Gram Positive vs Gram Negative

Article Published: August 21, 2019 | Karen Steward, PhD

Credit: Technology Networks

Being able to differentiate bacterial species is important for a host of reasons, from
diagnosing infection or checking food safety, to identifying which species it is that
gives a cheese it’s fantastic character. Bacterial species, and even specific strains
can be differentiated using a number of molecular techniques such as PCR,
quantitative PCR, genome sequencing and mass spectrometry. But even without
getting into the molecular nitty gritty, there are phenotypic differences between
groups of bacteria that can be used to differentiate them. This includes
characteristics like their shape (bacilli vs cocci for example), growth in particular
nutrients and preference for high or low oxygen environments. Depending on the
characteristic being studied, bacterial species may be broken down into broad
groups, but taken together this information can narrow the possible identities
greatly. One such useful classification – if a bacterium is Gram positive or Gram
negative - is based on the structure of bacterial cell walls.

Contents

Gram positive vs gram negative

Difference in structure of Gram positive vs Gram negative bacteria  

Gram positive vs Gram negative stain

-    Gram stain procedure - Preparing a sample

-    Gram stain procedure - Gram staining a sample

Gram positive vs Gram negative color

-    Gram positive bacteria
-    Gram negative bacteria

Gram positive vs gram negative

 Gram positive bacteria are surrounded by a single thick peptidoglycan cell
wall and are therefore termed monoderms. Gram negative bacteria have
a much thinner peptidoglycan cell wall, but in addition they have an outer
membrane containing lipopolysaccharides surrounding the cell and are
consequently termed diderms.

Gram positive bacteria Gram negative bacteria

Distinctive purple appearance after

Pale reddish color after gram staining
gram staining

Bacteria include all staphylococci, all Bacteria include enterobacter species,

streptococci and some listeria salmonella species and pseudomonas
species species

Thick peptidoglycan layer Thin peptidoglycan layer

No outer lipid membrane Outer lipid membrane present

No O-specific side chains present O-specific side chains present

Teichoic and lipoteichoic acids Teichoic and lipoteichoic acids not

present present

Difference in structure of Gram positive vs Gram negative

bacteria  

The diagram below illustrates the differences in the structure of Gram positive and
Gram negative bacteria. The two key features that lead to the differing visualization
properties of Gram positive and Gram negative species are the thickness of the
peptidoglycan layer and presence or absence of the outer lipid membrane. This is
because the wall structure affects the cell’s ability to retain the crystal violet stain
used in the Gram staining procedure which can then be visualized under a light
microscope.

Credit: Technology Networks

Gram positive bacteria have a thick peptidoglycan layer and no outer lipid
membrane whilst Gram negative bacteria have a thin peptidoglycan layer and have
an outer lipid membrane.

Continue reading below...

Article

An Introduction to Culturing Bacteria

Read More

As Gram positive bacteria lack an outer lipid membrane, when correctly referring
to their structure rather than staining properties, are termed monoderms. The
outer lipid membrane possessed by Gram negative bacteria means that, when
referring to their physical structure, they are termed diderms.

The Gram staining technique was developed in 1884 by Danish bacteriologist Hans
Christian Gram.1 Whilst a Gram stain will not tell you the specific species you are
looking at, it can be a quick way to narrow down greatly the list of potential
candidates and direct follow-up testing where necessary.
Gram positive vs Gram negative stain

Credit: Technology Networks

Gram stain procedure - Preparing a sample

1. Label a clean glass microscope slide with your sample identification. Ensure you
use a pencil as many inks are removed by the reagents used in the staining
procedure.

2. If preparing your slide from a liquid bacterial culture:

Dab a small drop culture onto the slide using a sterile loop. Gently smear the
droplet in a circular motion into an area of approximately 1 cm diameter. For very
dense cultures it may be necessary to pre-dilute your culture to ensure individual
bacterial cells can be seen under a microscope following staining.

If the source material is from a bacterial plate:

Resuspend a loop of colony material in sterile phosphate buffered saline (PBS) and
then proceed as for a liquid culture.

3. Once the smear has air dried, pass the smeared slide through a flame two or
three times.

This kills the microbes in the smear and fixes the sample to the slide, be careful not to
overheat the sample however as this can distort cellular morphology.

Gram stain procedure - Gram staining a sample

1. Gently flood the smear with crystal violet and leave for 1 minute. Tilt the slide
slightly and gently rinse with tap water or distilled water.

Crystal violet is a water-soluble dye which enters the peptidoglycan layer in the
bacterial cell wall.

2. Gently flood the smear with Gram’s iodine and leave for 1 minute. Tilt the slide
slightly and gently rinse with tap water or distilled water. The smear will now
appear purple.

Gram's iodine solution (iodine and potassium iodide) is added to form a complex with
the crystal violet, which is much larger and is insoluble in water.

3. Decolorize the smear using 95% ethyl alcohol or acetone. Tilt the slide slightly
and apply the alcohol drop by drop until the alcohol runs almost clear (5-10
seconds). Immediately rinse with water to avoid over-decolorizing.

Decolorizer dehydrates the peptidoglycan layer, shrinking and tightening it. In Gram
positive bacteria, the large crystal violet-iodine complexes are then unable to penetrate
and escape the thick peptidoglycan layer, resulting in purple stained cells. However, in
Gram negative bacteria, the outer membrane is degraded, the thin peptidoglycan layer
is unable to retain the crystal violet-iodine complexes and the color is lost.
4. Gently flood with safranin counterstain and leave for 45 seconds. Tilt the slide
slightly and gently rinse with tap water or distilled water.

Safranin is weakly water soluble and will stain bacterial cells a light red, enabling
visualization of Gram negative cells without interfering with the observation of the
purple of the Gram positive cells.

5. Blot the slide dry on filter paper then view the smear using a light-microscope
under oil-immersion.

Gram positive vs Gram negative color

Gram positive bacteria

Gram positive bacteria have a distinctive purple appearance when observed under
a light microscope following Gram staining. This is due to retention of the purple
crystal violet stain in the thick peptidoglycan layer of the cell wall. Examples of
Gram positive bacteria include all staphylococci, all streptococci and some listeria
species.
Credit: Technology Networks

Gram negative bacteria

Gram negative bacteria appear a pale reddish color when observed under a light
microscope following Gram staining. This is because the structure of their cell wall
is unable to retain the crystal violet stain so are colored only by the safranin
counterstain. Examples of Gram negative bacteria include enterobacter species
[June 6 2022], salmonella species and pseudomonas species.

Gram staining cannot be used reliably to assess bacterial phylogenetic

relationships. The single membrane of Gram positive species is thought to be the
ancestral state. Historically it had been thought that the second membrane found
on Gram negative bacteria evolved just once and so all Gram negative species
were more closely related to one another than they were to Gram positive species.
However, genetic analysis has since shown this not to be the case and it is likely to
have evolved multiple times in different lineages - a product of convergent
evolution.
 References (Click to expand)
-

1. Gram, H.C. (1884). "Über die isolierte Färbung der Schizomyceten in Schnitt-
und Trockenpräparaten". Fortschritte der Medizin (in German). 2: 185–189.

Correction: The article erroneously stated that enterococci were examples of Gram negative bacteria, this was

updated on June 6, 2022 to correctly identify enterobacter species as examples of Gram negative bacteria.

©2023 Technology Networks, all rights reserved, Part of the LabX Media Group