BIOL 2034 Microbiology Bacterial Cell Surface

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BIOL 203-4 MicrobiologyBacterial Cell Surface
Dr. Bob Melamede E-mail rmelamed_at_uccs.edu Webpage
http//www.uccs.edu/rmelamed/
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The Bacterial Cell Wall The bacterial cell wall
is a unique structure which surrounds the cell
membrane. Although not present in every bacterial
species, the cell wall is very important as a
cellular component. Structurally, the wall is
necessary for Maintaining the cell's
characteristic shape- the rigid wall compensates
for the flexibility of the phospholipid membrane
and keeps the cell from assuming a spherical
shape Countering the effects of osmotic
pressure- the strength of the wall is responsible
for keeping the cell from bursting when the
intracellular osmolarity is much greater than the
extracellular osmolarity Providing attachment
sites for bacteriophages- teichoic acids attached
to the outer surface of the wall are like landing
pads for viruses that infect bacteria
Providing a rigid platform for surface
appendages- flagella, fimbriae, and pili all
emanate from the wall and extend beyond it
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Atypical Cell Walls The genus Mycoplasma have
no cell walls. This genus is the cause of
"walking pneumonia", a subacute form of
pneumonia. L-forms are mutated bacteria with
defective cell walls. Because of the lack of
shape (normally created by a cell wall) atypical
bacteria are spherical and flexible and filter
along with viruses through biofilters.
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Different cell wall compositions result in
different staining properties
Gram-positive Gram-negative Acid-fast
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GRAM STAINING Who could have guessed that a
staining procedure devised more than a century
ago would still serve as one of the most
widespread methods of bacterial classification?
Microbiologists of this high-tech age are still
indebted to Danish physician Christian Gram, who
invented the gram-staining method in 1884. The
differences in the cell wall are more than simply
a classification tool. Cell wall characteristics
are intimately related to the disease-causing
potential of the bacterium. In fact, medical
researchers have found that an extremely
effective way to combat bacterial pathogens is by
interfering with cell wall formation. Because the
eukaryotic cell has no analog to the prokaryotic
cell wall, medicines which target bacterial cell
walls have little or no effect on plant or animal
cells.
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GRAM STAINING To gram stain, an investigator
smears a sample of bacteria on a slide, soaks it
in a violet dye and then treats it with iodine.
The slide is then rinsed with alcohol and
counterstained with a pink dye called safranin.
The cell walls of gram-negative bacteria have a
very low affinity for the violet stain, which is
rinsed out by the alcohol. Once counterstained
with safranin, the gram-negative bacteria appear
bright pink to red. Gram-positive cell walls have
a high affinity for the violet stain, and retain
it even through the alcohol rinse. When the
process is complete, they appear dark purple to
brown. One difference between the two cell types
appears to be in the amount of peptidoglycan in
the cell wall. Gram positive cell walls are about
five times as rich in peptidoglycan as
gram-negative cell walls. In addition,
gram-negatives have a second membrane (chemically
different from the plasma membrane) external to
the cell wall, and may also have a gelatinous
sheath external to the second membrane.
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A type of acid polysaccharide
Teichoic acids. Are found exclusively in gram
positive organisms. Are formed as polymers of
glycerol or ribitol through phosphodiester
linkages
Gram positive cell walls are essentially pure
peptidoglycan i.e.the sugars proteins of
various sorts.
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• Peptidoglycan
• Rigid cell layer lies directly outside cell
  membrane
• Provides strength
• Composed of 2 sugar derivatives
• N-acetylglucosamine (NAG)
• N-acetylmuramic acid (NAM)
• Amino acids
• Alanine
• D-glutamic acid
• Lysine or diaminpimelic acid

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N-acetylglucosamine
N-acetylmuramic acid
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• Peptidoglycan Features
• 3-D polymeric macromolecule
• Formed from subunits by two types of covalent
  bonds
• B-1,4 glycosidic bonds between hexose sugars
• peptide bonds between amino acids
• Determines cell shape and prevents osmolysis
• Dynamic structure
• must grow as cell grows
• must be regulated to allow septation

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Teichoic Acids and Gram bacteria Acidic
polysaccharides Contain glycerophosphate or
ribitol phosphate residues Polyalcohols
connected by phosphate esters and have other
sugars and D-alanine attached Negatively
charged-effect passage of ions through cell wall
Some glycerol containing acids bound to
membrane lipids (lipoteichoic acids)
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Note how complex this cell wall is compared to
the Gram positive wall. Especially note how
little peptidoglycan is present and how there is
an outer membrane and a periplasmic space outside
of the cell membrane.
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Gram-Negative
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Penicillin
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Penicillins are antibiotics that interfere with
proper cell wall formation in bacteria. Would
penicillins be an effective therapy for someone
infected with Mycoplasma pneumoniae or with an
L-form of a different genus? Explain. Why would
you not want to use penicillin in conjunction
with a bacteriostatic antibiotic?
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Would penicillin be more effective against a
bacterial cell that is dividing, or against a
bacterial cell that is not dividing but that is
producing toxins? Explain. Hint Think about how
penicillin function.
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Acid Fast bacteria- Mycobacteria, have a very
large amount of lipid in their cell wall so that
traditional stains are less effective, requiring
acid-fast staining for visualization of these
bacteria in cultures or tissue specimens. Most
Mycobacteria grow at a relatively slow rate
therefore, the acid-fast smear plays an important
role in the early diagnosis of mycobacterial
infections.   Microscopy is the oldest,
easiest, most rapid, and inexpensive procedure
that can be performed in the laboratory to detect
the presence of acid-fast bacilli.   However, 
the acid-fast smear should not be used in place
of culture.   AFB smears require 105 AFB per mL
of sputum for recognition by direct microscopy
culture detects as few as 10 to 100 CFU/mL of
sputum. 
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In spite of quantitative discrepancy in
sensitivity, examination of stained smears of
sputum, or other clinical material, can be
helpful in several ways 1. It provides a
presumptive diagnosis of mycobacterial
disease. 2. Smear positive patients, the most
infectious cases, are rapidly identified. 3.
It may be used to follow the success of
chemotherapy of tuberculosis patients. 4. It
is of vital importance to the patient's discharge
from the hospital, or return to employment. 5.
It can confirm that cultures growing on media are
indeed acid- fast.
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The lipoid capsule of the acid-fast organism
takes up carbol-fuchsin resists
decolorization (dilute mineral acid).
Carbol-fuchsin is more soluble in the lipids
of the cell wall than in acid-alcohol, but is
readily removed from bacteria that lack the waxy
capsule. Staining is enhanced by the phenol and
the alcohol, and both of these chemicals also aid
in dissolving the basic fuchsin. Alcoholic,
rather than aqueous, solutions of acid are used
because more uniform decolorization is obtained
with alcoholic solutions. The lipoid capsule of
mycobacteria is of such high molecular weight
that it is waxy at room temperature and
successful penetration by the aqueous-based
staining solutions used in Gram's staining
procedures is prevented.
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Definitive diagnosis of tuberculosis is made
through Acid Fast Bacilli (AFB) staining and
culture of sputum, bronchoalveolar lavage, or
lung biopsy specimen. This photomicrograph
demonstrates the positive AFB staining
characteristics of Mycobacterium species. This
specimen is from the brain of a patient with
disseminated tuberculosis, but demonstrates the
characteristic positive acid fast bacilli stain.
Culture is essential for definitive diagnosis and
species identification, as "atypical"
Mycobacterium may infect immune compromised
individuals.
Acid-fast Stain