Unit 6

Prokaryotes
This lab uses the following hazardous chemicals:

I. Crystal Violet
II. Gram’s Iodine Solution
III. Ethanol

As a result students are required to wear, at minimum, Goggles & Gloves.

Abstract
Throughout the laboratory this semester, you have primarily discussed chemical and
physiological processes in the context of eukaryotic cells. Eukaryotic cells are cells that
contain a nucleus, which houses the organism or cell’s genetic material. In this unit
we will be discussing prokaryotic organisms, which are organisms that do not have a
nucleus. Specifically you will be focusing upon bacteria. These particular prokaryotes
are an important organism to study as they play various roles of importance in any
ecosystem. Some bacteria can perform the role of decomposers, some can live at
extremely high temperatures, some live in the gastro intestinal tract of humans, and
some are the source of human diseases. In this unit you will not only learn about the
differences between bacteria and eukaryotic cells, but you will also learn how to
distinguish different types of bacteria based upon their morphology and the properties
of the molecules on their exterior.

6.1 Classification of Bacteria Based Upon Morphology

Introduction
As stated previously, bacteria are classified based upon their morphology. Furthermore,
the bacteria can be further classified based upon they way in which they orient
themselves around other bacteria. This is to say that bacteria can occur in single
structures or in chains. Below you will find a list of bacteria classifications as well as
terms regarding their orientation to other bacteria.

Bacilli: elongated cigar or rectangular shaped microbes

Cocci: round, spherical or circular shaped microbes
Spirilla: spiral or corkscrew shaped microbes

Staphylo: Grape-like clusters

Strepto: Long chain-like structure

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So if a grape-like cluster of cocci bacteria were observed, you would refer to them as
Staphylococcus.

Use this knowledge of the basic morphologies of bacteria to observe, identify and draw
one of each of these types of bacteria.

Classification of Bacteria Based Upon Morphology:

Materials and Methods

1. In your slide box you will find a slide that is labeled “Bacteria 3 Types Smear.”
Place this slide on the microscope stage.

2. Using the 4X objective, scan the slide for one of three dots. Once you have found
one, you may move your magnification up to 10X, then 40X.

3. Once you have the slide focused at 40X, add microscope oil, and switch to the
100X objective.

4. Draw what you observe, and correctly label the type of bacteria that you observe
in the space provided below.

5. Once you have finished drawing one type of bacteria, switch back to the 4X
objective, find one of the two other circles on the slide, and repeat steps 2-4 until
you have drawn all three types of bacteria.

Figure  1.  Drawings  and  Labels  of  the  three  different  types  of  bacteria  based  upon  their  
morphology.  

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 Tip
Become very familiar with these different types of bacteria and be able to identify their
morphology using proper terminology.

6.2 Comparison of Bacteria to Eukarya

Introduction
As discussed previously, there are some observable differences between Bacteria
(prokaryotic cells) and Eukarya (eukaryotic cells). Below you have been provided with a
brief description of some of the processes needed for bacterial reproduction and survival.
Read through this information and use your knowledge about eukaryotes to fill in the
table provided below.

Figure  2.  Schematic  of  a  bacterium  and  its  various  cellular  components.  

Bacteria lack the membrane-bound nucleus of eukaryotes, and their DNA forms a tangle
known as a nucleoid and is found in the cytoplasm of the cell. Additionally, while
proteins are bound to the DNA they do not keep it in a structured shape, and bacterial
chromosomes are circular, rather than linear. Also, additional DNA may be found within
bacteria that exist in loops known as plasmids. These plasmids can be ejected from the
cytoplasm of the cell into any extracellular space, and can also be transferred out of one

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cell and into another. This ability of bacteria to trade genes is what can eventually lead
certain strains of bacteria to become resistant to particular drugs or treatments.

Results
Characteristic Bacteria Eukarya

Nucleus

Chromosome

Organelles

Unicellular or
multicellular

Sexual reproduction

Plasma Membrane

Cell wall material

Simple or complex RNA

polymerases
Figure  3.  Identification  of  the  differences  that  exist  between  bacteria  and  eukarya.  

Points for Discussion:

1. In the picture of the bacterium (Figure 2), ribosomes were shown. Are ribosomes
membrane bound organelles? Why do you think bacteria have ribosomes?

6.3 Gram Staining Bacteria

Introduction:
Based upon your answers in Figure 3, it may seem as though all bacteria have a
peptidoglycan cell wall, and that the characteristics of this cell wall are the same in all
types of bacteria. However, this is not the case, and, as it turns out, this particular

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property of various types of bacteria may be exploited to differentiate between different
types.

A subset of bacteria have a very thick peptidoglycan cell wall composed of carbohydrates
crossed linked to proteins. Such a cell wall will retain a purple color when stained with a
substance known as crystal violet.

Another subset of bacteria have two cell walls: a thin inner wall composed of
peptidoglycans plus an outer wall composed of carbohydrates, proteins and lipids. These
bacteria do not stain purple when using crystal violet, and instead maintain a red stain
from the staining component safranin.

The variation in staining for these two particular types of bacteria allows them to be
categorized into two physiologically different groups: gram (+) and gram (-). The gram
staining technique was developed by Hans Christian Gram in 1884, and is still used
widely today to assist with the identification of various types of bacteria. More
importantly, some treatments will only work on gram (+) or gram (-) bacteria.

Reaction and appearance of bacteria

Solutions in order applied Gram Positive Gram Negative

1. Crystal Violet (CV) –
Cells stain violet Cells stain violet
primary stain
CV-I complex formed CV-I complex formed
2. Gram’s iodine (I) –
within cells; cells remain within cells; cells remain
mordant
violet violet
Cell wall dehydrated, pores Lipid extracted form cell
shrink, permeability walls; porosity increases,
3. Alcohol – decolorizer
decreases, CV-I complex CV-I removed from cells;
retained; cells remain violet cells colorless
Cells not affected, remain Cells take up the stain;
4. Safranin – counter stain
violet become red
Figure  4.  Effects  of  the  different  materials  used  in  gram  staining  on  gram  +  and  gram  –  cells.  
 
 
 

Tip
Become very familiar with each stain, its purpose, and how it affects both Gram + and
Gram – cells!

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 Gram Staining Bacteria

Materials and Methods

1. Set out a clean glass slide. Mark one edge with an identification code.

2. Place a small drop of deionized water on the slide.

During the remainder of the experiment, you will be working with hazardous
substances. Goggles and Gloves must be worn from this point on!

3. Sterilize an inoculating loop by placing it into the incinerator for a short period of
time. The loop should glow red. Allow the loop to cool.

4. Use the sterile inoculating loop to transfer a very small amount of bacterial colony
to the water drop. Mix well so that the bacteria/water mixture is about the size of
a nickel. Place the loop back in the incinerator until red, remove, and allow to air
dry.

5. Allow the water drop to air dry completely on your bench top before proceeding
to step 6.

6. Heat-fix the slide by passing it over the exterior of the incinerator at least 15
times (each time can be a very brief wave). Heat-fixing coagulates the bacterial
proteins to the slide so that the cells will not be washed off during the staining
process.

7. Place the slide on a staining tray and cover the bacterial smear with crystal violet
for 1 minute.

8. Hold the slide at a slight incline and rinse the crystal violet off with water.

9. Cover the smear with Gram’s iodine for 1 minute.

10. Rinse off the Gram’s iodine with water.

11. Destain the smear by dropping ethanol down the slanted slide 1 drop at a time.
Continue drop-wise until only a faint violet color is seen in the alcohol rinse.
Rinse the slide with water to stop the action of the alcohol.

12. Cover the smear with safranin for 1-2 minutes.

13. Rinse off the safranin with water and gently blot the slide dry with bibulous
paper.

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 14. Examine the slide under oil immersion. Record your observations in the table on
the next page.

Results

Sample Name Shape Gram Stain (+/-)

Figure  5.  Determination  of  shape  and  gram  stain  of  unknown  samples.  

6.4 Medical Microbiology

Introduction
External and internal organs of the body are covered with over one hundred trillion
bacteria, and these bacteria are required for healthy living. While we provide these
bacteria with food and space to live, they protect us from more pathogenic strains of
bacteria. Additionally, some of these bacteria can produce nutrients needed by the body
to maintain homeostasis. However, the introduction of virulence factors can result in
alterations in these bacteria that lead them to cause disease in humans. This delicate
balance means that it is extremely important for certain regions of the body to remain as
“sterile” as possible. Such sites are not typically exposed to the external environment.

Name four “sterile” sites for the human body that should not contain bacteria:

1. _________________________________
2. _________________________________
3. _________________________________
4. _________________________________

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When bacteria acquire virulence factors, are introduced into normally sterile sites, or are
exposed to immunocompromised patients, they can potentially lead to a disease
condition. In this case, the bacteria would be referred to as a pathogen.

In 1890 Robert Koch developed a means of identifying disease-causing bacteria based

upon a set of criteria he deemed “Koch’s Postulates.” These conditions are still utilized
in current day microbiology to determine if certain types of bacteria are pathogenic.

Koch’s postulates state that:

• The bacterium must be present in every case of the disease
• The bacterium must be isolated from the diseased host and grown in pure
culture
• The specific disease must be reproduced when a pure culture of bacterium is
injected into a healthy, susceptible host
• The bacterium must be recovered from the experimentally infected host

Conditional Elements of Koch’s Postulates:

1. A bacterium that is considered part of a the normal flora or common in the
environment may become pathogenic in certain situations
2. Not all those exposed to the pathogenic bacteria will develop clinically
obvious disease
3. Some pathogenic bacteria cannot be cultured outside the body
When a “new” disease outbreak is reported, what steps might be taken to:

a) Prevent the spread of the disease? ______________________________________

b) Identify the source of the disease? ______________________________________
c) Identify the transmission of the disease? _________________________________
d) Prevent re-occurrence of the disease? ___________________________________

Extra Credit (at the discretion of the instructor)

Take some time to observe the plates that you inoculated last week by swabbing various
surfaces in the laboratory.

1. Where do you observe bacteria growing?

2. Look at the sizes of the colonies growing on the plates. Are they all the same
size? Use a metric ruler to determine the smallest and largest sized colonies that
you observe. What is the range in sizes observed?

3. Are the colonies all the same color? What colors do you observe?

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Points for Discussion

1. How can gram staining be used effectively in the medical field when trying to
develop antibiotics for various pathogens?

2. You have a cough and go to the doctor. The doctor determines that you have a
bacterial infection that is causing the cough, and prescribes antibiotics. He
instructs you to take the antibiotics until the bottle is empty. You go home and
begin to take the antibiotics. After a week, your cough is gone, but you still have
about ten pills left. You decide that you are cured and do not take the additional
pills. Knowing what you know about bacteria and their ability to transmit DNA,
what complication could arise by you not completing your treatment as the doctor
requested?

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