Pre-Lab 3 Assessment: The Cell
Name:
Section:
Please read the lab in its entirety and answer the following questions to the best of
your ability.
1. Name three major differences between Prokaryotic and Eukaryotic cells?
2. Give an example of a prokaryotic organism.
3. What are the three forms (physical shapes) of bacteria?
4. When viewing gram-positive bacteria under a compound microscope, what
color(s) do they appear?
5. What is the major factor that determines if a bacteria is gram-positive or
gram-negative?
6. What is the function of choloroplasts?
7. List three membrane-bound organelle (found in the eukaryotes) and explain
the function of each.
Laboratory Three
The Cell
Purpose
The purpose of these experiments is to gain a better understanding of the Domains
Bacteria and Eukarya. Today, we will be observing bacteria using a compound
microscope. In order to properly view bacteria, we will be learning a staining technique
that allows us to differentiate bacteria. We will also be culturing various environmental
surfaces and analyzing the bacterial growth on the agar plates. At the end of this
experiment, you will realize that bacteria are everywhere. You will also be introduced to
the world of microbiology and learn the simplest method of bacterial identification. You
will also observe the eukaryotes by microscopic examination and analysis of various
specimens.
Introduction: Prokaryotes (Part One)
The cell is a highly complex biological structure that is found in all living organisms.
Constantly dividing, undergoing self-repair, it is considered to be the building block of
life. The human body contains trillions of cells that have a variety of essential functions.
Eating, drinking, breathing, walking, talking, thinking, and many more activities, all
require certain cellular involvement. For classification purposes, cells can be placed into
two major categories, prokaryotic (e.g. bacteria) and eukaryotic (e.g. humans) cells.
Prokaryotic cells (figure 3.1) include bacteria and are considered to be primitive,
evolving on the earth for 3 – 4 billion years. These cells contain no nuclei, do not have
membrane-bound organelles, are relatively very tiny (microscopic), unicellular, and
contain looped-DNA that is stored within the nucleoid region of the cell. The anatomy of
prokaryotic cells can be quite fascinating, as these cells contain many unique structures
not commonly seen in eukaryotes. Surrounding the cell is a massive encasing, known as a
capsule, which prevents desiccation by regulating water loss and protecting the bacterial
cell against the immune system (i.e. phagocytosis). Scattered on the surface of the
capsule, tiny projections known as pili, which allow the cell to reproduce and attach to
the hosts cells. The plasma membrane is a layer surrounding the prokaryotic cell that
aids in nutrient transport and further protection. Depending on the bacterium species, a
tail-like structure known as a flagellum might be present that aids in locomotion. Within
the prokaryotic cell, there are ribosomes that produce proteins for the cell. Collectively,
the prokaryotic cell is able to reproduce and function, as it has successfully for billions of
years.
26L.02001
Sss22dda2
(Pearson
Photo
Library)
Figure 3.1 The structure of a bacterial cell. Courtesy of Pearson Publishing.
Bacteria are extremely important organisms that allow for many forms of life to survive
and thrive. For instance, the bacteria in your intestine allow for proper absorption of
nutrients, release of essential vitamins, protection against pathogenic bacteria, and many
other key biological functions. However, bacteria are also pathogens and can cause
disease, such as pharyngitis (i.e. sore throat) caused by the bacteria Streptococcus
pyogenes. There are three basic forms of bacteria that allow scientists to categorize them
for better means of identification. The three forms of bacteria are cocci, bacilli, and
spirochete (figure 3.2).
Along with the bacteria’s shape, scientists also use an important staining technique to
identify the organism. This technique is known as Gram Stain, discovered by a Danish
bacteriologist Hans Christian Gram. The procedure involves a series of stains (e.g. crystal
violet, iodine and safranin), washings (e.g. water), and decolorization (e.g. typically
acetone) to better view the bacteria microscopically. After the staining process is
complete, the slide is ready to be observed using a compound microscope. Gram
discovered that this series of staining techniques could allow one to organize bacteria in
to two categories, gram-positive and gram-negative bacteria. To determine which
category a bacterial species falls under, one must observe the bacteria’s peptidoglycan
cell wall.
Figure 3.2 (from left to right) Cocci, bacilli, and spirochete bacterial shapes.
Courtesy of Vincent E. Piscitelli, MHS, M(ASCP)CM
Gram-positive bacteria have a thick peptidoglycan wall and therefore do not decolorize,
securely storing the crystal violet within its cell wall. Gram-negative bacteria have
thinner peptidoglycan wall and therefore decolorize easily, giving up the crystal violet
and absorbing the counterstain, safranin. When making observations under a compound
microscope, gram-positive bacteria will appear a purplish violet color (figure 3.3) while
gram-negative bacteria appear a reddish pink color (figure 3.3).
The ability to
differentiate the bacteria’s shape and determine whether it is gram-positive or gramnegative allows for clinicians to diagnose disease and begin treatment in a much quicker
manner.
Figure 3.3 Gram-positive bacteria (left) and gram-negative bacteria (right) observed
using a compound microscope (1,000X using oil immersion lens). Courtesy of
Vincent E. Piscitelli, MHS, M(ASCP)CM
Laboratory Part A:
Observing the Three Shapes of Bacteria
Materials for the lab group of 2 students:
- Prepared bacterial shape slide
- Lens paper
- Lens cleaner
- Immersion oil
- Compound microscope
Procedure
1. Obtain a “bacterial shape” slide and place the slide under the compound
microscope. Under low magnification, observe the characteristics of the bacteria
(including shape and staining category).
2. Repeat using medium and high magnifications.
3. Place a small drop of immersion oil on the slide and view under the oil immersion
lens.
4. Compare the observations you made while viewing the “bacterial shape” slide
under the microscope, to the images and descriptions provided in your
textbook/laboratory book.
5. Clean the microscope and place all equipment back to its designated area.
Laboratory Part 1B:
Inoculating Agar Plate.
Materials for the lab group of 2 students:
- Hot plate
- China marker
- Beaker with boiling stones
- Bottle of nutrient agar
- Biohazard waste bag (red)
- Three sterile (empty) agar plates
- Masking tape
- Disinfectant cleaner
- Paper Towels
- Three sterile swabs
- Latex/Nitrile gloves
- Hot mitt
Procedure
1. We will begin by melting the nutrient agar tube and placing its contents into the
three empty agar plates. First, place tap water into the beaker (that contains
boiling stones) so that the level of the water and the level of the agar are equal.
Turn the hot plate to its maximum heat level and allow the water to boil.
2. While the water is boiling, ponder the three locations/areas you will be swabbing
for culture.
3. Meanwhile, using a china marker, label (in detail) the three agar plates with the
following information (label the agar portion of the plate, not the lid) (see below).
Date & Time
Description of what you
swabbed for culture.
Type of Media
Incubation temp
Your name
+ your
partners
name
4. In order to minimize contamination, utilize the “clamshell” technique. This is
accomplished by opening the agar plate to the point where you are able to pour
the agar successfully without much room to spare (see below).
Open very
swiftly, and
avoid
breathing
over the agar
plate.
The “Clamshell” Technique
5. Once the nutrient agar is completely liquefied, remove from the beaker and dry
using a paper towel. Use a hot mitt to equally pour the contents of the nutrient
agar tube into each agar plate (*Note that each agar plate has a tiny “triangle” that
indicates how much agar should be placed into the plate, fill the plate with agar up
to the point of the triangle).
6. Allow the agar to solidify and cool-down (approximately 15 minutes).
7. When the nutrient agar plate is at room temperature, put on the latex/nitrile gloves
and inoculate your first agar plate. You have three sterile swabs, so you are
allowed to swab any three surfaces you desire (including your cheek/mouth,
skin, tables, handles, doorknobs, elevator buttons, etc.). Remember, ONE SWAB
PER AGAR PLATE.
8. In order to properly inoculate each agar plate, streak the plate with the sterile
swab and utilize the four-quadrant streaking method (see below).
A
B
Streak the agar plate with the swab to create a 1st quadrant (A). Streak the agar
plate via the four-quadrant streaking technique in order to spread out the bacteria
across entire agar plate and allow for colony isolation (B).
9. Once you have inoculated each agar plate, invert (to avoid moisture collection on
agar surface) each plate and tape together using the masking tape.
10. Place the inverted stack of three agar plates you inoculated into the 37° C
incubator (for 24-48 hours). If excessive drying of the agar is occurring, place the
agar plates into the refrigerator after 24 hours.
11. Clean and disinfect the lab bench and dispose of all waste material in
biohazard waste bag.
12. Remove the three plates from the incubator/refrigerator after 24-48 hours and
observe for bacterial growth. Please continue to part 2B.
Laboratory Part 2B:
Staining and Differentiating Bacteria.
Materials for the lab group of 2 students:
- Clean microscope slide
- Gram positive/Gram negative control slide
- Disposable pipettes
- Distilled water (via dropper bottle)
- Clothespin
- Hot plate
- Staining tray
- Microbiology stains: Crystal violet, iodine, and safranin.
- Acetone/ethanol decolorizer
- Lens cleaner
- Lens paper
- Compound light microscope
- Immersion oil
- Disinfectant spray
- Paper towels
- Sterile swab
- Biohazard waste bag (red)
- Sterile water
Procedure
1. Once the stack of three agar plates have been removed from the incubator/refrigerator,
locate a bacterial colony (i.e. raised, circular, colorful region on agar plate surface) and
use a sterile swab to gather a small sample of the bacterial colony (on the tip of the
swab).
2. Using a disposable pipette, place 1 drop of sterile water onto a microscope slide; take
the swab that contains the bacterial colony and smear it in the sterile water (located on the
microscope slide) very evenly and thin (spreading too thick makes viewing the bacteria
very difficult).
3. Turn on the hot plate on and set to 3-4 (temperature), allow hot plate to warm-up.
4. Use the clothespin to pick up the supplied gram positive/gram negative control slide
and heat-fix by slowly waving the slide over the hot plate three to five times.
5. Next use the clothespin to pick up the smeared microscope slide and slowly wave the
slide over the hot plate three to five times in order to heat-fix the smear on to the
microscope slide (this prevents the specimen from washing off the slide when we begin
the staining process).
6. Once the slides have been heat-fixed, allow the microscope slides to cool down for 2-3
minutes.
7. Stain each microscope slide individually. Once the slide is cooled, while hovering the
slide over a staining tray, flood the slide with crystal violet (Remember, if the bacteria
present are gram-positive, they will retain the crystal violet stain). Allow the stain to set
for 1 minute.
8. Rinse the slide off with distilled water.
9. Next, flood the slide with gram iodine stain for 1 minute.
10. Rinse the slide off with distilled water.
11. Decolorize the slide by applying the acetone/ethanol decolorizer continuously; once
the stain appears to have washed off the slide, stop immediately.
12. Rinse the slide with distilled water immediately (if the slide is not rinsed
immediately, over-decolorization might occur).
13. Finally, flood the slide with safranin stain (counterstain) for 30 seconds (Remember,
if the bacteria present are gram-negative, the bacteria will retain the safranin stain).
14. Rinse the slide with distilled water.
15. Once the slide has been properly stained, dry off the slide and place it under a
compound light microscope and observe the bacteria using low, medium, and high
magnifications.
16. Once all three powers have been observed, place a small drop of immersion oil
on the slide and rotate the rotating-objective turret, turning the power to the
highest magnification (oil immersion).
17. View the bacteria, noting gram-positive and gram-negative bacteria, shape, etc.
and record your observations in Lab Part 2B Observations table (below).
18. Clean and disinfect the lab bench, properly stow the microscope and dispose of
all waste material in biohazard waste bag.
Name:___________________________________
Section: _______________
Lab Part 2B: Staining and Differentiating Bacteria
Data Table
Specimen
Collected
Shape(s)
Observed
Gramneg./Gram-pos.
Number
of
Colonies
Present
Additional
Observations
1.
2.
3.
Introduction: The Eukaryotes (Part Two)
Eukaryotic cells appeared on Earth approximately 2.1 billion years ago. The first
eukaryotes were single-celled organisms resembling the present-day protists. Over time
they diversified, giving rise to various groups of protists and to the multicellular
eukaryotes in the Kingdoms Fungi (some fungi are single-celled, but most are
multicellular), Plantae and Animalia present on Earth today. While most protists are
single-celled organisms, some colonial and some multicellular (the seaweeds) species are
considered to be protists also.
Eukaryotic cells are larger and more complex than prokaryotic cells, and contain
membrane-bound organelles - membrane-enclosed compartments with specialized
functions within the cell. The DNA of a eukaryotic cell is contained within an organelle
called the nucleus (eu =true and karyon = kernel). The endomembrane system is made
up of a group of organelles that synthesize and then modify proteins and lipids. These
organelles, some of which are interconnected, include the rough and smooth
endoplasmic reticulum (ER), the Golgi apparatus, and various vacuoles (membraneenclosed sacs). Proteins and phospholipids are synthesized by the rough endoplasmic
reticulum (RER). Some of the products of the RER are sent to other locations in the cell
in transport vesicles. Lipids are synthesized in the smooth endoplasmic reticulum
(SER). The Golgi apparatus receives products from the ER, modifies them, and then
distributes them in vesicles to other locations in the cell. Other organelles found in
eukaryotic cells include mitochondria, the site of cellular respiration, where energy from
food molecules is harvested and converted into ATP. A network of fibers called the
cytoskeleton extends throughout the cytoplasm of eukaryotic cells. It provides support
and aids in the movement of organelles and cells. Plant cells contain chloroplasts, the
site of photosynthesis, a large central vacuole in which water and other substances are
stored, and cell walls made of cellulose. Animal cells contain lysosomes, membrane-
enclosed sacs containing digestive enzymes, and centrioles, which aid in mitosis and
meiosis.
Figure 3.4. A comparison of an animal and a plant cell. Note the structures they
have in common, and those that are unique to one cell type or the other. Courtesy of
Pearson Publishing.
All cells, whether eukaryotic or prokaryotic, are surrounded by a plasma membrane. The
cells of fungi and some protists also have cell walls made of various polymers
surrounding the plasma membrane. Animal cells do not have cell walls. A cell is an
incredibly busy place. Inside every cell there are thousands of chemical reactions going
on, including the synthesis and breakdown of various organic molecules. Materials are
being moved in and out across the plasma membrane, and organelles move around the
cell along tracks of the cytoskeleton. A cell’s activities are regulated in response to
signals from the cell’s external environment. Using the light microscope you will observe
some of the major organelles.
Laboratory Part C:
Observing Eukaryotic Cells
1. Observing Plant Cells
Plants are multicellular photosynthetic eukaryotes. They use the sun’s energy to convert
carbon dioxide into organic compounds. These organic compounds are then used by the
plant for maintenance, growth and for the storage of the sun’s energy.
Plants are
considered to be autotrophs (“self –feeders”) because they get their energy from the sun
rather than by feeding on other organisms.
Plants and other autotrophs (some
prokaryotes and protists are autotrophs) are the primary source of food and energy for all
heterotrophs (“other-feeders”), including humans. The energy we obtain from the food
we eat was originally solars energy that has been converted into chemical energy by
autotrophs. Plant cells have several structural features not found in animal cells. Plant
cells are surrounded by a cell wall, exterior to the plasma membrane, which provides
protection and structural support to the cell, and to the plant as a whole. The cell wall is
visible under the microscope as a clear, rectangular structure surrounding each cell. Plant
cells contain chloroplasts, the organelles in which photosynthesis (the production of
ATP) takes place. Chloroplasts are easy to observe within a plant cell – they are
numerous, fairly large, and green, due to the chlorophyll pigments they contain. Plant
cells also contain a large central vacuole in which water and various other substances are
stored. In addition to the cell wall, chloroplasts and central vacuole, which are unique to
plants, the plant cell also contains many structures common to all eukaryotic cells,
including the nucleus, rough and smooth endoplasmic reticulum, Golgi apparatus and
mitochondria. Take a look at the plant cell model on demonstration and identify these cell
components.
Materials for the lab group of 2 students:
Elodea leaf
plant cell model
slide and cover slip
dropper bottle of water
Procedure:
In this exercise you will observe cells in the leaves Elodea, an aquatic plant.
Prepare a wet mount of an Elodea leaf:
1. At the front of the room, obtain a leaf from the tip of an Elodea plant (where the
smallest leaves are located), using either forceps or your fingers to pull off a leaf.
2. Place the leaf on a slide, then add a drop of water and a cover slip to the slide, and
observe it under the light microscope, first with the 4X objective, then the 10X objective,
and finally, the 40X objective.
Draw a picture of an Elodea cell. Use the whole rectangle provided to draw the cell,
making it large and clear, and label the following structures: cell wall, chloroplasts,
central vacuole and if visible, the nucleus.
2. Observing Animal Cells
Animals are multicellular heterotrophic eukaryotes that ingest their food, which can
include other animals, prokaryotes, plants, protists, and fungi. In addition to the
organelles common to all eukaryotic cells (nucleus, rough and smooth endoplasmic
reticulum, Golgi apparatus and mitochondria) animal cells have several types of
organelles not found in plant cells. These include lysosomes, membrane-bound
organelles that contain digestive enzymes used to break down food molecules and
damaged organelles and centrioles which aid in the separation of chromosomes during
mitosis and meiosis. Take a look at the animal cell model on demonstration (or in the
biology atlas at your table) and identify these cell components.
Epithelial cells form a protective surface on the outside of an animal, and also line its
interior cavities and ducts. In this exercise you will examine the epithelial cells that line
the inner surface of your cheek.
Materials for the lab group of 2 students:
toothpicks
animal cell model
slide and cover slip
dropper bottle of water
dropper bottle of methylene blue
Procedure:
Prepare a sample of epithelial cells from the inner surface of your cheek:
1. Place a drop of water on a slide.
2. With a clean toothpick gently scrape the inside of your cheek several times, then swirl
the toothpick gently in the drop of water on the slide.
3. Dip the tip of a clean toothpick into the methylene blue, then swirl the tip gently in the
drop of water/ cheek cell mixture on the slide. Put a cover slip on top of the water/cheek
cell/ methylene blue mixture.
Put all used toothpicks in the biohazard waste bag at the front of the room.
4. Examine the epithelial cells under the light microscope, first with the 4X objective,
then the 10X objective, and finally, the 40X objective.
Draw a picture of several cheek cells. Use the whole rectangle provided to draw the cells,
making them large and clear. Label the plasma membrane, which forms the outer
boundary of the cell, and the nucleus, visible as a dark blue, circular structure near the
center of the cell.
Post-Lab 3 Assessment: The Cell
Name:
Section:
After completing the laboratory assignment, please re-read the lab in its entirety
and answer the following questions to the best of your ability.
A. Prokaryotes
1. The prokaryotes were the first living organisms to evolve over 3.5 billion years
ago, and are found in every type environment on Earth today, including in and on
other organisms. What can you conclude about the ability of prokaryotes to adapt
to Earth’s diverse and changing environments?
2. You have isolated bacteria from several patients with the same disease symptoms.
What are two methods you can use to help identify the bacteria you have isolated?
3. List two important beneficial functions of the bacteria that live in your intestine.
4. Why is it important to utilize the “clamshell” technique (microbiology)?
5. What is an advantage of the four-quadrant streaking method?
6. Out of the three surfaces that were swabbed, which contained the most bacterial
colonies? In your opinion, does this result make sense? Why?
B. Eukaryotes
7. Which organisms that you observed have chloroplasts? What is the function of
chloroplasts?
8. Name three types of organelles that are found in all eukaryotic cells:
9. Which types of eukaryotic cells have cell walls?
10. Which groups of eukaryotes contain both multicellular and single-celled
organisms?
11. Which groups of eukaryotes are made up entirely of multicellular organisms?
12. What is the dark blue structure visible in the center of cheek cells stained with
methylene blue? What is the function of this structure?
References
Commons.wikimedia.org
Rydene, H. (2010). Introduction to Biology Lab Procedures (and other important
information). (5th ed.). New York, NY: Freeman Custom Publishing.
Simon, E., Dickey, J., & Reece, J. (2013). Campbell Essential Biology with Physiology.
(4th Edition). Pearson Education.