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GRAM NEGATIVE VS.
GRAM POSITIVE
Physical differences & clinical signifigance.
Gram- positive bacteria
Figure 4.8b
Gram-negative bacteria
Figure 4.8a
Gram-Positive cell walls
• Teichoic acids:
• Lipoteichoic acid links to plasma membrane
• Wall teichoic acid links to peptidoglycan
• May regulate movement of cations
• Polysaccharides provide antigenic variation
Gram Negative: Outer Membrane (OM)
• A bilayer membrane found in gram-negative
bacteria
• Forms the outermost layer of the cell wall; is
attached to the peptidoglycan by a continuous
layer of lipoprotein molecules
• Proteins called porins form channels through the
OM (transports some proteins)
• OM has surface antigens and receptors
• Does prevent some molecules from entering (e.g.
some antibiotics), but generally isn’t that
selective
Gram-Negative Outer Membrane
Figure 4.13c
Damage to Cell Walls
• Lysozyme digests disaccharide in peptidoglycan.
• Penicillin inhibits peptide bridges in peptidoglycan. (effective
on Gram-positive cells)
•.
Bacterial Membranes
Gram-Positive
Cell Wall
• Thicker layers of
•
•
•
•
•
peptidoglycan
2-ring basal body
Teichoic acid
Disrupted by lysozyme
Penicillin sensitive
More penetrable
Gram-Negative
Cell Wall
 Thinner layer of
peptidoglycan
 4-ring basal body
 No teichoic acid; porin
proteins
 Endotoxin
 Tetracycline sensitive
 Less penetrable
Figure 4.13b–c
Exotoxins vs. Endotoxins
EXOTOXIN ANIMATION
ENDOTOXIN ANIMATION
Types of staining techniques:
Simple Staining:
use of a single stain
Differential staining:
Use of 2 contrasting
stains
Separation
into groups:
Visualizatio
n of various
structures:
gram stain
acid stain
flagella, capsule,
spore or nuclear
stain
The Gram Stain
(a) Gram-Positive
(b) Gram-Negative
Table 4.1
Techniques of Light Microscopy
• Wet mounts: A drop of medium containing
organisms is placed on slide and used to view
living microorganisms
• Smears: Microorganisms are spread onto the
surface of a glass slide and used to view
destroyed organisms
• Heat fixation: destroys the organisms, causes
organism to adhere to slide, and alters organism
to accept stains (dyes)
Step 1: applying primary stain
Outline of
Gram Stain procedure
Step 2: apply mordant
Step 3: apply decolorizing agent
Step 4: apply counterstain
animation
Gram Positive and Gram Negative Cells
Gram Stain
Color of
Gram-positive cells
Color of
Gram-negative cells
Primary stain:
Crystal violet
Purple
Purple
Mordant:
Iodine
Purple
Purple
Decolorizing agent:
Alcohol-acetone
Purple
Colorless
Counterstain:
Safranin
Purple
Red
Micrograph of Gram-Stained Bacteria
Figure 3.12b
The Gram Stain Mechanism
• Crystal violet-iodine crystals form in cell
• Gram-positive
• Alcohol dehydrates peptidoglycan
• CV-I crystals do not leave
• Gram-negative
• Alcohol dissolves outer membrane and leaves holes in
peptidoglycan
• CV-I washes out
Distinguishing Bacteria by Cell Walls
• Gram-positive Bacteria have a relatively thick layer
of peptidoglycan (60-90%)
• Gram-negative Bacteria have a more complex cell
wall with a thin layer of peptidoglycan (10-20%)
• Acid-fast Bacteria is thick, like that of gram-
positive bacteria, but has much less peptidoglycan
and about 60% lipid (mycolic acid)… Mycobacteria
(TB and leprosy)
Acid-Fast Bacteria
• Found in bacteria that belong to the genus,
Mycobacterium sp. (e.g. tuberculosis, leprosy)
• Cell wall is mainly composed of lipid, makes them very
hardy. Also has slow diffusion = slow growth rate for
many (eg: 20 days for M. leprae)
• Lipid component is mycolic acid
• Acid-fast bacteria stain gram-positive
Acid-Fast Stain
Color of
Acid-fast
Color of
Non–Acid-fast
Primary stain:
Carbolfuchsin
Red
Red
Decolorizing agent:
Acid-alcohol
Red
Colorless
Counterstain:
Methylene blue
Red
Blue
Acid-Fast Bacteria
Figure 3.13
Q&A
• Acid-fast staining of a
patient’s sputum is a rapid,
reliable, and inexpensive
method to diagnose
tuberculosis. What color
would bacterial cells appear
if the patient has
tuberculosis?
• Why doesn’t a negative stain color a cell? 3-7
• Why is fixing necessary for most staining procedures? 3-8
• Why is the Gram stain so useful? 3-9
Normal Microbiota
• Bacteria were once classified as plants, giving rise to use of the
term flora for microbes
• This term has been replaced by microbiota
• Microbes normally present in and on the human body are
called normal microbiota
Normal Microbiota on Human Tongue
Figure 1.7
Normal Microbiota
• Normal microbiota prevent growth of pathogens
• Normal microbiota produce growth factors such as folic acid
and vitamin K
• Resistance is the ability of the body to ward off disease
• Resistance factors include skin, stomach acid, and antimicrobial
chemicals
Biofilms
• Microbes attach to solid surfaces and grow into masses
• They will grow on rocks, pipes, teeth, and medical implants
Biofilms
Figure 1.8