My Classroom Material 117 - BIOLOGY JUNCTION

Microscope Lab

Learning to Use the Microscope

Introduction

“Micro” refers to tiny, “scope” refers to view or look at. Microscopes are tools used to enlarge images of small objects so as they can be studied. Microscopes range from a simple magnifying glass to the expensive electron microscope. The compound light microscope is the most common instrument used in education today. It is an instrument containing two lenses, which magnifies, and a variety of knobs to resolve (focus) the picture. It is a rather simple piece of equipment to understand and use. In this lab, we are going to learn the proper use and handling of the microscope.

Objectives

Demonstrate the proper procedures used in correctly using the compound light microscope.
Prepare and use a wet mount.
Determine the total magnification of the microscope.
Develop a checklist to insure the proper handling of the microscope.

Materials

Compound microscope
Glass slides
Cover slips
Eye dropper
Beaker of water
The letter “e” cut from newsprint
Scissors

Procedures

Proper Handling of the Microscope

Carry the microscope with both hands — one on the arm and the other under the base of the microscope.
One person from each group will now go over to the microscope storage area and properly transport one microscope to your working area.
The other person in the group will pick up a pair of scissors, newsprint, a slide, and a cover slip.
Remove the dust cover and store it properly. Plug in the scope. Do not turn it on until told to do so.
Examine the microscope and give the function of each of the parts found below.

a. Eyepiece –

b. Body tube –

c. Objectives –

d. Stage –

f. Diaphragm –

g. Coarse adjustment –

h. Fine adjustment –

i. Base –

j. Light source –

Preparing a wet mount of the letter “e”.

With your scissors cut out the letter “e” from the newsprint.
Place it on the glass slide so as to look like (e).
Cover it with a clean cover slip. See the figure below.

Using your eyedropper, place a drop of water on the edge of the cover slip where it touches the glass slide. The water should be sucked under the slide if done properly.
Turn on the microscope and place the slide on the stage; making sure the “e” is facing the normal reading position (see the figure above). Using the course focus and low power, move the body tube down until the “e” can be seen clearly. Draw what you see in the space below.

Describe the relationship between what you see through the eyepiece and what you see on the stage.

7. Offer an explanation of why this happened.

8. Looking through the eyepiece, move the slide to the upper right area of the stage.
What direction does the image move?

9. Now, move it to the lower left side of the stage. What direction does the image move?

10. Re-center the slide and change the scope to high power. You will notice the “e” is out of focus. Do Not touch the coarse focus knob, instead use the fine focus to resolve the picture.

11. Locate the diaphragm under the stage. Move it and record the changes in light intensity as you do so.

Determining Total Magnification:

1. Locate the numbers inscribed on the eyepiece and the low power objective and fill in the blanks below.

Eyepiece magnification ______________

(X)

Objective magnification ______________

Total Magnification _____________X

2. Do the same for the high power objective.

Eyepiece magnification ______________

(X)

Objective magnification ______________

Total Magnification _____________X

3. Write out the rule for determining total magnification of a compound microscope.

4. Remove the slide and clean it up. Turn off the microscope and wind up the wire so it resembles its original position. Place the low power objective in place and lower the body tube. Cover the scope with the dust cover. Place the scope back in its original space on the storage cart.

Summary:

Develop a procedure by which anyone can follow to demonstrate the proper handling of the microscope.

BACK

Microscope Lab

Microscope Lab – Using the Microscope and Slide Preparation

Examine the microscope and familiarize yourself with the parts of the microscope.

1. Magnification

The magnification written on the ocular lens (eyepiece) is _____________

The magnification on the Scanning objective ___________ Low Power Objective ___________
High Power Objective ___________

What is the total magnification for each lens (multiply ocular times objective)

Scanning _____________ Low Power ______________ High Power ________________

2. Diaphragm

Examine the diaphragm, what are the numbers written on it? ____________________

Which setting makes the specimen the lightest? ________________ The darkest? _______________

3. Lenses

Twist the ocular lens, does yours have a pointer? _____________ What is the purpose of the pointer? __________________________________________

Find out what happens to your viewing field if you do not have an objective fully clicked into place. ____________________________________________

4. Viewing a Slide

Obtain a prepared E slide. Focus the slide first with the scanning objective, then click to lower power and focus again. Finally, focus the slide under high power. Remember, at high power, you should ONLY use the fine adjustment knob.

Draw the E exactly as it appears in your viewing field for each magnification. The circles below represent your viewing field. The E should take up as much space in the drawing as it does in your viewing field while you’re looking at it.

Scanning

Low Power

High Power

5. Depth Perception

Obtain a prepared thread slide. You will only need to view it under scanning at this point. Your task is to figure out which thread is on top, which is in the middle, and which is on bottom. You should notice that as you focus the thread, different thread will come into focus at different times. The one that comes into focus the first should be the top thread.

What is the color order of your threads?

6. Making a Wet Mount of a Slide

1. Gather a few strands of cotton from a cotton ball using forceps. If your specimen is too thick, then the cover slip will wobble on top of the sample like a see-saw, and you will not be able to view it under High Power.

2. Place ONE drop of water directly over the specimen. If you put too much water, then the cover slip will float on top of the water, making it hard to draw the specimen, because they might actually float away. (Plus too much water is messy)

3. Place the cover slip at a 45 degree angle (approximately) with one edge touching the water drop and then gently let go. Performed correctly the cover slip will perfectly fall over the specimen.

Draw the specimen as it appears in your viewing field under scanning, low and high power.

Scanning

Low Power

High Power

7. Staining a Specimen

1. Place one drop of stain (methylene blue) on the edge of the cover slip. Caution: Methylene Blue will stain clothes and skin!

2. Place the flat edge of a piece of paper towel on the opposite side of the cover slip. The paper towel will draw the water out from under the cover slip, and the cohesion of water will draw the stain under the slide.

3. As soon as the stain has covered the area containing the specimen, you are finished. The stain does not need to be under the entire cover slip. If the stain does not cover as needed, get a new piece of paper towel and add more stain until it does.

4. Be sure to wipe off the excess stain with a paper towel.

Draw your specimen as it appears under low power. Used color pencils to show how the stain appears. It may appear darker or lighter in spots. Use shading to show darker and lighter spots.

Scanning

Low Power

High Power

8. Investigation of Pond Water

1. Prepare a wet mount of pond water – a sample of pond water is provided in a jar. The best specimens usually come from the bottom and probably will contain chunks of algae or other debris that you can see with your naked eye. (Be careful that your slide isn’t too thick)

2. Use the microscope to focus on the slide – try different objectives, some may be better than others for viewing the slide..

3. Make three separate drawings below at different areas of the slide and at different magnifications. Label where appropriate.

Drawing Specimens

1. Use pencil – you can erase and shade areas
2. All drawings should include clear and proper labels (and be large enough to view details). Drawings should be labeled with the specimen name and magnification.
3. Labels should be written on the outside of the circle. The circle indicates the viewing field as seen through the eyepiece, specimens should be drawn to scale – i.e……if your specimen takes up the whole viewing field, make sure your drawing reflects that.

Scanning

Low Power

High Power

9. Investigation of Large Specimen

Light microscopes are only useful for viewing small thin specimens. In biology, you will perform dissections on larger specimens an may need to magnify the area of interest. In this situation, a stereoscope may be the best instrument. Stereoscopes present a larger field of viewing and handle depth much better than the light microscope. The drawback of the stereoscope is that it does not have a high magnification. Examine one of the stereoscopes in the room. They will be positioned around the room with specimens.

Practice changing the light source and the focus on the stereoscope. For each specimen determine which light and magnification is best for viewing.

Name of specimen ____________________________________________

Magnification _________ Light ____________ Reasons _____________________________________________________________

10. Measuring with a Microscope

Use a clear ruler to determine the width of the viewing field under the scanning objective. Position the ruler so that the millimeter marks are visible in your viewing field. Remember that there are 1000 micrometers in a millimeter.

Estimate the length (diameter) of your viewing field in micrometers _____________________

You cannot use this method to determine the diameter under high power (try switching objectives). Instead you can use a mathematical proportion method to determine the diameter under high power.

High power field diameter = low power field diameter x low power magnification / high power magnification

What is the diameter (in micrometers) of your high power field _____________________

Fill out the table below after viewing prepared specimens.

Name of Object

Measurement of Object

BACK

Microscope Notes

MICROSCOPES

One of the most widely used tools in Biology
An instrument that produces an enlarged image of an object
Magnification – the increase in an object’s apparent size
Resolution – the power of a microscope to clearly show detail

TYPES OF MICROSCOPES

Compound Light Microscope

LM
With this type of microscope the thin sliced (enough to be transparent) and sometimes stained specimen is mounted on a glass slide to be viewed
The slide is placed on the stage and a light source (a light bulb or mirror in the base) directs the light upward
Light passes through the specimen and through the objective lens, which is positioned directly above the specimen
A set of objective lenses is located on the rotating nosepiece enlarges the image of the specimen with different powers of magnification
The most powerful objective lens produces an image 40 times (40X) the actual size of the specimen
From the objective lens, the magnified image is projected up through the body tube to the ocular lens in the eyepiece where it is magnified further (10X)
To compute the total magnification of a microscope, multiply the power of magnification of the lens being used (40X, 100X) by the power of magnification of the ocular or eyepiece lens (10X) example: 40 X 10 = 400X total power of magnification
The Resolution power of LM’s is limited by the physical characteristics of light (At powers of magnification beyond about 2000X, the image of the specimen becomes blurry.)

LIGHT MICROSCOPE

Electron Microscopes

Used to view extremely small objects
Beam of Electrons, rather than light, produces an enlarged image
Electron microscopes are more powerful than LM’s
There are several types of electron microscopes

Transmission Electron Microscope

Can magnify objects up to 200,000 times
Projects image onto a screen or photographic plate
Used to produce greatly magnified images of internal details of a specimen
Can not be used to view living specimens

TEM MICROSCOPE

Scanning Electron Microscope (SEM)

Produces a 3 dimensional image
Specimens aren’t sliced but are sprayed with a fine metal coating
A beam of electrons is passed over the surface of the metal coating to emit a shower of electrons
Showered electrons are projected onto a fluorescent screen or photographic plate
SEM’s produce greatly magnified image of surface details of specimens
Can magnify up to 100,000 times
Can not be used to view living specimens

SEM MICROSCOPE

BACK

Mitosis Activity

Stages of Mitosis

Introduction

Mitosis, also called karyokinesis, is division of the nucleus and its chromosomes. It is followed by division of the cytoplasm known as cytokinesis. Both mitosis and cytokinesis are parts of the life of a cell called the Cell Cycle. Most of the life of a cell is spent in a non-dividing phase called Interphase. Interphase includes G1 stage in which the newly divided cells grow in size, S stage in which the number of chromosomes is doubled and appear as chromatin, and G2 stage where the cell makes the enzymes & other cellular materials needed for mitosis.

Mitosis has 4 major stages — Prophase, Metaphase, Anaphase, and Telophase. When a living organism needs new cells to repair damage, grow, or just maintain its condition, cells undergo mitosis.

During Prophase, the DNA and proteins start to condense. The two centrioles move toward the opposite end of the cell in animals or microtubules are assembled in plants to form a spindle. The nuclear envelope and nucleolus also start to break up.

Prophase

During Metaphase, the spindle apparatus attaches to sister chromatids of each chromosome. All the chromosomes are line up at the equator of the spindle. They are now in their most tightly condensed form.

Metaphase

During Anaphase, the spindle fibers attached to the two sister chromatids of each chromosome contract and separate chromosomes which move to opposite poles of the cell.

Anaphase

In Telophase, as the 2 new cells pinch in half (animal cells) or a cell plate forms (plant cells), the chromosomes become less condensed again and reappear as chromatin. New membrane forms nuclear envelopes and the nucleolus is reformed.

Telophase

Objective:

In this lab, you will determine the approximate time it takes for a cell to pass through each of the four stages of mitosis. You may use your textbook and class notes to help you identify the stages of mitosis as seen under the microscope.

Materials:

Microscope, prepared slide onion root tip or whitefish blastula, textbook, lab worksheet, pencil

Procedure:

Set up a compound light microscope and turn on the light.
Place a slide containing a stained preparation of the Allium (onion root tip) or Whitefish blastula.
Locate the meristematic or growth zone, which is just above the root cap at the very end of the tip or
Focus in on low power, and then switch to medium or high power. Below find micrographs of the four stages of mitosis. Use them to help you identify the stages on the microscope slide.

Prophase (onion)

Metaphase (onion)

Anaphase (onion)

Telophase (whitefish)

Now count the number of cells found in each stage of mitosis and place the data in the chart below.
Determine the percentage of time each cell will spend in each stage of mitosis. Divide the number of each cell by the total number of cells and multiply by 100 to determine the percentage. Place these values in the chart below.

Stage of Mitosis	Number of Cells	Percent of time in each stage = # of cells in stage X 100% Total # of Cell
Prophase		%
Metaphase		%
Anaphase		%
Telophase		%
Interphase (Not a Mitotic Stage)		%
Total # cells		100%

Line graph the data you have just collected. Be sure to label the X and Y axis & include the units of measurement.

Title: __________________________________________________

Graph Legend:

Questions:

1. Of the four stages of mitosis, which one takes the most time to complete?

2. Which is the shortest stage in duration?

3. What would happen if the process of mitosis skipped metaphase? telophase?

Further Study:

Normal Cell Division may be observed in onion root tips. Many of the processes are similar to those in animal cells. However, in plant cells, the cell plate between daughter cells forms from the Golgi.

Find all of the stages of mitosis and interphase in the above picture. Make a sketch of each stage and briefly describe what is occurring. Count and record the number of cells you see in each stage.

	Projects
	Notes

Mitosis and Meiosis

Mitosis and Meiosis

Introduction
All new cells come from previously existing cells. New cells are formed by the process of cell division which involves both replication of the cell’s nucleus (karyokinesis) and division of the cytoplasm( cytokinesis).

There are two types of nuclear division: mitosis and meiosis. Mitosis typically results in new somatic (body ) cells. Formation of an adult organism from a fertilized egg, asexual reproduction, regeneration, and maintenance or repair of body parts are accomplished through mitotic cell division. Meiosis results in the formation of either gametes (in animals) or spores ( in plants). These cells have half the chromosome number of the parent cell. You will study meiosis in Exercise 3B. Where does one find cells undergoing meiosis? Plants and animals differ in this respect. In higher plants the process of forming new cells is restricted to special growing regions called meristems. These regions usually occur at the tips of stems or roots. In animals, cell division occurs anywhere new cells are formed or as new cells replace old ones. However, some tissues in both plant and animals rarely divide once the organism is mature.

Exercise 3A.1: Observing Mitosis in Plant and Animal Cells Using Prepared Slides of the Onion Root Tip and Whitefish Blastula

Figure 3.1 Close up view of different stages of mitosis in an onion root tip:

Figure 3.2 Whitefish Blastula

Procedure:
Examine prepared slides of either onion root tips or whitefish blastula. Locate the meristematic region of the onion, or locate the blastula with 10X objective, and then use the 40X objective to study individual cells. Identify one cell which clearly represents each phase of mitosis. Sketch and label the cell in the box provided.

1. The non dividing cell is in a stage called interphase. The nucleus may have one or more dark-stained nucleoli and is filled with a fine network of threads, the chromatin. During interphase, DNA replication occurs.

Interphase

2. The first signs of cell division occurs in prophase. There is a thickening of the chromatin threads, which will continue until it is evident that the chromatin has condensed into chromosomes. With somewhat higher magnification you may be able to see the two chromatids held together by the centromere. As prophase continues , the chromatids continue to thicken and shorten. The nuclear envelope disappears and the beginnings of the spindle apparatus begin to appear.

Prophase

3. At metaphase, the chromosome pairs have moved to the center of the spindle. One particular part of each chromosome, the centromere, attaches to the spindle. The centromeres of all the chromosomes lie about the same level of the spindle called the metaphase plate.

Metaphase

4. At the beginning of anaphase, the centromere regions of each pair of chromatids separate and are moved by the spindle fibers toward opposite poles of the spindle, dragging the rest of the chromatid behind them. Once each chromatid is separate it is called a chromosome.

Anaphase

5. Telophase, the last stage of division, is marked by a pronounced condensation of the chromosomes, followed by the formation of a new nuclear envelope around each group of chromosomes. The chromosomes gradually uncoil into the fine threads of chromatin, and the nucleoli reappears. Cytokinesis may occur. This is the division of the cytoplasm into two new cells. In plants, a new cell wall is laid down between the daughter cells. In animal cells, the old cells will pinch off in the middle along a cleavage furrow to form two new daughter cells.

Telophase

Analysis Questions:
1. Why is it more accurate to call mitosis “nuclear replication” rather than “cellular division”?

_____________________________________________________________________

2. Explain why the whitefish blastula and onion root tip are selected for study of mitosis.

_____________________________________________________________________

Exercise 3A.2: Time for Cell Replication
Procedure:
It is hard to imagine that you can estimate how much time a cell spends in each phase of cell replication from a slide of dead cells. Yet this is precisely what you are going to do in this part of the lab. Since you are working with a prepared slide, you cannot get any information about how long it takes a cell to divide. What you can determine is how many cells are in each phase. From this, you can infer the percent of time each cell spends in each phase.

1. Observe every cell in one high power field of view and determine which phase of the cell cycle it is in. This is best done in pairs. The partner observing the slide calls out the phase of each cell while the other partner records. Then switch so the recorder becomes the observer and visa versa. Count at least two full fields of view. If you have not counted 200 cells, then count a third field of view.

2. Record your data in Table 3.1.

Table 3.1

	Number of Cells				Percent of Total Cells Counted	Time in Each Stage
	Field 1	Field 2	Field 3	Total
Interphase
Prophase
Metaphase
Anaphase
Telophase
Total Cells Counted

3. Calculate the percentage of cells in each phase.

Consider it takes, on average, 24 hours (or 1,440 minutes) for onion root-tip cells to complete the cell cycle. You can calculate the amount of time spent in each phase of the cell cycle from the percent of cells in that stage.

Percent of cells in stage X 1,440 minutes = ___________ minutes of cell cycle spent in stage.

Questions:
1. If your observations had not been restricted to the area of the root tip that is actively dividing, how would your results have been different?

_____________________________________________________________________

2. Based on the data in Table 3.1, what can you infer about the relative length of time an onion root-tip cell spends in each stage of cell division?

_____________________________________________________________________