Fundamentals of Clinical

Microbiology
MAU: Bsc Lab sc
Lecture 12
The Bacterial Cell Wall

Martin Kalumbi
 Aims
• To describe the general properties of the
cell wall
• To draw and label the Gram positive and
Gram negative cell walls
• Identify bacteria using staining techniques
• To understand potential problems with
staining
 Bacterial Cell
General property:
•Typical prokaryotic cell
•Contain both DNA and RNA
•Most grow in artificial media
•Replicate by binary fission
•Almost all contain rigid cell wall
•Sensitive to antimicrobial agent
 STRUCTURE OF BACTERIA
Bacterial structure is considered at three levels.
1. Cell envelope proper: Cell wall and cell
membrane.
2. Cellular element enclosed with in the cell
envelope: Mesosomes,
ribosomes, nuclear apparatus, polyamies and
cytoplasmic granules.
3. Cellular element external to the cell envelope:
Flagellum, Pilus and Glycocalyx.
 Functions of the cell wall
1. Provide rigidity, including defining the
shape of the cell
2. Protect against osmotic damage
3. Contains receptor sites for phages
/complements
4. Contains toxic components to host
 General properties of the cell wall
• These properties are common to both
Gram positive and Gram negative cell
walls:
– 10-25nm thick
– Strong and relatively rigid
– Openly porous (freely permeable)
• It is the cytoplasmic membrane beneath that controls what
passes in and out of the cell
General properties of the cell wall
– Supports the weak cytoplasmic membrane
against the pressure inside the cell

– Maintains the characteristic shape of the cell

(coccus/ bacillus)

– Survival of the cell relies on the cell wall

remaining intact
• If the wall is weakened or ruptured, the cell may lyse due to
the inflow of water by osmosis
 Cell wall chemistry
• The strengthening component of the cell
wall is common to all species of bacteria
• This component is peptidoglycan
• Peptidglycan is a peptide, composed of
alternating molecules in a chain
– N-acetylglucosamine (NAG)
– N-acetylmuramic acid (NAMA)
 Cell wall chemistry
• The strength of the cell wall is due to
cross-links between strands

NAMA – NAG – NAMA – NAG – NAMA

NAG – NAMA – NAG – NAMA – NAG

 The Gram positive and Gram
negative cell walls
Components of cell wall of Gram negative
bacteria
1. Peptidoglycan
2. Lipoprotein
3. Phospholipid
4. Lipopolysaccharide
Bacterial cell wall-Gram negative
 The Gram positive and Gram
negative cell walls
Components of cell wall of Gram positive
bacteria
1. Peptidoglycan
2. Teichoic acid
Bacterial cell wall-Gram positive
Gram positive cell wall
Present in some
Polysaccharide capsule
bacteria only

Cross-linked strands
of peptidoglycan in
a thick layer

Cytoplasmic membrane Containing

porins
Gram negative cell wall
Present in some
Polysaccharide capsule
bacteria only
Outer membrane Lipopolysaccahride

Periplasmic space
Cross-linked
strands of
peptidoglycan in a
Periplasmic space thin layer

Cytoplasmic membrane
 Differences to note
• The main differences to note between
the Gram positive and negative cell walls
are:
– Thickness of peptidoglycan layer
– Periplasmic space in Gram negatives
– Additional outer membrane in Gram
negatives
 Functions of the outer membrane
• Protects the peptidoglycan from lysozyme
– Lysozyme is a defence enzyme in the body
that cleaves the bond between NAG and
NAMA, causing cell lysis
• Prevents entry of many antibiotics
 The Gram stain
• The Gram stain is a reaction used to help
identify pathogenic bacteria in clinical
specimens and cultures
• We can begin the process of identification by the
Gram reaction (positive or negative) and it also
tells us the shape of the bacterial cell (coccus or
bacillus)
• We can then carry out further tests to finally
establish the identity of the species
 The Gram stain
• Differences in the Gram reaction between
bacteria is thought to be due to differences
in the permeability of the cell wall during
the staining process
• Which is more permeable, the Gram
positive cell wall or the Gram negative cell
wall? Why?
 Reagents used in the Gram stain
1. Crystal violet

2. Iodine

3. Acetone-alcohol decolouriser

4. Counterstain: neutral red, safranin or

dilute carbol fuchsin
The Gram staining procedure
Gram staining procedure - 1
Gram staining procedure - 2
Gram staining procedure - 3
Reporting results from the Gram stain
 Problems you may encounter
• Gram positive bacteria may lose their ability to retain the crystal violet-iodine
complex, so they appear falsely Gram negative

Action Effect
Over heat slide when fixing smear Damages cell wall, making it more
permeable

Leave decolouriser on slide for too Decolouriser will have some effect on
long Gram positive cell walls

Use iodine that is too old Iodine cannot form a complex so well
with the crystal violet, so the dye will
not be retained so well by the Gram
positive wall

Use old culture to prepare smear Cell wall will be weaker and so more
permeable
 Problems you may encounter
• Gram negative organisms may appear
Gram positive if the smear is prepared too
thickly, so the decolouriser cannot work
ACID-FAST BACILLI MICROSCOPY (AFB)
EXAMINATION: AURAMINE O STAIN
AFB: Auramine procedure
AFB: Auramine procedure
AFB: Auramine procedure
AFB: Auramine procedure
AFB: Auramine procedure
AFB: Auramine procedure
AFB: Auramine procedure
 REMENBER THAT
AURAMINE O STAINED
SAMPLES ARE OBSERVED
UNDER FLOURESCENCE
MICROSCOPE