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•Shapes:
•cylindrical (rod-shaped)
• spherical (coccoid)
•a single twist (vibrios)
•many twists like a corkscrew (spirochaetes).
Classification
• The major function of the cell wall is to prevent bacterial cells from rupturing when
the water pressure inside the cell is greater than that outside the cell.
• It also helps maintain the shape of a bacterium and serves as a point of anchorage for
flagella.
• The amino acids occur in an alternating pattern of D and L forms (see Figure 2.
13, page 43)
Structure of the Gram-positive Cell
Wall
The Gram-negative outer membrane(1)
The Gram stain procedure
Developed in 1884 by the Danish physician
Hans Christian Gram
Gram's
Crystal iodine
violet
Decolorise with
acetone
Gram-positives
appear purple
Counterstain with
e.g. methyl red Gram-negatives
appear pink
Gram-positive cocci Gram-positive rods
Anaerobic Anaerobic
Gram-negative cocci Gram-negative rods
Gram staining
• Enteric Bacteria
– E. coli
– Salmonella
– Shigella
– Yersinia
– Pseudomonas
– Proteus
– Vibrio cholerae
– Klebsiella pneumoniae
Gram-Negative Rods
• Fastidious GNRs
– Bordetella pertussis
– Haemophilus influenzae
– Campylobacter jejuni
– Helicobacter pylori
• Anaerobic GNRs
– Fusobacterium
Gram-Negative Cocci
• Neisseria gonorrhoeae
– The Gonococcus
• Neisseria meningitidis
– The Meningococcus
• Both Gram-negative
intracellular diplococci
Gram-positive Cocci
• Staphylococci
– Catalase-positive
– Gram-positive cocci in
clusters
• Staphylococcus aureus
– coagulase-positive
• Staph. epidermidis
– and other coagulase
negative staphylococci
Gram-Positive Cocci
• Streptococci
– Catalase-negative
– Gram-positive cocci in
chains or pairs
• Strep. pyogenes
• Strep. pneumoniae
• Viridans-type streps
• Enterococcus faecalis
Gram-Positive Rods
• Clostridia
– Anaerobes
– C. tetani
– C. botulinum
• Bacillus cereus
– Aerobe
Non-Gram-stainable bacteria
• Unusual gram-positives
• Spirochaetes
• Obligate intra-cellular bacteria
Unusual Gram-positives
• Mycoplasmas
– Smallest free-living
organisms
– No cell wall
– M. pneumonia, M.
genitalium
• Mycobacteria
– Acid-fast bacilli, stained
by Ziehl-Neelsen stain
– M. tuberculosis
– M. leprae
– M. avium
Appendages
1. flagella
Some rods and spiral form have this.
a). function: motility
b). origin : cell membrane flagella attach to the cell
by hook and basal body which consists of set(s) of rings
and rods
Gram - : 2 sets of ring and rods, L, P, S, M rings
and rods
e.g. E. coli
Gram + : S, M rings and rods
e.g. B. megaterium
Organ of bacterial locomotion
Structure of the flagellum
Flagella movement(1)
Flagella movement(2)
Flagella movement(3)
b).Origin (continued)
– The structure of the bacterial flagella allows it to spin
like a propeller and thereby propel the bacterial cell;
clockwise or counter clockwise ( Eucaryotic , wave
like motion.
– Bacterial flagella provides the bacterium with
mechanism for swimming toward or away from
chemical stimuli, a behavior is knows as
CHEMOTAXIX, chemosenors in the cell envelope
can detect certain chemicals and signal the flagella to
respond.
c). position
monotrichous
lophotrichous
peritrichous
d). structure
2. Ribosome:
• Bacteria contain a group of ribosomes called polyribosomes present
in the cytoplasm of the cell. It is 70S type in bacterial cell having two
subunits—
• the large unit is 50S and the smaller unit 30S. Ribosomes help in
protein synthesis.
Function: protein synthesis
The bacterial chromosome and
supercoiling
4. Mesosomes
* Endospores
Spore former: sporobactobacilli and sporosarcinae - no
medical importance. bacillus and clostridium have
medical importance.
• Rickettsia
• Coxiella burneti
• Chlamydias
– C. pneumoniae
Outline
• Importance of bacteria
• Nature of bacteria
• Classification of bacteria
– Gram-positive versus Gram-negative
– Rods and Cocci
– Aerobic versus anaerobic
Monochromatic staining
• Principle:
• Staining in single dye solution contains salts.
• Auxochrome and chromophore.
• Responsible for dissociation and colour resp.
• In basic dye chromophore is positively charge
and the cell have negative charge.
• Ex. Methylene blue, crystal violet etc.
Procedure
• Smear preparation
• Crystal violet for 1 min
• Excess stain was washed with d. water
• Drying of slide
• Observe under oil immersion lens
• This is used to study morphological
arrangement.
• What do you mean by monochromatic
staining.
• What is the role of auxochrome and
chromophore
• Which stain you have used?
• How chromophore will colour the cell.
Motility
• Smear preparation
• Add drop of tannic acid and warm gently
• Add 5 % solution of crystal violet (1 min)
• Wash the slide with water
• Add 5% congo red solution
• Dry and observe under oil immersion lens.
• What is the main component of bacterial cell
wall.
• Which mordant you have used in cell wall
staining.
• Which primary stain you have used in cell
wall staining.
• What was counter stain.
Preparing Specimens for Viewing – Differential Stains
m u s
Acid-Fast Stain
This stains the cells of the genera
Mycobacterium and Nocardia, which
cause many diseases in humans,
including tuberculosis, leprosy, and Create a smear of the organism that
you are testing. Cover the smear
other lung and skin infections. with a strip of blotting paper.
Blotting paper : Ziehls carbolfuchsin (3 – 5 min heat) : rinse: Acid Alcohol (10 – 15 sec) : rinse : crystal violet (1 min)
OBSERVATION OF MICROORGANISMS
Differential Stains
Acid-Fast Stain
Distinguishes between two large groups of
microorganisms:
Is there a mordant?
Endospore Stain
Some bacteria produce endospores, dormant, highly-
resistant cells inside the cytoplasm of the bacteria, that
can survive environmental extremes (desiccation, heat,
harmful chemicals)
Endospore Stain
Is there a mordant?
Is there a decolorizer?