Question 1

You are reading culture plates today and identifying pathogens that grow from patient samples.

Which two pieces of information are absolutely critical in determining which additional tests must be done to identify the pathogen?

a) Appearance on charcoal agar

b) Endospore Stain

c) Gram Stain

d) Oxygen sensitivity

Question 2

A 10 year old has a wound on the arm that the physician suspects is infected.

Upon culture, you see small white colonies growing on blood agar as well as chocolate agar.

You gram stain the colonies to find that they are gram positive cocci.

Which test will you perform first?

a) catalase

b) indole

c) lactose fermentation

d) coagulase

Question 3

You have isolated catalase positive gram positive cocci from a wound culture.

Which test will you perform next?

a) coagulase

b) P disc, containing optichin

c) A disc, containing bacitracin

Question 4

You are working on a sputum culture.

You see mucoid alpha hemolytic colonies that number many more than the normal flora present.

The gram stain of the colonies shows gram positive cocci that are in lancet shaped pairs.

Which test will you do?

a) P disc, containing optichin

b) A disc, containing bacitracin

c) motility

d) acid fast staining

Question 5

You are working up a throat culture.

Standard procedure in plating throat cultures in your lab includes dropping an A disc near the initial inoculum in the first quandrant when streaking the specimen.

You examine the blood agar plate and see moderate normal flora and many beta hemolytic colonies that do not grow up to the A disc.

The beta hemolytic colonies are catalase negative gram positive cocci.

Which pathogen is in this throat culture?

a)Streptococcus agalactiae

b) Staphylococcus aureus

c) Streptococcus pyogenes

d) Staphylococcus epidermidis

Question 6

You are working up a male genital culture.

You see no growth on the blood agar plate but small colonies growing on the chocolate agar plate. The gram stain shows gram negative cocci in pairs.

Which test will you do next?

a) oxidase

b) motility

c) catalase

d) indole

Question 7

You are working up a cerebrospinal fluid culture.

You find colonies growing on blood agar as well as chocolate agar.

The colonies are oxidase positive gram negative cocci.

The colonies ferment glucose and maltose but not sucrose or lactose.

You identify the pathogen as

a) Neisseria meningitidis

b) Haemophilus influenzae

c) Streptococcus pneumoniae

d) Neisseria gonorrhoeae

Question 8

You are working up a urine culture.

You see >100 colonies that are gray and flat on the blood agar plate and >100 colonies that are bright pink on the MacConkey agar.

The IMViC results are Indole positive, Methyl Red positive, Vogues-Proskauer negative, Citrate negative.

You have identified the pathogen as

a) Citrobacter freundii

b) Escherichia coli

c) Enterobacter aerogenes

d) Proteus vulgaris

Question 9

You are working up a stool culture.

On MacConkey agar you see many bright pink colonies and many clear colonies.

Which colonies are potential pathogens that require further testing?

a) Clear colonies, non lactose fermenters

b) Bright pink colonies, non lactose fermenters

c) Bright pink colonies, lactose fermenters

d) Clear colonies, lactose fermenters

Question 10

DNA technology is useful in the identification of :

a) pathogens that are unable to be grown readily on artificial lab media.

b) pathogens that are no longer alive in the patient sample,

c) species that cannot be differentiated by conventional testing.

d) All of the above.

Question 11

You are preparing a sample of DNA from an unknown colony of bacteria.

After adding digestion buffer and incubating for the time suggested by the manufacturer, you centrifuge the sample.

The DNA is found:

a) stuck to the gel in the tube.

b) stuck to the sides of the tube.

c) in the pellet in the bottom of the tube.

d) in the supernatant in the tube.

Question 12

Which of the following is not true of the Polymerase Chain Reaction?

a) PCR is facilitated by a heat labile DNA polymerase.

b) PCR is a method of replicating DNA in a test tube.

c) PCR can facilitate the detection of DNA that is too low to detect by other methods.

Question 13

Why are dATP, dCTP, dTTP and dGTP added to a PCR reaction tube?

a) They catalyze the polymerase.

b) They buffer the mixture.

c) They allow the DNA in the sample to anneal.

d) They provide the building blocks of DNA.

Question 14

Why are universal 16S rDNA primers used in your experiment?

a) They will anneal to highly conserved areas of the gene that encodes bacterial 16S rRNA.

b) They will anneal to unique sequences of genes encoding 16S rRNA in specific bacteria.

Question 15

If universal primers are used to amplify DNA in a PCR reaction, then the PCR product must be sequenced to determine the bacteria that the DNA belongs to.

True

False

Question 16

How is the PCR product separated from the PCR mixture at the completion of the reaction?

a) Perform electrophoresis in an agarose gel, stain the gel and cut the band corresponding to the PCR product from the gel.

b) Pour the PCR mixture into a commercially prepared DNA microconcentrator column and follow the manufacturer’s directions to adhere and elute the PCR product from the column.

c) Both of the above procedures may be used.

d) Neither of the above procedures may be used.

Question 17

Your PCR product was sequenced by a method known as Cycle Sequencing.

Which of the following statements is false?

a) An automatic sequencer performs electrophoresis and reads the tagged DNA pieces, providing a read out of the nucleotide bases comprising the DNA sequence of the fragment being tested

b) Cycle sequencing is done in a PCR machine.

c)Tagged terminator nucleotides facilitate the creation of a series of nested DNA sequences of different length.

d) Cycle sequencing can be completed in just one test tube.

Question 18

The National Library of Medicine has a databank called GenBank that has deposited in it the DNA sequences of numerous genes isolated from known bacterial species.

True

False

Question 19

You obtained the following BLAST data from your sample:

99.9% Enterobacter sakazakii

95.2% Enterobacter aerogenes

93.7% Enterobacter cloacae

The pathogen in your sample is:

a)Enterobacter sakazakii

b)Enterobacter aerogenes

c)Enterobacter cloacae

d)Enterobacter species

 

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BIOL 102: Lab 2

Solutions, Acids and Bases

PRE-LAB ASSIGNMENT:

Students are expected to read pages 1 to 3 before coming to the lab to complete the experiments.

Print this entire lab packet and bring it to the laboratory. You must submit the completed lab worksheet for credit. Please provide a FULL lab report for this experiment.

Objectives:

After completing this laboratory assignment, students will be able to:

· Measure the pH of various liquids.

· Demonstrate that buffers stabilize the pH of a liquid.

· Measure the ability of commercial antacids to buffer the pH of a liquid.

Acids:

Acids are ionic compounds (compounds with a positive or negative charge) that break apart in water to form hydrogen ions (H+). The strength of an acid is based on the concentration of H+ in the solution.

The greater the concentration of H+, the stronger the acid.

1

1

1

Characteristics of Acids:

· Acids taste sour.

· Acids react strongly with metals (Zn + HCl).

· Strong acids are dangerous and can burn your skin.

Examples of Acids:

· Vinegar

· Stomach Acid (HCl)

· Citrus Fruits

Bases:

Figure 1: Hydrochloric acid (HCl) in water

Bases are ionic compounds that break apart to form a negatively charged hydroxide ion (OH-) in water. The strength of a base is determined by the concentration of hydroxide ions (OH-). The greater the concentration of OH-, the stronger the base. Solutions containing bases are often called alkaline .

Characteristics of Bases:

· Bases taste bitter.

· Bases feel slippery.

· Strong bases are very dangerous and can burn your skin.

Examples of Bases:

· Sodium hydroxide (lye)

· Ammonia

Figure 2: Sodium

Hydroxide (NaOH) in water

pH Scale and Indicators
The strength of an acid or a base in a solution is measured on a pH scale. The pH scale is a measure of the hydrogen ion (H+) concentration. It spans from 0 to 14 with the middle point (pH 7) being neutral (neither acidic nor basic). Any pH number greater than 7 is considered a base and any pH number less than 7 is considered an acid. 0 is the strongest acid and 14 is the strongest base. An indicator is a special type of compound that changes color as the pH of a solution changes, thus telling us the pH of the solution.

Figure 3: A pH scale indicating the pH of common substances.

Measuring pH
A convenient way of measuring the pH of a solution is with pH paper. pH paper is treated with a chemical indicator that changes color depending on the concentration of H+ in the solution that it has contacted. The color chart on the container of the pH paper is used to compare the color of the pH paper to determine the pH of the solution (see Table 1).

Table 1: Examples of pH indicators.

Indicator

Range

Color Change

Methyl violet

0.2 – 3.0

Yellow to blue-violet

Bromphenol blue

3.0 – 4.6

Yellow to blue

Methyl red

4.4 – 6.2

Red to yellow

Litmus

4.5 – 8.3

Red to blue

Bromcresol purple

5.2 – 6.8

Yellow to purple

Phenol red

6.8 – 8.0

Yellow to red

Thymol blue

8.0 – 9.6

Yellow to blue

Phenolphthalein

8.3 – 10.0

Colorless to red

Buffers
Buffers are mixtures of two chemicals that stabilize the pH of a solution by resisting changes in the pH.

· If the pH is too low, one chemical will bind some of the hydrogen ions and raise the pH.

· If the pH is too high, the other chemical will donate some hydrogen ions to lower the pH.

pH in the body
The blood pH must be maintained very close to 7.4 which is slightly alkaline. A change of 0.2 pH units in either direction is considered dangerous and potentially fatal. Hydrogen ions are extremely reactive and affects many molecules which regulate physiological processes.

The body regulates pH in several ways including eliminating CO2 by the lungs and eliminating other acids and bases by the kidneys. The most important way to minimize pH changes in the body is using buffers. All body fluids, inside or outside of the cells, have buffers which defend the body against pH changes. The most important buffer in extracellular fluids, including blood, is a mixture of carbon dioxide (CO2) and bicarbonate anion (HCO3). CO2 acts as an acid because it forms carbonic acid when it is dissolved in water and donates hydrogen ions when they are needed. HCO3 is a base, soaking up excess hydrogen ions. There are also other buffers in the blood, such as proteins and phosphates, but they are less important in maintaining the overall pH of the blood. Blood pH is established by a balance between bicarbonate and CO2.

LAB DATASHEET
Exercise 1: Determining the pH of common substances
1. Use pH paper to measure the pH of the following liquids.

2. Be as accurate as possible

3. Use a fresh piece of pH paper or pH dipstick for each test.

4. Record your data.

Baking soda

___7____________

0.1M HCl

___0____________

Vinegar

___2____________

Tap water

___6___________

Seltzer water

___5____________

Exercise 2: Testing the buffering capacities of various solutions
1. Obtain and label four test tubes according to the four solutions listed in Table 2.

2. Place 5 ml of each solution into its appropriately labeled tube.

3. Measure the pH of each of the solutions in the tubes and record these initial values in Table 2.

4. Add 5 drops of acid (0.1 M HCl) to the first tube. Cover the tube with parafilm and invert the tube gently to mix the contents.

5. Measure the pH of the acidified solution and record it in Table 2.

6. Repeat steps 4 and 5 for each of the remaining tubes. Record your results in Table 2.

7. Compare the initial pH and the pH after acid addition for each sample.

Table 2: Buffering Capacity Data

Solutions

Initial pH

Final pH (after HCl)

Comparison of pHs

Water

5

2

PH dropped by 3

0.1 M NaCl

5

3

PH dropped by 2

Skim milk

6

5

PH dropped by 1

0.1 M phosphate buffer

7

7

PH stayed the same

Analysis Questions:

1. What is the biological importance of using a buffer?

Buffers prevent changes in PH, and maintain balance.

2. Which of the solutions is the most effective buffer? Least effective?

The most effective buffer is the solution who’s PH changed less, (in this case will be 0.1M phosphate buffer). The least effective solution will have a significant change in PH (in this case it will be H2O with an original PH of 5, and a final PH of 2 after addition of 0.1M of HCL)

Exercise 3: Testing the effectiveness of commercial antacids and other products
Commercial antacids such as Alka Seltzer, Rolaids, and Tums claim to “neutralize stomach acid” by absorbing excess hydrogen ions (produced as hydrochloric acid in the stomach).

To test the abilities of these products to absorb acids, do the following:

1. Using a pipet or 10 mL graduated cylinder, add 1 ml of the antacid solution to a test tube.

2. Add 1 drop of the indicator Bromcresol purple to the tube.

3. Cover the tube with parafilm and invert the tube to mix the contents.

4. Add one drop of 0.1 M hydrochloric acid (HCl) to the tube; mix after each drop.

a. Continue this process until the solution turns yellow, indicating an acidic solution.

5. Record the number of drops of acid needed to generate the change of color.

a. This number of drops is an index to the amount of acid (H+) that the solution neutralizes before the pH drops below the yellow end-point of Bromcresol purple.

Table 3: Effectiveness of Antacids Data

Antacids

Drops of Acid

Alka – Seltzer

33

Rolaids

1

Tums

1

Analysis Questions:

1. Which antacid neutralizes the acid best? Which neutralizes the acid least?

Rolaids and Tums neutralized acid best while Alka-Seltzer neutralized acid the least.

2. Examine the package of the products you tested. What are the active ingredients of each product?

Tums – Calcium carbonate 50mg

Rolaids – Calcium Carbonate

Alka- Seltzer – Aspirin 325mg, citric acid 1000mg, and sodium bicarbonate 1916mg

 

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