Cells and Their Membranes

Cells and Their Membranes

Cells, small membrane-bound compartments of chemicals and water, are truly remarkable. All organisms consist of one or more cells, thus cells are the fundamental units of life. All cells arise from the division of pre-existing cells. In complicated organisms, such as ourselves, intricate systems of communication link together specialized groups of cells performing various functions. The plasma membrane that surrounds each cell creates a division between the living (biotic), and nonliving (abiotic) worlds. In this laboratory you will learn the structure and function of cells and their membranes.

Objectives

When you have finished this lab, you will be able to:

describe the processes of diffusion and osmosis.
predict the effect of isotonic, hypertonic, and hypotonic solutions on plant and animal cells.
recognize cell structures in living cells, models, and electron micrographs.
distinguish between prokaryotic and eukaryotic cells.
distinguish between plant and animal cells.
define all boldface terms.

How Small Molecules Enter and Leave Cells

Diffusion is the movement of molecules from an area of greater concentration to an area of lesser concentration. To demonstrate the process of diffusion, your instructor has prepared two petri dishes of agar through which certain ions will diffuse (see Figure 1). Four holes (A, B, C, and D) are filled with equal concentrations of the following solutions:

A:silver nitrateAgNO3

B:sodium chlorideNaCl

C:potassium bromideKBr

D:potassium ferricyanideK3Fe(CN)6

One petri dish will be kept at room temperature while the other will be refrigerated.

Figure 1. Petri dish prepared to demonstrate diffusion of ions through a gel.

The positive Ag+ ion will react with the negative Cl-, Br-, and [Fe(CN)6]-3 ions resulting in a colored combination (a chemical precipitate.) At the end of the experiment record the distance each ion moved in mm. Measure from the edge of the filled holes to the center of the colored band. Record your data in Table 1 below. As you might expect, the larger an ion is (meaning that it has a higher molecular weight, MW), the slower it will diffuse through the agar.

Given the MW of the ions in Table 1, predict which ion should move the fastest.______

Which ion should move the slowest? ______

Did your results agree with your predictions? ______

What effect did cold temperatures have on diffusion? ______

Table 1. Results of Diffusion of Liquids in Agar

Ion / Weight / Reaction / Band Color / Distance Traveled (mm)

Spring 2006

Ag+ / 108 / ----- / ------/ Warmer / Cooler
Cl- / 35 / AgCl

Br-

/ 80 / AgBr
[Fe(CN)6]-3 / 212 / Ag3Fe(CN)6

Spring 2006

Osmosis:

Osmosis is the diffusion of water across a selectively permeable membrane. A selectively permeable membrane,like the cell's plasma membrane, permits some substances through but not others. To demonstrate osmosis, you will perform only one of the following two experiments.

Option 1. Artificial cells.

In this experiment you will use dialysis tubing to simulate the selectively permeable membrane of an ‘artificial cell’. Dialysis tubing permits the passage of water but obstructs larger molecules like sucrose (table sugar).

Procedure:

Obtain 4 pieces of dialysis tubing that have been soaked in distilled water (dH20).
To form a dialysis bag, fold over one end of the tube and tie it tightly with a string. See Figure 2. Any leakage will spoil your results!

Figure 2. Procedures for filling dialysis bags.

Attach one tag labeled #1 to #4 at the end of each tube.
Slip the open end of the bags over the bottom of a funnel and fill the bags with the following solutions. Use a clean graduated cylinder to measure the amounts. See Figure 2C.

Bag / Contents
#1 / 15 mL of dH2O
#2 / 15 mL of 10% sucrose
#3 / 15 mL of 40% sucrose
#4 / 15 mL of dH2O

Force the excess air out of each bag by gently squeezing the bottom end of each bag. Be careful not to squeeze out any of the liquid!
Fold over the open end of the bag and tie it securely with a piece of string.
Rinse the filled bag in dH2O and gently blot off any excess water with paper toweling.
Weigh each bag to the nearest 0.01 g. and record the weights in the column labeled “O min” in Table 2.
Label 4 beakers with a wax pencil. Fill beakers #1 - #3 with 400 mL of dH2O; fill beaker #4 with 400 mL of 40 % sucrose solution.
Place each bag in the correspondingly numbered beaker and set your timer for 40 minutes. Proceed with the next section and return here for the rest of the directions after your timer goes off.

……………………………………………………………………………………………

After 40 minutes remove each bag from its beaker, blot off the excess fluid, and weigh each bag. Record the weights in the “40 min” column in Table 2.
Calculate the % change in the weight of each bag using the following equation:

% weight change = weight at 40 min – weight at 0 min

weight at 0 min

Table 2. Change in weight of dialysis bags as a result of osmosis.
Bag # / Bag Contents / Beaker Contents / Bag Weight (g)
0 min / 40 min / + % wt. change
1 / dH2O /

dH2O

2 / 10% sucrose / dH2O
3 / 40% sucrose / dH2O
4 / dH2O / 40% sucrose

Was the direction of the net movement of water into or out of . . .

Bag 1? ______Why? ______

Bag 2? ______Why? ______

Bag 3? ______Why? ______

Bag 4? ______Why? ______Which bag gained the most weight? ______Why? ______

Option 2. Eggs as model cells.

You will be given 4 chicken eggs from which the shell has been dissolved away. In this state, they are quite rubbery, but you don’t want to try bouncing them! What holds them together is one remaining membrane that is fairly tough, yet selectively permeable. We will use the eggs as models of large “cells”. The eggs have been stored in a solution that has the same concentration as the contents of the egg.

Procedure

Rinse the egg in dH2O and gently blot off any excess water with paper toweling.
Weigh each egg to the nearest 0.01 g. and record the weights in the column labeled “O min” in Table 3.
Label 4 beakers. Fill them with 400 mL of the following solutions:

Beaker 1:dH2OBeaker 3:20% sucrose

Beaker 2:10% sucroseBeaker 4:30% sucrose

At the same time place each egg in the correspondingly numbered beaker and set your timer for 15 minutes. Proceed with the next section and return for the rest of the directions after your timer goes off.

……………………………………………………………………………………………

After 15 minutes remove each bag from its beaker, blot off the excess fluid, and weigh each egg. Record the weights in the “15 min” column in Table 3.
Record the weights of the eggs 30 and 60 minutes from the time the eggs were immersed.
Calculate the % change in the weight of each egg using the following equation:

% weight change = weight at 60 min – weight at 0 min

weight at 0 min

Table 3. Change in weight of eggs as a result of osmosis
Beaker / Beaker Contents / Egg Weight (g)

Was the direction of the net movement of water into or out of . . .

Egg 1? ______Why? ______

Egg 2? ______Why?______

Egg 3? ______Why? ______

Egg 4? ______Why? ______

Which egg gained the most weight? ______

Why? ______

What would you expect to happen if an egg were put into a fifth breaker containing an 80% sucrose solution? ______

Osmosis in living cells:

Tonicity refers to the concentration of dissolved molecules in water compared to the concentration of another solution. A solution which has a lower concentration of dissolved substances than those inside the cell is said to be hypotonic compared to the cell. If a cell is placed in a hypotonic solution water passes through the plasma membrane into the cell. This movement of water may cause the cell to swell and burst. If the concentrations of dissolved substances are equal, the solutions are said to be isotonic to each other, and a cell would maintain its normal appearance. In hypertonic solutions, where the concentration of dissolved substances is greater outside than inside the cell, cells lose water and shrivel.

Procedure

Elodea Cells:

Remove two young leaves from the top of an Elodea plant.
Place the leaves on separate slides. Place a drop of distilled water on one leaf and a drop of 10% NaCl on the other. Place a coverslip on both.
Observe the leaf mounted in distilled water under high power. Draw one cell as it appears in the space below labeled hypotonic solution. Note that the cell is swollen or turgid, but the rigid cell wall has prevented it from bursting. If possible locate the following structures then draw and label them: cell wall, chloroplasts (small green structures), and the central vacuole.
Observe the leaf mounted in 10% NaCl using high power. Draw one affected cell as it appears in the space below labeled hypertonic. Label the same structures if possible. Note the shrunken appearance of the cell. This plasmolysis is due to water loss.

Where do the chloroplasts appear in the Elodea cell when it is placed in an:

hypotonic solution? ______

hypertonic solution? ______

Elodea leaf cells:

Hypotonic solution / Isotonic Solution / Hypertonic solution

Red Blood Cells

(Modified from Perry, J. and D. Morton. 1987. Laboratory manual for Starr & Taggart’s Biology, The Unity and Diversity of Life.)

Animal cells lack the rigid cell wall found in plant cells. Consequently when animal cells are placed in a hypotonic solution the swelling breaks the plasma membrane and lysis, or bursting, occurs. The cells of your body live in an environment where the internal concentration of salt is about 0.9%. This concentration is known as a normal saline solution.

Red blood cells will be used in this procedure to demonstrate the effects of osmosis on animal cells.

Procedure:

Obtain 3 clean screw-cap test tubes and label them 1, 2 and 3. Fill each tube as follows:

Tube 125 mL of normal saline solution (0.9%)

Tube 225 mL of 10% salt solution

Tube 325 mL of dH20.

Place 2-3 drops of animal blood into each tube trying not to get any on the

sides of the tube.

Replace the caps and mix the contents of each tube by inverting it gently

several times.

Hold each tube flat against the printed page of this lab handout.
Record your observations on Table 3 under the column “Print Visible?” Only if the blood cells have burst (hemolyzed) should you be able to read the print.
Label 3 clean microscope slides.
With three separate disposable pipettes place a drop of blood solution from each tube on the corresponding slide (blood from Tube 1 goes on slide 1, etc.). Cover each drop with a coverslip.
Observe the slides with a microscope using the high power.
Record your observation in Table 3 (under the column “Microscopic Appearance”) indicating whether the cells are normal, shrunken (crenated), or burst (hemolyzed). Usually hemolyzed cells cannot be seen because they have blown up!
Record in Table 3 the tonicity of the NaCl solution (external solution) added to the test tubes. Is it isotonic, hypotonic, or hypertonic?

Table 3. Effect of salt solutions on red blood cells.
Tube
Number / Contents of Tube / Print Visible?
(yes or no) / Microscopic Appearance / Tonicity of External Solution
1
2
3

Why did the red blood cells burst in the hypotonic solution whereas the Elodea cells didn’t? ______

After completing this exercise immediately clean up all equipment contaminated with blood. Put all bloody materials into the hazardous waste bag.

CELL STRUCTURE

In this section of the laboratory the light microscope will be used to observe living cells and prepared slides of cells. You will examine both prokaryotic and eukaryotic cells. A prokaryotic cell has no nucleus whereas a eukaryotic cell does.

Prokaryotic Cells

Procedures:

A prepared slide of the 3 morphological types of bacteria is available. Observe it and draw a picture of each type of bacteria in the circle below. Note: these cells are very small and have been stained.

Coccus Bacillus Spirillum

(round cells) (rod-shaped cells) (spiral-shaped cells)

Eukaryotic Cells

Protists are unicellular eukaryotic organisms. The protist available for this exercise isParamecium. Paramecium moves constantly by the rowing action of hairlike projections called cilia.

Procedures:

Place a small drop of the Paramecium culture on a clean slide and add a drop of

Protoslo. Stir the drop with a toothpick and add a coverslip.

View the slide under low power and then switch to high power.
Make a drawing of Paramecium in the space provided below. If your specimen won’t hold still, obtain a prepared slide. Using the protist guide provided, see if you can locate and draw the following structures: oral groove, large nucleus, contractile vacuoles.

Eukaryotic Plant Cells: Elodea

You have already observed a eukaryotic plant cell. Look back to the picture which you have already drawn to familiarize yourself of its eukaryotic nature.

Eukaryotic Animal Cells: Human Cheek epithelium

Procedure:

Use the flat edge of a clean toothpick to gently scrape the inside of you cheek.
Stir the scrapings in a drop of water on a clean slide. Add a drop of methylene blue stain and a coverslip.
Observe the slide under low power. Draw a cheek cell in the space provided below. Label the nucleus and plasma membrane.

Paramecium /
Your cheek cells

List two differences you have observed between plant and animal cells.

1. ______

2. ______

List two differences you have observed between prokaryotic and eukaryotic cells.

1. ______

2. ______

Electron Micrographs

Electron micrographs of labeled cell organelles are shown below. Use the chapter on cell structure in your textbook to identify the organelles and your answers the questions.

Sketch of an animal cell /
a) Name of this organelle ______
Found in: plants? ______animals? ______

b) Name of this organelle ______
Found in: plants? ______animals? ______/
c) This is an entire cell. Name these organellesa) ______b) ______
Found in: plants? ______animals? ______

d) Name of this organelle ______
Found in: plants? ______animals? ______/
d) Name of this organelle ______
Found in: plants? ______animals? ______

Spring 2006