Exercise 2: Microscopy and Cells

Figure 2.1 HeLa cells are a cell line used for cell culture experimentation from malignant cervical cancer of a women who lived in the 1950s named Henrietta Lacks. In this image, cells are stained with blue, red, and green-fluorescent dyes. The DNA appears blue, while the actin protein appears green.

Exercise 2 Learning Goals

After completing this lab, you should be able to:

• Define the term histology
• Calculate the total magnification of each objective lens used in the laboratory
• Identify the parts of the light microscope and describe their basic function
• Identify parts of eukaryotic cells
• Be able to demonstrate use of the light microscope using low and high-power magnification
• Explain how the process of osmosis can affect cell tonicity
• Define and differentiate between hypertonic, isotonic, and hypotonic solutions
• Identify the extracellular (interstitial) space; describe the free edges and lumen in histological images
• Identify and define the 4 basic tissue types: epithelial, connective, muscular, and nervous

Pre-Lab Activity for Exercise 2Pre-Activity 2.1: Metric Conversions

Complete the following conversions and answer the questions below.

• 1. 10 mm= ______µm
  2. 0.1 mm= ______dm
  3. 1 dm= _______ m
  4. 1 m= ________ µm
  5. 1 µm= _______nm
  6. What would be an appropriate unit of measurement for human height? ________
  7. A human kidney? ______
  8. Human red blood cell? ______
  9. A ribosome? _______

Pre-Activity 2.2: The Light Microscope and Total Magnification

Histology, or microanatomy, is the study of cells and tissues at the microscopic level. There are distinct types of microscopes used for studying cells and tissues; these include Transmission and scanning electron microscopy, confocal, and light microscopes. These types differ because they use different mediums to observe objects. The Transmission and scanning electron microscopes, for example, use beams of electrons to produce an image of the object. The confocal microscope uses a laser to produce an image of the object and can give you a three-dimensional view of an object. These types of microscopes are used to identify surface proteins, organelles/pathogens or to tag parts of the cell.

Light microscopes are useful for observing objects in the millimeter-micrometer range. Objects in this size range can still be seen using light waves. This includes the cells and tissues you will be studying in this course. Review the parts of the light microscope in the figure below.

Figure 2.2 Binocular light microscope depicted with all the major components labeled. Photograph by Gina Profetto

In this lab, you will use the light microscope to examine eukaryotic cells and tissues. The eyepiece has a magnification of 10x. The objective lenses have magnifications of 4x (scanning lens), 10x (low power), and 40x (high power). There is a 100x (oil immersion lens) which we will not use. To find the total magnification of the object being viewed under the microscope, multiply the magnification of the eyepiece lens by the magnification of the objective lens being used.

Calculating total magnification

1. Calculate the total magnification when the 4x objective is used:

Eyepiece ______ X 4x= Total Magnification ________

1. Calculate the total magnification when the 10x objective is used:

Eyepiece ______ X 10x= Total Magnification ________

1. Calculate the total magnification when the 40x objective is used:

Eyepiece ______ X 40x= Total Magnification ________

Matching the parts of the microscope with the appropriate description (Number) below:

______ a. stage

______ b. carrying handle

______ c. light

______d. objective lens

______ e. slide holder/ stage clips

______ f. condenser

______ g. iris diaphragm

______ h. coarse adjustment

______ I. fine adjustment knob

______ j. ocular lenses

______k. base

______ l. stage adjustment knobs

______ m. revolving nosepiece

1. Lenses that rotate for use of different magnifications (4x, 10x, 40x, 100x)

2. The broad platform of the microscope that supports it

3. Allows for control of the amount of light that passes through the condenser; is useful for adjusting the contrast of the image

4. Supports the objective lenses

5. Knob that moves stage in the z-direction (up and down) in a course (high incremental) fashion

6. Knob that moves the stage in the z-direction (up and down) in a fine (low incremental) fashion, improving clarity/ resolution of image

7. Knob that moves the stage in the xy direction (front and back, left, and right)

8. The platform that holds the slide

9. The metal clips/ prongs that hold the slide in place

10. Illuminates the object from below

11. Circular region which concentrates the light on the object

12. Handle for carrying the microscope

13. Lenses found with the eyepiece that have a magnification of 10

Critical Thinking

What is histology? Why is histology important to the study of anatomy and physiology?

Pre-Activity 2.3: Parts of the Eukaryotic Cell

Using your textbook, label the cell parts on the figure below with the appropriate letter/name.

Figure 2.3 Representative eukaryotic cell showing cellular compartments and organelles.

1. Golgi Apparatus
2. Mitochondrion
3. Nucleolus
4. Ribosome
5. Nucleus
6. Rough Endoplasmic Reticulum
7. Smooth Endoplasmic Reticulum
8. Cytoplasm
9. Centrosome (2 centrioles)
10. Plasma membrane
11. Nuclear Pore
12. Vesicle
13. Chromatin
14. Lysosome
15. Endosome
16. Microtubules

Pre-Activity 2.4: Questions

1. What is the plasma membrane? What is its basic function?
2. Where do you find the nucleus and the nucleolus? What is found in the nucleus of eukaryotic cells?
3. Describe the membranes within the cell including the smooth endoplasmic reticulum, the rough endoplasmic reticulum, and the Golgi apparatus. What is the basic function of each?
4. There are three types of proteins that help support the plasma membrane and have other essential functions in cells. List all three and give their major function?
5. Define the cell cycle? What is the purpose of mitosis and how does it fit in with the cell cycle? What are the stages of mitosis?

Figure 2.4 The Steps of somatic cell division also known as mitosis (out of sequence).

1. What is the purpose of meiosis? What are the stages of meiosis?
2. Define the following terms:
   1. Diploid:
   2. Haploid:

Pre-Activity 2.5: Osmosis and Diffusion

Define and label the solutes and the solvent represented in the diagram below.

Figure 2.5 Flask with aqueous solution containing two types of solute particles equally distributed in the flask.

Define diffusion. Label the direction of movement, using arrows, of the solute from high concentration to low concentration in the diagram below.

Figure 2.6 Flask with aqueous solution containing two types of solute particles. High concentration gradient of diamond solutes on the left and high concentration of circle solutes on the right. Diffusion will take place until equilibrium is reached where all solutes are evenly distributed in the flask.

Define osmosis. Label the direction of movement, using arrows, of water (solvent) from low solute concentration to high solute concentration across the semipermeable (permeable to water) membrane.

Figure 2.7 Flask with semipermeable membrane down the middle separating two aqueous solutions with different solute concentrations. The membrane only allows water to pass through but prevents the solutes from diffusing across. Only diffusion of water will occur.

Pre-Activity 2.6: Tonicity

Solutions are made up of solute, (such as sodium chloride) and solvent (liquid it is dissolved in). Due to the process of osmosis, molecules will randomly move across a semipermeable membrane (like the plasma membrane of a cell) to equalize the concentration of solutes on each side of the cell. Tonicity describes the ability of the extracellular concentration to influence water to move from the inside to the outside of the cell and or vice versa. The states of tonicity, known as hypertonic, isotonic, and hypotonic describe the concentration of solute compared across a semipermeable membrane (hyper= too much solute; iso=the same amount of solute; hypo=too little solute).

Figure 2.8 Blood cells placed in three types of tonic solutions with diffusion of water represented by arrows.

Human blood is a liquid connective tissue made up of red blood cells (erythrocytes), plasma (interstitial fluid), white blood cells, and platelets. The concentration of solutes, for example, sodium chloride, in the fluid surrounding red blood cells should be in balance with the concentration of sodium chloride on the inside of the cell. This condition is called _____________. When there is too much sodium chloride (hypertonic) in the fluid surrounding the cell, water will leave the cell causing it to shrink, or shrivel. When there is too little sodium chloride in the fluid surrounding the cell, water will move into the cell, causing the cell to first swell, and then burst. Both the hypertonic and hypotonic conditions are dangerous to the body. It is important for the body to regulate the cell’s extracellular environment and maintain fluid balance. The kidneys remove excess water, salt, potassium, and wastes from the blood plasma. When the kidneys stop working properly, a person must go on dialysis, a procedure that filters waste from the blood.

Figure 2.9 Patient undergoing dialysis to filter wastes out of the blood.

Lab Exercise 2: In Class Activities

Activity 2.1: Using the Light Microscope

Before you begin, your instructor may review the following basic rules for microscope use and slide care:

1. Always carry the microscope by its arm in an upright position. The free hand should support the base of the microscope.
2. When storing the microscope, make sure that slides are removed, and the lowest objective (4x) is in position and elevated relative to the stage.
3. Do not allow the lens of the objective to touch the slide. Doing so may break the slide and scratch the lens.
4. Never use the coarse adjustment knob with anything other than the scanning 4x or low power 10x objective.
5. Hold the glass slides along their edges to avoid oils from fingers.
6. You can clean both slides and objective lenses using lens paper and lens cleaning solution found in the lab.
7. Always treat microscopes with care and respect! They were expensive!

Using the light microscope at your lab bench, identify the microscope parts, and their functions. Next match the letters on the microscope images to the terms on the table below and fill in the functions. Some letters may be used more than once, or not at all.

Figure 2.10 Light microscope indicating movements of

the stage x, y, and z directions.

1. Which knobs move the stage right and left? How is this movement described in terms of an x, y, z, axis?
2. Which knobs move the stage up and down? How is this movement described in terms of an x, y, z, axis?

Activity 2.2: Observe the Slide “e” Using the Following Steps.

1. Make sure the lowest objective lens (4x) is in position and use the coarse knob to move the stage as far up as it will go down.
2. Place the slide on the stage, making sure it is stabilized by the stage clips (slides should not move unless the stage clips are moved). Stage clips can work differently: by being positioned on top of the slide, or by spreading apart, so that the slide can be wedged between. Check with the instructor if your slide is not stable or positioned with the stage clips.
3. Position the object on the slide over the light source/ under the objective lens. Then, using the stage adjustment knobs, look through the eyepiece to further position the object on the slide over the light source so that you can see it through the eyepiece. The object will be blurry.
4. Once the object is positioned correctly over the light source, use the coarse focus knob to move the stage in the z direction (up and down), until the object becomes a bit clearer.
5. Use the fine focus knob to improve the clarity of the image.
6. Once you have used the 4x to focus the slide, then you may move to the next highest objective (10x).
7. At the next highest objective, it may or may not be necessary to adjust the fine focus knob to improve the clarity/ focus of the image.
8. Once the image is clear, you may switch to the 40x objective and adjust the fine focus knob to gain clarity of the image.

• Follow these steps with the slide labeled “e.” What do you notice about the “e” when looking at it through the microscope? What is its orientation? What do you notice when changing objectives?

1. Repeat the process with the micrometer slide. Approximate the distance across the field of view for each objective.

Activity 2.3: Estimating the Size of the Letter “e.”

Specimens viewed with a light microscope are typically measured using millimeters (mm) or micrometers (µm). The field of view, or diameter that spans the length of the viewing field using the scanning objective is around 4 mm.

Fill in the graph and calculate the size of your letter “e.”

Considering the objective used, approximate the size of the letter “e” in mm: ______; in µm: ______.

Activity 2.4: Observe the Slide “Colored Threads” Using the Following Steps.

1. Make sure the lowest objective lens (4x) is in position and use the coarse knob to move the stage as far up as it will go down.
2. Place the slide on the stage, making sure it is stabilized by the stage clips (slides should not move unless the stage clips are moved). Stage clips can work differently: by being positioned on top of the slide, or by spreading apart, so that the slide can be wedged between. Check with the instructor if your slide is not stable or positioned with the stage clips.
3. Position the object on the slide over the light source, under the objective lens. Then, using the stage adjustment knobs, look through the eyepiece to further position the object on the slide over the light source so that you can see it through the eyepiece. The object will be blurry.
4. Once the object is positioned correctly over the light source, use the coarse focus knob to move the stage in the z direction (up and down), until the object becomes a bit clearer.
5. Use the fine focus knob to improve the clarity of the image.
6. Once you have used the 4x to focus the slide, use the stage adjustment knobs.
7. Then you may move to the next highest objective (10x).
8. At the next highest objective, it may or may not be necessary to adjust the fine focus knob to improve the clarity/ focus of the image.
9. Once the image is clear, use the stage adjustment knobs to practice moving and navigating around the slide. Choose a color and follow the color from one end to the other end. Repeat each color.

Activity 2.5: The Effects of Varying Tonicity on Red Blood Cell Morphology

Recall that tonicity refers to how a solution affects the shape of the human cell through the process of osmosis. In your body, red blood cells have a standard salt concentration of 0.85%. In this experiment, you will expose erythrocytes (red blood cells) to saline solutions with varying concentrations: 0.2%, 0.45%, 0.85%, 3.5%, and 10%. Using your microscope, you will observe the effects of extracellular tonicity on the shape of the red blood cells for each solution added.

• Which concentration(s) do you predict will be hypertonic to the blood cells? ____________________
• Which concentration(s) do you predict will be hypotonic to the blood cells? _____________________

Procedure

1. Obtain a clear microscope slide and a coverslip
2. Using a clean pipette, drop one drop of blood onto the center of the microscope slide
3. Gently place the cover slip over the blood. Try to avoid trapping large bubbles underneath the coverslip.
4. Observe the blood cells morphology without any saline solution
5. Record what you see in the table below
6. Obtain a new microscope slide and coverslip
7. Using a new pipette, drop one drop of blood onto the center of the microscope slide
8. Using a new pipette, place a drop of 0.2% saline solution on the slide over the blood
9. Gently place the coverslip over the blood/saline mixture. Try to avoid trapping large bubbles underneath the coverslip
10. Observe red blood cell morphology under the microscope
11. Describe in the table what you observed about the shape of the cells
12. Repeat steps 6-11 for each of the saline solutions
13. Fill in the table below with your observations from the experiment.

1. As you increase the saline solution concentration, what happened to the cell shape?
2. Was there any change in cell morphology between the control and the 0.85% solution? Explain your answer.
3. Hemolysis: splitting of the red blood cell because the cell membrane was too “tight”

Crenation: Cell membrane of the red blood cell is loose and “spikey”

Using these words to describe the events happening to the red blood cells;

• • Which saline concentration(s) represents the act of Hemolysis?
  • Which saline concentration(s) represents Crenation?

Activity 2.6: Representative Staining Techniques

Why are cells and tissues stained? Cells and their parts appear translucent/clear and colorless under the light microscope. Staining cells and tissues help to make cells, cell parts, and tissues more visible. Diverse types of staining include H&E (Hematoxylin and Eosin), PAS (Periodic Acid-Schiff Reaction), Masson’s Trichrome, Alcian Blue, van Gieson, Giemsa, Toluidine Blue, Silver Stain, Nissl, Methylene Blue, and there are various types of fluorescent dyes can be used as well.

In the following images, label the cell parts and structures that are visible below. Note the differences in color that are based on the several types of stains used.

Figure 2.11 In this image, tissue from the thyroid gland has been stained using H&E staining.

H&E Staining

In an H&E stain, the nucleic acids within the cell are stained purple/blue by hematoxylin. The eosin stains proteins pink. Spaces, or lumen, appear white.

Figure 2.12 Another example of an H&E stain. This is lung tissue from a patient with Emphysema. In this image you can see red blood cells, which appear bright pink.

Giemsa Stains

The purple nuclei are from cells that line the bronchioles and are more generally known as simple squamous epithelial cells. These cells are good at absorption and diffusion for gas exchange. The light pink lines are protein fibers. Lung tissue has a lot of elasticity to expand with each breath and return to its original shape; wavy pink lines indicate elastic fibers.

Figure 2.13 Normal blood smear with a Giemsa stain. Red blood cells (erythrocytes) are abundant throughout this type of connective tissue and appear pink/red. White blood cells and platelets stain purple because they have a nucleus. White blood cells are large and contain a dark purple mass or smaller dark purple globules within the cell. Platelets are smaller than erythrocytes and appear purple as well.

Sum it up:

1. In an H&E stain, nuclei appear _______________in color, while proteins appear ______________ in color.
2. In a Giemsa stain, cells containing DNA appear _______________ in color, while cells without DNA (red blood cells/ erythrocytes) and proteins appear ______________ in color.

Silver Stain

Figure 2.14 In this histological image above, a silver stain is used to show the structure of reticular tissue (a type of connective tissue). This tissue is found in bone marrow, lymphatic organs, around individual muscle cells, and beneath most epithelia (for example, skin). Lymphocytes appear light purple and reticular fibers can be seen that are visible as dark purple/black lines. Reticular fibers are a type of protein that help provide structure and support to tissues.

Critical Thinking:

On each image below, label one or more of the following: lumen, interstitial region, cell, nucleus, and protein fiber.

Identify the cell, the protein, and the type of stain used.

Figure 2.15 Pancreas. Photograph by Gina Profetto

What type of staining is used here?

Figure 2.16 Parotid Gland. Photograph by Gina Profetto

What type of staining is used here?

Figure 2.17: Elastic Connective Tissue. Photograph by Gina Profetto

What type of staining is used here?

Activity 2.7: Introduction to Tissues

Tissues are formed by groups of cells that are linked by intercellular junctions. The cells of tissue have a common origin and function together to perform specialized tasks. Histology is the term used for an area of science that deals with the study of tissues and pathology is the study of abnormal tissues. Body tissues are divided into 4 basic types: epithelial tissue, connective tissue, muscular tissue, and nervous tissue.

Your instructor will provide representative examples of the 4 tissue types. Using the table above draw what you see in your field using the light microscope at 400X magnification.

*Note some of the basic differences you see between the 4 types of tissue.

Post-Lab 2 Review Questions

1. What is the total magnification when the scanning objective lens is being used? 

A) 10X  B) 40X  C) 100X D) 4X 

1. Which of the following is the magnification of the high-power objective?

A) 4X  B) 10X  C) 40X  D) 100X 

1. What part of the microscope helps to move the stage in the x-y (back and forth) direction?

A) Condenser  B) Coarse/fine adjustment knob  C) Scanning objective lens D) Stage adjustment knob 

1. During osmosis, water will move through a semipermeable membrane towards _______ concentration.

A) High solute   B) Low solute  C) High solvent  D) Balanced solvent 

1. Which type of staining allows you to see reticular fibers?

A) H&E  B) Silver  C) Giemsa  D) Methylene blue  

1. In an H&E stain, nuclei appear ______.

A) Pink   B) Purple C) Green  D) Red E) Brown  

1. Gatorade and other sports drinks are hypotonic solutions. How could these drinks rehydrate someone with dehydration? 

1. One of the liver’s main functions is to detoxify potentially toxic substances. Given this information, which organelle (s) would you expect liver cells to have in abundance?

A) Mitochondria B) Peroxisomes C) Ribosomes D) Lysosomes E) Centrioles

1. What are the four basic tissue types?
2. The cell in the black box is indicating what phase of mitosis?

Figure 2.18 Whitefish blastula mitosis. Photograph by Gina Profetto

1. Define crenation and hemolysis. Why are these terms important in tonicity?
2. Match the term to its definition.