The Gram Stain: Background and Example Organisms

Every microbiology student should be familiar with the Gram stain. In this rst of two lessons on this technique,
we will look at some of the background information before describing and viewing some relevant example
organisms.

Step One
Ask any scientist, doctor, technician, or researcher who works with microorganisms what is the rst
step in any bacterial investigation. I bet that nearly every one of them will tell you 'the classic Gram
stain.' This technique is always one of the rst things taught to new students in the previously
mentioned disciplines - a technique that can often prove not just useful but crucial to identifying
the type of bacteria causing a particular disease, contaminating an experiment, growing on a
medium, or causing a foodborne illness. But, if you are new to the world of microbiology, you might
not know what a Gram stain is. Hopefully, we can shed some light on this important technique. If
you're interested in step-by-step instructions, see the lesson 'The Gram Stain: Theory & Procedure.'

Gram Stain Background

Christian Gram was a Danish bacteriologist who was determined to nd a procedure to make
bacteria more visible under the microscope. He was a major pioneer in the staining of bacteria to
color them and increase their visibility. In 1884, he developed a staining procedure that would
actually di erentiate, or distinguish, the di erent types of bacteria in a sample containing several
species. Today, this procedure is called the Gram stain after its inventor, and it continues to be a
staple of microbiology labs and hospitals worldwide.

The Gram stain is a di erential staining technique that allows scientists to detect di erences in the
bacteria based on the cell wall. Remember, a di erential staining procedure is one that allows
the observer to visually distinguish between di erent types of bacterial cells based on the idea that
not all cell types stain equally. If we look at these two de nitions together, a clearer picture will
emerge, I promise. Putting them together: performing a Gram stain on a mix of bacterial cells
allows the observer to visually distinguish two di erent types of bacteria based on the di erent
ways the two types of cell walls take up the stain.

The Bacterial Cell Wall

With that in mind, let's brie y review the two types of bacterial cell walls. The major structural
component of the bacterial cell wall is peptidoglycan, a complex structure that resembles a chain-
link fence and provides rigidity to the cell wall. Some bacteria have a very thick peptidoglycan wall
composed of many individual layers stacked on top of each other. These are called Gram-positive
bacteria, and they appear purple after staining.

Other species of bacteria have only one or two layers of peptidoglycan. This thin layer is covered by
an outer membrane, similar to the typical cell membrane that encloses all bacterial cells, giving
these bacteria a double membrane. These are called Gram-negative bacteria and they appear red
after staining. The Gram stain procedure takes advantage of the di erent ways the cells with a thick
peptidoglycan layer and the cells with a thin peptidoglycan layer and an outer membrane take up
and hold the stain, thus di erentiating the two di erent cell wall types.

Of course, things in science can never be so black and white - or in this case, purple and red. Some
bacteria can be Gram-variable, staining either Gram-positive or Gram-negative depending on the
staining procedure, the bacteria's growth conditions, or the age of the bacterial sample. Then there
are the bacteria that lack cell walls altogether. Mycoplasma, a common cause of pneumonia, has no
cell wall to stain!

Classic Example Species

For this lesson, we'll disregard these oddballs and take a look at several classic examples of Gram-
positive and Gram-negative bacteria. We'll start with the Gram-positives. You've probably heard of
many of these bacteria.

Staphylococcus is a Gram-positive, coccus-shaped bacteria that tends to grow in clusters. Notice the
grape-like clusters of purple spheres. The particularly nasty Staph is Staphylococcus aureus, a
pathogen that can cause a wide range of illness from mild food poisoning to a life-threatening skin
infection. You have probably heard of MRSA, which stands for 'methicillin-resistant Staphylococcus
aureus.'

The next example is Gram-positive bacteria in the genus Bacillus. These spore-forming, rod-shaped
bacteria run the gamut from harmless lab organism to deadly bioterrorism threat. This is an image
of Gram-positive Bacillus anthracis, the causative agent of anthrax. Note the long chains of purple
rods.

The next genus is Clostridium. These Gram-positive, rod-shaped bacteria include Clostridium
botulinum, a common food-poisoning agent but also the source of the toxin used for Botox. In this
image you can clearly see the purple-colored rods. The thickening on the poles of many of the cells
are endospores, a structure that allows the bacteria to survive during harsh, unfavorable
conditions.
Let's move on to the Gram-negatives. Possibly the most famous Gram-negative bacteria, and
possibly the most famous bacteria in general, is Escherichia coli, better known as E. coli. Notice the
rod shape and the red color. E. coli is most famous for causing foodborne illness, but nearly every
major microbiological advance in the past 50 years has used E. coli as a model organism or lab
workhorse.

Salmonella is another Gram-negative rod. This bacteria is infamous for causing disease from
undercooked chicken. There are even stories of children getting Salmonella from baby turtles after
deciding to pop the little buggers in their mouths! Not all Gram-negatives are rods, though.
Neisseria is Gram-negative cocci that causes the sexually transmitted disease gonorrhea. Notice the
small, round, red cells characteristic of Gram-stained Neisseria.

Lesson Summary
Let's brie y review the Gram stain. The Gram stain is a di erential staining technique that allows
scientists to detect di erences in bacteria based on the variable structure of the bacterial cell wall.
A di erential staining procedure is one that allows the observer to visually distinguish between
di erent types of bacterial cells based on the idea that not all cell types stain equally.

Gram-positive cell walls have a thick layer of peptidoglycan that, when Gram-stained, makes the
cells appear purple. Gram-negative cell walls have a thin peptidoglycan layer and an outer
membrane. Gram staining makes these cells appear red.

Several classic examples of Gram-positive bacterial genera include Staphylococcus, Bacillus, and
Clostridium. These cocci and rod-shaped bacteria all appear purple after staining. Several classic
examples of Gram-negative bacterial genera include Escherichia, Salmonella, and Neisseria. These
pathogens can be rods or cocci, but all appear red after staining.

Learning Outcomes
After the completion of the lesson, you could be able to:

Emphasize the importance of testing for bacterial agents

Give information about the Gram stain test

Provide classic examples of bacteria

Detail aspects of certain bacterial species