Virtual Lab

Bacterial Identification Virtual Lab Student Handout

BACTERIAL IDENTIFICATION LAB HANDOUT

INTRODUCTION
Go to https://www.hhmi.org/biointeractive/explore-virtual-labs. Scroll down and click on
“The Bacterial Identification Virtual Lab.” Maximize the screen if you wish. Answer the
following questions in the spaces provided. Prepare a sample from patient and isolate
whole bacterial DNA.Make many copies of the desired piece of DNA.Sequence the
DNA.Analyze the sequence and identify the bacteria.

1. What is the overall purpose of this virtual lab?

The overall purpose of this virtual lab is to learn about the science and
techniques used to identify different types of bacteria based on their DNA sequence.

2. What are the four basic steps involved in this bacterial identification lab?
The four basic steps involved in this bacterial identification lab are the following:
* Prepare a sample from patient and isolate whole bacterial DNA.
* Make many copies of the desired piece of DNA.
* Sequence the DNA.
* Analyze the sequence and identify the bacteria.

3. What is "16S rDNA," and how is it used to identify species of bacteria?

The piece of DNA used for identifying bacteria is the region that codes for a small
subunit of the rRNA.

Click to Enter the Lab. (Click the window on the left-hand side of the screen to enter the
lab.) As you enter the lab, follow the instructions in the lab (left-hand window). Using the
information in the Notebook window on the right, answer the following questions.

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PART 1: SAMPLE PREPARATION

4. As the pathology lab technician, what is your task in this virtual lab?
As a pathology lab technician my task would be to identify a bacterial sample
received from a clinician.

5. Extracting DNA involves which initial step?

Picking up a single colony and drop it into a microcentrifuge tube.

6. What is the wire ring used for?

The wire ring is used to scoop bacteria.

7. Why are the proteolytic enzymes necessary?

Proteolytic enzymes are necessary to lyse the bacteria cell wall.

8. Why do you then need to inactivate the proteolytic enzymes and how do you do it?
We need to inactivate the proteolytic enzyme, because we need to introduce a
new enzyme. The proteolytic can be denatured by heating the sample in a water
bath at 100°C.

9. After removing the enzymes, why do you spin the sample in the centrifuge?
Spinning the sample in the centrifuge would separate the DNA from other cellular
debris.

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10. a. What is the pellet?

Pellet talks about the cellular debris

b. What is the supernatant?

It is the liquid that is suspended above the pellet.

c. Where is the DNA?

The DNA is in the supernatant

PART 2: PCR AMPLIFICATION

Go on to Part 2 and work through the PCR steps. Be sure to read the information in the
notebook, including “What is PCR?”
11. What does “PCR” stand for and what is the purpose of PCR?
PCR stand for Polymerase Chain Reaction. The purpose of PCR is to multiplying
DNA copies.

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12. Summarize the process of PCR in a diagram. Include all the steps, labeled and in
the right order. (If you are completing this handout online, draw the diagram on a
piece of paper, take a photo, save the image as a PDF, and upload it in the space
below.)
Add the Master Mix and answer the following questions:

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13. What does the Master Mix contain?

Master Mix contains, water, a buffer, large quantities of the ATCG, large
quantities of oligonucleotide DNA primers, and a heat-stable DNA polymerase.

14. What are primers? Why is a primer added?

A primer is a short nucleic acid sequence that provides a starting point for DNA
synthesis. A primer is added for it to bind to the 16S rDNA region to initiate the
replication process.

15. Once the primers bind, what occurs next?

Once the primers bind, the DNA polymerase attaches to it and begin adding ATCG
in the 3’ to 5’ direction.

16. What does "highly conserved" mean?

Highly conserved means a Part of genes that are extremely similar in related
species.

17. Why are highly conserved regions important in this lab?

Highly conserved regions are important in this lab so that the primers bind to the
highly conserved regions of genes so that they can be used to copy DNA from a variety
of species of bacteria.

18. What does "highly variable" mean?

Highly variable means that a section of genome that adapt to each bacteria
environment. Where it is changing as the bacteria needs changes

19. Why are highly variable regions important in this lab?

Highly variable regions are important in this lab for identifying bacteria.

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20. What is missing in the negative control tube?

DNA sample is missing in the negative control tube.

21. What is present in the positive control tube that is not in the negative control tube?
Positive control DNA is present in the positive control tube that is not in the
negative control tube.

Now run the PCR. Be sure to watch the virtual lab animation before proceeding to the
questions.
22. List each step of a PCR cycle, the temperature, and the duration (time).

a. Denaturation: 95°C 30 seconds

b. Anneal: 60°C 30 seconds

c. Extend: 72°C 45 seconds

23. Describe what happens during each of the steps in one or two sentences.
a. In denaturation it separates each strand for the DNA by breaking the hydrogen
bonds.

b. In annealing it allows the primer to attach to each of the strand.

c. In extending it allows the DNA polymerase to elongate each strand.

24. After eight cycles, how many copies of the desired DNA have been synthesized?
After 8 cycles, 256 copies of desired DNA have been synthesized.

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25. After 30 cycles?

After 30 cycles, there are approximately 1 million copies of DNA have been
synthesized.

PART 3: PCR PURIFICATION

26. Approximately how long is the 16s rDNA (bp)?
16s rDNA is long as it has 1500 base pairs.
27. Why would it be useful to run an electrophoresis gel at this point?
It is useful to run an electrophoresis at this point to confirm if the PCR works and
to confirm its results.

28. If you were to run a gel, it would have three lanes. What would each lane contain,
and what would you see in each lane after running the gel?

a. Each lane should contain water and not any product that is contaminated.

b. Each lane should contain PCR product of known DNA and you would see it
moving through the lane after running the gel.

c. Each lane should contain a sample and you would see it moving through the lane
but different from the PCR product’s location after running the gel.

29. The gel is not run in this virtual lab. In order to purify the PCR product, you use a
microconcentrator column. (Proceed through the virtual lab steps.) What should the
final collection tube contain?
The final collection tube should contain many pieces of 1,500 bp-long 16S rDNA.

PART 4: SEQUENCING PREPARATION

Click on "Learn about cycle sequencing before proceeding."
30. Read the first two paragraphs and list the steps in cycle sequencing in the space
provided.
First step is to use a thermocycler to create many copies of the target DNA to

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terminate the replication process at random places, so the copies are all partial
sequences with different lengths. The reaction mixture contains normal DNA as well
as some special Dideoxynucleotide that are tagged with fluorescent markers. The
fluorescent markers differ for each base and are designed to fluoresce with different
colors. Dideoxynucleotide can substitute for normal DNA during replication, but if it
happens the chain can no longer be extended. Dideoxynucleotides are thus called
terminators.

Click to go back to Part 4.

31. What do the green and blue tubes contain? Describe the “sequencing brew” to which
you added your purified PCR.
The green and blue tubes contain a sequencing brew. Whereas sequencing brew
is the one that consist of buffers, primer a different one for each tube, DNA
polymerases, nucleotides, and fluorescence-tagged terminators in suitable
proportions

32. The purpose of the second PCR is not to create identical copies like the first PCR
you ran. What is the purpose of this PCR?
The purpose of this PCR is to produce many copies of variable length of DNA.

33. Where do scientists obtain primers to be used in PCR and in this technique?
Scientist obtain primers in the DNA polymerase and then used it in PCR and
other techniques.

Watch the virtual lab animation before proceeding to Part 5.

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PART 5: DNA SEQUENCING

34. What is the final PCR product, the stuff contained in your 12 tubes?
The final product of PCR is the final copies of target DNA.

35. What is the purpose of gel electrophoresis?

The purpose of gel electrophoresis is to separate molecules based on different
sizes.

36. How do DNA molecules move in relation to charge? Why?

DNA molecules can move because the DNA molecules are negatively charge, and
they would move through the tube toward the positively charged syringe end.

37. What is the purpose of the laser beam in determining a DNA sequence?
The purpose of the laser beam in determining a DNA sequence is to collect
information from the tubes by light refraction.

Be sure to watch the virtual lab animation before proceeding to Part 6.

PART 6: DNA SEQUENCE ANALYSIS

Click on "Learn about the science behind sequence
matching." 38. What is the ultimate goal of the
sequence matching analysis?

39. What is "homology"?

Homology refers to the similarity of DNA or amino acid sequences.

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40. What is BLAST and how is it used?

BLAST or the Basic Local Alignment Search Tool offer a good combination of
speed, sensitivity flexibility, and statistical rigorousness for matching rDNA sequence.

41. What’s a major assumption when drawing evolutionary relationships between

organisms based on DNA sequences?
The number of positions that is different in the nucleotide sequence is proportional
to the time elapsed since the two species formed their own lines of going down from
a common precursor.

Click to go back to Part 6 and click on "Learn more about BLAST search results."
42. Explain what the "Score (bits)" means on an actual BLAST search result.
The score measures the information, two for each matching pair. The higher the
score, the better is the match.

43. What does an E-value of 3 or less represent?

AN E-value or less represents an acceptable match.

Click to go back to Part 6 and proceed through the instructions in the right-hand
notebook window.
• Hints: "Ctrl A" will select all the data in the pop-up window, "Ctrl C" will copy it,
and "Ctrl V" will paste it into the NCBI website (large yellow box at the top of the
BLAST search page).
• Follow the steps listed on the page and be patient. BLAST data can take a while
to search.
• When the BLAST results appear, scroll down below the color key to the
significant alignments, and then go back to the virtual lab window (left) and follow
the instructions.

44. What is the scientific name of the bacterium you sequenced?

The scientific name of the bacterium that we sequenced was Barnotella Henselae.

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45. Write a brief description of this bacterium in the space provided.

Bartonella henselae is a proteobacterium that is the causative agent of cat-scratch
disease bartonellosis. B. henselae is a member of the genus Bartonella, one of the
most common types of bacteria in the world. It is a facultative intracellular microbe that
target red blood cells.
Reference: CDC.(n.d). Barnotella. Retrieved from: https://www.cdc.gov

After completing Sample A, perform DNA sequence analysis on three of the other five
samples.
46. Write in the letter of the samples you choose, the scientific name of the bacterium
(after doing a BLAST search), and a brief description of each.
Sample Letter Bacteria Scientific Brief Description
Name
A Escherichia coli is a type of bacteria that normally lives in your
intestines. It's also found in the gut of some
animals. Most types of E. coli are harmless
and even help keep your digestive tract
healthy. But some strains can cause diarrhea if
you eat contaminated food or drink fouled
water.

B Salmonella is a pathogenic Gram-negative bacteria

typhimurium predominately found in the intestinal lumen. Its
toxicity is due to an outer membrane consisting
largely of lipopolysaccharides (LPS) which
protect the bacteria from the environment. It
causes less serious gastrointestinal diseases,
collectively known as Salmonellosis. The
bacteria invade the cells of the intestinal tract
of the host, multiply, and sometimes escape to
the bloodstream and the lymphatic system.
Symptoms include fever, nausea, abdominal
pain, and diarrhea.

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D Pseudomonas a common encapsulated, Gram-negative,

aeruginosa facultatively aerobic, rod-shaped bacterium
that can cause disease in plants and animals,
including humans. P. aeruginosa has been
known to cause problems in debilitated
patients and is a common cause of disease in
children with cystic fibrosis.
Reference: Delost, M.D. (2014). Introduction to diagnostic microbiology for the
laboratory Sciences.

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