Lecture 2: Bacterial Anatomy

Bacterial cells are characterized by complicated structure and are composed of different elements.

Structural elements of bacterial cell: Constant Cell wall Cytoplasmic membrane Cytoplasm Ribosomes Mesosomes Nucleoid Inconstant Capsule Flagella Spores Intracellular inclusions Pilli Plasmids

All structural elements of bacteria are sub classified into 2 groups: 1. Group of constant elements- elements of this group are always present in bacterial cell and may be detected in structure of bacteria belonging to different species and genera. Constant elements are the cell wall, cytoplasmic membrane, cytoplasm, ribosomes, mesosomes and nucleoid. 2. Group of inconstant elements includes- elements which may present or absent in bacterial cell. Some of them appear under unfavourable environment conditions such as dessication, starvation, changing of tempersature. Some structural

elements such as the flagella and fimbria are detected only in structure of certain genus and species of microorganism. Inconstant elements are capsule, spores, intracellular inclusions, flagella, fimbria/pili and plasmids.

Structures enveloping the bacteria.

The outer layer covering the bacteria cell consists of 2 components- the rigid cell wall and the cytoplasmic membrane. The cell wall encloses the protoplasm, comprising the cytoplasma and the single circular chromosome of DNA. Some bacteria may possess additional structures such as the proctective, gelatinous layer known as capsule.

Chemical Structure of the Cell Wall

The cell wall is a tough and rigid structure surrounding the bacteria like a shell. It is elastic, porous and freely permeable layer. The cell wall takes part in cell division by forming an ingrowth from the cell wall. The chemical composition for cell wall of Gram (+ve) and Gram (-ve) bacteria is different. 1. Cell wall of Gram (-ve) bacteria.

The Gram(-ve) bacteria cell wall is a complex structure which containing 3 component layers outside the peptidoglycan layer. The lipoprotein layer connects the outer membrane to the peptidoglycan layer. The outer membrane is a phospholipids bilayer, it contains specific proteins. These specific proteins form porines and hydrophilic molecules are transported through these porines. The layer of lipopolysacchariedes is the third layer and it consist of a complex lipid called lipid A. This layer is attached to polysaccharides. The lipopolysaccharides layer is the endotoxin of Gram (-ve) bacteria. The toxicity is associated with the lipid portion. The polysaccharide represents a major surface antigen, the O-antigen. Also, the structure of the Gram (-ve) bacteria is characterized by the presence of periplasmic space. It is a space between the inner and outer membranes and contains a number of important proteins and oligosaccharides playing an important role in osmoregulation of cells. 2. Cell wall of Gram (+ve) bacteria. In Gram (+ve) bacterias cell wall, the peptidoglycan layer is much thicker than those in Gram (-ve) bacteria. The thickness of the peptidoglycan layer is 18-80 nm in Gram (+ve) bacteria while it is only 2 nm in Gram (-ve) bacteria. The periplasmic space is absent and the peptidoglycan layer is closely associated with cytoplasmic membrane. Most Gram (+ve) bacteria cell wall contains a significant anount of teichoic and teichuronic acids. These acids are water soluble polymers. The teichoic acids constitutes the major surface antigen of Gram (+ve) bacteria. Functions of the cell wall: maintenance of the cell shape; protection of cell from mechanical and osmotic action of the environment; taking part in the regulation of growth and division of cells; connect bacteria with the environment through channels and pores; determines the antigenic characteristics of the bacteria; contains receptors for bacteriophages attachment (viruses of bacteria).

But sometimes the bacteria may lose cell wall and form derivatives: spheroplasts, protoplasts and L-forms Spheroplasts are derived from Gram (-ve) bacteria. These derivatives lose their cell wall partially. They are produced by growth of bacteria with penicillin. They are osmotically fragile and maintain in hypertonic medium/culture. Protoplasts are derived from Gram (+ve) bacteria. They lose their cell wall completely. They are also unstable and osmotically fagile. They are produced artificially by lysosome/ penicillin action. Hypertonic media are necessary for their maintenance.

L-forms are cell wall deificient forms of bacteria (the cell wall is destroyed by the action of antibiotics and lytic enzymes) and they are more stable than spheroplasts and protoplasts. They develop either spontaneously or artificially under the action of penicillin or other agents. They may multiply by binary fission while spheroplasts and protoplasts can not. L-forms, spheroplasts and protoplasts are able reverse back to their initial forms. The main properties of L-forms: similarity of morphological changes; similarity of cultural changes; transformation of Gram (+ve) in Gram (-ve); formation of stable and unstable L-forms; changes in antigenic properties; decreasing of virulence; ability to long persistence (able to circulate in the body for a long time and cause chronic infection); ability to return to initial form.

Methods for cell wall demonstration: plasmolysis antigen-antibody reaction differential staining ( Grams staining or acid-fast staining) electron microscopy techinique

Cytoplasmic membrane
It is a thin, elastic layer lying beneath the cell wall, separating it from the cells cytoplasm. Electron microscopy shows 3 layers forming the cytoplasmic membrane: the central layer is composed of protein molecules and on its both sides are lipid molecules. The plasma membrane is visible in ultra thin section under the electron microscope. The cytoplasmic membrane is characterized by semi-permeability and selective transport. Functions of the cytoplasmic membrane: transport of chemicals through the cytoplasmic membrane (active and passive). the chemicals concentration. Cytoplasmic membrane separates the cytoplasm components from the external environment. takes part in the cell division. takes part in bacterial oxydation.

Methods for demonstration of the cytoplasmic membrane: plasmolysis electron microscopy

Mesosomes
Mesosomes are derivatives of cytoplasmic membrane. They are convoluted, multilaminated, membranous sac-like bodies. They are formed by convoluted unvaginations of the plasma membrane into the cytoplasm. Mesosomes are principle centers of respiratory enzymes storage in bacteria. There are 2 types of mesosomes: lateral and septal. Lateral mesosomes are centers of respiratory enzymes storage. Several lateral mesosomes are detected in the structure of bacteria. There is only 1 septal mesosome in a bacterial cell. The septal mesosome is attached to bacterial chromosomes (nucleoid) and involved in DNA segregation and in formation of cross over walls during cell division. Method for demonstration of mesosomes: electron microscopy.

Ribosomes
Ribosomes are complicated structures 10-20 nm in size. They have sedimentation coefficient - 70s. The unit of sedimentation equal to 70s (consist of 2 units- 30s unit and a 50s unit). They are distributed throughout the cytoplasm. Ribosomes are the sites of protein synthesis. Method for ribosome demonstration: electron microscopy.

Cytoplasm
The cytoplasm is a colloidal watery solution containing organic and inorganic dissolved chemicals. It lacks mitochondria and chloroplasts and contains large number of ribosomal granules, few membranous bodies of mesosomes, vacuoles and inconstant elements (e.g. intracellular inclusions). It is also the place of localization of the bacterial nucleoid. The biological role is that it is the medium for chemical reactions to take place in bacterial cell. Methods for demonstration of the cytoplasm: simple method of staining complicated method of staining

plasmolysis electron microscopy

Nucleoid
The bacterial nucleoid is a thin fiber of double stranded DNA tightly coiled inside the cytoplasm. Its structure resembles the nucleus, but there is no nuclear membrane and small nucleus (micronucleus). The nuclear DNA doesnt contain basic proteins, the double stranded DNA is haploid. Its function is the maintenance of genetic information/ characteristics. Methods for demonstration of the nucleoid: electron microscopy special method of staining PCR- polymerase chain reaction , DNA-DNA hybridization

Inconstant Structural Elements

The inconstant structural elements are not as important as constant elements, bacteria still alive without inconstant elements.

Capsule
The capsule is an outer covering of thick material surrounding the bacterial cell wall. It is the most external layer. It consist of 98% water and 2% of solids. The solid component is complicated of polysaccharides or polypeptides. Functions of the capsule: It serves as a protective covering against antibacterial substances such as bacteriophages, phagocytes and enzymes. It protects the bacteria from phagocytosis and penetration of lytic enzymes. It enhances the virulence of the bacteria. (virulence is the measure of pathogenicity) It act as an antigent.

Methods for demonstration of the capsule: electron microscopy special staining (negative method of staining, Burvy-Gience staining)

Pathogenic micro-organism representatives: Encapsulated bacteria (always with capsule layer):

Klebsiella pneumoniae (pathogen of pneumonia) Klebsiella ozaenae (pathogen of ozena ) Klebsiella rhinoscleromatis (pathogen of rhinoscleroma).

Capsule forming bacteria (form capsule only in human / animal body): Bacillus anthracis (pathogen of anthrax) Streptococcus pneumoniae ( pathogen of lobal pneumonia) Yersinia pestis ( pathogen of plague) Francisella tularensis (pathogen of tularemia) Staphylococcus aureus (pathogen of skin lesion, internal organ infection, mucous membrane infection, infection of the GIT). Streptococcus pyogenus ( pathogen of pyogenus infection of different localization).

Flagella
Flagella are filamentous, cytoplasmic appendages protruding through the cell wall. They are unbranched, long structures composed of the protein- flagellin. Flagella are the organs of locomotion and have characteristics patterns of distribution in the bacterial cells.

According to the localization of flagella, flagellated bacteria may be classified into 4 groups: monotrichous- single polar flagellum located at one end. ( the fastest microorganisms);

peritrichous- numerous flagella surrounding the body of the bacterium; lophotrichous- a tuft of flagella at one or both ends; amphitrichous- flagella located on both sides (at both polar ends of bacteria).

The structure of the Gram (+ve) and Gram (-ve) bacteria are different. The main substructures are namely flagellum/filament, hook and basal membrane. In Gram (+ve) bacteria, the basal membrane is localized in cytoplasmic membrane and the number of basal membrane is only one. In Gram (-ve) bacteria, the flagella has 2 basal membrane because its cell wall is thinner and has hook and filament. Method for flagella demonstration: direct methods: Lofflers staining, staining by silver impregnation technique (Morosovs stain), and electron microscopy. indirect method: method of motility detection (i) non-fixed smear preparation: crush drop technique and hanging drop technique. [Examination (x40)] (ii) study growth of microorganism in semi-solid medium. ( 2 tubes; bacteria inoculated by injection; then incubate; and we will see that in 1 tube, the bacteria grow everywhere and while in the other tube, the bacteria grow in the line of inoculation.)

Bacterial spores
Bacterial spores are highly resistant dormant (resting) forms of bacteria. Bacterial spores are formed under unfavourable environmental conditions. (e.g. during starvation,

dessication, change in temperature, evaporation of water, action of chemicals, action of penetrative radiation and action of UV light.) The spores are formed within the bacterial cell are named endospores. Endospores are small spherical or oval bodies. Only Bacillus and Clostridium are able to form bacterial spores. So, only rod shaped bacteria are sporing.

In Bacillus, the diameter of the spore is smaller than the diameter of the vegetative part. In Clostridium, the diameter of the spore is bigger than the diameter of the vegetative part. Bacteria form endospores and never form exospore (e.g. fungi) Thus, the function of spores is protection form unfavourable conditions, but NOT replication. N.B! Spores are not forms of replication. The duration of sporulation process is 18-20 hours. The sporulation process occurs in 4 stages: 1. preparatory stage, 2. forespore stage, 3. Stage of cell wall formation, 4. maturation stage. Spore may localize in different parts of the vegetative form: centrally subterminal between septal and end of the vegetative form. termial- at the end of the vegetative form.

Methods of bacterial spore demonstration:

Grams staining- vegetative form stain violet for Gram (+ve) bacteria and the spore is still colourless. Auesky staining (modification of acid-fast staining)

Spore forming bacteria: Bacillus anthracis ( pathogen of anthrax). Clostridium botulinum ( pathogen of botulism). Clostridium tetani (pathogen of tetanus). Clostridium perfringens (pathogen of gas gangrene infection) Clostridium novyi (pathogen of gas gangrene infection) Clostridium septicum (pathogen of gas gangrene infection) Clostridium histolyticum (pathogen of gas gangrene infection)

Fimbria/ Pilli
Fimbria are thin, short filamentous appendages extruding from the cytoplasmic membrane. They are typical structures of Gram (-ve) bacteria. The number of fimbria is more than the number of flagella. Fimbria are arranged peritrichously. Fimbria are composed of the protein- pillin There are 2 types of fimbria: common fimbria and sex fimbria. Common fimbria are responsible for attachment of the micro-organism to the substrate (host cells or tissue.) Each bacteria possesses 100-500 common fimbria. Sex fimbria take part in conjugation and is responsible for transmission of genetic information from 1 cell to another. Each bacteria possess around 1-4 sex fimbria. Methods for demonstration of fimbria: electron microscopy.

Intracellular inclusion
Intracellular inclusions are localized in the cytoplasm. They are the sources of stored energy and are only present in some species of bacteria. They are chemically composed of lipids, volutin, polysaccharides and element sulphur. These granules are found in large quantities when the bacteria are grown in abundance of nutrient, and the energy that it stores is used during starvation. The braight representative of bacteria with intracellular inclusions is Corynebacterium diphtheriae where it has volutin granules. Methods for demonstration of intracellular inclusions: electron microscopy;

Neissers method of staining.