Experiment 2: Concentration Gradients and Membrane Permeability

In this experiment, you will dialyze a solution of glucose and starch to observe:

The directional movement of glucose and starch.
The effect of a selectively permeable membrane on the diffusion of these molecules.
An indicator is a substance that changes color when in the presence of a specific substance. In this experiment, IKI will be used as an indicator to test for the presence of starch.

Materials

(5) 100 mL Beakers
10 mL 1% Glucose Solution, C6H12O6
4 Glucose Test Strips
(1) 100 mL Graduated CyliStructure and Microscopy

Lab 4: Structure and Microscopy (100 points)

Student Name:

Student ID:

Course ID:
-Each question on the lab worksheet must be answered completely, thoroughly, in complete sentences and correctly in order to be considered for full credit
-If the question asks you to do research or find a source, a reputable, credible and/or scholarly source citation must be included in order to be considered for full credit
-If a math formula is required to arrive to an answer, work must be shown otherwise, no credit will be awarded
Pre-Lab Questions
1. What determines if a bacterial cell is Gram-positive or Gram-negative? (5 points)

Amount and location of the peptidoglycan molecule in the prokaryotic cell wall determines whether a bacterial cell is Gram-positive or Gram-negative.

2. In this lab, both viruses and prions were introduced as acellular organisms. Do some research and describe one other type of acellular organism. What characteristics about this organism classify it as acellular? (5 points)

Viroids are another type of acellular organism along with viruses and prions. They are plant pathogens, which consist only of a short strand of circular RNA capable of self-replication.

3. Bacteria have many different shapes that often determine their class. Research and form a hypothesis on the evolutionary reasons for so many different bacterial morphologies. (5 points)

Each bacterial morphology may be a selectable feature to aid survival and may have affected by different physical, environmental, and biological forces to contribute to natural selection.

Reference:

Young, K. D. (2006, September). The Selective Value of Bacterial Shape. Retrieved September 30, 2018, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1594593/

4. Do a search online or look in your textbook for 1-2 antibiotics that affect Gram-positive bacteria and list them. On what part of the cell do the antibiotics usually work? List one or two antibiotics that affect Gram-negative bacteria? On what part of the cell do the antibiotics usually work? (Be sure to cite your sources in your answer.) (5 points)

5. Why do you think it is important to identify a bacterial disease in a patient before prescribing any antibiotic treatments? (Be specific.) (5 points)

d

Experiment 1 Results Tables
Table 1: Experiment 1 Staining Observations (5 points)

Stain used:

Crystal Violet

Observations:

Purple rod-shape bacteria with white background were observed

Experiment 1 Post-Lab Questions
1. How does crystal violet enhance the visualization of microbial features? (5 points)

Crystal violet enhances the contrast between the microorganism itself and the slide, making the bacteria appear as purple.

2. What are some of the limitations of simple staining? (5 points)

3. Give an example of a situation in a lab or medical setting in which simple staining would be utilized. (5 points)

Simple staining is used to obtain basic information about morphology of one type of microorganism through clear visualization.

Experiment 2 Results Tables
Table 2: Experiment 2 Staining Observations (5 points)

Stain used:

Nigrosin

Observations:

Background is stained, bacteria shows up as clear spiral.

Experiment 2 Post-Lab Questions
1. After visualizing the stained samples either using your microscope or by looking at the sample images provided, describe what physical/visual characteristics you were able to observe after performing the negative staining vs. after performing the simple stain. (5 points)

After looking at the sample images provided, negatively stained bacteria showed up as clear straight spirals against a dark background. Bacteria that are simple stained showed up as dark purple rods-shaped with white background.

2. So far in this lab, you have used one type of simple stain and one type of negative stain, yet there are many other simple and negative dyes available. Pick one simple dye and one negative dye, and discuss how those dyes differ from the ones you used in this lab. Give a scenario in which their use would be appropriate. (5 points)

Methylene blue is another dye that can be used for negative stain.

India Ink is another type of negative stain.

Experiment 3 Results Tables
Table 3: Experiment 3 Staining Observations (5 points)

Stain used:

Crystal violet (primary stain) & Safranin (counterstain)

Observations:

Gram-positive appeared as purple and Gram-negative showed up as pink.

Experiment 3 Post-Lab Questions
1. What color are the Gram-positive bacteria after Gram staining? Gram-negative bacteria? (5 points)

Gram-positive bacteria appear as dark purple or blue due to retaining the primary dye (Crystal Violet) in the cell wall.

Gram-negative bacteria appear as red or pink due to decolorizing to accept the counterstain (Safranin).

2. What different characteristic(s) exist between the two groups that account for the different staining conditions? (5 points)

Gram-positive bacteria are stained purple, and gram-negative bacteria stain as pink. They are two distinct morphological groups of bacteria.

3. Why was the Gram iodine added to the Gram staining procedure? (5 points)

Gram iodine is added as a mordant to stabilize the crystal violet iodine complex so that the dye cannot be removed easily.

4. Why is a counterstain (safranin) added to the Gram staining procedure? (5 points)

A counterstain is used to help identify gram-negative bacteria. Gram-negative bacteria lose the crystal violet and stain red.

5. What are the advantages of performing a Gram stain vs. a simple stain for visualizing bacteria? (5 points)

Gram stain contains two or more different stains and can differentiate the species of bacteria into two main groups (gram-positive and gram-negative) by looking at the color of cells (pink or purple). Simple stain involves single stain and it is used to easily determine cell shape, size, and arrangement.

6. Using either a textbook or a reputable online resource, research some of the typical characteristics of bacteria, and discuss why it might be important for a researcher or a hospital technician to be able to differentiate between Gram-positive and Gram-negative bacteria. (5 points)

7. Did you experience any technical difficulties or atypical results during this experiment? If so, what happened, and how could you avoid these issues in the future? (5 points)nder
4 mL 1% Iodine-Potassium Iodide, IKI
5 mL Liquid Starch, C6H10O5
3 Pipettes
4 Rubber Bands (Small; contain latex, handle with gloves on if allergic)

Permanent Marker
*Stopwatch
*Water
*Scissors

*15.0 cm Dialysis Tubing

*You Must Provide
*Be sure to measure and cut only the length you need for this experiment. Reserve the remainder for later experiments.

Attention!

Do not allow the open end of the dialysis tubing to fall into the beaker. If it does, remove the tube and rinse thoroughly with water before refilling it with the starch/glucose solution and replacing it in the beaker.

Note:

If you make a mistake, the dialysis tubing can be rinsed and used again.

Dialysis tubing must be soaked in water before you will be able to open it up to create the dialysis “bag.” Follow these directions for this experiment:

1. Soak the tubing in a beaker of water for ten minutes.

2. Place the dialysis tubing between your thumb and forefinger, and rub the two digits together in a shearing manner. This motion should open up the “tube” so that you can fill it with the different solutions.

Procedure

1. Measure and pour 50 mL of water into a 100 mL beaker using the 100 mL graduated cylinder. Cut a piece of dialysis tubing 15.0 cm long. Submerge the dialysis tubing in the water for at least ten minutes.

2. Measure and pour 82 mL of water into a second 100 mL beaker using the 100 mL graduated cylinder. This is the beaker you will put the filled dialysis bag into in Step 9.

3. Make the glucose/sucrose mixture. Use a graduated pipette to add 5 mL of glucose solution to a third 100 mL beaker and label it “dialysis bag solution.” Use a different graduated pipette to add 5 mL of starch solution to the same beaker. Mix by pipetting the solution up and down six times.

4. Using the same pipette that you used to mix the dialysis bag solution, remove 2 mL of the dialysis bag solution and place it in a clean beaker. This sample will serve as your positive control for glucose and starch.

a. Dip one of the glucose test strips into the 2 mL of glucose/starch solution in the third beaker. After one minute has passed, record the final color of the glucose test strip in Table 3. This is your positive control for glucose.

b. Use a pipette to transfer approximately 0.5 mL of IKI into the 2 mL of glucose/starch solution into the third beaker. After one minute has passed, record the final color of the glucose/starch solution in the beaker in Table 3. This is your positive control for starch.

5. Using a clean pipette, remove 2 mL of water from the 82 mL of water you placed in a beaker in Step 2, and place it in a clean beaker. This sample will serve as your negative controls for glucose and starch.

a. Dip one of the glucose test strips into the 2 mL of water in the beaker. After one minute has passed, record the final color of the glucose test strip in Table 3. This is your negative control for glucose.

b. Use a pipette to transfer approximately 0.5 mL of IKI into the 2 mL in the beaker. After one minute has passed, record the final color of the water in the beaker in Table 3. This is your negative control for starch.

Note:The color results of these controls determine the indicator reagent key. You must use these results to interpret the rest of your results.

6. After at least ten minutes have passed, remove the dialysis tube and close one end by folding over 3.0 cm of one end (bottom). Fold it again and secure with a rubber band (use two rubber bands if necessary).

7. Test to make sure the closed end of the dialysis tube will not allow solution to leak out. Dry off the outside of the dialysis tube bag with a cloth or paper towel. Then, add a small amount of water to the bag and examine the rubber band seal for leakage. Be sure to remove the water from the inside of the bag before continuing.

Using the same pipette that was used to mix the solution in Step 3, transfer 8 mL of the dialysis bag solution to the prepared dialysis bag.
Figure 4: Step 9 reference.

Figure 4:Step 9 reference.

9. Place the filled dialysis bag in the 100 mL beaker filled with 80 mL of water, leaving the open end draped over the edge of the beaker as shown in Figure 4.

10.Allow the solution to sit for 60 minutes. Clean and dry all materials except the beaker holding the dialysis bag.

11.After the solution has diffused for 60 minutes, remove the dialysis bag from the beaker and empty the contents of the bag into a clean, dry beaker. Label the beaker “final dialysis bag solution.”

12.Test the final dialysis bag solution for the presence of glucose by dipping one glucose test strip into the dialysis bag. Wait one minute before reading the results of the test strip. Record your results for the presence of glucose in Table 4.

13.Test for the presence of starch by adding 2 mL IKI. After one minute has passed, record the final color in Table 4.

14.Use a pipette to transfer 8 mL of the water in the beaker to a clean beaker. Test the beaker water for the presence of glucose by dipping one glucose test strip into the beaker. Wait one minute before reading the results of the test strip, and record the results in Table 4.

15.Test for the presence of starch by adding 2 mL of IKI to the beaker water. Record the final color of the beaker solution in Table 4.

Table 3: Indicator Reagent Data

Indicator

Starch Positive
Control (Color)

Starch Negative
Control (Color)

Glucose Positive
Control (Color)

Glucose Negative
Control (Color)

Glucose Test Strip

n/a

n/a

IKI Solution

n/a

n/a

Table 4: Diffusion of Starch and Glucose Over Time

Indicator

Dialysis Bag After 60 Minutes

Beaker Water After 60 Minutes

IKI Solution

Glucose Test Strip

Post-Lab Questions

1. Why is it necessary to have positive and negative controls in this experiment?

2. Draw a diagram of the experimental set-up. Use arrows to depict the movement of each substance in the dialysis bag and the beaker.

3. Which substance(s) crossed the dialysis membrane? Support your response with data-based evidence.

4. Which molecules remained inside of the dialysis bag?

5. Did all of the molecules diffuse out of the bag into the beaker? Why or why not?

Experiment 1: Diffusion through a Liquid

In this experiment, you will observe the effect that different molecular weights have on the ability of dye to travel through a viscous medium.

Materials

1 60 mL Corn Syrup Bottle, C12H22O11
Red and Blue Dye Solutions (Blue molecular weight = 793 g/mole; red molecular weight = 496 g/mole)
(1) 9 cm Petri Dish (top and bottom halves)

Ruler
*Stopwatch
*Clear Tape

*You Must Provide

Procedure

1. Use clear tape to secure one-half of the petri dish (either the bottom or the top half) over a ruler. Make sure that you can read the measurement markings on the ruler through the petri dish. The dish should be positioned with the open end of the dish facing upwards.

Carefully fill the half of the petri dish with corn syrup until the entire surface is covered.
Develop a hypothesis regarding which color dye you believe will diffuse faster across the corn syrup and why. Record this in the post-lab questions.
Place a single drop of blue dye in the middle of the corn syrup. Note the position where the dye fell by reading the location of its outside edge on the ruler.
Record the location of the outside edge of the dye (the distance it has traveled) every ten seconds for a total of two minutes. Record your data in Table 1 and use your results to perform the calculations in Table 2.
Repeat the procedure using the red dye, the unused half of the petri dish, and fresh corn syrup.

Table 1: Rate of Diffusion in Corn Syrup

Time (sec)

Blue Dye

Red Dye

Time (sec)

Blue Dye

Red Dye

10

70

20

80

30

90

40

100

50

110

60

120

Table 2: Speed of Diffusion of Different Molecular Weight Dyes

Structure

Molecular Weight

Total Distance
Traveled (mm)

Speed of Diffusion
(mm/hr)*

Blue Dye

Red Dye

*Multiply the total distance diffused by 30 to get the hourly diffusion rate

Post-Lab Questions

Record your hypothesis from Step 3 here. Be sure to validate your predictions with scientific reasoning.

Which dye diffused the fastest?

Does the rate of diffusion correspond with the molecular weight of the dye?

Does the rate of diffusion change over time? Why or why not?

Examine the graph below. Does it match the data you recorded in Table 2? Explain why, or why not. Submit your own plot if necessary.

https://nuonline.neu.edu/bbcswebdav/pid-9451339-dt-content-rid-14232100_1/courses/BIO1101.90155.201714/BIO1101.90155.201714_ImportedContent_20160930044714/CourseRoot/html/lab006s001.html

https://nuonline.neu.edu/bbcswebdav/pid-9451340-dt-content-rid-14232401_1/courses/BIO1101.90155.201714/BIO1101.90155.201714_ImportedContent_20160930044714/CourseRoot/html/lab006s002.html

https://nuonline.neu.edu/bbcswebdav/pid-9451341-dt-content-rid-14232402_1/courses/BIO1101.90155.201714/BIO1101.90155.201714_ImportedContent_20160930044714/CourseRoot/html/lab006s003.html

 

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