic information between cells of the same generation.
true
You begin time 0 for the experiment (record the start time) when you add the 2.9 mL E. coli to the ADS tube.
true
When creating your PCR reaction mixture, how many reactions worth are to be created at once?
two
What will happen if you get liquid from Wash Buffer 2 on the bottom of your silica column and do not remove it?
your reaction will likely fail.
match the terms
- Transduction: The transfer of DNA between bacterial cells that requires a bacteriophage. - Transformation: The uptake of naked DNA from the environment. - Transfection: The uptake of bacteriophage DNA from the environment. - Conjugation: The transfer of DNA between two bacterial cells that requires cell-to-cell contact.
Which of these is a mechanism that can facilitate horizontal gene transfer among prokaryotes?
- all of the above: Plasmids Transposons Bacteriophage
There are 229 colonies on the 10-5 dilution plate. This is equivalent to...
2.29 X 107 CFU/g
Which of these is the correct way to express 235 x 10^3 in scientific notation?
2.35 x 10^5
If you counted 289 colonies on the 10^-4 dilution plate, this is equivalent to...
2.89 x 10^6 CFU/g
At what time point do you first plate a sample from the DIL tube onto a plate with E. coli B and soft agar?
25 minutes*
What is the acceptable range for counting colonies on plates in the bacterial enumeration exercise?
30 to 300 colonies per plate
In the United States, about how many people die each year due to antibiotic resistant infections?
35,000
You have counted 42 colonies on a 10-6 plate. Which of these correctly states the bacterial titer of this sample?
4.2 x 107 CFU/g
For the image below of an agarose gel run with PCR reaction samples, answer the two questions. Note that the ladder is a 100 base-pair ladder as was used in our lab exercises. The ladder is in the first well on the left. Ladder bands are a series of DNA fragments in 100 bp increments from 100 - 1000 base pairs. The smallest band size is 100 base pairs. A: Which lanes were positive for the PCR reaction? Give all the lanes that had a positive result based on the lane numbers above each well. B. What is the size of the band for the positive samples?
A) Lanes 1, 2, 3, 4, and 6 were positive for the PCR reaction. B) The size of the band for the positive samples is about 500 base pairs
Cilantro sample A had 102 colonies on the 10^-5 plate. Cilantro sample B had 31 colonies on the 10^-6 plate. A.) Give the titer for each sample (separately). Remember to include units. B.) Which cilantro sample has a greater concentration of bacteria?
A) Sample A had 1.02x10^7 PFU and sample B had 3.1x10^7 PFU. B) Sample B has a greater concentration of bacteria.
Indicate if the following actions are examples of aseptic technique or if they are not examples of aseptic technique. A. This is an example of aseptic technique. B. This is NOT an example of aseptic technique.
A- Changing the pipette tip if it touches the benchtop B- Opening serological pipettes at the tip side A- Passing open test tubes through the flame B - Placing caps and lids on the benchtop B - Transferring different liquids with the same pipette tip A - Working with multiple test tubes to limit the amount of time each tube is opened and exposed
What is the main difference(s) between spread plates and pour plates
Both the first and second answers: - Spread plates have bacteria only on top - Pour plates have bacteria throughout the agar and on top
When diluting 0.5 mL of any sample into 4.5 mL of diluent (for a total of 5 mL), by how much have we just diluted the sample?
By a tenth (10^-1)
In the transformation experiment of Frederick Griffith, what was the virulence factor that was contributed from the killed, pathogenic Streptococcus pneumoniae that resulted in lethal infection among the test mice?
Capsule formation
What is the definition of a direct method for enumerating bacteria?
Counting individual bacterial cells
______________ ______________ is the negative control organism for the Gram stain.
Escherichia coli
What is the definition of an indirect method for enumerating bacteria?
Estimating the number of bacteria based on metabolic tests or other growth characteristics
Complete the correct steps of the Gram stain by using the words below. Ensure that you type them correctly. 95% ethanol, crystal violet, dry mount, Gram's iodine, heat fix, safranin
Prepare a of your dry mount cells and heat fix the slide. Cover the cells with crystal violet for 2 minutes. Rinse the slide with water. Cover the cells with Gram's iodine for 2 minutes. Shake off or rinse this reagent. Apply several drops of95% ehtanol and rinse immediately. Cover the cells with safranin for 5 minutes. Rinse, blot dry and view the slide.
Which of these attaches to the DNA during the annealing stage of PCR?
Primers
In this exercise, we will be transferring 1 mL from the dilution tube to the spread plate.
false
It is not that important to label the various carbohydrate fermentation test tubes because each one has a unique color.
false
Observations of colony morphology, cell morphology, and Gram stain reaction will provide enough data to identify the genus and species of your unknown environmental isolate.
false
When phenol red turns yellow, this indicates a negative carbohydrate fermentation test result.
false
Brightfield microscopy is used to view live, unstained organisms.
false; live and fixed (stained)
A bacterial cell that divides and produces two daughter cells gives its genetic material to both daughter cells. This is an example of horizontal gene transfer.
false; vertical
Below is an image of stained bacteria on a slide. In the space provided, give: (a) the name of the stain method, (b) the full scientific names of organism(s) showing a positive reaction, (c) the full scientific names of organism(s) showing a negative reaction if any (if no negative organisms state "none"), (d) the name of the primary stain, (e) the name of the counter stain, if any (or enter "none"). Base your answer on the organisms and stains used in OUR lab activities at NAU. Organism names must be spelled and capitalized correctly, no abbreviations can be used. Do not worry about text formatting, assume organism names are italicized/underlined.
image: purple rods, purple spheres, pink rods - The stain method used is the gram stain method, the full scientific name of the organism showing a positive reaction is Streptococcus and negative is Escherichia coli, the name of the primary stain is crystal violet, and the counter stain is safranin.
