Laboratory
4
Cell Structure and Function
(LM pages 43–56)
Time Estimate for Entire Lab: 2.5 hours
Special Requirements
1. Living material (order in advance for timely delivery): whole sheep blood
2. Fresh material (obtain locally close to time of use): potato
Seventh Edition Changes
This was lab 3 in the previous edition. In section 4.2 Crossing the Plasma Membrane, the diffusion exercises were revised. The experimental procedure Osmosis, using the thistle tube, was removed.
New or revised figures: 4.1 Human (animal) cell; 4.7 Enzymatic action
MATERIALS AND PREPARATIONS1
4.2
Crossing the Plasma Membrane (LM page 45)
Diffusion Through a Semisolid (LM page 46)
_____
petri dish (Carolina 74-1156A)
_____
gelatin powder (Carolina 86-4658) or agar powder (Carolina 84-2131) for 1.5% solution
_____
potassium permanganate (KMnO4) crystals (Carolina 88-4130)
_____
wax pencils
_____
rulers, plastic millimeter (preferably transparent)
Diffusion demonstration through gelatin or agar. (Note: Agar allows faster diffusion than gelatin.) Prepare
one dish per student group. At least a day ahead, prepare a 1.5% gelatin solution in a beaker or flask by dissolving 1.5 g of gelatin powder or agar in 100 ml of boiling water; stir thoroughly until dissolved. Allow to
cool until the glassware can be handled with a hot mitt. Fill a petri dish 3–5 mm deep with gelatin solution.
Put a lid on dish until cool. After cooling, store the dish in a refrigerator. After gelling, make a small depression in the center of the dish. Using forceps, drop a crystal of potassium permanganate into the depression.
Diffusion Through a Liquid (LM page 46)
_____
potassium permanganate (KMnO4) crystals (Carolina 88–4130)
_____
container, wide-mouth, screw-capped, shallow, for potassium permanganate crystals
_____
microspatulas (Carolina 70-2702) or forceps, dissecting fine-point, chrome (Carolina
62-4024)
_____
rulers, plastic millimeter
_____
petri dishes (one per student group)
_____
water
_____
white paper
Potassium permanganate. Only 1−2 crystals are needed per student group. While wearing gloves, dispense
several crystals of potassium permanganate into a shallow, wide-mouth, screw-top container appropriately
labeled. (Note: Potassium permanganate diffuses very quickly.)
Diffusion Through a Selectively Permeable Membrane (LM page 47)
_____
dialysis tubing, approximately 15 cm per setup (Carolina 68-4202)
_____
plastic droppers or Pasteur pipettes
_____
thread or rubber bands for tying off end of dialysis tubing
_____
rubber bands that fit snugly around brim of 250 ml beaker
_____
1% glucose solution
_____
1–2% starch solution
_____
beakers, 250 ml
1 Note: “Materials and Preparations” instructions are grouped by exercise. Some materials may be used in more than one exercise.
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_____
_____
_____
_____
_____
_____
_____
_____
rubber bands that fit snugly around brim of 250 ml beaker
water, distilled
iodine (IKI) solution
test tubes
test-tube rack
wax pencils
Benedict’s reagent (Carolina 84-7091, -7111)
boiling water bath
_____ hot plate
_____ boiling chips, pumice
_____ thermometer, Celsius
_____ beaker
_____ beaker clamps
_____ test-tube clamp
Glucose solution. Prepare as described in the instructions for Laboratory 3 (page 14).
Starch solution. Prepare as described in the instructions for Laboratory 3 (page 13).
Iodine (IKI) solution. Prepare as described in the instructions for Laboratory 2 (page 6).
Benedict’s reagent. Prepare as described in the instructions for Laboratory 3 (page 14).
Boiling water bath. Place a large beaker of water on a hot plate. Adjust the dial on the hot plate so that the
water is maintained at a gentle rolling boil during the experiment. Thermometers are optional since students
should know that boiling water is 100°C.
Tonicity in Potato Strips (LM page 49)
_____
potato, fresh
_____
rulers, plastic millimeter
_____
razor blades, single-edged
_____
wax pencils
_____
cutting board for potato
_____
10% sodium chloride (NaCl) in wash bottles
_____
test tubes and racks
_____
water
Tonicity in Red Blood Cells (LM pages 49-50)
_____
test tubes, screw-capped (Carolina 73-1509); or test tubes, Pyrex 16 mm × 150 mm
(Carolina 73-0014) with stoppers (below)
_____
stoppers, rubber laboratory, solid, size 1 (Carolina 71-2402); four per group
_____
sheep blood, pooled, citrated (Carolina 82-8950, -8954, -8960)
_____
water, distilled
_____
0.9% and 10% sodium chloride (NaCl) solutions (Carolina 88-8880)
_____
dropping bottles, or bottles with droppers
_____
whole blood demonstration (test tubes on display, optional)
_____
microscopes, compound light
Whole blood. Blood should not be human blood. Use any available animal blood, other than human, to
remove the risk of transmission of the HIV virus to students. Blood is shipped in iced, insulated containers
and should be stored in the refrigerator. If kept refrigerated, sheep blood may be stored for up to 2 weeks.
Prepare the test tubes as follows:
Tube 1:
5 ml 0.9% NaCl plus three drops of sheep blood
Tube 2:
5 ml 10% NaCl plus three drops of sheep blood
Tube 3:
5 ml 0.9% NaCl plus distilled water and three drops of sheep blood
Cap or stopper the tubes.
To prepare the NaCl solutions (50 ml per student group is sufficient for all procedures):
0.9% NaCl: Add 9 g of NaCl to 1 liter of distilled water.
10% NaCl: Add 100 g of NaCl to 1 liter of distilled water.
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Slides of whole blood (optional). Prepare a demonstration slide of the 0.9% sheep blood solution (Tube 1)
and the 10% sheep blood solution (Tube 2) for student observation.
4.3 pH and Cells (LM pages 51-52)
_____
pH 7 buffer (inorganic) solution (Carolina 84-9380, -9683)
_____
protein solution, buffered (e.g., albumin—Carolina 84-2250, -2252)
_____
pH paper (range pH 1–12) (Carolina 89-3930)
_____
rods, glass stirring (Carolina 71-1303 to -1311)
_____
0.1 N hydrochloric acid (HCl) (see Carolina Chemicals, Hydrochloric Acid)
_____
beakers, 50 ml (two for each group) (Carolina 71-7900)
_____
droppers
_____
water, distilled
pH 7 buffer. 50 ml per student group is sufficient. If you wish to make it yourself, combine 50 ml 0.1 M
potassium dihydrogen phosphate (1.36 g per 100 ml distilled water) with 29.1 ml 0.1 M NaOH (0.4 g per 100
ml distilled water). Dilute this mixture to 100 ml with distilled water.
Buffered protein (e.g., albumin) solution. 50 ml per student group should be sufficient. Mix 1 g of albumin
with 100 ml of pH 7.0 buffer (buffer may be purchased).
0.1 N HCl solution. Mix 0.83 ml of concentrated HCl with 100 ml of distilled water. Place in dropper
bottles.
Effect of pH on Enzyme Activity (LM pages 53-55)
_____
3% H2O2 (hydrogen peroxide) solution (Carolina 86-8120, -8122)
_____
sand
_____
potato, fresh, cubed
_____
mortar and pestle
_____
5 M HCl (from concentrated HCl, see Carolina Chemicals, Hydrochloric Acid)
_____
2 straight-sided beakers, 1 liter (Carolina 72-1213A)
_____
5 M NaOH (from 200 g NaOH pellets, Carolina 88-9470)
_____
stirrer plate (see Carolina Apparatus, Laboratory Equipment and Supplies)
_____
magnetic spinbars (Carolina 70-1080, -1085)
5 M HCl. CAUTION—This solution will get HOT. Add 400 ml of distilled water to a 1-liter graduated
beaker. Place beaker with magnetic spinbar on a stirring plate. While stirring, slowly pour in 416 ml concentrated HCl. Add distilled water to bring the volume up to 1,000 ml.
5 M NaOH. CAUTION—This solution will get very HOT. In a 1-liter beaker with a magnetic spinbar, gradually add a total of 200 grams of NaOH pellets to 750 ml of distilled water, allowing the heat to dissipate
between additions of NaOH. After the solution cools, add distilled water to bring the volume up to 1,000 ml.
EXERCISE QUESTIONS
4.1 Human (Animal) Cell Structure (LM pages 44-45)
With the help of Figure 4.1 identify the following structures in a cell model, and give a function of each
from Table 4.1.
Structure
Function
Structure
Function
Plasma membrane
Vacuole and vesicle
Nucleus
Selective passage of molecules
into and out of cell
Storage of genetic information
Lysosome
Storage and transport of
substances
Intracellular digestion
Nucleolus
Ribosomal formation
Mitochondrion
Cellular respiration
Ribosome
Protein synthesis
Cytoskeleton
Shape of cell and
movement of its parts
Endoplasmic reticulum (ER)
Synthesis and/or modification of
proteins and other substances,
and transport by vesicle formation
Cilia and flagella
Movement of cell
Centriole
Formation of basal bodies
Rough
Protein synthesis
Smooth
Various functions; lipid synthesis
in some cells
Golgi apparatus
Processing, packaging, and
distributing molecules
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4.2 Crossing the Plasma Membrane (LM page 45)
Diffusion (LM page 45)
Experimental Procedure: Diffusion (LM page 46)
Table 4.2 Speed of Diffusion
Diffusion data will depend on room temperature, gelatin consistency, and the molecular weight of the dye used.
Conclusions (LM page 47)
• In which experiment was diffusion the fastest? diffusion through a liquid
• What accounts for the difference in speed? All molecules are in constant, random motion. Molecules in a
liquid state move more rapidly than those in a solid; thus, diffusion is faster through a liquid.
Diffusion Through a Selectively Permeable Membrane (LM page 47)
Table 4.3 Diffusion Through a Selectively Permeable Membrane
Bag
At Start of Experiment
At End of Experiment
Contents
Color
Color
Benedict’s Test
Conclusion
Glucose
Starch
Whitish
Black
Negative (–)
Glucose diffused from
bag to beaker.
Yellowish
Yellowish
Positive (+)
Iodine diffused from
beaker to bag.
Beaker Water
Iodine
Conclusions (LM page 48)
• Which solute did not diffuse across the dialysis membrane from the bag to the beaker? starch
Explain. Starch molecules are too large to diffuse across the dialysis membrane.
Osmosis and Tonicity (LM page 48)
Experimental Procedure: Tonicity in Potato Strips (LM page 49)
5. Which tube has the limp potato strip? tube 2 Why did water diffuse out of the potato strip in this
tube? The solution in tube 2 was hypertonic. Which tube has the stiff potato strip? tube 1 Why did
water diffuse into the potato strip in this tube? The solution in tube 1 was hypotonic.
Red Blood Cells (Animal Cells) (LM page 49-50)
Table 4.4 Effect of Tonicity on Red Blood Cells
Concentration (NaCl)
Tonicity
Effect on Cells
Explanation
0.9%
Isotonic
None
Normal tonicity
Higher than 0.9%
Hypertonic
Crenation
Cells have lost water.
Lower than 0.9%
Hypotonic
Swell to bursting (hemolysis)
Cells have gained water.
Table 4.5 Tonicity and Print Visibility
Tube
Tonicity
Print Visibility
Explanation
1
Isotonic
No
Cells are intact.
2
Hypertonic
No
Cells are intact.
3
Hypotonic
Yes
Cells have burst.
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4.3 pH and Cells (LM pages 51-55)
Why are cells and organisms buffered? to maintain pH of the cells
Experimental Procedure: pH and Cells (LM page 51)
Table 4.6 pH and Cells
Tube
Buffered?(Yes or No)
pH Before Acid
pH After Acid
Explanation
1 Water*
No
6–6.5
2–3
Not buffered
2 Inorganic buffer*
Yes
7
7
Buffered
3 Simulated cytoplasm*
Yes
7
7
Buffered
*These results are based on 1 ml of test solution.
Experimental Procedure: Buffer Strength (LM page 52)
Table 4.7 Drops of HCl and pH Values
Data will vary but will show that the buffers will hold the pH constant until an excess of HCl is added, at which time
the pH will be reduced considerably.
Graph Graphed data will vary. The graph should be labeled appropriately, as shown in the lab manual. The general
curve will show a steady pH until the buffers are overwhelmed by an excess of acid, at which time the pH will
begin to decrease with additional acid.
Conclusion (LM page 52)
• Do the graphs have a similar pattern? yes Explain. An inorganic buffer and an organic buffer behave similarly, and they can be overwhelmed by an excess of acid.
Effect of pH on Enzyme Activity (LM page 53)
Experimental Procedure: Catalase Activity (LM page 54)
Table 4.9 Catalase Experiment
Tube
Contents
Bubbling
Explanation
1
Hydrogen peroxide
Sand
0 (no bubbling)
This is the control tube.
2
Hydrogen peroxide
Potato, cubed
+ (moderate bubbling)
Catalase is present in the potato.
3
Hydrogen peroxide
Potato, macerated
++ to +++ (good to
very good bubbling)
Macerating the potato breaks open
cells, thereby making more enzyme
available.
Experimental Procedure: Effect of pH on Catalase Activity (LM page 55)
Table 4.10 Effect of pH on Catalase Activity
Tube
Contents
Bubbling
Explanation
1
Distilled water
Potato, macerated
Hydrogen peroxide
++ (good bubbling)
Neutral pH is preferred.
2
Hydrochloric acid
Potato, macerated
Hydrogen peroxide
0 (no bubbling)
Acid pH is not preferred.
3
NaOH
Potato, macerated
Hydrogen peroxide
0 (no bubbling)
Basic pH is not preferred
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Conclusion (LM page 55)
• What happens to an enzyme if the pH is too far removed from its preferred pH? The enzyme is
denatured and it loses its normal shape.
LABORATORY REVIEW 4 (LM page 56)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
What is the function of rough endoplasmic reticulum? protein synthesis
Which organelle carries on intracellular digestion? lysosome
What is the function of the nucleus? storage of genetic information
What term is used to describe the movement of molecules from an area of higher concentration to
an area of lower concentration? diffusion
What is the name for the movement of water across a selectively permeable membrane? osmosis
Is 10% NaCl isotonic, hypertonic, or hypotonic to red blood cells? hypertonic
What appearance will red blood cells have when they are placed in 0.0009% NaCl? swollen to
bursting
How does water move when cells are placed in a hypertonic solution? out of cells into the solution
If acid is added to water, does the pH increase or decrease? decrease
What type substance protects solutions and cells from undergoing drastic pH changes? buffer
In general, what does the wrong pH do to the shape of an enzyme? changes the shape
If an enzyme reaction is exposed to an unfavorable pH, what happens to the speed of the
reaction? it slows or stops
What is a pH of 7 called? neutral pH
Thought Questions
14. If a dialysis bag filled with water is placed in a starch solution, what do you predict will happen
to the weight of the bag over time? The bag will lose weight. Why? Water will diffuse out of the bag and
enter the starch solution.
15. Distinguish between rough endoplasmic reticulum and smooth endoplasmic reticulum on the
basis of structure and function.
a. Structure: Rough endoplasmic reticulum has ribosomes; smooth endoplasmic reticulum does not.
b. Function: Rough endoplasmic reticulum is the site of protein synthesis; smooth endoplasmic reticulum has
various functions.