Human Nutrition lab

Microscopy Lab—Cells of the Gastrointestinal Tract A. Laboratory Objectives

Students will: ✓ Become familiar with the use of the compound light microscope ✓ Review the parts of the Gastrointestinal tract, particularly

absorptive surfaces like intestinal villi ✓ Record observations of prepared slides of different tissue/cell types

of the GI tract ✓ View and understand the structure of a typical human cell and its

organelles, particularly mitochondria responsible for cellular metabolism

✓ Compare and contrast animal and plant cells

Laboratory report due next class period: Data Record & Discussion Questions (Sections E & F)

B. Introduction Light Microscopy

Ordinarily, humans cannot see objects smaller than 0.1 mm in diameter, but the light microscope allows us to see objects as small as 0.2 microns (1 micron = 10-3mm). Most animal cells are extremely small, ranging in diameter from about 10 to 30 microns, so they are invisible without the aid of a microscope. The compound light microscope uses two lenses to magnify an object. An image seen in a microscope at a magnification of 100X means that its linear dimensions are 100 times those of the object giving rise to the image. Magnification is the ratio of the apparent size of the object as seen through the microscope (image size) to the real size of the object (object size). In addition to magnification, a microscope must also have resolving power. Resolution refers to the ability of the microscope to distinguish as separate and distinct objects that lie in close proximity (i.e. increase in visible detail).

! Cell Structure and Function The cell is the basic unit of life. Cells can take in nutrients, break them down to release energy, and get rid of wastes. They can reproduce, react to stimuli, and maintain an internal environment different from their surroundings. In multicellular organisms such as humans, cells work together in groups to maintain life and are organized into tissues! organs ! organ systems. Cells can be grouped into two broad categories: prokaryotic cells and eukaryotic cells. All prokaryotes are single-celled organisms while eukaryotes can be single-celled or multicellular organisms. Prokaryotic cells are less complex than eukaryotic cells in that they lack membrane-bounded nuclei and membrane-bound organelles. Two of the three domains of life—bacteria and archaeans —are prokaryotes. The third domain, the eukaryotes, comprises protists, fungi, plants, and animals. Animal cells are surrounded by a cell membrane which maintains homeostasis in the cell by controlling what enters and what exits the cell. The plasma membrane is a double-layered structure and has two main components: phospholipids and proteins. Inside the cell membrane is the cytoplasm that contains the cell organelles that perform functions necessary for cell survival. The largest organelle is the nucleus, which contains the cell’s genetic material and controls cell activities (“the control center of the cell”). Other organelles include the rough and smooth endoplasmic reticulum, the golgi apparatus, and lysosomes and peroxisomes. Of particular importance to nutrient utilization are mitochondria. The mitochondria are catabolic organelles where the process of cellular respiration breaks down carbohydrates, lipids, and proteins to obtain energy (“the powerhouse of the cell”). Mitochondria are able to convert the energy contained in the bonds of nutrients into ATP, the energy currency of the cell. Plant cells differ from animal cells in that they contain a rigid cell wall that surrounds the plasma membrane. They also contain distinct organelles such as chloroplasts and water-filled vacuoles that animal cells lack.

The Gastrointestinal Tract The macronutrients include protein, carbohydrate, and lipids. These

macronutrients can be absorbed only when they are broken down into their smallest units. The body prepares foods for absorption through the process of digestion. When food is ingested, it enters the gastrointestinal tract which can be thought of as a hollow tube. The inside of the tube is called the lumen. Food within the lumen has not been absorbed and is therefore technically still outside the body. Only after food is transferred into the cells that line the intestine by the process of absorption is it actually ‘inside’ the body. Once food is broken down into its smallest units—namely amino acids, simple sugars, and fatty acids—and absorbed by intestinal epithelial cells, these molecules can then be metabolized by individual cells to provide the cells with the energy they need to function properly.

Food consumed in the diet is digested and absorbed within the gastrointestinal (GI) tract. The upper GI tract is made up of the mouth, esophagus, and stomach. The lower GI tract is made up of the small intestine, large intestine, and anus. There are also a number of important organs involved in digestion that provide digestive enzymes and other secretions necessary for digestion to occur. These include the pancreas, liver, and gallbladder. The small intestine is the main site of digestion of food and absorption of nutrients. It is divided into three segments: the duodenum, the jejunum, and the ileum. The structure of the small intestine is specialized to allow for maximum absorptive surface area. Intestinal walls are arranged in special folds, which increase the surface area in contact with nutrients. Also, the inner surface is covered with finger-like projections called villi. Each of these villi is covered with tiny microvilli, referred to as the brush border. Nutrients must be absorbed by the intestinal cells of the brush border. Each villus contains a blood vessel and a lymph vessel; both are located only one cell layer away from nutrients in the intestinal lumen and are responsible for delivering nutrients to the tissues of the body.

This Laboratory Exercise In this exercise we will be using a compound light microscope to observe different cell types under the microscope. You will investigate the overall structure of a typical animal cell and identify organelles such as mitochondria. You will make a wet mount slide of onion skin to observe plant cells and of human cheek cells to compare and contrast animal and plant cells. You will journey through the gastrointestinal tract and observe similarities and differences among the different cell types you observe along the way. You will illustrate your observation of the major site of absorption in the body, the intestinal villi, under the microscope.

C. Materials Needed Student Microscope

Mounted specimens (available on supply table) Microscope slides (3 per student) Cover slips (3 per student) Wax pencil Dropper bottle with water Lugol’s Iodine Methylene Blue Janus Green B stain Toothpicks Onion skin sample

D. Procedure

A. Use of Microscope 1. Remove the dust cover from your microscope and carefully plug it in. 2. Obtain a size-12 font letter “e” from the supply table. 3. Carefully place the letter “e” in the middle of a clean microscope slide. 4. Place a cover slip over the specimen. Now, carefully mount the letter “e” slide onto the microscope stage. 5. Turn on the illuminator on your microscope by gently turning the knob on the right-hand side of the microscope to a level of 4-6.

6. Select the 4x objective to “scan” and locate the image of your specimen while looking through your eyepiece. Give everyone a chance to locate the specimen on the slide. 7. Now, switch to the 10x objective and use the coarse and fine adjustment knobs to bring the specimen into focus. Now, switch to the 40x objective and use the focus adjustments to bring the specimen into focus one last time. Show the focused image of the letter “e” to your TA to obtain the remainder of your specimens for today’s lab.

B. Typical Animal and Plant Cell Structure 1. Obtain a typical ‘animal cell’ slide from the supply table and mount it onto the microscope stage.

2. Follow the steps above to focus and obtain the most distinct image of the cell possible.

3. Illustrate what you see through the microscope in the space provided on your data record. Make sure to include any visible organelles and important features in the specimen and label them accordingly. 4. Give each student in your group a chance to do this. 5. Return the animal cell slide to the supply table and repeat steps #1-4 with the typical ‘plant cell’ slide from the supply table. 6. Return the ‘plant cell’ slide to the supply table when you are finished.

C. Mount a slide of human cheek cells 1. Obtain a clean microscope slide from your supply tray and a clean toothpick.

2. Label the slide on one end with your lab group station and initials. 3. Obtain a cover slip from your supply tray.

4. Use one straight-edge end of the toothpick (not the tip) to obtain human cheek cells from inside your mouth according to your TA’s demonstration. 5. Immediately after obtaining the sample, gently use your toothpick to smear the specimen onto the middle of the microscope slide. 6. Place the cover slip over your specimen smear. The cover slip is lowered on to the specimen at an approximately 45 degree angle gently. This reduces the number of air bubbles the specimen will have. 7. Mount the slide on the microscope and observe the specimen at the different objectives. Record your observations.

8. Then, remove the slide from the microscope stage. 9. Now place a drop or two of water on the specimen. Then, place a drop of the Methylene blue stain beside and under one corner of the cover slip. 10. Then, place a towel on the opposite side of the cover slip near the water. This will draw the stain through the entire specimen in a few seconds without removing the cover slip. This technique will also remove any air bubbles which have formed. The stained specimen may now be observed. 11. Mount the slide on the microscope and observe the specimen at the different objectives. Record your observations. 12. Repeat steps #1-11, but stain the slide with the Janus Green B stain instead of the Methylene blue stain. The Janus Green B stain will stain the mitochondria within the cells. Record your observations. 13. There should be two slides for each person in your lab group.

D. Mount a wet slide of onion skin to observe plant cells 1. Obtain a specimen of onion skin from the supply table for each person in your lab group. 2. Obtain a clean microscope slide and place the specimen on the slide using the forceps and/or spatula in your supply tray. 3. Place the cover slip over your specimen. The cover slip is lowered on to the specimen at an approximately 45 degree angle gently. 4. Mount the slide on the microscope and observe the specimen at the different objectives. Record your observations.

5. Then, remove the slide from the microscope stage. 6. Now place a drop or two of water on the specimen. Then, place a drop of the Iodine stain beside and under one corner of the cover slip. 7. Then, place a towel on the opposite side of the cover slip near the water. This will draw the stain through the entire specimen in a few seconds without removing the cover slip. This technique will also remove any air bubbles which have formed. The stained specimen may now be observed. 8. Mount the slide on the microscope and observe the specimen at the different objectives. Record your observations. 9. There should now be three slides for each person in your lab group: (1) cheek cell slide stained with Methylene Blue, (2) cheek cell slide stained with Janus Green B, and (3) onion skin slide stained with Iodine. Show your TA your mounted slides before moving on to sections E and F.

E. Observation of different tissue types of GI tract 1. Obtain a prepared slide from the supply table that contains a section of the stomach, small intestine, and large intestine on a single slide. 2. Mount this slide carefully onto your microscope stage. Observe the section of each type of tissue and record any similarities and/or differences you see in the different tissue types. 3. Make sure that each person in your lab group has an opportunity to record their observations. Put this slide to the side for now—you will look at this slide again to complete section F. 4. Obtain a prepared slide from the supply table that contains a section of mitochondria in liver. Record your observations in detail so that you can answer discussion questions that follow. 5. Now, as a group, choose a particular part of the GI tract you are interested in observing under the microscope. Tell your TA which organ or section of the GI tract you have chosen. Your TA will provide you with the corresponding specimen. 6. Record your observations carefully and be prepared to draw what you see on your data record. Answer the corresponding questions about the role of that organ/tissue in digestion.

F. Illustration of intestinal villi as seen through microscope 1. Now, revisit the slide you observed for section E1 (slide containing a section of stomach, small intestine, and large intestine). 2. Mount and orient the slide on your microscope so that the small intestine section is in view. Illustrate the intestinal villi as you observe them through your microscope in the space provided on your data record. 3. Now that you have illustrated what you see under the microscope for intestinal villi, draw a diagram of an individual villus containing microvilli (brush border). Include any and all important structural features of an intestinal villus. You may use your lecture textbook or any other materials to help you in accurately depicting an intestinal villus.

Laboratory Report for Microscopy Laboratory

Student Name: Date:2/4/2019

TA Name:_________________________________

E. Data Record and Lab Write-up (20 points) 1.) In the space provided below, illustrate what you expect to see in typical animal cells and typical plant cells, with as many features/organelles identified as possible. (3 pts)

2a.) Illustrate below the image you see when you observe the onion skin with the 4x and 40x objectives. How does the addition of iodine change what you observe? (2 pts) _______________________________________________________ _______________________________________________________

2b.) Illustrate below the image you see when you observe the cheek cell sample with the 4x and 40x objectives. How does the addition of methylene blue and Janus green B stain change what you observe? (2pts) _______________________________________________________ _______________________________________________________

3a.) In the space provided below, illustrate the image you see when observing intestinal villi under the microscope. 3b.) In the space provided below, illustrate the image you see when observing the section/organ of the GI tract you selected from the supply table. (6 pts)

3.) Now, diagram below the internal structure of each villus from what you’ve learned in lecture and lab. Make sure to carefully depict the microvilli brush border and the system of vessels within each villus. Identify in your drawing the path taken by water-soluble versus fat-soluble nutrients once absorbed in the intestinal villi. (7 pts)

F. Discussion Questions (30 points) 1. When observed under the microscope, what is the most striking difference between plant and animal cells? What features are they likely to have in common? (5 pts)

________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 2. The Janus Green B stain should allow you to see mitochondria within the cells obtained from your cheek sample. Describe below the observations you made about the number of mitochondria per cell and their relative distribution and size within the cytoplasm of cheek cells. Compare and contrast what you observed with the prepared slide you observed in Section E4 for Liver mitochondria. What function do mitochondria have within cells? What processes take place within this organelle? (10 pts) ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 3. In the space provided below, please identify the section and/or organ of the GI tract you chose in Section E5. Is it part of the upper or lower GI tract? What is its primary function in digestion and what occurs in this section of the GI tract? Be specific. (5 pts) ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ 4. Explain in detail below the process of digestion and absorption that occurs in the small intestine. Provide a detailed description of how different types of nutrients are taken up by intestinal epithelial cells that line the gut. How does the brush border behave for different types of nutrients and how are they transported to the liver and the bloodstream following absorption. Please identify in your answer those structures you illustrated in your data record for a typical intestinal villus. (10 pts) ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________

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