MICROBIOLOGY PRACTICAL

Gram + and Gram

Typical Bacteria Cell:
Plasma Membrane (Inner most membrane):
o Directs what enters and exits
o Phospholipid Bilayer
Cell Wall (Middle layer):
o Provides structural support to the cell
o Made of by a polysaccharide called Peptidoglycan
Capsule (Outer layer):
o Sticky outer layer for protection and attachment

Gram Positive vs. Gram Negative

Gram +
Inner most plasma
membrane
Thick peptidoglycan cell wall

Outer capsule
More easily treatable with
antibiotics Because of the
lack of the extra outer
plasma membrane layer
Stains purple/violet after
Gram stain

Gram Inner most plasma

membrane
Thin peptidoglycan cell wall
Another plasma membrane
Outer plasma membrane
Outer capsule
Harder to treat with
antibiotics
Stains red/pink after Gram
stain

Gram Stain:

The purpose of the Gram stain is to help identify whatever infectious

bacteria is getting us sick.
The reason why we do this is because the treatment will differ based
upon the outcome of this Gram stain.
Steps:
1) Smear bacteria sample across a glass slide
2) Fix the bacteria to the slide with heat
3) Apply Crystal Violet to the bacteria
This molecules crystal violet will pass through all
layers of the bacteria into the actual bacteria cell.

4) Apply add iodine to the bacteria (Lugol)

The iodine molecules pass through all the layers of the
bacteria as the crystal violet did.
The iodine molecules binds with the Crystal Violet
molecules making the molecule bigger.

5) Wash with alcohol

In Gram +
The cell wall is going to shrink and the capsule will
be dissolves and washed away
The crystal violet and iodine (lugol) molecules will
be retained inside the bacteria cell
In Gram
The cell wall is going to shrink and the capsule will
be dissolved and washed away, but the outer
plasma membrane disappears also; this is because
of the dehydrated nature of the alcohol
Because the remaining cell wall is so Thin, the
crystal violet and iodine (lugol) molecules will get
out of the cell => making the Gram- bacteria to
lose its color

6) Apply Safranin or Fuchsine to the bacteria

Reddish colored molecule dye
The red/pink molecules pass through the cell wall of
peptidoglycans and binds to the lipids of the
Phospholipid bilayer (PM)

Results:
o Gram Positive = Purple/Blue/Violet in appearance
o Gram Negative = Pink/Red Appearance

Proper treatment can begin once the results of a Gram Stain are
known:
o Gram Positive are more easily treatable with antibiotics,
because of its 1 phospholipid bilayer, compared to the Gram
Negative bacteria which has 2 phospholipid bilayers.

Bacillus Cerus Bacilli

Gram +

Escherichia Coli Bacilli

Gram

Staphylococcus Aureus Coccus

Klebsiella
Coccus
Gram +
Gram

All with positive preparation (Crystal Violet)

Pneumoniae