PreLabs 7-11

Which tool will you use to inoculate the semi-solid medium when conducting the motility test?

1. Toothpick

2. Inoculating loops

3. Inoculating needle

4. Pipette tip

Inoculating needle

Which of the following describes a characteristic of motile bacteria in the motility test?

1. They form a compact growth pattern around the stab line.

2. They disperse from the inoculation line, producing a diffuse growth pattern.

3. They exhibit no growth in the semi-solid media.

4. They produce a distinct color change in the medium.

They disperse from the inoculation line, producing a diffuse growth pattern.

What is the role of cytochrome c oxidase in aerobic respiration in bacteria?

1. It synthesizes ATP

2. It transfers electrons from the Electron Transport Chain to oxygen

3. It accepts electrons from water

4. It synthesizes hydrogen peroxide

It transfers electrons from the Electron Transport Chain to oxygen

In the image below, which of the strips indicates a species that is positive for cytochrome c oxidase?

1. A

2. B

B

What do you add to the oxidase reagent strip before adding a bacterial sample?

1. Alcohol

2. Crystal Violet Die

3. Water

4. HCl

Water

In the image below, bacteria are grown on MacConkey Agar. Which species is Gram-negative?

1. A

2. B

3. Both

4. Neither

Both (because of them grew on the agar)

In the image above, bacteria are grown on MacConkey Agar. Which species produce an enzyme that ferments lactose?

1. A

2. B

3. Both

4. Neither

B

MacConkey Agar was used by Alfred MacConkey to test for:

1. Fecal contamination of drinking water

2. The pH needed to eliminate bacteria growth

3. The effectiveness of a course of antibiotics

4. Sulfide, motility and indole production

Fecal contamination of drinking water

How did MacConkey select for the growth of enteric (think gut) bacteria?

1. He increased the pH of the agar.

2. He included lactose in the agar.

3. He included bile in the agar.

4. He added mammalian blood to the agar.

He included bile in the agar.

10. Examine the image above, what do you expect to observe where you have streaked E. coli?

1. No bacteria grow

2. Bacteria grow and pink/red agar

3. Bacteria grow and agar remains the same color

Bacteria grow and pink/red agar

Examine the image above, what do you expect to observe where you have streaked E. raffinosus?

1. No bacteria grow

2. Bacteria grow and pink/red agar

3. Bacteria grow and agar remains the same color

No bacteria grow

Examine the image above, what do you expect to observe where you have streaked P. putida?

1. No bacteria grow

2. Bacteria grow and pink/red agar

3. Bacteria grow and agar remains the same color

Bacteria grow and agar remains the same color

Let's say that you determined through the Gram staining procedure that a soil isolate is Gram-positive. Do you expect to see any bacteria grow when you streak this isolate on MacConkey agar?

1. Bacteria should not grow

2. Bacteria should grow

Bacteria should not grow

Why does a clear zone surround beta-hemolytic bacteria?

1. The zone forms because the bacteria are producing antibiotics.

2. The zone forms because the bacteria are being killed by an antibiotic.

3. The zone forms because the bacteria are motile.

4. The zone forms because the bacteria are breaking down red blood cells.

The zone forms because the bacteria are breaking down red blood cells.

What type of hemolysis do you see in FIG. 10 below?

1. Alpha

2. Beta

3. Gamma

Alpha

What type of hemolysis do you see in FIG. 2 below?

1. Alpha

2. Beta

3. Gamma

Beta

Examine the image above, what do you expect to observe where you have streaked E. coli?

1. No bacteria grow

2. A clear zone

3. Green coloration

4. Agar remains the same color

A clear zone

Examine the image above, what do you expect to observe where you have streaked E. raffinosus?

1. No bacteria grow

2. A clear zone

3. Green coloration

4. Agar remains the same color

A clear zone

Examine the image above, what do you expect to observe where you have streaked P. putida?

1. No bacteria grow

2. A clear zone

3. Green coloration

4. Agar remains the same color

Green Coloration

How many Blood Agar plates will you use this coming week?

1. One

2. Two

3. Three

4. Four

Two

What do you find in the bubbles produced by Catalase-positive bacteria?

1. Water

2. Carbon dioxide

3. Oxygen

4. Hydrogen peroxide

Oxygen

Where will you conduct your Catalase Test?

1. At your table

2. In the fume hood

In the fume hood

How many drops of hydrogen peroxide will you add to your sample?

1. 1-2 drops

2. 5 drops

3. 10 drops

4. 20 drops

1-2 drops

Why is catalase an important enzyme?

1. It breaks down antibiotics.

2. Hydrogen peroxide can damage cell components.

3. It produces oxygen bacteria need for respiration.

4. It allows aerobic bacteria to live in anaerobic environments.

Hydrogen peroxide can damage cell components.

E. raffinosus grown on blood agar would test Catalase-positive, giving a false-positive result. Why?

1. E. raffinosus produces hydrogen peroxide only when grown on blood agar.

2. E. raffinosus transcribes the Catalase gene only when grown on blood agar.

3. Catalase is produced by mammals and found in mammalian blood.

4. Mammalian blood releases hydrogen peroxide when treated with catalase.

   1. A

   2. B

   3. C

   4. D

C

Match the test to its description:

1. Tests for the presence of aerobic respiration

2. Tests for the breakdown of hydrogen peroxide

3. Tests for the breakdown of lactose

4. Tests for the presence of flagella

5. Tests for the breakdown of red blood cells

1. Oxidase

2. Catalase Test

3. MacConkey Agar

4. Motility Test

5. Hemolysis Test

Ribosomes are made of:

1. Just DNA

2. DNA and protein

3. Just RNA

4. RNA and protein

RNA and protein

Where is the 16S rRNA gene found?

1. The gene is encoded by bacterial ribosomes

2. The gene is found encoded in the nucleus of a bacterium

3. The gene is encoded by a bacterium's circular chromosome

4. The gene is found encoded in the cytoplasm of a bacterium

The gene is encoded by a bacterium's circular chromosome

The 16S rRNA gene is a sequence of:

1. Nucleotides

2. Amino acids

3. Ribosomes

4. rRNA's

Nucleotides

Which of the following best describes the function of the 16S rRNA gene?

1. Encoding proteins for ribosome assembly

2. Serving as a sequence of nucleotides on a bacterium's chromosome

3. Regulating gene expression within the bacterial chromosome

4. Provide instructions for synthesizing ribosomal RNA

Provide instructions for synthesizing ribosomal RNA

Which of the following statements about the 16S rRNA gene sequence is correct?

1. It acts as a blueprint for synthesizing bacterial lipids.

2. It serves as a marker for identifying the presence of bacteria in a sample.

3. It codes for enzymes involved in bacterial metabolism.

4. It regulates the expression of antibiotic resistance genes within bacteria.

It serves as a marker for identifying the presence of bacteria in a sample.

True for False. The polymerase chain reaction (PCR) is a process that makes millions of copies of an entire chromosome.

1. True

2. False

False

True or False: PCR is a process that makes millions of copies of a targeted sequence of nucleotides on a chromosome.

1. True

2. False

True

What targeted sequence will you amplify using PCR?

1. The entire bacterial chromosome

2. 16S rRNA gene

3. RNA

4. The ribosome

16S rRNA gene

What is the name of the custom-made DNA molecules that help us find the beginning and end of the 16S rRNA gene?

1. DNA polymerase

2. Nucleotides

3. Primers

4. Ribosomes

Primers

Place in order the three PCR steps described in the video:

Step 1:

Step 2:

Step 3:

1. Open up the DNA (Denaturation)

2. Find the target DNA with Primers (Annealing)

3. Copy the target DNA by DNA polymerase (Extension)

Which enzyme in the Master Mix adds nucleotides to the growing complementary strand of DNA?

1. The 16S rRNA gene

2. The 27F Primer

3. The 1492 Primer

4. Taq polymerase

Taq polymerase

True or False: The 27F and 1492R Primers ONLY blind to the 16S rRNA gene.

1. True

2. False

True

How long (in base pairs) is the region of the 16S rRNA gene that we will amplify with PCR?

1. 16 base pairs

2. 20 base pairs

3. 1,465 base pairs

4. The entire bacterial chromosome

1,465 base pairs

This week we will provide you with a colony of Escherichia coli bacteria. Next week you will repeat these procedures with your own soil isolates. Why should you see the same region amplified in your colony next week?

1. Your soil isolate is also E. coli.

2. All species of bacteria have the 16S rRNA gene.

3. All living organisms have the 16S rRNA gene

All species of bacteria have the 16S rRNA gene.

What will be present in your E. coli Colony PCR tube at the end of the reaction?

1. Millions of copies of the 16S rRNA gene

2. Millions of copies of the bacterial chromosome

3. Millions of copies of the ribosomal subunit

4. Millions of copies of bacteria

Millions of copies of the 16S rRNA gene

What is the difference between the E. coli Colony PCR tube and the Negative Control tube?

1. Only the Colony PCR tube has Master Mix

2. Only the Colony PCR tube has the Primer Mix

3. Only the Colony PCR tube has a colony sample

4. Only the Colony PCR tube is placed in the thermal cycler

Only the Colony PCR tube has a colony sample

Why is Agarose Gel Electrophoresis a fundamental technique in biology?

1. It allows you to determine whether the DNA has a negative or positive charge

2. It allows you to determine the size of DNA fragments

3. It provides you with the DNA sequence

4. It creates copies of the DNA molecules

It allows you to determine the size of DNA fragments

True or False: Shorter pieces of DNA move more quickly toward the far side of the gel (the side with the positive charge).

1. True

2. False

True

When you are pouring a gel, what is the purpose of the comb?

1. It carries the current

2. It stains the DNA

3. It creates wells to add a sample

4. It creates a positive charge to attract DNA

It creates wells to add a sample

What does one band in the gel represent?

1. One DNA molecule

2. DNA molecules of different lengths

3. Millions of DNA molecules of the same size

Millions of DNA molecules of the same size

What is a DNA ladder?

1. A series of DNA fragments of known lengths

2. The sugar-phosphate backbone that forms a ladder in DNA

3. The piece of equipment used to pour the gel

4. The piece of equipment used to create wells in the gel

A series of DNA fragments of known lengths

When do you add a DNA stain that allows you to visualize the location of the DNA?

1. When making the gel

2. When adding the buffer solution

3. When applying the electrical current

4. When turning on the blue light

When making the gel

Which of the following describes the band in the Colony PCR lane.

1. Millions of copies of the 16S rRNA gene

2. Evidence for the presence of bacteria in the colony sample

3. Amplicons measuring about 1,500 base pairs in size

4. Products of PCR

5. All of the above

All of the above

When using the plunger to pipette your PCR sample into a well, you should only go to the:

1. First stop

2. Second stop

First stop

When placing your gel in the electrophoresis rig, you will place the wells of the gel closest to the black electrode. Your DNA will then migrate toward the red electrode or "run to red." What is the charge of the red electrode?

1. The red electrode is positive

2. The red electrode is negative

The red electrode is positive

What is the purpose of this coming week's lab?

1. To verify that your soil isolates are bacteria

2. To determine whether your soil isolates are Gram-positive or Gram-negative

3. To determine the DNA sequence of the four soil bacteria isolates

4. To detect a band on the Negative Control lane

To verify that your soil isolates are bacteria

Which techniques will you complete this week?

1. Gram staining and Microscopy

2. PCR and Gel electrophoresis

3. Serial dilution and mobility test

4. Catalase and oxidase tests

PCR and Gel electrophoresis

Which enzyme is essential for PCR amplification?

1. DNA polymerase

2. RNA polymerase

3. Ligase

4. Helicase

DNA polymerase

Which of the following is NOT a step in a typical PCR cycle?

1. Denaturation

2. Annealing

3. Sequencing

4. Extension

Sequencing

Which temperature is commonly used for the denaturation step in PCR?

1. 37 °C

2. 55 °C

3. 72 °C

4. 95 °C

95 °C

Which component is typically NOT included in PCR Master Mix?

1. DNA polymerase

2. Nucleotides

3. Primers

Primers

Which of the following terms is analogous to 'amplify'?

1. Copy

2. Delete

3. Denature

4. Sequence

Copy

The 27F and 1492R primers will anneal to _______, providing a starting point for Taq polymerase.

1. bacterial DNA

2. bacterial RNA

3. bacterial proteins

4. bacterial ribosomes

bacterial DNA

The DNA sequence amplified when the 27F and 1492R primers are used measures approximately ...

1. 20 base pairs in length.

2. 100 base pairs in length.

3. 1,000 base pairs in length.

4. 1,500 base pairs in length.

1,500 base pairs in length.

By using 27F and 1492R primers, we ensure that...

1. only the 16S rRNA gene found on bacterial chromosomes is copied.

2. the bacteria are lysed, and the DNA is denatured.

3. contamination of the Negative Control is minimized.

4. if any type of DNA is present in the sample (bacterial and eukaryotic), it is detected.

only the 16S rRNA gene found on bacterial chromosomes is copied.

What happens during the denaturation step of PCR?

1. Bacteria are lysed

2. Double-stranded DNA separates

3. Primers anneal to complementary sequences

4. Taq polymerase synthesizes complementary strands

Double-stranded DNA separates

True or False: Taq polymerase synthesizes primers.

1. True

2. False

False

True or False: Taq polymerase can only synthesize complementary DNA if it's given a primer.

1. True

2. False

True

What happens during the coolest stage of PCR?

1. Bacteria are lysed

2. DNA is denatured

3. Primers anneal to complementary sequences.

4. Taq polymerase synthesizes complementary strands of DNA.

Primers anneal to complementary sequences.

At which temperature is Taq polymerase active?

1. 95 °C

2. 72 °C

3. 55 °C

4. 32 °C

72 °C

What is in the PCR tube after the reaction ends IF a bacterial chromosome IS in the tube and the reaction occurred as expected?

1. Millions of copies of the 16S rRNA gene sequence

2. Millions of copies of the entire bacterial chromosome

3. Millions of copies of the ribosomes

4. Millions of bacteria

Millions of copies of the 16S rRNA gene sequence

What is in the Negative Control PCR tube after the reaction ends if there is NO contamination?

1. Millions of copies of the 16S rRNA gene sequence.

2. No copies of the 16S rRNA gene sequence.

No copies of the 16S rRNA gene sequence.

True or False: The 16S rRNA gene should be amplified in the Positive Control PCR Tube if the reaction occurs as expected.

1. True

2. False

True

How will you know that the 16S rRNA gene has been successfully amplified when looking at the gel?

1. You will not see a band in the lane.

2. You will see two bands in the lane.

3. You will see a ladder in one lane.

4. You will see a band with amplicons measuring 1,500 bp in size.

You will see a band with amplicons measuring 1,500 bp in size.

How will you know that bacteria have not contaminated your reagents when looking at the gel?

1. You will not see a band in the Negative Control lane.

2. You will see two bands in the lane.

3. You will see a ladder in one lane.

4. You will see a band with amplicons measuring 1,500 bp in size in the Positive Control lane.

You will not see a band in the Negative Control lane.

True or False: You should see a band measuring 1,500 bp in size in the Positive Control lane in the gel if the reaction occurred as expected.

1. True

2. False

True

Let's say you were unable to lyse the bacteria, and the PCR reagents could not access the bacterial chromosome. Will amplification occur? Will there be a band in the gel?

1. Yes, Yes

2. No, No

3. Yes, No

4. No, Yes

No, No

What is the purpose of the agarose gel used in Agarose Gel Electrophoresis?

1. It keeps the samples from evaporating.

2. It separates DNA molecules based on their size.

3. It produces the current.

4. It amplifies the DNA

It separates DNA molecules based on their size

Place the following Agarose Gel Electrophoresis procedures in the correct order-

Step 1:

Step 2:

Step 3:

Step 4:

Step 5:

Step 6:

Step 1: Make a solid agarose gel

Step 2: Place the gel in the electrophoresis chamber

Step 3: Add running buffer

Step 4: Load the DNA Ladder and PCR Tube samples

Step 5: Apply an electrical current

Step 6: Measure the presence and size of the DNA molecules using a DNA ladder

What lane do you look at to determine whether the reagents are contaminated?

1. DNA Ladder lane

2. Negative Control lane

3. Positive Control lane

4. Any soil isolate lane

Negative Control lane

Examine the gel image above. Does it appear that the reagents, thermocycler, and overall PCR process functioned as expected?

1. Yes

2. No

Yes

What lane do you look at to determine whether the process functioned as expected?

1. DNA Ladder lane

2. Negative Control lane

3. Positive Control lane

4. Any soil isolate lane

Positive Control lane

Examine the gel image above. Does it appear that Soil Isolate One was a colony of bacteria?

1. Yes

2. No

No

Examine the gel image above. Does it appear that Soil Isolate Two was a colony of bacteria?

1. Yes

2. No

Yes

Which of the following is an example of higher (or better) resolution?

1. When (r) = 200 nanometers

2. When (r) = 1000 nanometers

When (r) = 200 nanometers

What is the resolution (r) of your microscope, when the 40X objective is in alignment? The Numerical Aperture of the 40X objective is equal to 0.65.

1. 40 nm

2. 65 nm

3. 385 nm

4. 400 nm

385 nm

What is the relationship between magnification and the number of micrometers per reticle unit?

1. As magnification increases the number of micrometers per reticle unit increases.

2. As magnification increases the number of micrometers per reticle unit decreases.

As magnification increases the number of micrometers per reticle unit decreases.

You measure the width of a Paramecium with the 10X objective in alignment. You determine it measures 5 reticle units. How many micrometers does this equal?

1. 5 µm

2. 12.5 µm

3. 50 µm

4. 75 µm

50 µm

With which objective will you have the biggest field of view?

1. 4x

2. 10x

3. 40x

4. 100x

4x

With which objective will you have the largest depth of field remain in focus?

1. 4x

2. 10x

3. 40x

4. 100x

4x

You can use the coarse knob to focus on the specimen when which objective is in alignment?

1. 4x

2. 10x

3. 40x

4. 100x

4x

You must use immersion oil when which objective is in alignment?

1. 4x

2. 10x

3. 40x

4. 100x

100x

Should the 100X objective lens come into direct contact with the immersion oil?

1. Yes

2. No

Yes

What should you use to clean the objective lens and slide after using immersion oil?

1. Ethanol

2. Paper towels

3. Water

4. Lens cleaner and Lens paper

Lens cleaner and Lens paper

Let's say that you use the Gram staining procedure to stain an L-form bacterium (a bacterium that lacks a cell wall). What color will the bacterium be after the staining procedure is finished?

1. Purple

2. Pink

Pink

What is one purpose of the cell wall in bacteria?

1. Moves the cell through the environment

2. Protects the cell from lysis

3. Carries genetic information

4. Produces proteins

Protects the cell from lysis

What structure do you find in Gram-negative bacteria that you do not find in Gram-positive bacteria?

1. Teichoic acid

2. Peptidoglycan

3. Outer membrane

4. Cell wall

Outer membrane

What does the Crystal Violet dye bind to?

1. Cell membrane

2. Phospholipids

3. DNA

4. Peptidoglycan

Peptidoglycan

What is produced when the Iodine solution is added?

1. Large crystals

2. Alcohol

3. Peptidoglycan

4. LPS

Large crystals

Why is the violet color lost in Gram-negative bacteria when a decolorizer is used?

1. Because the thicker peptidoglycan layer is dehydrated and shrinks

2. Because the crystals are trapped in the thicker cell wall

3. Because the crystals are trapped in the plasma membrane

4. Because the crystals are washed away by alcohol from the thinner cell wall

Because the crystals are washed away by alcohol from the thinner cell wall

Which stain is used to turn Gram-negative bacteria a pink or red color?

1. Teichoic acid

2. Safranin

3. Crystal violet

4. Alcohol

Safranin

What setting should you use on the hot plate?

1. 1

2. 1.5

3. 2

4. 2.5

2.5

What do you watch for after you place the slide on the hot plate?

1. Smoke

2. A color change

3. Evaporation of water

4. A spark

Evaporation of water

After the water evaporates, how long should the slide remain on the hot plate to heat fix the cells?

1. One minute

2. Five minutes

3. 10 minutes

4. 30 minutes

One minute