cell transport lab 6 - my anatomy and physiology mentor · pdf filelab 6 cell transport | 37...

OBJECTIVES

● To identify, demonstrate, and explain the various processes utilized for transport of substances inthe cell.

● To understand and explain the factors influencing movement of substances into and out of the cell.

● To understand and explain various tonicities and their effect on the cell.

● To demonstrate and explain the factors that affect the rate of diffusion.

DIFFUSION● Diffusion is defined as movement of solute from high to low concentration. The rate at which

diffusion takes place is dependent upon three main factors.

● Conduct the following exercises to determine the factors that affect the rate of diffusion.

PART A—EFFECT OF TEMPERATURE ON THE RATE OF DIFFUSION1. Obtain two equal sized test tubes. Using tape, label one tube “KMnO4” and the other “CuSO4.”

2. Fill both tubes with equal amounts of water.

3. Allow the water to become still.

4. Before starting, measure equal amounts of both salts in the small dropper bottles provided andrecord the amounts in the table below next to each.

5. Start the timer as soon as you add the salts to the center of each test tube.

6. Observe the distance the salt has moved in each beaker and record those observations at each timeinterval on the table below.

7. Observe how the dye spreads every 15 s for the first 30 s, then 1 min thereafter for 3 min. Recordthese observations in the second table.

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Cell Transport

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36 | Lab 6 Cell Transport

8. When you are DONE with this experiment:

● DISPOSE of both solutions in the waste container

● WASH the beakers with soap and water and then DRY with paper towel

QUESTIONS

1. How did the diffusion rate in each of the beakers compare? Which one diffused the fastest?

2. How long did it take for the dye to completely diffuse in the hot water? In the cold water?

3. What effect does temperature have on the rate of diffusion?

PART B—EFFECT OF PARTICLE SIZE ON THE RATE OF DIFFUSION1. Obtain an agar plate with 1 cm wells scooped out of the middle.

2. Using a marker, label the lid of the agar plate well #1, well #2, well #3, and well #4.3. Fill the wells as follows:

● Well #1 with 0.02 M Alizarin Yellow (MW = 287.23 g/mol)

● Well #2 with 0.02 M Janus Green (MW = 511.19 g/mol)

● Well #3 with 0.02 M Congo Red (MW = 696.67 g/mol)

● Well #4 with 0.02 M Aniline Blue (MW = 737.74 g/mol)

4. Replace the lid and place the agar plate on a sheet of white paper.

KMnO4 CuSO4

TIME ObSErvaTIONS ObSErvaTIONS15 s 0.5 mm 2.0 mm30 s 1.3 mm 3.8 mm1 min 2.1 mm 5.7 mm2 min 2.9 mm 6.9 mm3 min 3.7 mm 8.5 mm

TablE a—Diffusion results for Salts in Water

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KMnO4 diffused at a much slower rate than CuSO4. CuSO4 diffused fastest.

It took 3 minutes to completely diffuse on the hot water. The salt in the cold water did not even diffuse through half of the distance of the salt in the hot water.

It is clear that temperature greatly increases the rate of diffusion.

Lab 6 Cell Transport | 37

5. Measure with a ruler the distance (in mm) that each dye has diffused in 10 min intervals for thenext 50 min. Record the results in the table below.

TIME (MIN)

WEll 1(0.02 M Alizarin

Yellow) Diffusion Distance (mm)

WEll 2(0.02 M Janus Green)

Diffusion Distance (mm)

WEll 3(0.02 M Congo Red) Diffusion Distance

(mm)

WEll 4(0.02 M Aniline Blue Diffusion Distance

(mm)10

20

30

40

50

TablE b—Diffusion results for 0.02 Mdyes in agar


● Discard the agar plate in the BIOHAZARD WASTE bag.

QUESTIONS

1. All four wells contained dyes with a concentration of 0.02 M. Which one had the fastest rate ofdiffusion? Why? Explain the results.

2. What factor caused the difference in the rates of diffusion in all the wells?

PART C—EFFECT OF CONCENTRATION ON THE RATE OF DIFFUSION1. Obtain an agar plate with 1 cm wells scooped out of the middle.

2. Using a marker, label the lid of the agar plate well #1, well #2, and well #3.3. Fill the wells as follows:

● Well #1 with 0.01 M Potassium permanganate, KMnO4

● Well #2 with 0.05 M KMnO4

● Well #3 with 0.10 M KMnO4

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2.0 mm 1.5 mm 1.0 mm 0.5 mm

4.0 mm

6.0 mm

8.0 mm

10.0 mm

3.0 mm

4.5 mm

6.0 mm

7.5 mm

2.0 mm

3.0 mm

4.0 mm

5.0 mm

1.0 mm

1.5 mm

2.0 mm

2.5 mm

The dye with the fastest rate of diffusion was the yellow dye. The reason that the yellow dye diffused the fastest was because it contained the smallest particles within any of the dyes as seen in its molecular weight. The larger the size of the particles in a solution, the slower the rate of diffusion.

The size of the solutes that were within the dye solution caused the difference in the rate of diffusion.


TIME (MIN)WEll 1

(0.01 M KMnO4 )Diffusion Distance (mm)

WEll 2(0.05 M KMnO4 )


WEll 3(0.10 M KMnO4 )


10

20

30

40

50

TablE C—Diffusion results for Different Concentrations of KMnO4 in agar

4. Measure with a ruler the distance in mm that each concentration of KMnO4 has diffusedin 10 min intervals for the next 50 min. Record the results in the table below.

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5. When you are DONE with this experiment:● Discard the agar plate in the BIOHAZARD WASTE bag.

QUESTIONS

1. How did the rates of diffusion compare between wells? Which one had the fastest rate of diffusion?Why? Explain the results.

2. What factor caused the difference in the rate of diffusion between the wells?

3. Name 2 places in your body where each of the following occur:

a. Diffusion of a solute into a liquid

b. Diffusion of a gas into a liquid

c. Diffusion of a solute or liquid into a colloid

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1) Digestive system (mouth); 2)Urinary system (kidneys)

1) Respiratory system (lungs); 2) Nervous system (blood-brain barrier)

1) Cells (phagocytosis of bacteria); 2) Cells (pinocytosis)

2 mm

4 mm

6 mm

8 mm

10 mm

1 mm

2 mm

3 mm

4 mm

5 mm

0.5 mm

1 mm

1.5 mm

2 mm

2.5 mm

The rate of diffusion increased as the concentration of the solution increased (directly proportional). The 0.1 M solution was the fastest because it was the most concentrated solution. The greater the concentration of a solution, the more solutes present and the faster the rate of diffusion.

The amount of solutes dissolved in the solution (concentration).


OSMOSISPART A—THISTLE TUBE OSMOMETER1. Set up the osmometer as follows:

a. Cut a 4-in. piece of dialysis membrane tubing and soak it in a dish-containing distilled water (dH2O) until it is soft and pliable.

b. Open the tubing with your fingers.

c. With scissors, cut dialysis membrane along one edge in order to make ONE longpiece of membrane.

d. Have one person fill the bulb of the tube with molasses while the other personcloses off the stem of the thistle tube with his/her finger.

e. Place the wet dialysis membrane over the bulb of the thistle tube and secure it TIGHTLY in placeby winding rubber bands over the bulb several times. Make sure that the syrup will not leakthrough the sides!

f. Invert the tube and allow the syrup in the stem of the thistle tube to settle in the bulb beforeMARKING the level of the syrup with a grease pencil.

g. Fill the plastic cup half full with distilled water and lower the thistle tube bulb in the cup. (Seediagram)

2. Observe the level of the molasses for the next 30 to 40 min and write your observations below.

● Observations:


● Dismantle the osmometer and thoroughly RINSE the thistle tube.

● Wash and dry the plastic cup.

● DISCARD dialysis membrane in trash bin.

QUESTIONS

1. What happened to the level of the molasses?

2. Explain what caused the movement of the molasses. Be specific.

3. Why didn’t the molasses leave the thistle tube and enter the water in the beaker?

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It increased up the thistle tube at a specific rate.

Osmosis occurred (diffusion of water across a selectively semi-permeable membrane). The osmotic pressure (amount of water) outside of the tube was much greater than the osmotic pressure (amount of water) inside the molasses and this caused water to move through the osmotic membrane from high to low amount.

Molasses is a large carbohydrate and the osmotic membrane was selectively semi-permeable to only water.


4. If the thistle tube were a cell and the water in the beaker were the solution surrounding the cell:

a. Is the solution isotonic, hypotonic, or hypertonic? Explain.

b. Are there more solutes inside or outside the cell?

c. How long would water move into the cell? What might result from this?

5. Give some examples of where osmosis is taking place in your body.

6. Is osmosis diffusion? Explain.

PART B—OSMOSIS IN POTATO CELLS1. Fill one culture dish with distilled water (dH2O) and the other with 10% NaCl.2. Using a cork borer, make SIX plugs of potato. Three plugs will be

soaked in distilled water; the other three will be soaked in 10%NaCl.

3. Measure the original volume of each set of three plugs as follows:

a. Place 25 mL of dH2O in BOTH 50 mL graduated cylinders.[This is the initial water volume]

b. In the first cylinder, put the first set of three potato plugs andrecord the final volume of water in the table below. [This setof plugs will be soaked in the dish-containing dH2O]

c. Determine the ORIGINAL volume of these plugs by subtract-ing the initial volume of water from the final. Record thisvolume in the table below.

d. Repeat this procedure for the other set of three potato plugs.[This set of plugs will be soaked in the dish- containing 10%NaCl]

4. After determining the volume of the plugs, place EACH set ofplugs into their designated solution and allow soaking for 30min.

5. After 30 min, remove the potato plugs with the forceps providedand observe the texture, color, and flexibility of the plugs in eachset. Record these observations in the data table.

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Hypotonic- because the water is less concentrated than the molasses inside the cell.

There are more solutes inside the cell.

Water would continue to move into the cell until the concentration of solutes is equal on either side of the membrane when equillibrium is established.

In our cells and in our blood stream.

Yes; it is the diffusion of water from where there is a lot of water to where there is less water down its gradient.


6. Re-measure the volume of each set of plugs and record the results below. If the mass increased overthe 30 min, place a (+) in the data table next to the amount changed. If the mass decreased, placea (−) in the space.


● WASH glassware with soap and water and then DRY the outside surfaces with paper towel.

OSMOSIS IN POTaTO CEllS

Distilled Water (ml) 10% NaCl (ml)

1. Volume of H2O before adding plugs 50 mL 50 mL

2. Volume of H2O after adding plugs

3. Original Volume of Potatoes(Volume in #2—Volume in #1)

4. Volume of H2O 30 min later(2nd measurement) before addingplugs

50 mL 50 mL

5. Volume of H2O after adding plugs(2nd measurement) after 30 min inSolution

6. Volume change (+) or (−) (negativechange represents volume losswhile positive change representsvolume gain) (Volume in #5—Volume in #4)

7. Observations

QUESTIONS

1. Which group of plugs gained volume? Explain.

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(+) (-)

The plugs placed into water gained volume because there was less water (lower osmotic pressure)inside the cell compared to the solution it was placed in. This established a concentration gradient and caused diffusion of the water through the process of osmosis.

SEE RESULTS

SEE RESULTS


2. Which group of plugs lost volume? Explain.

3. What type of solution was the 10% salt solution? The distilled water?

4. How did the rigidity of the plugs in the two solutions compare? Why the difference?

5. In order to maintain the normal osmolarity of a potato cell, in what type of solution would needto put the potatoes?

PART C—OSMOSIS IN HUMAN CELLS1. Clean your finger with an alcohol pad. Let it dry before you prick your finger.

2. Prick your finger with the lancet at the SIDES of the finger.

3. Prepare THREE blood slides as follows:

a. Slide 1: Add one drop of distilled water + one drop of blood.

b. Slide 2: Add one drop of 0.9% NaCl solution + one drop of blood.

c. Slide 3: Add one drop of 5% NaCl solution + one drop of blood.

4. Cover each slide with a cover slip and observe under the microscope on high power.

5. Draw and record your observations below.

SlIDE 1 [DISTIllED H2O] SlIDE 2 [0.9% NaCl] SlIDE 3 [5% NaCl]

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The plugs that were placed into the salt solution lost volume because of osmosis of water

HYPERTONIC HYPOTONIC

The plugs placed into the hypotonic (water) solution were more rigid than the plugs placed into the hypertonic(10% salt) solution because the osmotic pressure inside of the plugs versus the surrounding solution caused osmosis to occur of water from where there was more water to where there was less water.

In order to maintain the osmolarity of the plugs, they would need to be placed in an isotonic solution


6. IMPORTANT SAFETY DISOSAL INSTRUCTIONS:

● DISCARD the used blood lancets in the REDSharps Container.

● DISCARD the used slides in the BiohazardousDisposal Pouch.

● DISCARD the used alcohol pads and anyother materials containing blood in the largereceptacle.

QUESTIONS

1. Which slide contained cells in a hypertonic solution? Hypotonic? Isotonic?

2. Describe and explain the cause of the appearance of the cells in each slide.

3. What is a physiological saline solution?

4. Define the following terms:

a. crenation—

b. hemolysis—

PART D—DIALYSIS1. Cut approximately 15 cm (6-in.) of dialysis tubing and soak in dishpan filled

with distilled water to soften.

2. Remove the tubing from the water; fold over one end and tie off with string.

3. Slip the stem of a funnel into the open end of the bag and fill the bagapproximately half full with the solution labeled “Dialysis Solution.”

Contents of test solution in dialysis tubing:

● Water ● Glucose ● NaCl

● Albumin (protein) ● Starch

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Slide 3 Slide 1 Slide 2

An isotonic solution that contains 0.9 % NaCl solution (like blood plasma) and contains cells with the same concentration of NaCl inside of them (like red blood cells or erythrocytes).

cell shrinking

rupturing of blood cells


4. Remove the funnel; fold and tie off the open end of the bag.

5. Place the dialysis tube “sausage” into a beaker of distilled water and let sit for 30 min.

6. After 30 min, test the water in the beaker for the following substances to determine if they have leftthe tubing and entered the water. Record your results in the table.

a. Test for NaCl

i. Place several milliliter of water from the beaker in a test tube.

ii. Add several drops of silver nitrate solution (AgNO3).

iii. Formation of a white, cloudy precipitate indicates the presence of NaCl.

b. Test for Glucose

i. Place several mL of water from the beaker in a test tube.

ii. Add a squirt or 10 drops of Benedict’s solution to the tube.

iii. Warm the tube in a hot water bath for 5 to 10 minutes.

iv. Look for a color change. Any change to green, yellow, orange or red indicates the presenceof glucose.

c. Test for Starch


ii. Add several drops of iodine solution.

iii. A bluish or purplish-black color indicates the presence of starch.

d. Test for Albumin (protein)


ii. Carefully add several drops of nitric acid (HNO3) to the tube.

iii. Formation of a white, cloudy precipitate indicates the presence ofprotein.

rESUlTS OF TESTS FOr SUbSTaNCES IN bEaKEr

Test for Presence of

Substance added

Observations following Test

Substance Present? (+) or (–)

NaCl Silver nitrate

Glucose

Starch

Albumin

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white, cloudy precipitate forms (+)

Benedicts solution

Nitric acid

Iodine

blue to green color change in hot waterbath

(+)

(-)

no white cloudy ppt formed (-)

no visible color change to purple



● WASH ALL glassware with soap and water and then DRY the outside surfaces withpaper towel.

● INVERT test tubes in the rack and allow to air dry.

QUESTIONS

1. Define dialysis—

2. Which of the four substances left the dialysis tube and entered the water?

3. Why did some substances get through the membrane while others didn’t?

4. What physical characteristic of albumin affected its ability to pass through the membrane?

5. When solutes move, what usually follows?

FILTRATIONPART A—FILTRATION OF AN UNKNOWN SOLUTION1. Assemble the filtration apparatus shown on diagram.

2. Fold a piece of filter paper per diagram.

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The diffusion of small solutes and water ("solutes suck") across a selectively semi-permeable membrane.

NaCl and Glucose

Because of their size and the permeability of the membrane that they were moving through.

It is the size of it since it is a large protein made of more than 50 amino acids linked together as a polypeptide chain.

"Solutes suck" water with them to maintain balance if possible.


3. Open filter paper in a cone and place it inside the funnel.

4. Fill the funnel with the solution labeled “Filtration Solution” to determine what will pass throughthe filtration membrane.

5. If the filter paper gets clogged, place a new piece of filter paper in the funnel. You might have torepeat this process a few times.

6. Continue adding the solution to the filter until there is enough filtrate in the beaker to fill fourseparate test tubes about 1 in. high.

7. Use one test tube of filtrate to test for each of the following substances.

a. Test for Salt

i. See Part D 6a (dialysis experiment) for the procedure to test for NaCl. Record the results inthe table.

b. Test for Starch

i. See Part D 6c (dialysis experiment) for the procedure to test for starch. Record the resultsbelow.

c. Test for Charcoal

i. Observe the filtrate in the test tube. Any gray or black particles indicate presence of charcoal.

ii. Indicate that the charcoal passed through the filter.

iii. Record the results below.

d. Test for Copper II Sulfate (CuSO4)

i. Observe the filtrate in the test tube for a bluish or aqua color.

ii. A blue color indicates that copper sulfate has passed through the filter.

iii. Record the results below.

RESULTS OF FILTRATION TESTS

TestSubstance Present? (+) or (–)

NaCl

Starch

Charcoal

Copper II sulfate

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(+)

(-)

(-)

(+)



● WASH ALL glassware with soap and water and then DRY the outside surfaces witha paper towel.

● INVERT test tubes in the rack and allow to air dry.

QUESTIONS

1. Name three things that will determine whether a substance will pass through a filtration membrane.

2. In a closed system, what force is necessary for filtration to take place?

3. How could you increase this force?

4. Where does filtration take place in the body?

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1) Membrane permeability2) Size of the solutes 3) Amount of hydrostatic pressure vs. resistant osmotic pressure

Hydrostatic pressure must overcome the resistant osmotic pressure.

Add more fluid (increase the pressure )

In the glomerulus (filtration units) of the kidney in the Urinary system.

Michael Cryder

Sticky Note

Accepted set by Michael Cryder

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(of glucose)

cell transport lab 6 - my anatomy and physiology mentor · pdf filelab 6 cell transport | 37...

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