Biology 141
Cells & Cell Fractionation

CELLS

Today you will work with several types of cells and cell organelles. You have studied prokaryotic cells in lecture, so this lab will focus on eukaryotic cells.

Use this chart to keep track of the differences in components between the different types of cells.

Eukaryotes

Animal

Plant

Plasma membrane

+

+

+

Cell wall

+

-

+

Ribosomes

+

+

+

Circular DNA strands

+

-

-

Linear DNA strands

-

+

+

Endoplasmic reticulum

-

+

+

Golgi complex

-

+

+

Lysosomes

-

+

+

Microbodies

-

+

+

Nuclear envelope

-

+

+

Nucleolus

-

+

+

Mitochondria

-

+

+

Chloroplasts

-

-

+

"9+2" cilia/ flagella

-

+

+

Other flagella

+

-

-

Microtubules

-

+

+

Microfilaments

-

+

+

Intermediate filaments

-

+

-

 

Label the prokaryotic cell with its components listed in table.

 

 

 

 

 

 

 

 

 

 

Label the plant cell:

a) Rough endoplasmic reticulum

b) Central vacuole

c) Cell wall

d) Golgi apparatus

e) Plasmodesmata

f) Chloroplast

g) Mitochondrion

h) Smooth endoplasmic reticulum

i) Plasma membrane

j) Ribosome

k) Nucleus

l) Nucleolus

m) Cytoplasm

 

 

 

 

 

 

 

 

 

 

Label the animal cell:

a) Rough endoplasmic reticulum

b) Golgi apparatus

c) Centriole

d) Nucleus

e) Nucleolus

f) Peroxisomes

g) Lysosomes

h) Flagellum

i) Mitochondrion

j) Smooth endoplasmic reticulum

k) Plasma membrane

l) Ribosome

m) Cytoplasm

 

 

CHLOROPLASTS IN ELODEA

Usually the organelles are too small to observe well with a microscope. However, chloroplasts can be seen easily in Elodea, a plant that grows immersed in water. Because chloroplasts contain a pigment, they are "prestained" and you do not need to stain them as you do other organelles too see them. Obtain a glass slide and an Elodea leaf. Place the leaf on the slide with a drop of tap water and examine it under the compound microscope. You will need to look at a region that is thinner because there are several layers of cells in an Elodea leaf.

Focus on the chloroplasts. What color are they?

 

Why are they this color?

 

 

Explain what the function of the chloroplast is.

 

 

 

 

Can you find chloroplasts in animal cells?

 

Are organisms that have chloroplasts heterotrophic or autotrophic?

 

Can you see them moving around the cells? Why are they doing this?

 

 

 

 

 

Why do the chloroplasts in the cells in the middle of the leaf appear to be pushed to the outer portions of the cytoplasm (as in the picture below)?

 

 

 

 

MITOCHONDRIA IN CELERY

 

Mitochondria may be observed in living cells with the aid of a vital stain called Janus Green B. This stain is colored when it is an oxidizing agent. When the mitochondria are actively engaged in cellular respiration (oxidzation of sugar), the stain acts as a hydrogen acceptor and becomes reduced and colorless. The Janus Green B will stain the mitochondria right away, but as you watch over a few minutes, the stain will fade.

Obtain 2 razor blades and 2 coverslips. Place one razor blade on the lab bench and place one coverslip on each end so the coverslips barely stick out past the sharp edge of the blade. Place the other razor blade on top of the coverslips so you have a space between the sharp edges that is one coverslip thick.

 

 

 

 

 

 

 

 

 

Tape the two razor blades together securely with white tape.

Obtain a celery stalk, and place it on the lab bench so it is stable.

Position the razor apparatus firmly over it and make a slice. You should have a thin slice of celery that is no thicker than the coverslip.

Place the slice on a glass slide with two drops of sucrose solution, and put a clean coverslip over it.

Observe the celery cells under the compound microscope. Find a section of the celery thin enough to observe the chloroplasts, and even a clear nucleus (you can stain the celery with methylene blue, another vital stain to see the nucleus clearer, but do not do this until you have finished this exercise!)

Place a piece of filter paper on one side of the coverslip so it begins to soak up the sucrose. At the same time, place 2-3 drops of Janus Green B at the opposit side of the coverslip. The filter paper will draw the stain across the celery. Watch the cells, and the mitochondria (very small spots) in the cells should take on a blue/ green color. Observe what happens for a few minutes.

 

Explain what the function of the mitochondrion is.

 

 

 

 

Can you find mitochondria in animal cells too?

What happens to the mitochondria after a few minutes of observing?

 

 

 

What would happen to the color of the mitochondria (a few minutes after you added Janus Green B) if you added another oxidizing agent such as sodium bisulfite? Explain your answer.

 

 

 

 

 

 

 

Now create a null hypothesis and a research hypothesis regarding this experiment.

HO:

 

 

 

HA:

 

 

 

 

Place a piece of filter paper along the side of the coverslip and add two drops of sodium bisulfite. Do you see any changes? What happens to the color of the mitochondria?

 

 

 

 

Which hypothesis do you accept?

 

 

 

 

CELL FRACTIONATION

Introduction

You will work with peas today. A pea is the seed of this plant. The pea consists of a seed coat (a waxy coating around the seed), two cotyledons (tissue that stores food for the growing embryo and begins photosynthesis), and an embryo (young plant). Each of these tissues is made up of cells. Each cell in the pea contains organelles. Some organelles that are common in most eukaryotic cells are the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, and ribosomes. Other organelles specific to the plant cell that you need to be concerned with today are the cell wall made of cellulose, chloroplasts, and amyloplasts.

Amyloplasts are a type of plastid that stores starch for use by the growing and reproducing cells of the plant. Some cells in the pea are specialized for storage, and thus, primarily contain amyloplasts.

You are going to use a technique called differential cell fractionation to break open pea cells and separate the contents based upon weight. The centrifuge spins at a high rate and the force of that spin, called centrifugal force (away from an axis), pulls the heaviest organelles away from the center of the spinning centrifuge. Organelles will end up in layers based upon their density and weight. If you have a high-powered centrifuge, you can separate each specific organelle into a narrow band as illustrated below. However, we have a low-powered centrifuge and can only separate organelles into general bands.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Once a substance has been centrifuged, the heaviest particles will make a pellet at the bottom of the centrifuge tube. The remaining liquid portion is called the supernatant. The color and composition of the layers, and the results of staining with methylene blue and IKI will allow you to determine whether the amyloplasts are heavy or light.

 

Procedure 1: AGetting a Feeling for the Organism@

1. Obtain a pea seed that has been soaked in water for 24 h.

2. Cut the pea open with a razor blade. Find the seed coat, the cotyledons, and the embryo.

3. Using a toothpick, gently scrape the interior of the seed coat until you obtain a thin layer of tissue (the inner layer of the seed coat). Place this tissue on a glass slide. Place a drop of methylene blue on the slide. Methylene blue is a vital stain that will stain nuclei, cell membrane, and cell wall material. Observe the tissue under a compound scope. Record the results in the table on page 2.

4. Repeat step #3, but stain the tissue with IKI rather than methylene blue and observe under a compound scope. Record the results in the table.

5. Using a toothpick, scrape some cotyledon tissue onto a slide. Stain this tissue with methylene blue and observe under a compound scope. Record the results in the table.

6. Repeat step #5, but stain the cotyledon tissue with IKI (IKI stains for the presence of starch) and observe under a compound scope. Record the results in the table.

- Now you should have an understanding of what you will look for in the layers of the pellet and the supernatant.

Tissue/ Stain Drawing What organelles can you see?
seed coat

methylene blue

 

 

 

 

 

 

 

 

seed coat

IKI

 

 

 

 

 

 

 

 

cotyledon

methylene blue

 

 

 

 

 

 

 

 

cotyledon

IKI

 

 

 

 

 

 

 

 

What is the magnification that the cells are most visible?

 

 

Which organelles can you see in which tissues?

 

 

 

What tissue contains cells that have amyloplasts?

 

What evidence do you have to support this?

 

 

Why do you think cotyledon tissue has amyloplasts?

 

 

Think about how amyloplasts may compare to other organelles in terms of their size and their composition. Do you think amyloplasts are light or heavy organelles? Make a research hypothesis based on this question.

 

HA:

 

Make a null hypothesis that corresponds with the research hypothesis.

 

HO:

 

Based on your hypotheses, explain where the amyloplasts might be once you centrifuge the cells. Where will the amyloplasts be when you centrifuge pea cells? In the supernatant or the pellet? What layer of the pellet will they be in?

 

 

 

 

 

 

 

 

 

 

 

 

Procedure 2: ALysing the Cell and Separating the Components@

 

1. For your group, place 4 peas, 6 ml of sucrose buffer, and a TINY pinch of sand in mortar. Grind the peas with the pestle until the homogenate is very smooth.

2. Using a plastic Pasteur pipette, put 1 ml of homogenate into 1 centrifuge tube (there are marks on the side of the tube). Do this for one more tube so you have two tubes.

3. Label the tubes, indicating your group.

4. Put the tubes in the centrifuge so the

tubes are balanced across the centrifuge.

5. Once the centrifuge is full, centrifuge on

speed 7 for 10 minutes.

6. Draw the tube and the layers of the

pellet in the picture. Label the colors you

see, consistency, etc.

 

 

 

 

 

 

 

 

 

 

7. Carefully decant or pipette the supernatant into another centrifuge tube, BEING VERY CAREFUL NOT TO DISTURB THE PELLETS!

8. Place a drop of the supernatant onto a glass slide. Label the slide with the name of the layer. Place one drop of methylene blue onto the supernatant. Place a coverslip over the stain. On a separate slide, place a small amount of ONE pellet layer, label the slide, stain the tissue with methylene blue and cover with a coverslip. Do this for each layer in your pellet. Observe each slide with a compound scope and record the results in the table below. Draw a picture of what you see on the slide and whether amyloplasts are present. Indicate the number of amyloplasts using a (+) for a few, (++) for many, and (+++) for a great number.

9. Repeat step #8, but this time stain the supernatant and each pellet layer with IKI. Record your results in the table.

 

 

 

Layer

Drawing- methylene blue

Drawing- IKI

Amyloplasts? yes/no (+++)

supernatant  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Answer the following questions about your results:

 

 

Do you accept your research hypothesis?

 

 

Do you accept your null hypothesis?

 

 

In what centrifuged layer are the majority of amyloplasts found?

 

What evidence do you have to support this?

 

 

 

 

 

What do you conclude about the weight of amyloplasts?

 

What evidence do you have to support this?

 

 

 

 

 

Can you compare the weight of amyloplasts to any other organelles?

Why or why not?

 

 

 

 

 

 

How might this technique be used to study cell structure and organelle functions?