The Cell Walls of Gram Positive and Gram Negative Bacteria.
Bacteria are unique among all organisms in the composition of
their cell walls. Composed of a material called peptidoglycan,
the bacterial cell wall is essentially a tremendously long polysaccharide
which wraps around the bacterial cell membrane. The parallel strands
of polysaccharide are cross linked by short peptides to provide
additional structural strength. The peptidoglycan may be present
in a single layer (Gram negative bacteria)or in multiple layers
(Gram positive bacteria), sometimes as many as 12 layers. Gram
positive and Gram negative bacteria also have many fundamental
differences in their physiology and ecology. Furthermore, the
medical treatment of Gram positive and Gram negative bacterial
infections is also frequently different. For example, many of
our antibiotics target cell wall formation and drugs which work
against Gram positive cell wall formation are generally ineffective
against Gram negatives. For these reasons, whenever bacteria are
being characterized, we start by finding their Gram's stain characteristics
and cellular morphology.
Bacteria are an excellent model system for studying physiological
processes. Most metabolic pathways proceed in the same fashion
in all organisms. Because bacteria are easy to work with and grow
so rapidly they are ideal for many types of metabolic studies.
In addition, discerning which metabolic pathways a particular
species of bacteria uses is an important differential tool for
identification purposes. In this exercise we will look at several
important physiological processes such as whether the bacteria
use oxidative respiration or fermentation (or both), the production
of particular waste products and the utilization of particular
One key physiological determination to be made when working with
bacteria is to assess their requirement for oxygen. Some bacteria
absolutely need oxygen to live (aerobes), others are killed by
a faint whiff of oxygen (obligate anaerobes) while others can
grow with oxygen but can also grow (albeit more slowly) in its
absence (facultative anaerobes). There are also aerotolerant anaerobic
bacteria like the streps which can live in the presence of oxygen
but don't use it.
Anaerobic bacteria (either obligate or facultative) produce ATP
without the assistance of Krebs Cycle or the oxidative electron
transport system. Many will therefore get all their ATP from fermentation,
meaning they produce lots of acidic waste products. We can therefore
differentiate between aerobes and facultative bacteria based on
detecting these acidic waste products. Obligate anaerobes can't
grow without special media to remove all the oxygen so we will
not be working with any of these. Beside, anaerobic bacteria really
The SIM media is extremely useful for looking
at several physiological characteristics. SIM is an acronym for
sulfur, indole and motility. This media allows us to simultaneously
determine whether these two metabolic waste products are being
produced and whether the bacteria is motile. Sulfur and indole
are produced when amino acids are used for energy sources. The
amino acids methionine and cysteine contain sulfur. When they
are used for energy, the bacteria excrete excess sulfur in the
form of hydrogen sulfide (H2S).
reacts with iron in the SIM agar and forms a black precipitate.
Indole (part of the amino acid side chain) is produced as a waste
product when bacteria use the amino acid tryptophan.
Part One. Gram Stain Characteristics.
Once we have isolated colonies of bacteria,
the next step need to characterize the bacteria by using the Gram
stain. Practice the gram stain technique by staining the known
gram positive and gram negative bacteria provided by your instructor.
Once you are comfortable with the technique, Gram stain your unknown
pure culture isolate. and determine its Gram stain characteristics
and verify its cellular morphology.
Part Two: Oxygen Utilization.
On a PER TABLE basis, inoculate one known
aerobe and one known facultative anaerobe to phenol
red broth tubes. These are your
control tubes. In addition, each student will inoculate his/her
unknown to a separate phenol red broth. Record your results after
24 hour incubation
Part Three: The SIM media.
Each table of students should inoculate
SIMs with the two control bacteria provided by the instuctor
and each of the unknowns. After 24 to 48 hours, check for the
- Motility. Motile bacteria
will make the whole media turbid while non-motile bacteria will
grow only along the stab line.
- Sulfur. Excretion of sulfur
will turn the media black.
- Indole. Excretion of indole
is detected by adding Kovac's Reagent to the top of the media
AFTER the incubation period. The presence of indole will turn
the Kovac's Reagent bright red.
Part four. Exoenzyme production.
Some bacteria produce exoenzmes to help them degrade macromolecules
from the environment so that the simple nutrients can be transported
across the cell membrane. One example of an exoenzyme is amylase,
the enzyme responsible for breaking down starch into smaller units,
primarily glucose and maltose, the glucose disaccharide. Each
student should inoculate their unknown bacteria along with a known
starch positive and negative bacteria onto a starch
agar and obtain results after 24
- 48 hours.