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Bacterial Cell

Objectives:
State the shapes and arrangements of named
examples of bacteria
Describe the appendages and inclusions found in
various examples of bacteria
Distinguish between simple, differential and
special stains
State the differences between the cell walls of
Gram positive and Gram negative bacteria and
how these relate to staining characteristics.
Describe the bacterial endospore and explain its
role and describe its role in the survival of the
bacteria.

Bacteria come in many shapes.


most are spherical (called cocci, sing. Coccus),
Or rod shaped (called bacilli, sing. Bacillus and all

enteric bacteria eg. E. coli, Clostridium spp.)

Or spiral-shaped (called spirilla, sing. Spirillium-

Treponema palladium, Leptospira interrogans).

There are some intermediate shapes. The most

common are short bacillus (called coccobacillus), and a


short comma shaped spirillium (eg. vibrio-vibrio

The Cocci or round


Division in one plane produces one of two

arrangements:

diplococci: cocci arranged in pairs

(genera Neisseria eg. N. meningitidis and N.


gonorrhoeae)

streptococci: cocci
arranged in chains
(genera
Streptococcus
eg. S. pneumoniae,
S.mutans, S.
Pyogenes)

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Division in two
planes produces
squares of 4 called
tetrads
arrangement.
(genera micrococcus

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c. Division in three planes

produces a sarcina arrangement.


sarcinae: cocci in arranged cubes of

8
(Genera Sarcina eg. S. ventriculi
and S. lutea)

d. Division in random planes

produces a irregular, often


grape-like clusters called
Staphylococci.
(Genera staphylococcus: eg S.
aureus and S. epidermidis)

Bacilli or rod shaped

Bacilli are rod-shaped bacteria.

Bacilli all divide in one plane


producing a bacillus,
streptobacillus, or coccobacillus
arrangement.

a. bacillus: single bacilli

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b. streptobacillus: bacilli arranged

in chains

C. coccobacillus: oval and similar to

a coccus
(eg. Haemophilus influenzae and
Chlamydia trachomatis )

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Spiral
a. vibrio: a curved or comma-

shaped rod
(eg. V. cholerae and V.
parahaemolyticus)
b. spirillum: a thick, rigid spiral
(eg. Spirillum minus)
c. spirochete: a thin, flexible spiral

(eg. Treponema pallidum and


Borrelia recurrentis)
Spiral bacteria usually remain as

single micro organisms however


they vary in the number of
corkscrew turns.

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Exceptions to the above


shapes
Sheathed (actinomyces,

Sphaerotilus)
Stalked (Gallionella ferruginea)
Filamentous (Herbidospora
cretacea)
square (Walsbys square
bacterium)
star-shaped
spindle-shaped
lobed, and (hyphomicrobium)
pleomorphic

http://en.wikipedia.org/wiki/Bacterial_cellular_morphologies

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Pilli
are straight hair-like

appendages which tend to


be short.
They are made of the

protein pillin which is


arranged helically around a
central hollow core.
Pilli function to attach

bacterial cells to other


cells.

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The protein called adhesions

found in either the tip or side of


pilli make the connection
possible.
Sex pilli attach one bacterial cell

to another during mating.


While others attach them to

plant or animal cells or generally


anchor bacteria in a favorable
environment.

Flagellum

The function of the flagella is


locomotion

Flagella structure has three distinct


parts:

1. An outer helical-shaped filament-

Composed of subunits of the


protein flagellin arranged in a
helical manner around a hollow
core.
2. A hook- the filament is attached to

a hook which allows the filament to


move in different directions. The
hook is attached to the basal body.

3. A basal body- Anchors the flagella to the

envelope and causes it to rotate.


The part of the basal body that penetrates

the envelope has four (in gram negative)


or three (in gram positive) rings.

In gram negatives the L ring is embedded


in the outer membrane however Grampositives lack this ring.

The other rings are the P (peptidoglycan), and the

inner
S(superficial) and M (membrane) rings.

The motor that rotates the flagellum is a bell-

shaped structure that sticks into the cytoplasm.

The core of the flagellum (the rod) rotates inside the

rings which act as anchors to the envelope.

Bacterial Flagella

(copied from the internet)

Flagellum

(copied from the internet)

A-Monotrichous; B-Lophotrichous; C-Amphitrichous;


D-Peritrichous;

Different types of
bacteria have
different numbers of
flagella:
Monotrichous
(genera
pseudomonas),
amphitrichous,
lophotrichous and
peritrichous
(genera
Escherichia).

A group of bacteria called the spirochetes

have flagella which are bundled into two


axial filaments which are trapped inside
the periplasm.

Capsule
Most bacteria secrete a slimy or gummy

substance that forms outermost layer of


the cell.

Capsules vary in thickness and

composition with the organism that


produces it.

Most are however made of

Functions of the Capsule:

Principally protect the cell against drying

out
Adhere cells to a surface where conditions

are favorable for growth


Protect disease causing bacteria against

phagocytosis thus play an important role in


infection.
Stains of S. pneumoniae that lack a capsule are

Capsules and slime layers can also

provide protection from the loss of


nutrients by holding them within the
layer.
These extra layers coating the surface

of the cell may also potentially mask


viral receptors making it more difficult
for viruses to attach

Bacteria Surrounded by Capsule

Sheath
Some bacteria develop within sheaths

which are long transparent polysaccharide


tubes.
Cells divide and grow inside the tube

elongating the tube to fit the cells.


When growth conditions become

unfavorable the cells swim out leaving the


empty tube.

(sheathed bacteria)

Nucleoid
The nucleoid or nuclear region is well defined

even though it is not membrane bound.


It is a mass of DNA-carries the cells genetic

information.
Bacterial DNA (chromosomal) is usually

arranged in a single circular molecule.


Usually they also contain smaller circular

DNA molecules called plasmids.

Ribosomes
Found in the cytoplasm.
Their great number and small size give the

cytoplasm its characteristic grainy


appearance.
The ribosome is the site of protein synthesis.
A ribosome is composed of two subunits both

composed of protein and RNA. The large


subunit of prokaryotic cells is smaller than that
of Eukaryotic cells (80s).

Two complexes of RNA and protein make

up the prokaryotic ribosome, the 30S


subunit and the 50S subunit.
The 30S subunit is composed of 21

proteins and a single-stranded rRNA


molecule of about 1,500 nucleotides,
termed the 16S rRNA.
The 50S subunit contains 31 proteins

and two RNA species, a 5S rRNA of 150


nucleotides and a 23S rRNA of about
2,900 nucleotides.

Storage granules
Many bacterial species have several kinds

of storage granules.

Granules of carbon containing compounds

like glycogen and poly-betahydroxyalkanes.

Other granules containing reserves of

sulphur and nitrogen, and granules of


polyphosphate.

Other inclusions
Gas vacuoles: gas-filled regions

surrounded by a monolayer of a single


protein that allows the bacteria to float
at the water level with the conditions
best suited for photosynthesis.

Chlorosomes: Seen in photosynthetic

bacteria. These structures house


pigments necessary for photosynthesis.

Magnetosomes:
These magnet-like structures are needed

for magnetotaxis.
They allow the bacteria to follow magnetic

lines of force toward the bottom of bodies of


water- their optimum environment.

Heterocysts:
Nitrogen fixation and oxygenic

photosynthesis are incompatible since


nitrogen fixing systems are extremely
sensitive to oxygen.

Many cyanobacteria solve this problem by

carrying out nitrogen-fixation in specialized


cells called heterocysts.

All other cells photosynthesize.

Heterocyst

(diagram copied from internet)

Stains

Unstained bacteria are practically

transparent when viewed using the light


microscope and thus are difficult to see.

Stains serve several purposes:


1. Stains differentiate microorganisms from

their surrounding environment


2. They allow detailed observation of
microbial structures at high
magnification
3. Certain staining protocols can help to
differentiate between different types of
microorganisms.

Staining protocols can be divided into 3

basic types: simple, differential, and


specialized.

Simple stains react uniformly with all

microorganisms and only distinguish the


organisms from their surroundings.
Differentiation of cell types or structures is
not the objective of the simple stain.

However, certain structures which are not

stained by this method may be easily seen,


for example, endospores and lipid inclusions

Differential stains discriminate between

various bacteria, depending upon the


chemical or physical composition of the

The Gram stain is an example of a differential stain.


Because of the waxy substance (mycolic acids) present on

the cell walls, cells of species of Mycobacterium do not stain


readily with ordinary dyes.

Acid fast Stain


However, treatment with cold carbol fuchsin for several
hours or at high temperatures for five minutes will dye the
cells.
Once the cells have been stained, subsequent treatment

with a dilute hydrochloric acid solution or ethyl alcohol


containing 3% HCl (acid-alcohol) will not decolorize them.
Such cells are thus termed acid-fast in that the cell will hold
the stain fast in the presence of the acidic decolorizing
agent.
This property is possessed by few bacteria other than

Mycobacterium.

Specialized stains detect specific structures of cells such as

flagella and endospores

Specialized stains detect specific structures of cells such

as flagella and endospores

The nature of the endospore requires a vigorous treatment

for staining, but once stained, the endospores are difficult


to decolorize.

In the endospore stain, water is the decolorizing agent

that removes the primary stain from the vegetative cells.


Endospore stains require heat to drive the stain into the
cells.
For an endospore stain to be successful, the temperature

of the stain must be near boiling and the stain cannot dry
out.

Most failed endospore stains occur because the stain was

allowed to completely evaporate during the procedure.

Differential Stain
The Gram Reaction
The Gram reaction is named after the Danish

physician, Christian Gram, who developed this staining


technique in 1884. It involves a series of simple steps.

Bacterial cells are dried onto a glass slide and stained

with crystal violet, then washed briefly in water.

Iodine solution (mordant) is added so that the iodine

forms a complex with crystal violet in the cells.

Alcohol or acetone is added to solubilise the crystal

violet - iodine complex.


The cells are counterstained with safranin, then rinsed

and dried for microscopy.


Gram-positive cells retain the crystal violet-iodine

complex and thus appear purple.

The thickness of this wall blocks the escape of the crystal

violet-iodine complex when the cells are washed with alcohol


or acetone.

Gram-negative bacteria have only a thin layer of

peptidoglycan, surrounded by a thin outer membrane


composed of lipopolysaccharide (LPS).

The region between the peptidoglycan and LPS layers is

termed the periplasmic space.

It is a fluid or gel-like zone containing many enzymes

and nutrient-carrier proteins.

The crystal violet-iodine complex is easily lost

through the LPS and thin peptidoglycan layer when


the cells are treated with a solvent.

Gram-negative cells are decolourised by the alcohol

or acetone treatment, but are then stained with


safranin so they appear pink

Thus, the essential difference between Gram-positive and

Gram-negative cells is their ability to retain the crystal violetiodine complex when treated with a solvent.

Gram-positive bacteria have a relatively thick wall composed

of many layers of the polymer peptidoglycan (sometimes


termed murein).

The thickness of this wall blocks the escape of the crystal

violet-iodine complex when the cells are washed with alcohol


or acetone.

Gram-negative bacteria have only a thin layer of

peptidoglycan, surrounded by a thin outer membrane


composed of lipopolysaccharide (LPS).
The crystal violet-iodine complex is easily lost

through the LPS and thin peptidoglycan layer when


the cells are treated with a solvent.

Cell wall
Bacterial wall is made mostly of a rigid

macromolecule called peptidoglycan.

Peptidoglycan is composed of N-

acetylglucosamine (NAG) and N-acetylmuramic


acid (NAM) joined by 1,4-glycosidic bonds

Chains of NAM and NAG are cross-linked by

peptide chains (differ among bacterial species).

Peptide chains are made of aa of D configuration.


The most common peptides are four amino acid

long: L- alanine, D- alanine, D-glutamic acid and


lysine or diaminopimelic acid (DAP).

NAM and NAG molecules form a repeating

structure. The strength of the bacterial cell wall is


proportional to the extent of cross-linkages.

Covalently bound to the thick peptidoglycan

are teichoic acid

Differences between Gram-negative and Grampositive cell wall

In Bacteria the reaction to Gram stain

reagents is explained by different cell wall


structures.

Gram-positive microbes have a much thicker

cell wall, while that found in Gram-negative


microbes is thinner or nonexistent.

http://www.google.com.jm/imgres?q=gram+positive+and+gram+negative+bacterial+cell+wall+structure&um=1&hl=en&sa=N&tbo=d&biw=1137&bih=570&tbm=isch&tbnid=UtTPQoAW33fD9M:&imgrefurl=http://tommytoy.typepad.com/tommy-toy-pbtconsultin/2011/11/antibacterials-startups-draw-venture-capital-for-drugs-targeting-gram-nevagive-bacteria-and-infectio.html&docid=Xq4WMe8QAdqnVM&imgurl=http://tommytoy.typepad.com/.a/6a0133f3a4072c970b0162fc6173d0970d550wi&w=445&h=312&ei=O6oPUeO5MoT49QTQwoDQCQ&zoom=1&ved=1t:3588,r:1,s:0,i:82&iact=rc&dur=1681&sig=102320833935158558452&page=1&tbnh=182&tbnw=267&start=0&ndsp=12&tx=114&ty=61

The cultures to be stained should be young -

incubated in broth or on a solid medium until


growth is just visible (no more than 12 to 18 hours
old if possible).

Old cultures of some gram-positive bacteria will

appear Gram negative.

This is especially true for endospore-forming

bacteria, such as species from the genus Bacillus.

When feasible, the cultures to be stained should

be grown on a sugar-free medium.

Many organisms produce substantial amounts of

capsular or slime material in the presence of


certain carbohydrates.

This may interfere with decolorization, and certain

Gram-negative organisms such as Klebsiella may


appear as a mixture of pink and purple cells.

Endospores
Endospores are dormant alternate life forms produced by the

genus Bacillus (obligate aerobes found in the soil) and

the genus Clostridium (obligate anaerobes often found as

normal flora of gastrointestinal tract of animals)

and several other less common genera

function: An endospore is not a reproductive structure but

rather a resistant, dormant survival form of the


organism.

Endospores are quite resistant to high temperatures

(including boiling), most disinfectants, low energy


radiation, drying, etc.
The endospore can survive possibly thousands of

years until a variety of environmental stimuli trigger


germination, allowing outgrowth of a single
vegetative bacterium

Formation Of Endospores
Under conditions of starvation, especially the lack of carbon

and nitrogen sources, a single endospores forms within some of


the bacteria.

The process is called sporulation : First the DNA replicates

and a cytoplasmic membrane septum forms at one end of the


cell forming a forespore.

The remainder of the vegetative cell engulfs the forespore.

Then there is synthesis of peptidoglycan in the space

between the two membranes surrounding the forespore to


form the first protective coat, the cortex.

Calcium dipocolinate is also incorporated into the forming

endospore.

A spore coat composed of a keratin-like protein then forms

around the cortex. Sometimes an outer membrane


composed of lipid and protein and called an exosporium is
also seen.

Finally, the remainder of the bacterium is degraded and the

endospore is released. Sporulation generally takes around


15 hours.

Endospore Structure:
The completed endospore consists of multiple layers of

resistant coats including:


a cortex
a spore coat

and sometimes an exosporium

These layers surround a nucleoid, some ribosomes, RNA molecules, and


enzymes.

Exosporium- thin covering made of protein

and lipids

Spore coat- highly cross-linked keratin and

layers of spore-specific proteins

Cortex- loosely cross-linked peptidoglycan


Innermost spore cell- components of the

vegetative cell

Bacterial endospores are resistant to antibiotics, most

disinfectants, and physical agents such as radiation,


boiling, and drying.

resistance of endospores is due to A variety


of factors:
Proteinaceous spore coat- confers resistance to

lysozyme and harsh chemicals

Calcium-dipicolinate, abundant within the endospore,

may stabilize and protect the endospore's DNA.

Specialized DNA-binding proteins saturate the

endospore's DNA and protect it from heat, drying,


chemicals, and radiation.

Critical functions of the cell Membrane


Separates contents of the cells from their external

environment.

Membranes allow for the compartmentalization of

the cell.

Membrane-bound compartments keep certain reactive

compounds away from other parts of the cell which might


be affected by them.
Also reactants that are located in a small space are far
more likely to come into contact and hence the reaction
can be dramatically increased.

References
http://www.merck.com/media/mmhe2/figures/MMHE_17_190_01_eps.gif
http://www.microbiologytext.com/index.php?module=Book&func=displayart

icle&art_id=70
http://www.arn.org/docs/mm/flag_labels.jpg
http://answers.yahoo.com/question/index?qid=20090420112848AATic8k