BACTERIAL CELL

Objectives:
1.State the shapes and arrangements of named examples of bacteria 2.Describe the appendages and inclusions found in various examples of bacteria 3.Distinguish between simple, differential and special stains 4.State the differences between the cell walls of Gram positive and Gram negative bacteria and how these relate to staining characteristics. 5.Describe the bacterial endospore and explain its role and describe its role in the survival of the bacteria.

BACTERIAL CELL Bacteria come in many shapes.

most are spherical (called cocci, singular Coccus), or rod shaped (called bacilli, singular. bacillus and all enteric bacteria eg. E. coli, Clostridium spp.) or spiral-shaped (called spirilla, singular. 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 cholerae).

BACTERIAL MORPHOLOGY

Coccus

Spirochete

Rod

Budding and appendage

Spirillium Filamentous

BACTERIAL MORPHOLOGY The Cocci or round

A. Division in one plane produces one of two arrangements:
1. diplococci: cocci arranged in pairs (genera Neisseria eg. N. meningitidis and N. gonorrhoeae) 2. streptococci: cocci arranged in chains (genera Streptococcus eg. S. pneumoniae, S.mutans, S. Pyogenes)

B. Division in two planes produces squares of 4 called tetrads arrangement.

(genera micrococcus eg. M. luteus and M. roseus)

BACTERIAL MORPHOLOGY 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)

BACTERIAL MORPHOLOGY

BACTERIAL MORPHOLOGY

Bacilli or rod shaped

Bacilli all divide in one plane producing a bacillus, diplobacillus, streptobacillus, palisades or coccobacillus arrangement.
a) bacillus: single bacilli b) streptobacillus: bacilli arranged in chains c) Palisades: vertical side by side d) coccobacillus: oval and similar to a coccus (eg. Haemophilus influenzae and Chlamydia trachomatis )

BACTERIAL MORPHOLOGY

OTHER BACTERIAL CELL STRUCTURES

BACTERIAL MORPHOLOGY
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.

BACTERIAL MORPHOLOGY

BACTERIAL MORPHOLOGY 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

BACTERIAL CELL STRUCTURES

CAPSULE

Flagellum

Pili

PILLI 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.
The protein called adhesions found in either

the tip or side of pilli make the connection possible.

PILLI

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. Escherichia coli and Neisseria gonorrhoeae both have pilli.

FLAGELLUM The function of the flagella is LOCOMOTION Flagella structure has three distinct parts:
1. An outer helical-shaped filamentComposed 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.

FLAGELLUM

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 Gram-positives lack this ring.

FLAGELLUM 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.

FLAGELLUM

 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.

OTHER BACTERIAL CELL STRUCTURES

Different types of bacteria have different numbers of flagella:

Monotrichous
eg (pseudomonas)

Lophotrichous Amphitrichous Peritrichous

eg Escherichia

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 polysaccharide and a few of protein.

CAPSULE

CAPSULE

Functions of the Capsule:

1. Principally protect the cell against drying out 2. Adhere cells to a surface where conditions are favorable for growth 3. Protect disease causing bacteria against phagocytosis thus play an important role in infection.
Stains of S. pneumoniae that lack a capsule are harmless because they are quickly consumed.

CAPSULE

Functions of the Capsule: 4.Capsules and slime layers can also provide protection from the loss of nutrients by holding them within the layer. 5.These extra layers coating the surface of the cell may also potentially mask viral receptors making it more difficult for viruses to attach

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. These organisms have a free-swimming stage where they exit sheath and move by flagella Sheathed bacteria are found in contaminated streams and sewage treatment ponds. Sheath serves as protection against debris Allows survival over a wide pH range

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).

RIBOSOMES

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.

BASIC STRUCTURE OF BACTERIA

Ribosome

STORAGE GRANULES Many bacterial species have several kinds of storage granules. Granules of carbon containing compounds like glycogen and poly-beta-hydroxyalkanes. 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 accessory pigments necessary for photosynthesis.

OTHER INCLUSIONS 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.

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.

STAINS
3 basic types: simple, differential, & 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 microorganism.

STAINING BACTERIA

Types of dyes and stains used

Simple Stains
cationic + charged

Differential Stains Gram Stain Acid-fast stain

Structural Stains Endospore Stain Capsule Stain Flagella Stain

Basic Dyes

anionic, - charged

Acidic Dyes

Indifferent Dyes

ACID FAST STAIN Because of the waxy mycolic acids present on the cell walls, cells of species of Mycobacterium do not stain readily with ordinary dyes. However, treatment with cold carbol fuchsin for several hours or at high temp for 5 mins 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.

ACID FAST STAIN

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.

STRUCTURAL STAINS Specialized stains detect specific structures of cells such as flagella and endospores Endospore Stain 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.

STRUCTURAL STAINS Endospore Stain Endospore stains require heat to drive the stain into the cells. For an endospore stain to be successful, the temp 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.

GRAMS STAIN

Named after the Danish physician, Christian Gram, who developed this staining technique in 1884.
1. Bacterial cells are dried onto a glass slide and stained with crystal violet, then washed briefly in water. 2. Iodine solution (mordant) is added so that the iodine forms a complex with crystal violet in the cells.

GRAMS STAIN 3. Alcohol or acetone is added to solubilise the crystal violet - iodine complex. 4. The cells are counterstained with safranin, then rinsed and dried for microscopy. Gram-positive cells retain the crystal violetiodine 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.

GRAMS STAIN

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.

GRAMS STAIN The PERIPLASMIC SPACE 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

GRAMS STAIN Thus, the essential difference between Grampositive and Gram-negative cells is their ability to retain the crystal violet-iodine 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.

GRAMS STAIN

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.

GRAMS STAIN

+Positive

-Negative

GRAMS STAIN

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 may appear Gram negative . This is especially true for endosporeforming bacteria, such as species from the genus Bacillus.

GRAMS STAIN

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.

BACTERIAL CELL WALLS

Made mostly of a rigid macromolecule called PEPTIDOGLYCAN. Peptidoglycan is composed of Nacetylglucosamine (NAG) and Nacetylmuramic acid (NAM) joined by 1,4-glycosidic bonds Chains of NAM and NAG are cross-linked by peptide chains (differ among bacterial species).

BACTERIAL CELL WALLS 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 crosslinkages. Covalently bound to the thick peptidoglycan are teichoic acid

BACTERIAL CELL WALLS

BACTERIAL CELL WALLS

BACTERIAL CELL WALLS

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

ENDOSPORES 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

ENDOSPORES
Formation of Endospores Under conditions of starvation, especially the lack of carbon and nitrogen sources, a single endospores forms within some of the bacteria. This process is called sporulation : 1. First the DNA replicates and a cytoplasmic membrane septum forms at one end of the cell forming a forespore. 2. The remainder of the vegetative cell engulfs the forespore.

ENDOSPORES Formation of Endospores continued

3. Then there is synthesis of peptidoglycan in the space between the two membranes surrounding the forespore to form the first protective coat, the cortex. 4. Calcium dipocolinate is also incorporated into the forming endospore. 5. 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. 6. Finally, the remainder of the bacterium is degraded and the endospore is released. Sporulation generally takes around 15 hours.

Formation of Endospores

ENDOSPORE STRUCTURE The completed endospore consists of multiple layers of resistant coats including: a cortex
loosely cross-linked peptidoglycan

a spore coat
highly cross-linked keratin and layers of spore-specific proteins

and sometimes an exosporium

thin covering made of protein and lipids

These layers surround components of the vegetative cell

a nucleoid, some ribosomes, RNA and enzymes.

ENDOSPORE STRUCTURE

ENDOSPORE RESISTANCE
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.

CELL MEMBRANE Critical functions:

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.