POSTIVE AND NEGATIVE GROUP 7

GRAM STAINS
Presented By:
Marissa Woolcock
Novia Stephenson
Jahnessa Duncan
Romeo Larcher
GRAM POSTIVE VS GRAM
NEGATIVE
GRAM POSITIVE AND NEGATIVE ENVELOPES
GRAM POSITIVE AND NEGATIVE ENVELOPES
CHARACTERISTICS AND COMPARISON
STEPS IN GRAM STAINING
 THE GRAM STAINING PROCESS

The Gram-Staining Process includes ‘4’ – basic steps:

1. Apply crystal violet, a (primary stain), then rinse briefly with

water.

2. Add iodine solution (mordant), forming a complex with crystal

violate.
 THE GRAM STAINING PROCESS

4. Apply alcohol, ethanol or a mixture of both, to decolorize the

solution.

5. Counterstain cells with safranin, rinse and dry for observation.

GRAM-POSITIVE CELLS STAINS PURPLE

GRAM-NEGATIVE CELLS STAINS PINK

THE GRAM STAINING PROCESS
 HOW DOES THE
CELL ENVELOPE
STRUCTURE
INFLUENCE GRAM
STAIN?
GRAM POSITIVE BACTERIA

The cellular wall of gram-positive

bacteria is relatively thick and is
made up of numerous layers of
peptidoglycan polymers.

When the crystal violet-iodine

complex is added to these cells the
thickness of the walls blocks the
escape of the solvent when the cells
are washed with alcohol or acetone.
GRAM POSITIVE BACTERIA

Seeing that the gram–positive cells

retain the solvent when washed,
they exhibit the color purple, which
is the color of the primary stain.
 GRAM NEGATIVE BACTERIA

Within gram negative bacteria only a thin

layer of peptidoglycan and a thin
lipopolysaccharide outer membrane are
present.

When the cells are exposed to the crystal

violet-iodine compound solvent it is easily
lost through the thin LPS and
peptidoglycan layers.
 GRAM NEGATIVE BACTERIA

Due to the lack of thickness in these layers in

comparison to the gram-positive cells, the
negative cells cannot retain the solvent as
well as it’s counterpart.

Gram negative cells are decolorized by the

alcohol/acetone treatment, but are then
stained with “SAFRANIN”, so that they
appear pink.
TROUBLESHOOTING
GRAM STAINS
 TROBLESHOOTING…

A. – Smear Preparation:
Bacterial smear: If the smear is applied in too thick of a layer,
Gram-negative bacteria appear darker and could be mistaken for
Gram-positive bacteria.

Fixation: If bacteria are overheated during fixation, the cell wall is

destroyed and now incapable of retaining the crystal violet solvent.
All bacteria will, as a result, appear Gram-negative.
 TROBLESHOOTING…

Expired culture smears: If culture smears are too old, the bacteria cell
wall might have already broken down, rendering them incapable of
retaining the crystal violet solvent.
All bacteria will, as a result again, appear Gram-negative.
 TROBLESHOOTING…

• B. – Staining:
Under-decolorization: If alcohol (the decolorizer) is washed away
before it had any effect on the cell wall, the Crystal Violet
molecules will not escape the Gram-negative cell wall.

As a result, all bacteria appear Gram-positive (purple) after

counterstaining with Safranin.
 TROBLESHOOTING…

Over-decolorization: Leaving alcohol (the decolorizer) on the slide

for too long disrupts the cell wall of both Gram-positive and Gram-
negative bacteria.

The Gram-positive bacteria can not retain Crystal Violet, and

therefore, all bacteria appear Gram-negative (pink) after
counterstaining with Safranin.
 TROBLESHOOTING…

Many organisms produce substantial amounts of capsular or slime

material in the presence of certain carbohydrates. This may
interfere with decolorization, and certain Gram-negative organisms
such as Klebsiella may appear as a mixture of pink and purple
cells.
A SHORT VIDEO TO SUMMERIZE…
 TEST YOUR KNOWLEDGE, Q&A:

1. Which cell envelope has a single peptidoglycan layer?

2. (T/F) Lipopolysaccharides can be found in the outer-membrane of

gram-positive bacteria.

3. What is the key component of gram-positive bacteria that allows

them to retain the crystal violet solvent?
 TEST YOUR KNOWLEDGE, Q&A:

4. What is the name of the stain used on gram-negative bacteria to

give its pink color?

5. Why is it important to use fresh culture when gram staining?

References

• https://theory.labster.com/gram-stain-troubleshooting/

• https://youtu.be/AZS2wb7pMo4?si=mEyaPlahEZaVBYt4

• https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_Laboratory_Manual_(Hartline)/
01%3A_Labs/1.10%3A_Gram_Stain

• https://instr.bact.wisc.edu/book/displayarticle/89

• https://byjus.com/biology/difference-between-gram-positive-and-gram-negative-cell-wall/