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Basic Microscopy
Figure 1. Marine Diatoms Viewed through a Microscope Lens
(Image source: Wipeter.)
Are There Aliens Among Us?
If you ever needed proof that organisms from other worlds live among us, diatoms (Figure 1) are your answer. The extreme
shapes and structures of diatoms are pretty out-of-this-world!
Each organism begins life as a single, tiny cell. Some organisms remain a single cell, such as diatoms, whereas others divide
and replicate to become multicellular organisms, such as yourself. Being able to view objects too small to see with the naked
eye, such as single cells, allows you to enter a whole new world.
Diatoms are a unique group of phytoplankton that are entirely enclosed in a cell wall made out of silica. This cell wall material
allows the cells to form geometrically shaped structures that are usually symmetrical bilaterally, although not perfectly. There
are so many intriguing and beautiful creatures to see if you just look at some "ordinary" water using a microscope.
Studying Microscopic Life
Cells are the building blocks of life. The size of a skin cell is about 30 m (30 X 10-6 or 0.000030 m), which is too small for the
human eye to see. It took the invention of the microscope to be able to study cells. Anton van Leeuwenhoek prepared the first
simple microscope.
There are two basic types of cells: prokaryotes and eukaryotes. Prokaryotes, which consist of the bacteria and archaea, are
mostly unicellular organisms and are the smallest and the simplest form of living things. Eukaryotes, which consist of the
protists, fungi, plants, and animals, can be found as both unicellular and multicellular organisms. They are larger and more
complex than prokaryotes in both structure and function.
Cell Structure
All cells, both prokaryotic and eukaryotic, have a plasma membrane (cell membrane) and contain cytoplasm. More
specifically, all cells are surrounded by a plasma membrane, which is a phospholipid bilayer with pores. Selective permeability
to various molecules allows the movement of substances into and out of the cell, thus regulating what is inside versus what is
outside the cell.
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The cytoplasm is a thick, jellylike substance that is enclosed in the plasma membrane. It contains all the dissolved and
suspended ions and other molecules needed to keep the cell alive. The cytoplasm is constantly in motion and actually helps
in projecting forward some cells, such as amoebas, as a form of movement.
Differences between Prokaryotic and Eukaryotic Cells
Eukaryotic cells have membrane-bound organelles, such as a nucleus or mitochondria (Figure 2). The nucleus contains most
of the genetic material of the cell and the mitochondria supply most of the energy for eukaryotes.
Figure 2. Diagram of Typical Cellular Structures of Eukaryotes
(Image source: Hayden-McNeil, LLC.)
Plant cells are eukaryotic and have organelles called chloroplasts. Chloroplasts are the functional organelle for
photosynthesis and contain chlorophyll. Chlorophyll absorbs light energy and converts it to food for the plant. The different
parts of cells enable organisms to perform different functions.
Prokaryotic cells do not contain membrane-bound organelles. Prokaryotes do contain a nucleoid, which is a region where the
circular chromosome of DNA is located (Figure 3). Prokaryotic cells that perform photosynthesis have structures called
thylakoids where the photosynthetic chlorophyll molecules are located.
Figure 3. Diagram of Prokaryotic Cell
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Prokaryotic cells are also much smaller than eukaryotic cells, so they are more difficult to visualize using a microscope.
Cytoplasm is transparent, but eukaryotes have membrane-bound organelles that provide visual markers within the cell.
Prokaryotes do not have membrane-bound organelles, making it difficult to see these cells without some sort of manipulation.
Common Cellular Structures
Cell Wall
A cell wall is a rigid layer outside the plasma membrane that gives the cell a more structured shape and strengthens the cell
by resisting osmotic pressure. Some protists, most bacteria, and all plants and fungi have cell walls.
Vacuoles
Both animal and plant cells have vacuoles within the cell. Vacuoles are membrane-bound storage areas that store fluid and
nutrients as well as wastes. Plant cells have a central vacuole that takes up about 75% of the total volume of the cell and
helps to maintain the plant cell's rigid structure. Plant cells make their own food and need these massive storage areas to
store it.
Flagella and Cilia
Many eukaryotic and prokaryotic cells have flagella or cilia. Flagella are long hair-like extensions that extend from the cell and
aid in locomotion. The single-celled plant algae, protozoans, many bacteria, and animal sperm cells are some cell types that
have flagella. Cilia are shorter and more numerous than flagella. Rows of cilia move in waves to propel the cell or to move
fluids around the cell and remove particles from around them.
Using a Microscope
Magnification
Microscopes are used to see very small objects that cannot be seen with the naked eye by magnifying an image of the object.
Magnification is achieved with two optical elements, the ocular lens (eyepiece) and the objective lens (objective), shown in
Figure 4 below.
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Figure 4. Objective and Ocular Lenses
The ocular lens is the lens at the top of the microscope that is looked through to see the image. It usually has a magnification
power of 10X, which means it enlarges the image of an object ten times its original size. The objective lens is the one closest
to the slide and further magnifies the object on the slide. Some common objective lens magnifications are 4X, 10X, 40X, and
100X. The total optical magnification is the product of the power of the ocular lens and the objective lens.
total optical magnification = ocular power objective power
For example, if a 10X ocular lens and a 40X objective lens are used, the total magnification of the object being viewed is
400X (400 times) its normal size.
Focusing the Image
The microscope's optics are similar to a digital camera in that only one depth of field can be focused on at a time. With a
digital camera, objects that are closer or farther from the depth of field than the one in focus appear fuzzy. The same is true
with a specimen on a microscope slide. However, the slides in the virtual lab have been digitized into one focal plane, so after
initially focusing on the specimen, all focal planes can be viewed at once. The coarse and fine focus knobs move the stage up
and down and set the fixed focus of the lenses at different depths within the specimen.
Some Methods for Easier Visualization of Prokaryotic Cells
Because bacteria are so small and therefore difficult to see using the bright field setting on a typical light microscope, two
special features have been developed to better visualize bacteria. These techniques are also very useful for examining live
eukaryotic specimens.
The first technique is called dark field microscopy. This technique uses a special condenser so that only the light reflected off
the specimen is seen. Dark field microscopy highlights the specimen so it appears brightly lit against a dark background,
almost the reverse image of the standard bright field image. This technique is particularly useful for providing contrast around
the edges of a specimen.
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The second technique is called phase contrast microscopy. This technique takes advantage of contrast produced when light
waves combine. Light waves are bent when they enter the specimen. Those bent waves overlap in many different ways,
producing varying levels of interference and a great deal of contrast. In phase contrast microscopy, the background appears
gray and the specimen appears as various degrees of light and dark.
About This Lab
In this lab, you will study the small world of the individual cell by using a virtual microscope. The cell is considered to be the
building block of all organisms. You will look at different types of cells and see how they vary in shape, size, function, and
other ways.
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Procedures
Experiment 1: Introduction to the Virtual
Microscope
1. Place the microscope from the Instruments shelf onto the right side of the workbench so that it does not hide the shelf
tabs. Notice that the microscope has a view-screen attached to it. This view-screen is connected to the lens and shows
you the view through the lenses on top.
2. Familiarize yourself with the location of the stage, x-axis adjust dial, y-axis adjust dial, coarse focus dial, and fine
focus dial (Figure 1).
Figure 1. Stage, Adjustment Knobs, and Focus Knobs
3. Place an Amoeba slide from the Materials shelf onto the microscope stage. Once the slide is loaded, you will see a fuzzy
image on the view-screen.
4. Working comfortably with the microscope requires getting up close to it. Click on the Zoom In button in the lower right
hand side of the lab to get a better view of the microscope.
5. To make the image clearer, start with the coarse focus dial. Click and slowly drag the dial up or down to focus in on the
specimen. Then do the same with the fine focus, if necessary, to get the best image possible.
6. Once the Amoeba is in focus, you may want to look at different areas of the slide. The x-axis adjust and y-axis adjust
dials move the stage in small, finely tuned increments to the right and left or in and out. Click on the x-axis adjust dial and
drag slightly upward or downward to move the stage in the desired direction. Click on the y-axis adjust dial and drag
slightly to the right or left to move the stage in the desired direction.
7. The objective lens that is in use at any time is the one in front, pointing straight down (Figure 2). It has the magnifying
power engraved on it, for example, 4X in the image below. Click on the lens to the right of the current one to rotate the
lens to the one with the next higher magnification. Click the lens to the left of the current one to reduce the magnification.
You may have to refocus the image slightly after changing objectives.
Figure 2. Objective Lenses
8. Look at the buttons at the bottom of the view-screen (Figure 3). The set of buttons in the lower left hand side of the view
screen allows you to adjust the brightness and contrast of the image.
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Figure 3. Brightness, Contrast, and Micrometer Options
9. To adjust either feature:
Click the Brightness or Contrast button.
Click the + or buttons to increase or decrease the amount.
When you are satisfied with the image quality, click the Brightness or Contrast button again.
10. Click the Reset button to return the image to its original brightness and contrast levels.
11. Click the Toggle Micrometer button to show a grid that you can use to estimate the sizes of objects. Click it again to hide
it.
12. On the right is a pair of overlay buttons that you will use to label and draw on the images. There are two buttons in this
set. Clicking each will open an overlay panel.
13. Annotation overlay allows you to add text to an image. To use this feature, click the Annotation Overlay button (Figure
3).
Figure 3. Annotation Overlay Button
14. Locate the word "Sample" on the display screen, which shows you the settings that are currently in place. There are two
features that can be adjusted while using the annotation overlay:
Size of the font: To change the size of the font, click the drop down arrow to the right of the font size number and
select the desired font size number.
Color of the font: To change the color of the font, click the colored box to the right of the font size and select your
desired color.
15. Drag the text icon indicated by the capital letter T onto the image and drop it in the desired location.
16. Click within the statement "Enter your text here" begin typing your text. Add the label "Paramecium" to your image.
17. You can delete a single text entry by clicking on the X above the text, or you can delete all annotations by clicking on the
trash can icon.
18. Delete your existing label and add the correct label of "Amoeba" to your image.
19. The Draw Overlay button allows you to add free form drawing, straight lines, and shapes to an image. Click on the Draw
Overlay button (Figure 4).
Figure 4. Draw Overlay Button
20. The four overlay options are brush, line, ellipse, and rectangle overlays. The brush overlay is selected by default. Click on
other options to change the type of overlay.
21. Once you have selected the type of overlay, you can adjust several features:
Color of the stroke (line) or fill: To change the color, click the colored box next to the option and select your desired
color.
Width of the stroke: To change the width of the stroke, click and drag the triangle next to the option and select your
desired width.
Transparency: To change the transparency of a stroke or shape, find the option labeled "Alpha". Click and drag the
triangle next to the option and select your desired transparency.
Not all types of overlays will allow for changing all features.
22. Add one example of each type of overlay to your Amoeba image.
23. Once text or annotation has been added to an image, do not adjust the image by using the x-axis and y-axis adjust dials
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or switching objectives. The text and annotation will not travel with the image.
24. Click the Snapshot button (Figure 5) located above the objective lenses on the microscope, to the left of the slide name,
to save a snapshot of the image you see on the view-screen.
Figure 5. Snapshot Button
25. Name and describe the snapshot in the pop-up window and click OK. The snapshot will be placed into your media player.
Experiment 2: Looking at Cells Using a Microscope
Part 1: Amoeba
An Amoeba is a single-celled eukaryote that can change shape often due to pseudopodia (singular: pseudopodium), a
temporary protrusion of the cytoplasmic body that aids in locomotion.
1. Switch to the 10X objective, if you are not currently using it.
2. Move the Amoeba to the center of your view-screen using the x-axis and y-axis adjust dials. Record a description of the
shape and color of the Amoeba in your Lab Notes. Remember to press Save Notes.
3. Find and label the following structures using the overlay tools:
Nucleus
Plasma membrane
Cytoplasm
Pseudopodium
4. Use the annotation tools to record the total magnification you are using onto the corner of the image.
5. Take a snapshot of the labeled Amoeba and save it to your media player.
6. When you are done, take the slide off the microscope stage and discard the slide in the recycling bin. Change the
objective back to the lowest setting.
Part 2: Spirogyra
Spirogyra is a filamentous algae that grows in almost every pond or body of water. At the end of the summer, Spirogyra can
grow so profusely that they form a thick scum on the surface of the water.
1. Take the Spirogyra slide from the Materials shelf and place it on the microscope stage.
2. Use the coarse and fine focus dials to bring your image into focus.
3. Observe the cells using multiple objectives. Record the shape and color of the cells in your Lab Notes. Remember to
press Save Notes.
4. Label the following structures:
Cell wall
Nucleus
Plasma membrane
Cytoplasm
Chloroplast
5. Put text in one of the corners to record the total magnification you are using.
6. Take a snapshot of the Spirogyra cells with labels of the structures. If you cannot find a single cell with all of the
structures, use multiple cells and take snapshots of each one.
7. When you are done, take the slide off the microscope and discard the slide in the recycling bin. Change the objective
back to the lowest setting.
Part 3: Cardiac Muscle Cells
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The cardiac muscle cells of a human heart are often branched and contain only one nucleus. A prominent and unique feature
of cardiac muscle is the presence of irregularly-spaced dark bands between cardiac muscle cells, or myocytes. These bands
are known as intercalated discs and are due to areas where the membranes of adjacent myocytes come very close together.
1. Take the cardiac muscle slide from the Materials shelf and place it on the stage of the microscope.
2. Use the coarse and fine focus dials to bring your image into focus.
3. Observe the cells using multiple objectives. Describe the shape and color of the cells in your Lab Notes. Remember to
press Save Notes.
4. Label the following structure:
Plasma membrane
Cytoplasm
Nucleus
Intercalated discs
5. Put text in one of the corners and record the total magnification you are using.
6. Take a snapshot of the slide with labels and save it to your media player.
7. When you are done, take the slide off the microscope and discard the slide in the recycling bin. Change the objective
back to the lowest setting.
Part 4: Dark Field Microscopy with Bacteria
1. Take the bacteria slide from the Materials shelf and place it on the stage of the microscope.
2. Record what you observe in your Lab Notes. The bacteria should be very difficult to visualize using bright field
microscopy. Remember to press Save Notes.
3. Switch to the 100X objective. Even at this high magnfication, you should only be able to see faint outlines of cells.
4. Find the Change Mode button (Figure 6) at the front of the microscope as shown below. It should be set on bright field.
When you click on it, it should switch to dark field.
Figure 6. Change Mode Button
5. Describe the shape of the cells in your Lab Notes. Describe any general differences between the appearance of the
overall field in dark field and bright field in your Lab Notes as well. Remember to press Save Notes.
6. Observe and label the following structures:
Plasma membrane
Cytoplasm
7. Put text in one of the corners and record the total magnification you are using.
8. Take a snapshot of the slide with labels and save it to your media player.
Part 5: Phase Contrast Microscopy with Bacteria
1. Click on the Change Mode button again to switch to phase contrast.
2. Does the shape of the bacteria change in this different field? Describe their shape in your Lab Notes. Describe any other
differences between phase contrast, dark field, and bright field modes in your Lab Notes. Remember to press Save
Notes.
3. Observe and label the following structures:
Plasma membrane
Cytoplasm
4. Put text in one of the corners to record the total magnification you are using.
5. Take a snapshot of the slide with labels and save it to your media player.
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Notes
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