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Identification of an unknown quiz
You will be using information and techniques you have learned to identify an unknown.
In the real
world, identification of a bacteria requires a number of biochemical tests and media (or genetic
methods).
But since we are going to narrow it down to a few bacteria that differ in at least one
characteristic, we will be able to do this.
This quiz is worth 50 points, so be very thorough in your
answers.
You will need the Table of Bacteria to complete this assignment.
1.
You have been provided with a broth culture containing an unknown (the unknown will come
from the table).
The instructor tells you that the broth culture is pure, but since so much of your
grade is riding on this, you want to make sure.
What would be the first thing you do to obtain a
pure culture, and why?
(4 points)
To obtain a pure culture of bacteria I would spread the bacteria on the surface of a solid medium so
that
a single cell occupies an isolated portion of the agar surface. Than I would inoculate the
culture to transfer the culture to another medium while using the aseptic technique. After this, I
would spread the bacterium on an agar medium using the quadrant streak method to obtain
isolated colonies. Using the aseptic technique for the retrieval and placement of the suspected
pure culture helps to ensure that no other microorganisms compromise the integrity of the original
broth culture and the later placed agar medium. Using the quadrant streak method to obtain
isolated colonies for this suspected pure culture is to identify the bacterium, spread it across the
quadrants to purify it by diluting the strain. This single cell will go through repeated multiplication
to produce a visible colony of similar cells, or clones
2.
Below is an image of your unknown growing on TSA. (5 points)
a.
Describe the growth of your unknown on TSA, using information you learned in Lab 4.
Make
sure to provide enough information to help you determine what unknown you have.
I believe
this growth of this unknown has isolated coloniesthe bacterium pigment is white/beige, has round
shape, has entire margins, and opacity is opaque, the colonies also seem smooth.
3.
Below are your Gram stain results. (8 points)
a.
What does this result tell you (consider gram reaction, shape, morphology)?
This result
of the gram stain tells me the bacterium is gram negative the cell shape and morphology
are
bacillus single and the cell arrangement is irregular opaque and rough
b.
Using your Table of Bacteria, which bacteria can you exclude?
Explain your answer, and
list your evidence. (Please provide the specific names of the bacteria you can cross off
your list.) I can exclude staphylococcus aureus, micrococcus luteus due to this being
gram negative.
3.
You determine the temperature requirements of your unknown, and get the following results.
‘-‘ means no growth; ‘+/-‘ means there may be slight growth; ‘+’ means slight amount of growth; ‘++’
means moderate amount of growth; and ‘+++’ means heavy growth.
(9 points)
Bacteria
4C
refrigerator
10
C
25
C
37
C
55
C
Unknow
n
-
-
-
-
+++
a.
Is this bacteria a psychrophile, psychrotroph, mesophile, or thermophile?
How do you know?
This bacteria is a thermophile because it thrives in higher temperatures and has more growth in higher
temperatures.
b.
Based on your results, what is the optimum temperature of your bacteria? 45-80 degree
celcius
c.
Based on this answer, are there any other bacteria you can exclude?
Why, or why not? I can
exclude all in the table except bacillus stearothermophilus due to the there only being one thermophile
listed.
4.
You determine the oxygen requirements of your unknown bacteria and receive the following results.
(12 points)
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Related Questions
TRY TO KEEP IN SHORT AND USE OWN WORD FOR THIS QUESTION
You are studying a type of bacteria isolated from the acidic water runoff of a mining operation. You subject two batches of the same bacteria type to different environmental growth conditions. One batch is grown at pH 2, while the other is grown at pH 7. All other environmental parameters are kept identical between the two batches. You then collect their proteins and run a Western blot using an antibody that binds to a proton efflux pump protein (which actively expends energy to pump protons out of a cell). How would you characterize the information obtained in this experiment? What does it tell you, and why is that potentially valuable information?
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DNA Extraction by Alkaline Lysis
Procedure:
1. Spin 1.5 ml of cells in a microcentrifuge at maximum speed (12,000 rpm) for 20s to
pellet. Remove the supernatant completely with a Pasteur pipet or a plastic pipettor tip.
The spins can be performed at FC or at room temperature. Longer spins make it difficult
to resuspend cells.
2 Resuspend pellet in 100pul GTE solution and let sit 5 min at room temperature. Be sure
cells are completely resuspended.
3. Add 200ul NaOH/SDS solution, mix by tapping tube with finger, and place on ice for 5
min.
4.
Add 150ul potassium acetate solution and vortex at maximum speed for 2s to mix.
Place on ice for 5-15 min. Be sure mixing is complete.
5. Spin 3 min at 12,000 rpm to pellet cell debris and chromosomal DNA
6. Transfer 0.4 ml supernatant to a fresh tube, mix it with 0.8 ml of 95% ethanol or 0.4
ml isopropanol, and let sit 2 min at room temperature precipitate nucleic acids.
7. Spin at 12,000rpm for 3 min at room temperature to pellet plasmid DNA and…
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Drag and drop the appropriate text in the gaps below.
Firstly, the plasmid DNA in the E. coli is propagated through
Then the bacterial cells are
pelleted by centrifugation at 10,000 RPM. The media supernatant is
removed and the bacterial cells are
The bacterial cells are then
lysed via
|. Contaminating
macromolecules such as protein and chromosomal DNA is removed by
Overnight incubation of the bacterial culture
washed in cell resuspension buffer
addition of lysis buffer
addition of neutralisation buffer
washing of the spin column
separation via agarose gel electrophoresis
arrow_forward
Pipetting
1. Explain why a micropipettor is an important instrument in biochemistry labs.
2. Describe the basic components (and function) of a micropipettor.
Bacterial Techniques
1. Define the following.
(a) Serial Dilution
(b) Streak Plating
(c) Spread Plating
Transformation
1. Define bacterial transformation and explain why it is an important method in
biochemistry labs.
2. Describe (figure, narrative) a plasmid and describe the basic components of a
plasmid.
3. What role does CaCl2 play in bacterial transformation?
4. What role does heat shock play in bacterial transformation?
Plasmid Isolation
1. Describe the roles the following play in plasmid purification.
(a) Lysis Buffer
(b) Neutralization Buffer
(c) Elution Buffer
arrow_forward
4. Look at the gel image and answer the questions below and be specific.
a) Based on your calculations of the DNA concentrations, how much DNA was
loaded into each well? Do you see DNA for each of your samples? If not, why do
you think that is so?
b) Is the DNA in a single sharp band, multiple bands or a smear? What would each
of these scenarios be due to, and why would you see them for your samples?
c) Do you see multiple bands in your plasmid DNA sample? What are they?
arrow_forward
You are given the following DNA fragment to sequence:5'-GCTTAGCATC-3'. You first clone the fragment in bacterialcells to produce sufficient DNA for sequencing. You isolate theDNA from the bacterial cells and carry out the dideoxy-sequencingmethod. You then separate the products of the polymerizationreactions by gel electrophoresis. Draw the bands that shouldappear on the gel from the four sequencing reactions.
arrow_forward
Complete the following tasks.
You discovered that a species of bacteria can break down StyrofoamT (polystyrene) products due to an
enzyme it produces, polystyrenase. You wish to study the gene that codes for this enzyme.
Task 1: DNA Extraction
To begin work on the bacterium, you begin by extracting its genomic DNA (GDNA). What is the purpose of
the following procedures? Answer briefly but completely.
Using sodium dodecyl sulfate, a detergent
Answer:
а.
b. Adding RNase A and Proteinase K during extraction
Answer:
c. Adding ethanol before recovering the DNA extract
С.
Answer:
Task 2: Polymerase Chain Reaction
After purifying the gDNA extract, you want to isolate and amplify the polystyrenase gene. You perform PCR
using the appropriate gene-targeted primers. What is the purpose of the following PCR components? Answer
briefly but completely.
DNA polymerase isolated from Thermus aquaticus
Answer:
а.
b. Deoxynucleotide triphosphates (DNTPS)
Answer:
С.
Forward and reverse primers
Answer:
Task…
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You are tasked with cultivating the following microorganisms:A recombinant strain of E. coliA wild strain of Streptomyces clavuligerus• Suggest a storage method for each microorganism and justify your choice. You have at your disposal a refrigerator and an ultrafreezer, in addition to materials and conditions for handling microorganisms.• Propose a culture medium for each (choose a complex medium for one organism and a synthetic medium for the other)As a complement of nutrients, they must be indicated on the following scale: +++ : above 5 g/L++ : from 1 to 5 g/L+ : below 1 g/L• Describe the procedure for preparing and sterilizing the components of the culture media for a volume of 5.0 L• Make a bench process selection scheme, up to the step of selecting a benchtop reactor 5 L.
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MULTIPLE CHOICE Read the questions below carefully. There is only one correct answer for each question. (no explanation only correct letter needed)
1. In a DNA sample of Escherichia coli (a bacteria), 23.6% of the nitrogenous bases are thymine. What percentage of cytosine nitrogen bases is present in this sample?
a) 76.4%
b) 23.6%
c)26.4%
d) 52.8%
2. If the adenine is located on the Z strand as shown (see attachement 3) , then on the W strand, in the same place, we should find:
a) Uracil
b) Adenine
c) Thymine
d)Cytosine
3. Which of these deletions best represents a chromosomal deletion? (see options in attachment 3)
4. It is known that RNA is a nucleic acid responsible for the synthesis of proteins. However, there is another nucleic acid alongside RNA: DNA. What role does the latter play in relation to RNA?
a) RNA is synthesized in the cell in case too large a mutation damages the DNA.
b) DNA has the reproductive genetic code of all cells and passes it on to RNA.
c) DNA directs the…
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Which of the following gels is the preferred choice for separation of DNA fragments?
Agarose
Polyacrylamide
SDS PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis)
Agar
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Could you please help me with the following questions? I am struggling tremendously. Thanks so much!
Why are cell membranes disrupted by soap?
Some organisms can live in hot springs. What does this imply about their DNA?
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explain how the following genotypic andphenotypic methods of microbe identification is done. Use diagrams or schematic representations to explain yourwork. (1) Restriction Fragment Length Polymorphism (RFLP)(2) Pulsed-Field Gel Electrophoresis (PFGE)(3) Whole Genome Sequencing (WGS)(4) Gram Staining(5) Biochemical Reactions: Multiple Tube Fermentation Technique(6) Biochemical Reactions: IMViC Test
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Transcribed Image Text:Complete the following tasks. You discovered that a species of bacteria can break down StyrofoamT (polystyrene) products due to an enzyme it produces, polystyrenase. You wish to study the gene that codes for this enzyme. Task 1: DNA Extraction To begin work on the bacterium, you begin by extracting its genomic DNA (GDNA). What is the purpose of the following procedures? Answer briefly but completely. Using sodium dodecyl sulfate, a detergent Answer: а. b. Adding RNase A and Proteinase K during extraction Answer: c. Adding ethanol before recovering the DNA extract С. Answer: Task 2: Polymerase Chain Reaction After purifying the gDNA extract, you want to isolate and amplify the polystyrenase gene. You perform PCR using the appropriate gene-targeted primers. What is the purpose of the following PCR components? Answer briefly but completely. DNA polymerase isolated from Thermus aquaticus Answer: а. b. Deoxynucleotide triphosphates (DNTPS) Answer: С. Forward and…
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You are provided with cultures of E. coli cells that contain the plasmid pUC118. Each student should carry out the plasmid isolation procedure. Note the appearance of the suspension after steps (v), (vi) and (vii).
The steps are
(v). Resuspend the bacterial pellet in 300 μl solution A [25 mM Tris-HCl (pH 8.0) 10 mM EDTA. Ensure that the suspension is homogeneous.
(vi). Add 300 μl solution B [1% (w/v) SDS 0.2 M NaOH] and mix thoroughly.
(vii). Add 300 μl solution C [3M potassium acetate, (pH 4. 3)], mix thoroughly, and incubate on ice for 5 minutes.
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PART C (cont'd)
Which of the following steps (on this page and the next page) would be necessary to
determine the most effective antibiotic for a bacterial infection using the Disk Diffusion
Assay? Select three necessary steps and three unnecessary or incorrect steps, and
explain why it is or is not necessarv/correct.
Collect a sample of bacteria from the infected patient.
Collect samples of bacteria from the bathroom taps in the patient's room.
Collect samples of bacteria from other patients who seem to have the same
infection.
Soak disks in many different types of antibiotic.
Soak disks in the antibiotic you have the biggest supply ot.
Label the disks with the type of antibiotic you soaked them in.
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PART C (cont'd)
Which of the following steps (on this page and the next page) would be necessary to
determine the most effective antibiotic for a bacterial infection using the Disk Diffusion
Assay? Select three necessary steps and three unnecessary or incorrect steps, and
explain why it is or is not necessarv/correct.
Collect a sample of bacteria from the infected patient.
Collect samples of bacteria from the bathroom taps in the patient's room.
Collect samples of bacteria from other patients who seem to have the same
infection.
Soak disks in many different types of antibiotic.
Soak disks in the antibiotic you have the biggest supply ot.
Label the disks with the type of antibiotic you soaked them in.
• Leave the petri dishes uncovered while the bacteria grow.
, cover the petri dishes while the bacteria grow.
Treat the bacterial culture with a disinfectant before spreading on the agar plate
Wear personal protective equipment (gloves, mask, lab coat) while performing the
procedure
Wear…
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DNA EXTRACTION
Why do scientists isolate DNA?
From what part of the subject’s body were cells collected?
Give the specific purpose of each experimental step:
Lysis solution & heat:
Salt & centrifugation:
After centrifuging, where in the tube is the DNA?
What is the purpose of adding alcohol?
arrow_forward
Could you please help me with the following questions? I am struggling tremendously. Thanks so much! Please form a brief answer, if possible.
Some organisms can live in hot springs. What does this imply about their DNA?
arrow_forward
BIOTECHNOLGY
Date:
Name:
Instructor:
Section/Group:.
POST-LAB QUESTIONS
1. In one or two sentences, summarize the technique of gel electrophoresis.
2. How does the process of gel electrophoresis separate DNA fragments?
3. Why is the fact that DNA has a negative charge so important in the gel electrophoresis process?
Biotechnology 165
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Electrophoretic Analysis of DNA via Agarose gel Electrophoresis
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Looking at the model data i upload and given the question how could i analyse tboth experimental results (antibiotic selection and agarose gel). Including one clearly labelled image of the agarose gel and with an accompanying legend. And how could i interpret the findings of the results and draw a conclusion i.e. what is in each tube? Also is there any further experiments that I could perform to confirm the identity of the plasmid stocks?
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Why is Nucleic Acid Testing a desirable testing method to use to screen donated
blood for viral infections
O a. It is the test legally required by the Health Insurance Portability and
Accountability Act (HIPAA)
O b. It is easy to perform, even without training
O c. The procedure has virtually no opportunities for error
O d. It can amplify a small amount of DNA or RNA, making it sensitive to detect
viruses
Next page
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Could you please help me with the following questions? I am struggling tremendously. Thanks so much!
Some organisms can live in hot springs. What does this imply about their DNA?
arrow_forward
Helping tags: Biology, bacterial count, dilution, serial dilution
WILL UPVOTE, just pls help me answer the following questions and explain them clearly. Pls
show complete solutions also for the computation part. Thank you.
1. A bacterial culture was grown for 9 hours. At 3-hour interval, the culture was sampled to
determine the population of the culture, by transferring 25 ml of the suspension to 225
ml 0.85% NaCl. Three consecutive dilutions were further made by using 1 ml aliquot in 9
ml of 0.85% NaCI. One ml from each dilution was plated in each of duplicate plates. The
following table shows the results of the plating method.
Sampling
COUNTS
2nd dilution
3rd dilution
4th dilution
0,0
30: 35
250;245
1st dilution
1st
2nd
3rd
0; 0
240; 235
TNTC
55; 60
5; 6
TNTC
TNCT
TNTC
TNTC
a) Illustrate the dilution series used and label the final dilution of each dilution.
b) Determine the bacterial count (CFU/ml) every 3 hours of incubation for 9 hours.
Show all computations.
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Currently I am working with qPCR quantification specific for Lactobacillus bacteria. I found in some cases, the sample showing multiple peaks of melting curves while others looks perfect, so do the amplification plot duplication. Any one have an opinion about this issues? Does it caused by sample DNA quality, the omount of the bacteria detected, or primers problem? I am condisering that this condition reflecting the low abundance of the specific bacteria inside its sample.
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Question 3
Listen
Which of the following statements describe disadvantages of the classical Sanger sequencing
approach when compared to the modern Sanger sequencing method?
|Radioactivity must be used, representing an added hazard
Fluorescence is used, representing an added hazard
Four reaction are used, requiring more reagents and escalating cost
OGel electrophoresis is used to separate the various products by size but slowing overall
completion of sequencing
OA single reaction is used, complicating the analysis
O Capillary electrophoresis is used to separate the various products by size but slowing
overall completion of sequencing
Question 4
4 Listen
Electrophoresis of polynucleotides can be done using either agarose or polyacrylamide gels.
What is the primary consideration when choosing which polymer gel to use?
O Overall charge on polynucleotide sample, greater overall negative charge requires agarose
gel with smaller pores to slow the migration of the highly charged sample
O…
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biotechnology lab class :1. The bacterial streaking on antibiotic containing agar plates has been completed and the plates will be available for you to inspect in the lab class.2. You will complete a restriction enzyme digest on the four available plasmid stocks and then examine the products by agarose gel electrophoresis.
Photographs of the gel are attached to identify the plasmid present in each analysed sample.Now consider the following questions :- For each of the four plasmids used in the practical, identify the size (in kb) of the linear DNA fragment(s) that you would expect to obtain if the EcoRI digest is complete.- Using the provided negative image of the gel, which shows the bands present in the ND and D samples for each plasmid, identify the plasmid that was present in each of the four plasmid stock.- Explain how you were able to identify plasmid in each sample using the gel, considering how linear DNA fragments of different length are separated by agarose gel…
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the image is the steps for help. Not writing assignment!!!!
What is the intended outcome of the lysozyme-based ion exchange chromatography described above? Will the protein be effectively separated using the materials listed in the image, or will it fail? Give a brief explanation for your decision.
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Topic: Isolation of E. coli bacteriophage
What would happen if:
- the enrichment method in the isolation of bacteriophage was omitted?
- the chloroform was not added to the enrichment?
- the 0.1 ml lysate-E. coli mix was plated directly on top of the bottom agar?
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The following image is of an agarose gel. If DNA samples were loaded to this gel and the electrophoresis experiment was started, explain what would happen and why.
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researcher runs an agarose gel
IIl ladder
one läñé ând
100 ng of a 500 bp DNA fragment in another lane. The gel is 0.5% agarose,
and it is run for 45 minutes time at 200 V. When he checked the gel, he saw
three bands in the Hind III lane and no other bands on the gel. What can be
the reason behind this. What additional information we may need to be sure
about the reason.
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A description of the principles of agarose gel electrophoresis of DNA.
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Electro blotting techniques are routinely used in diagnostic microbiology for detection of a specific DNA sequence in the given bacterial culture. Explain.
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What is the genus and species of your bacteria? And What led you to confirm this result and why? Please discuss.
☆My bacteria is Alcaligenes faecalis
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DNA is visualized during agarose gel electrophoresis by ______________ .
the fact that DNA fluoresces when illuminated with UV light
the binding of a fluorescent dye that is easily detectable
using radioactive antibodies that specifically bind to DNA
the fact that DNA is blue and can be seen when millions of copies are present in a band
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