ULTRA-STRUCTURE OF BACTERIA (MICROBIOLOGY NOTES)(SECOND YEAR B.PHARMACY NOTES) PCI SYLLABUS

 ULTRASTRUCTURE OF BACTERIAL CELL

ULTRA-STRUCTURE OF TYPICAL BACTERIAL CELL:-

Under electron microscope the structure of bacterial cell is look like a capsule. It has various components.  Some of these are outside the cell membrane; others are inside of cell membrane.

The outer layer or cell envelope consists of two components such as cell wall and a cytoplasmic or plasma membrane. Inside the plasma membrane, there is protoplasm comprising the cytoplasm, cytoplasmic inclusions such as ribosomes, mesosomes, granules, vacuoles, and nuclear body.The cell may be enclosed in a viscid layer which is termed as capsule. Many bacteria have filamentous appendages called fimbriae or pili (organ of adhesion). Many bacteria also posses flagella which are organs of locomotion.

Components outside the cell membrane:-

1.     Capsule 

2.     Pilli

3.     Flagella

4.     Cell wall

Components inside the cell wall:-

1.     Cytoplasmic membrane (Plasma membrane)

2.     Cytoplasm

3.     Ribosomes

4.     Mesosomes

5.     Intracytoplasmic inclusion

6.     Nucleoid & nucleus

7. Spores

1.     CAPSULE:- It is the outer layer of the bacterial cell.  Many prokaryotic micro organisms synthesize amorphous organic exopolymers which are deposited outside the cell wall called capsules or slime layer or glycocalyx or sugar coat.

Capsule refers to the layer tightly attached to the cell wall.  While the

Slime layer is the loose structure that often diffuses into the growth medium.  

Depending on the chemical nature capsule are thick or thin.

Microcapsule:- Capsule layer may be thin of a size less than 0.2 µm called microcapsule. 

Macrocapsule:- Thick layer of size more than 0.2 µm to 10 µm called macrocapsule.

Capsules may be composed of a complex polysaccharide (Klebsiella pneumoniae) polypeptide (Bacillus anthrocis)  hyaluronic acid (Streptococcus pyogenes).   Water (98%) is the main component of bacterial capsule. Capsule or slime layer has less affinity for battle dyes and is not visible in Gram staining. Special capsule staining techniques are used by using copper salts as mordants. Capsules may be easily observed by negative staining in wet films with lndian ink.

Functions of capsule:   

1.     They may provide protection against mechanical injury, temperature & temporary drying by binding water molecules.  

2.     They may promote attachment of bacteria to surfaces. E.g. Streptococcus mutant that cause dental carries attach on teeth surface by its capsule.

3.     They may promote the stability of bacterial suspension by preventing the cells from aggregation and settling.    

They inhibit phagocytosis (antiphagocytic) and contribute to the virulence of pathogenic bacteria.                                                                             

PILLI/FIMBRIAE: -

Many Gram negative bacilli contain short, thin, hair-like microfibrils called Pilli.  The size of the pilli is 0.5 to 2 um in length and 5 to 7 nm in diameter.                                                                                

They are more numerous than flagella.  Fimbriae are composed of protein known as pillin and its molecular weight is 18000 daltons.  Fimbriae can be seen only under the electron microscope. They are best developed in freshly isolated strains and in liquid cultures. They may disappear after sub culturing on solid media.  

FUNCTIONS OF PILI:-      

1.     Pilli are non motile but adhesive structure. They enable the bacteria to stick firmly to other bacteria & to a surface hence pili is also called an organ of adhesion.                                                                          

      Pili is used for the transfer of genetic material from the donor to the recipient cell (bacterial conjugation).   

FLAGELLA:-

Flagella are long, slender, thin hair like structure. The size of the flagella is about 0.01 µm to 0.02 µm in diameter & 3 to 20 µm in length. Flagella are made up of a protein (flagellin) similar to keratin or myosin & they are responsible for the motility of bacteria hence it is called as organs of locomotion.                                                                  

Flagella are found in both Gram positive and Gram negative bacteria. A large no of bacteria such as spirilla, vibrios, most of bacilli & few coccal forms, are motile by means of flagella.

Flagella can be seen by an ordinary light microscope by special staining techniques in which their thickness is increased by mordanting.

Flagellum has three basic parts                                                                                                                   (1) Filament, (2) Hook and (3) Basal body.                                                                                                  Filament is the thin, cylindrical, long outermost region with a Constant diameter. The protein in the filament is made up of monomers called ‘flagellin' with molecular weight ranging between 20,000 to 40,000 The filament is attached to a slightly wider hook, consisting of a different protein . The basal body is composed of a small central rod inserted into a series or rings.

Gram-negative bacteria contain four rings as L-ring, P-ring, S -ring and M ring.                                        L-ring is embedded in Lipo-polysaccharide layer of outer membrane membrane,                                      P-ring in Peptidoglycan layer,                                                                                                                      S-ring in just above cytoplasmic membrane (Semi-position of membrane) and                                      M-ring within cytoplasmic Membrane. Gram-positive bacteria have only S and M rings in basal body. 

      The number and arrangement of flagella are characteristics of each bacteria. Flagella may be seen on bacterial body in the following manner.

1. MONOTRICHOUS:- These bacteria have single polar flagellum. E.g. vibrio cholera, pseudomonas aeruginosa, spirillum.
2. LOPHOTRICHOUS:- Bacteria have two or more flagella only at one end of the cell. E.g. pseudomonas fluorescens.
3. AMPHITRICHOUS:- Bacteria have single polar flagella at both poles. E.g. Alcaligenes fecales, Aquaspirillum serpenes.
4. PERITRICHOUS:- Several flagella present all over the surface of bacteria. E.g. E.coli, Salmonella typhi.

CELL WALL:-

Cell wall is a rigid structure which gives definite shape to the cell and protect from osmotic lysis.  

They are situated between the capsule and plasma membrane.  

SIZE:- It is about 10 20 nm in thickness and constitutes 20-30%  of the dry Weight of the cell. The wall can protect a cell from toxic substances and is the site of action of several antibiotics.

Peptidoglycan-the most important molecule in the cell walls of bacteria. Peptidoglycan or murein is an enormous polymer composed of many identical subunits. The polymer contains two sugar derivatives, N-acetylglucosamine and Nacetylmuramic acid (the lactyl ether of Nacetylglucosamine), and several different amino acids, three of which—D-glutamic acid, D-alanine, and meso-diaminopimelic acid        

Gram + cell wall:- The gram-positive cell wall consists of a single 20 to 80 nm thick. They are homogeneous peptidoglycan or murein layer lying outside the plasma membrane. Homogeneous cell wall of gram-positive bacteria is composed primarily of peptidoglycan, which often contains a peptide interbridge . The teichoic acids are connected to either the peptidoglycan by a covalent bond with Nacetylmuramic acid or to plasma membrane lipids are called lipoteichoic acids.

Gram – cell wall:-  The gram-negative cell wall is quite complex. It has a 2 to 7 nm peptidoglycan layer surrounded by a 7 to 8 nm thick outer membrane. The outer membrane lies outside the thin peptidoglycan layer • The most abundant membrane protein is Braun’s lipoprotein, covalently joined to the underlying peptidoglycan and embedded in the outer membrane by its hydrophobic end. • Constituents of the outer membrane are its lipopolysaccharides • outer membrane is more permeable than the plasma membrane due to the presence of special porin proteins 

Functions of cell wall:

1. Cell wall is involved in growth and cell division of bacteria.
2. It gives shape to the cell.
3.  It gives protection to the internal structure and acts as a supporting layer.
4.  It provides attachment to complement.
5. It shows resistance to the harmful effects of environment.  

                                                                                                           

CYTOPLASMIC MEMBRANE:-

Also called as plasma membrane, is the most dynamic structure of a bacterial cell. The cytoplasmic membrane is a thin (5 to 10 nm) layer lining the inner surface of the cell wall and separating it from the cytoplasm. It is composed Of phospholipids (20 to 30%) and proteins (60 to 70%).

The phospholipids form a bilayer surrounding the cytoplasm and regulate the flow of substance in and out of the cell in which most of the proteins are tenaciously held and are called integral proteins. Other proteins are loosely attached are called peripheral proteins. The phospholipids molecules are arranged in two parallel rows. called a phospholipids bilayer. Each phospholipid molecule contains a polar head composed of a phosphate group and glycerol. The non-polar tails are in the interior of the bilayer and the polar heads are on the two surfaces of the phospholipids bilayer.

Functions of cytoplasmic (plasma) membrane:-

1.  Its main function is a selective permeability barrier that regulates the passage of substances into and out of the cell.
2. It provides mechanical strength to the bacterial cell.
3.  It helps in DNA replication, segregation with septum formation & cell division.
4.  It contains the enzyme, permease, which plays an important role in the passage of selective nutrients & ions through membranes.
5. It contains the enzymes involved in the biosynthesis of membrane lipids and synthesis of murein (cell wall peptidoglycan) & other macromolecules of the bacterial cell

CYTOPLASM:-

The bacterial cytoplasm is a Gel-like matrix composed of mostly water (4/5 th ), enzymes, nutrients, wastes, and gases The cytoplasm of bacteria differs from that of higher eukaryotic microorganisms as it not contain endoplasmic reticulum, Golgi apparatus, mitochondria and lysosomes. It contains ribosome, chromosomes, plasmids, proteins as well as the components necessary for bacterial metabolism. It carries out very important functions for the cell - growth, metabolism, and replication.

The main constituents of cytoplasm is Proteins including enzymes Vitamins, Ions, Nucleic acids and their precursors – Amino acids and their precursors – Sugars, carbohydrates and their derivatives – Fatty acids and their derivatives.

RIBOSOMES:-

The most notable structures in the bacterial cytoplasm are the ribosomes. They are involved in protein synthesis & translate the genetic code from the molecular language of nucleic acid to that of amino acids. Bacterial ribosome’s are similar to those of eukaryotes, but are smaller and have a slightly different composition and molecular structure. Bacterial ribosome’s are never bound to other organelles as they sometimes are bound to the endoplasmic reticulum in eukaryotes, but are free-standing structures distributed throughout the cytoplasm. Their number varies with the rate of protein synthesis {15000/cell). The greater the rate of protein synthesis the greater the number of ribosome’s.

The bacterial ribosomes are referred to as 70S ribosomes. (S-Svedberg unit, the unit of sedimentation).These ribosome’s when placed in a low concentration of magnesium, dissociate into two components as 5OS and 30S particles.

Each 50S particle contains one molecule of 23S-RNA, one molecule of 5S-RNA and 32 different proteins. The 30S subunit contains one molecule of 16S r-RNA and 21 different proteins. These ribosome’s, during active protein synthesis are associated with the m-RNA and such associations are called polysomes.

MESOSOMES:-  

Mesosomes are also called chondroids and are visualized only under an electron microscope. Mesosomes are the invaginated structures formed by the localized infoldings of the plasma membrane. The invaginated structures comprise of vesicles, tubules of lamellar whorls. In some bacteria particularly in gram-positive bacteria depending upon the growth conditions the membrane appears to be infolded at more than one point such infoldings are called as mesosomes. Generally mesosomes are found in association with nuclear area or near the site of cell division. They are absent in eukaryotes.

There are two types of mesosomes:-

Central mesosomes:-  Are present deep into the cytoplasm and located near the middle of the cell. This mesosome is attached to bacteria chromosome and is involved in DNA segregation and in the formation of cross walls during cell division.

Peripheral mesosomes:-  Are not restricted to a central location and are not associated with nuclear material.   

mesosomes are supposed to take part in respiration but they are not analogous to mitochondria because they lack outer membrane. In the vesicle of mesosomes the respiratory enzymes and the components of electron transport such as ATPase, dehydrogenase, cytochrome are either absent or present in low amount. 

Mesosomes might play a role in reproduction also. During binary fission a cross wall is formed resulting in formation of two cells. Mesosomes begin the formation of septum and attach bacterial DNA to the cell membrane.

In addition, the infoldings of mesosomes increase the surface area of plasma membrane that in turn increases the absorption of nutrients.

INTRACYTOPLASMIC iNCLUSIONS:- It is also called as inclusion bodies. Bacteria can produce within their cytoplasm a variety of small bodies which is called as inclusion bodies. Some are called granules and other are called vesicles. They are mainly used for storage of energy & reduce osmotic pressure by tying up molecules in particulate forms like polysaccharides granules, glycogen granules, metachromatic granules, lipid granules etc.

 Inclusions are considered to be nonliving components of the cell that do not possess metabolic activity and are not bounded by membranes. The most common inclusions are glycogen, lipid droplets, crystals, and pigments.

Granules:-

metachromatic or Babes~Ernst granules are highly refractive, basophilic bodies consisting of polymetaphosphate.   They appear reddish when stained with polychrome methylene blue or toluidine blue. Albert's or Neisser's special staining techniques are used for the study of metachromatic granules.

Lipid granules consist mainly of polymerised beta hydroxybutyric acid. They can be seen in unstained preparations and by staining with sudan black and by modified ziehl-Neelsen stain. Polysaccharide granules can be stained with iodine. They are made-up of either glycogen (red brown) or starch (blue).

Vesicles:-

 Some aquatic photosynthetic bacteria and cyano bacteria have rigid gas-filled vacuoles and it helps in floating at a certain level - allowing them to move up or down into water layers with different light intensities and nutrient levels.

NUCLEOID AND NUCLEUS:-

The nucleoid is a region of cytoplasm where the chromosomal DNA (genetic material) is located. Bacterial nucleus does not possess nuclear membrane, nucleolus and deoxyribonucleoprotein.

Most bacteria have a single, circular chromosome that is responsible for replication, although a few species do have two or more smaller circular auxiliary DNA strands, called plasmids, are also found in the cytoplasm.

Bacterial nucleus can be demonstrated by acid or ribonuclease hydrolysis. They may be seen by a light microscope after staining (Feulgen stain) or by electron microscopy.  They appear as oval or elongated bodies generally one per cell. The genome consists of a single molecule of double stranded DNA arranged in a circle. It may open under certain conditions to form a long chain about 1000 um in length. 

SPORES: -

Many bacterial species produce spores inside the cell (endospores) as well as outside the cell (exospores). eg. Bacillus anthracis, Bacillus subtilis, Clostridium tetani etc.

Endospores are thick-walled, highly refractile bodies that are produced one per cell. Each bacterial spore on germination forms a single vegetative cell. Therefore sporulation in bacteria is a method of preservation and not reproduction. Spores are extremely resistant to dessication, staining, disinfecting chemicals, radiation and heat.They help bacteria to survive for long periods under unfavourable environments.

All endospores contain large amount of dipicolinic acid (DPA) with 10 to 15 percent of the spores being dry weight

An endospore returns to its vegetative state by a process called germination, which has three distinct stages.

Activation: The activation process requires agents like heat, low pH, abrasion etc. These agents damage the coat of the spore and help in germination by growing in a nutritionally rich environment.

Initiation: Binding of the effector substance (L-alanine. adenosine, glucose etc.) to the. spore coat, activates an autolysin which destroys peptidoglycan of the cortex. releases calcium dipicolonic acid and absorbs water.

Outgrowth: Spore coat breaks and forms a single germ cell. It starts growing into a new vegetative cell and active synthesis takes place producing an outgrowth.

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1.      INTRODUCTION OF MICROBIOLOGY

2.      BRANCHES OF MICROBIOLOGY

3.      SCOPE OF MICROBIOLOGY

4.      HISTORY OF MICROBIOLOGY {PART 1}

5.       HISTORY OF MICROBIOLOGY {PART 2}

6.      HISTORY OF MICROBIOLOGY {PART 3}

7.        PROKARYOTES VS EUKARYOTES DIFFERENCES

8.      MORPHOLOGY OF BACTERIA

9.      ULTRASTRUCTURE OF BACTERIA

1.   NUTRITIONAL REQUIREMENTS OF BACTERIA

1.   RAW MATERIAL USED FOR CULTURE MEDIA

1.   TYPES OF CULTURE MEDIA IN MICROBIOLOGY

1.   PHYSICAL PARAMETERS FOR GROWTH

1.   GROWTH CURVE OF BACTERIA

1.   MEASUREMENT OF BACTERIAL GROWTH.

1.    ISOLATION OF PURE CULTURE