Bacteriology Introduction

Assistant Prof. Dr. Karreema Amine AL-Khafajii Babylon University, College of Medicine, Department of Microbiology.

Cells in our World come in two basic types; prokaryotic(has no nucleous) and eukaryotic (has nucleous). and the main difference between them are;Differences between Prokaryotic cells and Eukaryotic cells

Prokaryotic Cells Eukaryotic Cells

 They are very minute in size.  They are comparatively larger in size.

  Nuclear region (nucleoid) is not enveloped by a nuclear membrane.

  Nucleus is surrounded by a double membrane layer.

  Single chromosome present.   More than one chromosome are present.

 Nucleolus is absent.  Nucleolus is present. Membrane bound organelles are absent.

 Membrane bound organelles are present.

 Multiplication of cell is by fission or budding.   Cell division by mitosis or meiosis.

 Cell Walls preset, which are chemically complex.

 Cell walls seen in only plant cells, which are chemically simpler. 

 Cell type is usually unicellular. Usually multicellular cells.  Cell size is 1-10μm  Cell size 10 - 100µm.

 Example: Bacteria, archaea  Example: animal cells and plant cells.

Prokaryotes Prokaryotic cells are the smallest, simplest and most abundant cells on Earth.

Most prokaryotes range in size from 1-10 micrometers (µm).

Prokaryotes include bacteria and archaea (ancient bacteria)

Prokaryotes lack a nucleus and complex organelles. Bacteria are classified and named based upon their genetic structure,

physical structure, and metabolism.

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Bacterial Shapes & Arrangements Prokaryotes (bacteria) are smaller (1-10 micrometers, um) than Eukaryotic cells and can be distinguished by the lack of a membrane-bound nucleus. Prokaryotic cells

have 3 major shapes: cocci, bacilli or spirilla. Bacterial cells can be arranged in different groups or patterns. These cells can be arranged singly, in pairs (diplo), groups of 4

(tetrads), chains (strepto), clusters (staphylo), packets of 8 or 16 (sarcinae), or hinged together (palisades).

Prokaryotic Cell Structure

In general speech , Bacteria cell structure can be divided into 3 parts:   external (appendages & coverings), cell envelope (cell wall and membranes), and internal organelles. The external structures of a bacterial cell include appendages for movement (flagella) and adhesion (pili, fimbriae, and glycocalyx) to surfaces. Bacterial cells contain:   circular chromosome(s), ribosomes (protein synthesis), and internal storage compartments for nutrients or survival. 1. External structures (appendages & coverings): flagella, fimbriae, sex pilus and glycocalyx

 2. Cell envelope: cell membrane, peptidoglycan cell wall or ad an outer lipid membrane (only found in Gram-negative cells)

 3. Internal Structures: Cytoplasm, nucleoid, bacterial chromosome, plasmid, ribosomes, and storage granules

 External Structures

The external structures of a bacterial cell include appendages for movement (flagella) and adhesion (pili, fimbriae, and glycocalyx) to surfaces.

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Fimbria and (pili): are interchangeable terms used to designate short, hair-like structures on the surfaces of bactetial cells. Like flagella, they are compose of protein (called pilin). Fimbriae are shorter and stiffer than flagella, and slightly smaller in diameter. There are two types of pili:

common pili(often called Fimbriae): are usually involved in specific adherence (attachment) of bacteria to surfaces in nature. In medical situation they are major determinants of bacterial virulence. Second type is called;

 A sex pilus is a long, rigid tube made of protein called, pilin.   Most bacteria reproduce asexually through a process called binary fission. However, some bacteria use the sex pilus or pili (plural) to "mate" with other bacterial cells.   During conjugation (sexual reproduction), bacteria use the sex pilus to transfer genetic material between the two cells (see photo below).

 

 

Flagella (flagellum-singular) are long, hair-like appendages attached to the cell surface , function like propellers to allow bacteria to swim and move. The diameter of a prokaryotic (bacteria), flagellum is about 20 nanometers, well-below the resolving power of the light microscope. The flagellar filament is rotated by a motor apparatus in the plasma membrane allowing the cell to swim in fluid environments. About half of the bacilli and all the spiral and curved bacteria are motile by means of flagella. Very few cocci are motile, which reflect their adaptation to dry environments and their lack of hydrodynamic design. The number and arrangement of flagella on a bacterial cell can vary. Flagella can have one of the following arrangements:  axial filament, where the flagellum is located INSIDE the bacterial cell giving the bacteria cell a "corkscrew" appearance (found in spirochetes such as Treponema *monotrichous, where there is 1 flagellum located at one 1 end of the cell .*lophotrichous, where there are small tufts of flagella located at 1 end of the cell . *amphitrichous, where there are flagella on both ends of the cell. *peritrichous, where there a several flagella dispersed around the surface of the cell. 

   

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Bacterial capsuleThe cell capsule is a very large structure of some prokaryotic cells, such as bacterial cells. It is a layer that lies outside the cell wall of bacteria. It is a well organized layer, not easily washed off, and it can be the cause of various diseases. When the amorphous viscid secretion (that makes up the capsule) diffuses into the surrounding medium and remains as a loose undemarcated secretion, it is known as slime layer. Composition ;It usually consists of polysaccharides,  but can be composed of other materials (e.g., polypeptide in B. anthracis). Because most capsules are water soluble , they are difficult to stain using standard stains because most stains do not adhere to the capsule. For examination under the microscope, the bacteria and their background are stained darker than the capsule, which doesn't stain. When viewed, bacterial cells as well as the surface they are on, are stained dark, while the capsule remains pale or colorless and appears as a ring around the cell. Function :(1)The capsule is considered a virulence factor because it mediates adherence of cells to the surface of the host. (2) it enhances the ability of bacteria to cause disease (e.g. prevents phagocytosis). The capsule can protect cells from engulfment by eukaryotic cells, such as macrophages. (3) Capsules also contain water which protects bacteria against desiccation.(4) They also exclude bacterial viruses and most hydrophobic toxic materials such as detergents. There are 14 different capsule types, which each impart their own specific antigenicity .Immunity to one capsule type does not result in immunity to the other types. . Diversity :The capsule is found most commonly among Gram-negative bacteria: However, some Gram-positive bacteria may also have a capsule Glycocalyx: the surface of bacteria is covered with a sticky coating called a glycocalyx.   The glycocalyx is composed of polysaccharides (sugars) and proteins.   The bacterial glycocalyx has 2 forms, a rigid capsule and a loose slime layer.   Capsules are found on many pathogenic (disease-causing) bacteria including, Streptococcus pneumoniae, which causes a respiratory infection of the lungs. The glycocalyx has several functions including:   protection, attachment to surfaces, and formation of biofilms.  

A biofilm: is a living layer of bacteria that is attached to a surface.   For example, biofilms are commonly found in showers, toilets, catheters, medical equipment, and even in your mouth!   Dental plaque is an example of a biofilm commonly found in humans. The glycocalyx helps protect the bacteria cell by preventing immune cells from attaching to it and destroying it through phagocytosis.  

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Biofilms can have serious medical implications, because once they form on surfaces, they are difficult to eliminate.   They can form on damaged tissues, teeth, and medical devices (catheters, artificial hip joints, IUDs).

Bacterial Cell Wall

The bacterial cell wall surrounds and protects the fragile cell or plasma membrane, much like a bicycle tire protects the inner tube.   The cell wall provides protection and structural support for the cell.   It also determines the shape of the bacterial cell. The bacterial cell wall contains peptidoglycan, which is made from long glycan (sugar) chains cross-linked (held together) by peptides, much like a chain-link fence.  There are 2 types of glycan chains that make up peptidoglycan:   N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM).

Gram-Positive, Gram-Negative and Acid-Fast Bacteria

Bacteria can be classified into 3 groups based on differences in the thickness or composition of the cell wall structure: Gram-positive, Gram-negative, and Acid-fast. The Gram stain is a technique used to distinguish between Gram-positive cells that have a thick layer of peptidoglycan and stain purple and Gram-negative cells which have a thin layer of peptidoglycyan and stain pink. The Gram-positive cell wall consists of a thick (20-80nm) layer of peptidoglycan. This thick layer is porous making the cell wall absorbent like a sponge.   The Gram-positive cell wall also contains teichoic acid, which functions in cell wall maintenance and gives the cell surface an acidic (-) charge.

Picture of the Gram positive cell wall

 

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The Gram-Negative Cell Wall consists of thin (1-3nm) layer of peptidoglycan.   Because the Gram-negative cell wall is so thin, these bacterial cells require an extra layer of protection, called the outer membrane.  The outer membrane consists of a phospholipid membrane, similar to the cell or plasma membrane.   This membrane has tiny holes or openings called porins. Porins block the entrance of harmful chemicals and antibiotics, making Gram-negative bacteria much more difficult to treat than Gram-positive cells.In many antibiotic resistant bacteria, these porins are connected to drug pumps, which pump out any drugs or harmful chemicals that enter through the porins. Attached to the outer membrane is is a highly-branched fatty sugar called lipopolysaccharide (LPS).   LPS acts as an endotoxin because it induces fever and shock in the human host. The importance of LPS: 1-LPS is the major component of the outer membrane of Gram-negative bacteria, contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack.2- LPS also increases the negative charge of the cell membrane and helps stabilize the overall membrane

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structure. 3- It is of crucial importance to gram-negative bacteria, whose death results if it is mutated or removed. LPS is an endotoxin, and induces a strong response from normal animal immune systems. It has also been implicated in non-pathogenic aspects of bacterial ecology, including 4-surface adhesion, 5- bacteriophage sensitivity, and 6- interactions with predators such as amoebae.

LPS acts as the prototypical endotoxin because it binds the CD14/TLR4/MD2 receptor complex, which promotes the secretion of pro-inflammatory cytokines in many cell types, but especially in macrophages and B cells. In Immunology, the term "LPS challenge" refers to the process of exposing a subject to an LPS that may act as a toxin.

LPS is also an exogenous pyrogen (external fever-inducing substance).

Being of crucial importance to gram-negative bacteria, these molecules make candidate targets for new antimicrobial agents.

It comprises three parts: 1-O antigen (or O polysaccharide)

2-Core oligosaccharide 3- Lipid AO-antigen A repetitive glycan polymer contained within an LPS is referred to as the O antigen, O polysaccharide, or O side-chain of the bacteria. The O antigen is attached to the core oligosaccharide, and comprises the outermost domain of the LPS molecule. The composition of the O chain varies from strain to strain. For example, there are over 160 different O antigen structures produced by different E. coli strains.  The presence or absence of O chains determines whether the LPS is considered rough or smooth. Full-length O-chains would render the LPS smooth, whereas the absence or reduction of O-chains would make the LPS rough. Bacteria with rough LPS usually have more penetrable cell membranes to hydrophobic antibiotics, since a rough LPS is more hydrophobic.

O antigen is exposed on the very outer surface of the bacterial cell, and, as a consequence, is a target for recognition by host antibodies. Core oligosaccharideThe Core domain always contains an oligosaccharide component that attaches directly to lipid A and commonly contains sugars such as heptose and 3-deoxy-D-mannooctulosonic Acid (also known as KDO, keto-deoxyoctulosonate). The LPS Cores of many bacteria also contain non-carbohydrate components, such as phosphate, amino acids, and ethanolamine substitutents.

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Lipid A: Lipid A is, in normal circumstances, a phosphorylated glucosamine disaccharide decorated with multiple fatty acids. These hydrophobic fatty acid chains anchor the LPS into the bacterial membrane, and the rest of the LPS projects from the cell surface. The lipid A domain is responsible for much of the toxicity of Gram-negative bacteria. When bacterial cells are lysed by the immune system, fragments of membrane containing lipid A are released into the circulation, causing fever, diarrhea, and possible fatal endotoxic shock (also called septic shock). The Lipid A moiety is a very conserved component of the LPS.

Gram negative bacteria cell wall , the LPS continents.

جدا the differentiation between Gram positive and gram negativebacterial cellمهمwall

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In a Gram stain test, bacteria are washed with a decolorizing solution after being dyed with crystal violet. On adding a counterstain such as safranin or fuchsine after washing, Gram-negative bacteria are stained red or pink while Gram-positive bacteria retain their crystal violet dye. This is due to the difference in the structure of their bacterial cell wall. Gram-positive bacteria do not have an outer cell membrane found in Gram-negative bacteria. The cell wall of Gram-positive bacteriais high in peptidoglycan which is responsible for retaining the crystal violet dye. 

Acid-Fast Bacteria

Acid-fast bacteria contain a waxy substance called mycolic acid and a small amount of peptidoglyan.   Due to their waxy cell wall, these bacteria are highly resistant to staining and treatment.   Mycobacterium tuberculosis , the causative agent of TB, is one example of a bacterial cell with an acid-fast cell wall.   These bacteria must be heated and treated with an acid-alcohol in order to stain them in

the lab (See image below).

Another type of bacteria with an unusual cell wall is Mycoplasma pneumoniae.   This bacteria attaches to the epithelial cells in the lungs, causing pneumonia.   The cell wall of this bacteria contains large amounts of sterols (rigid lipids), which make it difficult to treat. Due to the waxy content of their cell wall, these bacteria are pleiomorphic and vary in shape from long and filamentous to round.  

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Cell or Plasma Membrane

The cell or plasma membrane is a thin, fragile membrane located just beneath the bacterial cell wall.   The plasma membrane consists of a phospholipid bilayer that contains negatively-charged phosphate heads that are hydrophilic and hydrophobic lipid tails that are insoluble in water.  The plasma membrane also contains proteins that aid in the transport of materials into and out of the cell.

  The plasma membrane is selectively permeable, which allows it to regulate the flow of materials into and out of the cytoplasm. Materials that dissolve in lipids (fat soluble) pass between phospholipids.   Materials that cannot dissolve in lipids must pass through the transport proteins.

The plasma membrane is often targeted and disrupted by detergents and antibiotics.

Picture shows the plasma membrane

 

Internal Structures

Inside the flexible plasma membrane is the cytoplasm or "guts" of the cell.   The cytoplasm is a gelatin-like substance made of water, protein , carbohydrates and

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salt.   The other internal structures, such as the nucleoid, plasmid, ribosomes, storage granules, and endospores are suspended in the cytoplasm. 

 Bacterial Chromosome & Nucleoid Region

Like all prokaryotes, bacteria do not have a nucleus.   Instead, the bacterial chromosome is found floating within a dense region of the cytoplasm called the nucleoid region.   Unlike humans, bacteria only have 1 or at most a few chromosomes, which are tightly-coiled, circular pieces of DNA that contain all of the information required for survival. In addition to the bacterial chromosome, some bacteria also contain plasmids.   Plasmids are small, circular pieces of DNA that are not required for survival.   Plasmids are transferred between bacterial cells during conjugation via the sex pilus.   Plasmids carry genes (information) for antibiotic resistance.   They also allow bacteria to produce a sex pilus for sexual reproduction (conjugation).

 

 

Ribosomes

Although bacteria lack many of the complex organelles found in Eukaryotic cells, they do contain up to 10,000 ribosomes per cell. Ribosomes consist of 2 subunits (50S/30S) or parts that resemble a hamburger bun.   The large subunit (top of the bun) is made of ribosomal RNA (rRNA) and has the molecular size of 50 Svedberg units (50S).   The smaller subunit (bottom of the bun) has a size of 30S.   These subunits join together to form a protein factory where messenger RNA (mRNA) is translated into long chains of amino acids to form proteins. 

Storage Granules

Bacteria use granules to store minerals and nutrients (lipids, carbohydrates, phosphates, sulfur or metals) for the cell to use when needed.

Endospores

Some bacteria have the ability to produce endospores.   Endospores are an internal storage compartment

that consists of 3 layers of protection: peptidoglycan, calcium (dipicolinic acid) and keratin.   Endospores provide long-term storage and protection of the genetic material when moisture

and nutrients are not available.   Sporulation, or the

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formation of an endospore, occurs when nutrients and moisture are low.   This process takes about 6-8 hours and begins with duplication of the bacterial chromosome. During the process of sporulation, the duplicated chromosome is sealed inside 3 thick layers of protection.   Once moisture and nutrients return, the endospore quickly uncoats during a process called germination.

Endospores are highly resistant to conditions that would kill most bacteria.   They can withstand extremely high and low temperatures, pH, treatment with chemicals, and exposure to ultraviolet (UV) radiation.   Endospores can survive these extreme conditions for up to 250 million years!   This makes endospore-forming bacteria the very difficult to treat. Not all bacteria are able to form endospores.   Bacillus anthracis (anthrax), Clostridium botulinum (botulism) and Clostridium tetani (tetanus) are examples of endospore-forming.

Methods of Bacterial Identification

Bacteria are classified based upon: cell wall structure, aerobes vs. anaerobes, shape, genetic structure . -Macroscopic morphology (appearance  of  bacterial colonies on petri dish. -Microscopic morphology (bacterial shape & arrangement under the microscope).-Physiological / biochemical characteristics (metabolism: aerobes vs anaerobes).-Chemical analysis (cell wall composition). -Serological analysis (antibodies).-Genetic & molecular analysis (DNA & rRNA sequence).

Good luck

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