Bacterial Ultrastructure

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Bacterial ultrastructure is concerned with the cellular and molecular construction of bacteria. The bulk of research in bacterial ultrastructure investigates the ultrastructure of the cell wall that surrounds bacteria.

The study of bacterial ultrastructure began with the development of the staining regimen by Danish pathologist Christian Gram (1853–1938) that classifies the majority of bacteria as either Gram-negative or Gram-positive. The latter

Light micrograph of Klebsiella bacteria showing "halo" created by the capsule. bacteria retain the crystal violet stain, while Gram-negative bacteria do not retain this stain and are stained by the second stain that is applied, safranin. While the basis for this difference was not known at first, scientists suspected that the structure of the wall surrounding the contents of the bacteria might be involved.

Subsequent to the time of Gram, scientists have discovered that the cell wall plays only a secondary role in the Gram stain reactions. However, the cell wall of Gram-positive bacteria is indeed much different than that of Gram-negative bacteria. The study of bacterial ultrastructure relates these constituent differences to the intact cell wall. In other words, ultrastructure explores the structure of each constituent and the chemical and other associations that exist between these constituents.

The exploration of bacterial ultrastructure requires samples that are as undisturbed as possible from their natural, or so-called native, state. This has been challenging, since much of the

information that has been obtained has come from the use of electron microscopy. The techniques of conventional transmission electron microscopy and scanning electron microscopy require the removal of water from the sample. Because the bulk of living things, including bacteria, are comprised of water, the removal of this fluid can have drastic consequences on the structure of the bacteria. Much effort has gone into the development of regimens that chemically "fix" bacteria, so that structure is maintained following the subsequent removal of water.

Techniques have also been developed that prepare bacteria for transmission electron microscopy without the necessity of removing water from the specimen. One technique uses an embedding resin (a substance in which the bacteria are immersed and, when the resin is hardened, allows thin slices of the bacteria to be cut) that mixes with water. This resin is harder to work with than the conventional resins that are not water-soluble. Thus, while valuable information can be obtained using water-soluble resins, a great deal of experience is necessary to produce high quality results.

A second technique of sample preparation relies on the instantaneous freezing of the bacteria. Freezing is so fast that the interior water does not extensively crystallize (which would be extremely damaging to structure). Again, an experienced analyst can produce samples that information concerning the native ultrastructure of the bacteria.

In the past several decades, other tools are increasing the ultrastructure information that can be obtained. For example, the technique of atomic force microscopy can produce information on the atomic associations between adjacent molecules on the surface of bacteria. Atomic force microscopy has been very useful in ultrastructure studies of the regularly structured surface layers on bacteria.

Modern techniques of molecular genetics can also yield ultrastructure information. Mutants can be selected or designed in which a particular gene or genes has been rendered incapable of producing a protein product. If the gene is involved with cell wall constituents, the analysis of the wall can reveal the alterations that have occurred in the absence of the gene product. An example are the many mutants that are defective in the construction or assembly of lipopolysaccharide, a carbohydrate and lipid constituent of the outer membrane of Gram-negative bacteria. The loss of the carbohydrate portion of lipopolysaccharide makes the outer membrane more hydrophobic.

One approach that has been known for decades still yields useful information concerning bacterial ultrastructure. This is the substitution of the metals present in the cell wall with other metals. Metals act like glue to hold various wall components in association with one another. Examples of such metallic species include calcium and magnesium. Out-competing these species by supplying large concentrations of another metal, the influence of the normal metallic species can be assessed. For example, replacement of metals in the Gramnegative outer membrane can cause the release of lipopolysaccharide and the formation of bubbles along the surface of the membrane, where the underlying attachment to the rigid peptidoglycan layer is disrupted.

The use of specific antibodies to determine the molecular arrangement of ultrastructural constituent targets greatly enhances the effectiveness of agents to be used in drug therapy.

See also Atomic force microscope; Bacterial appendages; Bacterial surface layers; Caulobacter; Electron microscope, transmission and scanning; Electron microscopic examination of microorganisms; Sheathed bacteria

CLASSES OF DISINFECTANTS

AND THEIR USESby Kathy Johnson

There are literally hundreds of disinfectants out on the market now. So--which disinfectant do you pick? I will offer an overview of the different classes of disinfectants, their uses, drawbacks and strong points, and an idea of approximate costs for each class. If you are still in doubt after reading this article, do not hesitate to consult with a qualified avian vet for assistance in deciding which disinfectant, or combination, is best for your situation. It is not uncommon to need more than one kind of disinfectant to combat different types of pathogens in the same aviary. Hospitals, on the average, have over 14 different types of disinfectants in use at any given time; your home may not need 14, but just one may not be enough!

DEFINITIONS USED TO DESCRIBE CONTROL OF MICROORGANISMS

ANTISEPTIC--An agent that can be used as directed to reduce the microbial population found on skin. The maximum useable concentration of antiseptic is limited by risk of skin and mucous membrane irritation.

DISINFECTANT--An agent that will destroy many of the disease-causing microorganisms present on the surface of an inanimate object. A disinfectant claim is granted by the EPA to any solution which will destroy the following three microorganisms using an official AOAC (AOAC: Association of Official Analytical Chemists) procedure: Staph aureus, Pseudomonas aeruginosa, and Salmonella choleraesuis. A disinfectant label does not imply or include efficiency against viruses, mycobacterium, protozoa or heat-resistant bacterial spores.

GERMICIDE--(Also called Bactericidal) An agent that kills certain specified types of pathogenic microorganisms when used as directed. The labels of these agents make minimal claims, such as excluding specific bacteria by name. The limitations of these claims can only be found by closely reading the labels. A germicide does not automatically kill spores, viruses, tuberculosis or fungi.

SANITIZER--An agent that reduces microbial contamination on the surface of an object to an acceptable level. Sanitizers must not leave a harmful residue.

STERILANT--An agent that destroys all microbial organisms including heat-resistant bacterial spores. Sterilization can be achieved by boiling, autoclaving or exposure to toxic chemicals. Solutions that contain chlorine or glutaraldehyde are frequently labeled as chemical sterilants.

SPORICIDE--An agent that kills two specific types of vacuum-dried bacterial spores, according to specific AOAC test requirements.

TUBERCULOCIDAL--An agent that kills mycobacteria and especially M. tuberculosis according to procedures recently defined by the EPA (Environmental Protection Agency). A disinfectant or germicide are not automatically considered to be tuberculocidal.

VIRUCIDE--An agent that kills certain specified types of viruses when used as directed. An EPA approved label claim must state which viruses the agent has been proved effective against.

CLASSES OF DISINFECTANTS

FIRE AND FREEZING

Freezing temperatures will deactivate some infectious organisms, but many, including viruses will survive. The longer the freezing time, the lower the survival rate for most organisms, but it won't kill everything.

Flame is an excellent cleaner. Gas torches will kill any known living organism; remember, the solution to cleaning up after epidemics has been to burn down anything that was contaminated! But flame is obviously limited in its uses--it's hard to disinfect wood nest boxes and plastic objects with flame, and it will often discolor metal surfaces.

STEAM

Pressurized steam directed into cracks and corners is an excellent sterilant. It is, however, quite disruptive to birds, especially during breeding, and it can be costly due to equipment rental/purchase charges. It is best to thoroughly wash all equipment prior to steaming it.

SOAPS/DETERGENTS

Soaps and detergents do not disinfect. But they help remove surface organic debris so it does not interfere with the function of disinfectants. Always rinse soap or detergent off completely before disinfecting, and never mix with disinfectant unless the disinfectant instruction specifically state that it is safe. Avoid oral ingestion of these products, as they can cause intestinal upset, and can irritate mucous membranes.

ALCOHOLS

Alcohols are the base ingredient for many other disinfectants--for example, Lysol spray contains 79% ethyl alcohol and only 0.1% orthophenylphenol. When used as a surface spray or solution on inanimate objects, alcohol is an excellent pathogen destroyer. But it must be left in contact with the item to be disinfected for long periods to do its job--20 minutes contact time is considered proper for disinfection with ethyl alcohol. The higher the "proof" of an alcohol product, the better disinfectant it is, but the more volatile and evaporative it will be. Isopropyl alcohol is not considered to be a disinfectant--it's main use is as a skin wipe to remove loose organic debris from the site of a wound or injection. ADVANTAGESLow cost; effective against many pathogens with correct contact time

DISADVANTAGESLong contact time required for disinfecting action; only certain types of alcohol contain true disinfectant properties; may dissolve synthetic surfaces; fumes may be irritating and contain a fire hazard risk; not effective against some viruses; evaporates quickly, so items being disinfected must be physically soaked in alcohol to obtain disinfection

CHLORHEXIDINE GLUCONATESBRAND NAMES: Nolvasan, Virosan, Hibitane, Hibistat

Chlorhexidine products are often used as disinfectants for inanimate objects or antiseptics for cleaning skin wounds. Some chlorhexidine compounds contain alcohol, and these have been found to have superior antimicrobial properties to those containing only chlorhexidine. Chlorhexidine is effective against many bacteria, and yeast (especially Candida). It is not effective against most viruses, mycobacteria spores and Pseudomonas. Hexachlorophene has been suggested to be a potent carcinogenic. Some aviculturists use chlorhexidine as a water additive for control of pathogens--this is not recommended by the manufacturers, as these products were never meant for ingestion, and long-term effects have not been studied. ADVANTAGESRecommended as a water pan additive in incubators and brooders for control of aspergillus fungus; effective against Newcastle virus; not corrosive to equipment; readily available, medium costDISADVANTAGESPoor efficiency against most viruses and many gram-negative bacteria including Pseudomonas (Virosan is the exception--it is effective against Pseudomonas); must be discarded and re-mixed daily; not effective in the presence of organic debris; not effective against bacterial spores or mycobacterium

CHLORINE BRAND NAMES: Clorox, Purex

The best known member of this class is sodium hypochlorite (bleach). Bleaches are very harsh but effective. They attack pathogens, organic debris and living tissues equally well. Bleach can create toxic fumes which can lead to chemical pneumonia, skin and eye irritation or burns. It is recommended to wear protective clothing and eye gear when using bleach. ADVANTAGES: Bleach is inexpensive; easily available without a license; depending on the concentration at which it is mixed it can kill most bacteria, viruses, and mycoplasmas; it is a potent deodorizer, and works best in the presence of sunlight which releases more free radicals (which destroy cells, including pathogens). DISADVANTAGESIt is very caustic to tissues and equipment; very rapidly inactivated by organic debris (any dirt left on the object being disinfected will interfere with the action of the free radicals, up to the point where no chlorine is left to act on the actual pathogens); it loses its effectiveness quickly while still on the shelf in the bottle; not all brands of bleach, and not all production lots are the same concentration, so the standard dilution of 1/2 cup to a gallon of water (5:25% concentrate) may not always turn out to be the same strength; prolonged contact may be required for heavy sterilization, and the solution may require freshening every few hours. Bleach produces carcinogenic by-products, and must be used in a well-ventilated area; all objects treated with bleach must be well rinsed and allowed to dry before birds are

allowed to contact them.

STABILIZED CHLORINE DIOXIDEBRAND NAMES: Oxyfresh Dent-a-gene (full strength stabilized chlorine dioxide), Oxyfresh Cleansing Gele' (detergent with stabilized chlorine dioxide added)

Stabilized chlorine dioxide is a chlorine derivative which is a powerful oxidizing agent. It can destroy many pathogens, including bacteria, viruses, fungi and protozoa. Many studies have suggested that stabilized chlorine dioxide is a superior disinfecting agent to sodium hypochlorite (bleach). It is used in Europe to treat drinking water because it does not form carcinogenic by-products like sodium hypochlorite does. Stabilized chlorine dioxide has been shown by Dr. Branson Ritchie DVM, to inactivate avian polyoma virus at a level of dilution of 1:200. A detergent product containing stabilized chlorine dioxide is a good washing/soaking product for syringes, dishes and other hard surfaces, and can also be safely used on the skin of avian caretakers. Chlorine dioxide is an excellent deodorizer; the oxidizing properties destroy odor-causing molecules.ADVANTAGESSafe for use around birds and humans at recommended working dilutions; deactivates avian polyoma virus in 1 minute contact time; diluted solution creates no harmful fumes and is safe to use on skin or other surfaces; diluted solution at 1:200 is good for 7 days once mixed if kept sealed and out of direct sunlight; when first mixing up solution, the fumes created may be used to fumigate brooders. Medium cost--1 pint makes up to 16 gallons of diluted solution. DISADVANTAGESIn undiluted form, fumes of stabilized chlorine dioxide may be toxic to living tissue; rapidly deactivated by organic debris and exposure to sunlight.

GLUTARALDEHYDESBRAND NAMES: Wavecide, Cidex, Sporcide, Banacide, Sterol

This is a relatively new class of disinfectants which has come out within the past 25 years. The chemical action is to deactivate DNA and RNA proteins. They will deactivate most bacteria (including mycobacteria), viruses, and chlamydia. They are very stable and most work well even in the presence of organic debris. When mixed up in solution, they last a long time, making the cost per use fairly low. But they are very expensive to purchase initially compared to other disinfectants, and have many possible side effects, including tissue toxicity, irritation to the eyes, mucous membranes, respiratory tract and skin. Some glutaraldehyde formulas are corrosive to metals, others are not; read the label of a particular product to find the corrosive properties of that product. Never, ever mix glutaraldehydes with any other cleaning or disinfectant product. ADVANTAGESEqually effective in water of any temperature or hardness; effective against essentially any pathogen, even in presence of organic debris; solutions are good for longer periods than any other disinfectant available which lowers cost per use; speed of killing pathogens is very fast compared to many other disinfectants; available in many forms, including sprays, concentrates and bulk volumesDISADVANTAGESMay require a medical license to purchase from some suppliers; EPA testing did not include all animal and bird pathogens--assumptions were made regarding those, based on results of human pathogen

testing; may irritate respiratory system if not used in extremely well-ventilated areas; may cause eye, skin or mucous membrane irritation or damage with some brands; must be well-rinsed before allowing birds contact with cleaned surfaces; may cause skin irritation, yellowing or peeling; concentrated forms not available in all states; some forms/brands of product may be corrosive/caustic than others--it is necessary to read all labels carefully before using these products.

IODINESBRAND NAMES: Vanodine, Betadyne, Povidone, Scrubodyne

Iodine solutions are frequently used as antiseptics for cleaning wounds and skin. Most iodine-containing disinfectants also contain a detergent, and are called "iodophors". Medium cost. ADVANTAGESLimited vapor production; not usually affected by hard water; long shelf life; works well in hot or cold water; are effective against many bacteria, some fungi and viruses. DISADVANTAGESMost require full-strength use which increases cost per use; may stain surfaces and tissues brown; toxic if ingested (may cause iodine overdose); may dry and crack skin; corrosive to metal surfaces with prolonged exposure; easily deactivated by contact with organic debris; is NOT effective against hydrophylic viruses such as polyoma and PFBD (Psittacine Beak and Feather Disease); not effective against all strains of Pseudomonas bacteria.

PHENOLSBRAND NAMES: Lysol, One Stroke Environ, O-Syl

Phenols are produced by coal distillation. Sodium orthophenol is the active ingredient in most phenol disinfectants. Phenols are effective against many bacteria, including Pseudomonas and mycobacteria, fungi and some viruses. They may not work well in the presence of organic material. Some phenols are inexpensive, and are easily available at the grocery store. ADVANTAGESKills many pathogens, including bacteria such as Salmonella and Pseudomonas, mycobacteria, fungi and lipophilic viruses; effective even in hard water; doesn't stain surfaces or leave residual odors, low cost; easy to rinse off objectsDISADVANTAGESToxic to many tissues including skin, eyes, and respiratory tract; VERY toxic to cats and reptiles; may not work well if organic debris is present; not effective against bacterial spores or hydrophilic viruses; must be used with adequate ventilation; must be rinsed off cleaned surfaces before allowing birds contact with them

QUATERNARY AMMONIUM COMPOUNDSBRAND NAMES: Roccal-D, Quintacide, Parvosol, Hitor, Omega, Barquat, Merquat, Cetylcide

"Quats" are a large class of disinfectants which add organic compounds to ammonia. Many quats also function as a detergent, and help remove organic debris from objects. The presence of organic debris, however, may deactivate the disinfectant in the quat compound. They are not recommended for use on objects that will be in direct contact with birds because they are very difficult to rinse off completely,

and residue can cause respiratory paralysis and death! May be diluted for lower cost per use, but initial purchase cost may be expensive. Quats are effective against many types of bacteria, some viruses, and chlamydia; they are not effective against spores, mycobacteria or fungi, Pseudomonas, and hydrophylic viruses such as Polyoma or PFBD. ADVANTAGESMay be used at very dilute solutions, allowing for lower cost per use; contains detergent for action against organic debris; pleasant scent in most forms; good disinfectant against many bacteria, a few viruses, and chlamydiaDISADVANTAGESNot effective against bacterial spores, Pseudomonas, fungi or mycobacteria, hydrophylic viruses; high levels of organic debris may inactivate the product; hard water may inactivate the quat product; may leave slimy residue on objects which won't rinse off; ingestion and inhalation of products or residue may cause respiratory paralysis and even death.

WOOD TAR DISTILLATESBRAND NAMES: Pine-Sol, Hexol

Wood tar distillates are a by-product of the lumber industry. They include such products as creosotes, turpentine and pine oils. Pine oils are the only member of this group with any disinfectant applications, and only when mixed with soap. They are very safe, but have very low levels of effectiveness against any pathogens. Very inexpensive, and available at many department, hardware and grocery stores. ADVANTAGESEasily available; low cost; pleasant fragrance; low toxicity; detergent ingredients make them good cleaning products for removing organic debrisDISADVANTAGESVery poor effectiveness against any pathogens; hard to rinse off surfaces, may leave floors slick.

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Bacterial Morphology

Bacterial Structure

:: Cytoplasmic Structures ::

      Gram positive and Gram negative bacteria have similar internal, but very different external structures. The cytoplasm of the bacterial cell contains the DNA chromosome, the mRNA, ribosomes, proteins, and metabolites). Unlike eukaryotes, the bacterial chromosome is a single, double-stranded circle that is contained not in a nucleus but in

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a discrete area known as the nucleoid. Histones are not required to maintain the conformation of the DNA, and the DNA does not form nucleosomes. Plasmids, which are smaller, circular, extrachromosomal DNAs, may also be present. Plasmids are most commonly found in Gram negative bacteria, and although not usually essential for cellular survival, they often provide a selective advantage: many confer resistance to one or more antibiotics.

Gram positive and Gram negative bacteria. A Gram positive bacterium has a thick layer of peptidoglycan (left). A Gram negative bacterium has a thin peptidoglycan layer and an outer membrane (right). Structures in () are not found in all bacteria.

      The lack of a nuclear membrane simplifies the requirements and control mechanisms for the synthesis of proteins. Without a nuclear membrane, transcription and translation are coupled; in other words, ribosomes can bind to the mRNA, and protein can be made as the mRNA is being synthesized and still attached to the DNA.       The bacterial ribosome consists of 30S + 50S subunits, forming a 70S ribosome. This is unlike the eukaryotic 80S (40S + 60S) ribosome. The proteins and RNA of the bacterial ribosome are significantly different from those of eukaryotic ribosomes and are major targets for antibacterial drugs.       The cytoplasmic membrane has a lipid bilayer structure similar to the structure of the eukaryotic membranes, but it contains no steroids (e.g., cholesterol); mycoplasmas are the exception to this rule. The cytoplasmic membrane is responsible for many of the functions attributable to organelles in eukaryotes. These tasks include electron transport and energy production, which are normally achieved in the mitochondria. In addition, the membrane contains transport proteins that allow the uptake of metabolites and the release of other substances, ion pumps to maintain a membrane potential, and enzymes. A coiled cytoplasmic membrane, the mesosome, acts as an anchor to bind and pull apart daughter chromosomes during cell division.

:: Cell Wall ::

      The structure, components, and functions of the cell wall distinguish Gram positive from Gram negative bacteria. The important differences in membrane characteristics are outlined in. The cytoplasmic membranes of most prokaryotes are surrounded by rigid peptidoglycan (murein) layers. The exceptions are Archaeobacteria organisms (which contain pseudoglycans or pseudomureins related to peptidoglycan) and

mycoplasmas (which have no cell walls at all). Because the peptidoglycan provides rigidity, it also determines the shape of the particular bacterial cell. Gram negative bacteria are also surrounded by outer membranes.

Bacterial Membrane Structures

Structure Chemical ConstituentsPlasma Membrane Phospholipids, proteins, enzymes for energy, membrane potential, transport.

Gram +ve BacteriaPeptidoglycan Glycan chains of GlcNAc and MurNAc cross linked by peptide bridge.Teichoic Acid Polyribitol phosphate or glycerol phosphate cross linked to peptidoglycan.Lipoteichoic Acid Lipid linked teichoic acid.

Gram -ve BacteriaPeptidoglycan Thinner version of that found in Gram positive bacteria.Periplasmic Space Enzymes involved in transport, degradation, and synthesis.Outer Membrane Phospholipids with saturated fatty acids.

Proteins Porins, lipoprotein, transport proteins.Lipid A, core polysaccharide, O antigen.

Other StructuresPolysaccharides (disaccharides and trisaccharides) and polypeptides.Pilin, adhesins.

Flagellum Motor proteins, flagellin.M proteins of streptococci (for example).

GlcNac=N-Acetylglucosamine; MurNAc=N-acetylmuramic acid; LPS=lipopolysaccharide.

Functions Of The Bacterial Envelope

Function Component(s)Structural Rigidity All.Packaging Of Internal Contents All.Permeability Barrier Outer membrane or plasma membrane.Metabolic Uptake Membranes and periplasmic transport proteins, porins, permeases.Energy Production Plasma membrane.Adhesion To Host Cells Pili, proteins, teichoic acid.Immune Recognition By Host All outer structures.Escape From Host Recognition Capsule, M protein.Antibiotic Sensitivity Peptidoglycan synthetic enzymes.Antibiotic Resistance Outer membrane.

Flagella.Pili.Pili.

:: Gram Positive Bacteria ::

      A Gram positive bacterium has a thick, multilayered cell wall consisting mainly of peptidoglycan (150 to 500 A) surrounding the cytoplasmic membrane. The peptidoglycan is a meshlike exoskeleton similar in function to the exoskeleton of an

insect. Unlike the exoskeleton of the insect, however, the peptidoglycan of the cell is sufficiently porous to allow diffusion of metabolites to the plasma membrane. The peptidoglycan is essential for the structure, for replication, and for survival in the normally hostile conditions in which bacteria grow. During infection, the peptidoglycan can interfere with phagocytosis, is mitogenic (stimulates mitosis of lymphocytes), and has pyrogenic activity (induces fever).       The peptidoglycan can be degraded by treatment with lysozyme. Lysozyme, an enzyme in human tears and mucus, is also produced by bacteria and other organisms. Lysozyme degrades the glycan backbone of the peptidoglycan. Without the peptidoglycan, the bacteria succumb to the large osmotic pressure differences across the cytoplasmic membrane and lyse. Removal of the cell wall produces a protoplast that lyses unless it is osmotically stabilized.       The Gram positive cell wall may also include other components such as teichoic and lipoteichoic acids and complex polysaccharides (usually called C polysaccharides). Proteins such as the M protein of streptococci and R protein of staphylococci also associate with the peptidoglycan. Teichoic acids are water-soluble polymers of polyol phosphates, which are covalently linked to the peptidoglycan. Lipoteichoic acids have a fatty acid and are anchored in the cytoplasmic membrane. These molecules are common surface antigens that distinguish bacterial serotypes and promote attachment to other bacteria as well as to specific receptors on mammalian cell surfaces (adherence). Teichoic acids are important factors in virulence. Lipoteichoic acids are shed into the media and host and, although weaker, can initiate endotoxic-like activities.

:: Gram Negative Bacteria ::

      Gram negative cell walls are more complex than Gram positive cell walls, both structurally and chemically. Structurally, a Gram negative cell wall contains two layers external to the cytoplasmic membrane. Immediately external to the cytoplasmic membrane is a thin peptidoglycan layer, which accounts for only 5% to 10% of the Gram negative cell wall by weight. There are no teichoic or lipoteichoic acids in the Gram negative cell wall. External to the peptidoglycan layer is the outer membrane, which is unique to Gram negative bacteria. The area between the external surface of the cytoplasmic membrane and the internal surface of the outer membrane is referred to as the periplasmic space. This space is actually a compartment containing a variety of hydrolytic enzymes, which are important to the cell for the breakdown of large macromolecules for metabolism. These enzymes typically include proteases, phosphatases, lipases, nucleases, and carbohydrate-degrading enzymes. In the case of pathogenic Gram negative species, many of the lytic virulence factors such as collagenases, hyaluronidases, proteases, and beta-lactamase are in the periplasmic space. This space also contains components of the sugar transport systems and other binding proteins to facilitate the uptake of different metabolites and other compounds. Some binding proteins can be components of a chemotaxis system, which senses the external environment of the cell.

Comparison of the Gram positive and Gram negative bacterial cell walls. A, a Gram positive bacterium has a thick peptidoglycan layer that contains teichoic and lipoteichoic acids. B, a Gram negative bacterium has a thin peptidoglycan layer and an outer membrane that contains lipopolysaccharide, phospholipids, and proteins. The periplasmic space between the cytoplasmic and outer membranes contains transport, degradative, and cell wasll synthetic proteins. The outer membrane is joined to the cytoplasmic membrane at adhesion points and is attached to the peptidoglycan by lipoprotein links.

      As mentioned previously, outer membranes are unique to Gram negative prokaryotes. The outer membrane is like a stiff canvas sack around the bacteria. The

outer membrane maintains the bacterial structure and is a permeability barrier to large molecules (e.g., proteins such as Lysozyme) and hydrophobic molecules. It also provides protection from adverse environmental conditions such as the digestive

system of the host (important for Enterobacteriaceae organisms). The outer membrane has an asymmetric bilayer structure that differs from any other biologic membrane in

the structure of the outer leaflet of the membrane. The inner leaflet contains

phospholipids normally found in bacterial membranes. However, the outer leaflet is composed primarily of an amphipathic molecule (meaning that it has both

hydrophobic and hydrophilic ends) called lipopolysaecharide (LPS). Except for those LPS molecules in the process of synthesis, the outer leaflet of the outer membrane is

the only location where LPS molecules are found.       LPS is also called endotoxin, a powerful stimulator of immune responses. LPS

activates B cells and induces macrophage and other cells to release interleukin-I and interleukin-6, tumor necrosis factor, and other factors. LPS causes fever and can cause shock. The Shwartzman reaction (disseminated intravascular coagulation) follows the

release of large amounts of endotoxin into the blood stream. LPS is shed from the bacteria into the media and host. Neisseria meningitidis sheds large amounts of a related compound, lipooligosaccharide (LOS), resulting in fever and symptoms.

      The variety of proteins found in Gram negative outer membranes is limited, but several of the proteins are present in high concentration, resulting in a total protein

content higher than that of the cytoplasmic membrane. Many of the proteins traverse the entire lipid bilayer and are thus transmembrane proteins. A group of these proteins

is known as porins because they form pores that allow the diffusion of hydrophilic molecules less than 700 Da in mass through the membrane. The outer membrane and the porin channel allow passage of metabolites and small hydrophilic antibiotics, but the outer membrane is a barrier for large or hydrophobic antibiotics and proteins such

as 1ysozyme.       The outer membrane also contains structural proteins and receptor molecules for

bacteriophages and other ligands. The outer membrane is connected to the cytoplasmic membrane at adhesion sites and is tied to the peptidoglycan by lipoprotein. The

lipoprotein is covalently attached to the peptidoglycan and is anchored in the outer membrane. The adhesion sites provide a membranous route for the delivery of newly

synthesized outer membrane components to the outer membrane.       The outer membrane is held together by divalent cation (Mg+2 and Ca+2) linkages between phosphates on LPS molecules and hydrophobic interactions betwecn the LPS and proteins. These interactions produce a stiff, strong membrane that can be disrupted by antibiotics (e.g., polymyxin) or by the removal of Mg and Ca ions (chelation with ethylenediaminetetraacetic acid [FDTA]). Disruption of the outer membrane weakens the bacteria and allows the permeability of large, hydrophobic molecules. The addition

of lysozyme to cells treated in this manner produces spheroplasts, which, like protoplasts, are osmotically sensitive.

:: External Structures ::

      Some bacteria (Gram positive or Gram negative) are closely surrounded by loose polysaccharide or protein layers called capsules. In cases in which it is loosely

adherent and nonuniform in density or thickness, the material is referred to as a slime layer. The capsule and slime layers are also called the glycocalyx. Bacillus anthracis,

the exception to this rule, produces a polypeptide capsule. The capsule is hard to see in a microscope but can be visualized by the exclusion of India ink particles.

Transmission electrom micrographs of Porphyromonas (formerly Bacteroides) gingivalis and Pseudomonas aeruginosa revealing the surface associated capsule. Both strains were isolated from human patients, P. gingivalis from an adult with peridontitis and P. aeruginosa from a patieng with cystic fibrosis. C = capsule; OM = outer membrane; PG = peptidoglycan; CM = cytoplasmic membrane; R = ribosome; PP = polyphosphate. Bar = 0.1 um.

      Capsules and slimes are unnecessary for the growth of bacteria but are very important for survival in the host. The capsule is poorly antigenic and antiphagocytic

and is a major virulence factor (e.g., Streptococcus pneumoniae). The capsule can also act as a barrier to toxic hydrophobic molecules, such as detergents, and can promote adherence to other bacteria or to host tissue surfaces. For Streptococcus mutans, the

dextran and levan capsules are the means by which the bacteria attach and stick to the tooth enamel. Synthesis of the capsule takes energy and will not be effected by the

bacteria after continued growth under laboratory conditions away from the selective pressures of the host.

      Flagella are ropelike propellers composed of helically coiled protein subunits (flagellin) that are anchored in the bacterial membranes through hook and basal body

structures and that are driven by membrane potential. Bacterial species may have one or several flagella on their surfaces, and they may be anchored at different parts of the

cell. Flagella provide motility for bacteria, allowing the cell to swim (chemotaxis) toward food and away from poisons. Bacteria approach food by swimming straight and then tumbling in a new direction. The swimming period becomes longer as the

concentration of chemoattractant increases. The direction of flagellar spinning determines whether the bacteria swim or tumble. Flagella also express antigenic and

strain determinants.       Fimbriae (pill) (Latin for "fringe") are hairlike structures on the outside of bacteria;

they are composed of protein subunits (pilin). Fimbriae can be morphologically distinguished from flagella because they are smaller in diameter (3 to 8 nm versus 15 to 20 nm) and usually are not coiled in structure. Generally, several hundred fimbriae

are arranged peritrichously (uniformly) over the entire surface of the bacterial cell. They may be as long as 15 to 20 nm, or many times the length of the cell.

      Fimbriae promote adherence to other bacteria or to the host (alternative names are adhesins, lectins, evasins, and aggressins). As an adherence factor (adhesin), fimbriae are an important virulence factor for E. coli colonization and infection of the urinary

tract, for Neisseria gonorrhoeae and other bacteria. The tips of the fimbriae may contain proteins (lectins) that bind to specific sugars (e.g., mannose). F pili (sex pill) promote the transfer of large segments of bacterial chromosomes between bacteria.

These pill are encoded by a plasmid (F).

:: Bacterial Exceptions ::

      Mycobacteria have a peptidoglycan layer (slightly different structure), which is intertwined with and covalently attached to an arabinogalactan polymer and

surrounded by a waxlike lipid coat of mycolic acid (large alpha-branched beta-hydroxy fatty acids), cord factor (glycolipid of trehalose and two mycolic acids),

waxD (glycolipid of 15 to 20 mycolic acids and sugar), and sulfolipids. These bacteria are described as acidfast staining. The coat is responsible for virulence and is anti phagocytic. Corynebacterium and Nocardia organisms also produce mycolic acid

lipids. The mycoplasmas are also exceptions in that they have no peptidoglycan cell wall and they incorporate steroids from the host into their membranes.

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Bacteriology – Science that deals with Bacteria.Bacteriology - A unicellular organisms without a true nucleus.Mycoplasmas are bacteria that have no cell wall and therefore have no

definite shape.

Internal StructureBacteria have a very simple internal structure, and no membrane-bound

organelles.

Nucleoid: DNA in the bacterial cell is generally confined to this central region. Though it isn’t bounded by a membrane, it is visibly distinct (by transmission microscopy) from the rest of the cell interior.

DNA- Deoxyribo Nucleic Acid is single long circular molecule. It contains all genetic information for the structure & function of a bacterium under its optimum conditions.

RibosomeRibosome gives the cytoplasm of bacteria a granular appearance in electron

micrographs. It is important in translating the genetic massage in messenger RNA ( Ribo Nucleic Acid) into the production of peptide sequences (proteins). It is responsible for protein synthesis.

Storage GranulesNutrients and reserves may be stored in the cytoplasm in the form of

glycogen, lipids, polyphosphate, or in some cases, sulfur or nitrogen.

EndosporeSome bacteria, like clostridium botulinum, form spores that are highly

resistant to drought, high temperature and other environmental hazards. Once the hazard is removed, the spore germinates to create a new population.

CapsuleThis layer of polysaccharide (sometimes proteins) protects the bacterial cell

and is often associated with pathogenic vacteria because it serves as a barrier against phagocytosis by white blood cells.

Outer membraneThis lipid bilayer is found in Gram negative bacteria and is the source of lipo-

polysaccharides (LPS) in these bacteria. LPS is toxic and turns on the immune system of, but not in Gram positive bacteria.

Cell wallThis is the complex & semi-rigid outermost layer. It gives shape to the

bacterial cell & protects it from external environment. It is composed of peptidoglycan (polysaccharides + protein); the cell wall maintains the overall shape of a bacterial cell.

Periplasmic Space

This cellular compartment is found only in those bacteria that have both an outer membrane and plasma membrane

(e.g. Gram negative bacteria). In the space are enzymes and other proteins that help digest and move nutrients into the cell.

Plasma membrane

This is a lipid bilayer much like the cytoplasmic (plasma) membrane of other cells. There are numerous proteins moving within or upon this layer that are primarily responsible for transport of ions, nutrients and waste across the membrane.

Appendages:Bacteria may gave the following appendages ( a tail or a limb attached to a

major part):

PiliPili: These are hollow, hair like structures made of protein allow bacteria to

attach to other cells. A specialized pilus, the sex pilus, allows the transfer from one bacterial cell to another. pili (sing; pilus) are also called fimbriae (sing; fimbria).

FlagellaThe purpose of flagella (sing; flagellum) is motility. flagella are long

appendages which rotate by means of a “motor” located just under the cytoplasmic membrane. Bacteria may have one, a few or many flagella in different positions on the cell.

Classification of BacteriaBacteria can be classified in many ways but three are important –

1. Classification on the basis of morphology (Shape & Size):

a) Coccus (Spherical) – round shaped. e.g. Streptococcus, Pneumococcus.

   b) Bacillus (Cylindrical) – Rod-Shaped. e.g. E. coli.

c) SPirillum (Spirocheate) – Spiral-shaped. e.g Treponema pallidum.

d) Vibrio – Comma-shaped. e.g. Vibrio cholerae

e) Filamentous – threadlike. e.g. Actinomyces israelli.

2. Classification on the basis of Gram staining:Taxonomists divide bacteria into various subgroups including –

a. Gram-positive bacteria     &      b. gram-negative bacteria

Most species of bacteria can be grouped into two categories based on their response to a laboratory technique called Gram Staining. (These are terms for the way bacteria respond to a procedure called Gram-staining).

Gram-staining procedure:Dr. Hans Christian Gram, a Danish Microbiologist, developed the Gram-stain

technique in 1884. The technique involves stainig bacteria with a Purple Dye (Crystal Violet), & Iodine, & Rinesd with Alcohol. Then Re-stained with a Pink Dye (Safranin). Depending on Structure of their Cell Walls, the Bacteria absorb either the Purple. Gram-Negative Bacteria will appear PINK from the pink dye.

Gram-positive bacteriaHave a thicker layer of Peptidoglycan in their cell walls, made of a Protein-

Sugar complex that takes on the Purple color during Gram Staining.

Gram-negative bacteriaHave an extra layer of lipid on the out side of the cell wall & appear Pink color

after Gram Staining.

Examples of Gram-Positive & Gram-Negative bacteria:Organisms Types of & or site of

infectionA. Gram-Positive

BacteriaStaphylococcus aureus Pneumonia, Boils, post-

operative bone/joints.Streptococcus

pneumoniaePneumonia, meningitis,

otitis media, sinusitis, septicemia

Streptococcus pyogenes

pharyngitis, impetigo

Streptococcus viridans EndocarditisB. Gram-Negative

BacteriaEscherichia coli (E. coli) Urinary tract,

pyelonephritis, peritonitis

Haemophilus influenzae Pneumonia, meningitis, otitis media.

Klebsiella pneumoniae Pneumonia, woundsNeisseria gonorrhoeae GonorrheaSalmonella typhi Typhoid feverVibrio cholerae Cholera

3. Classification on the basis of Oxygen requirement:a, Aerobes – can not survive without atmospheric oxygen.b, Anaerobes – can survive without atmospheric oxygen.c, Facultative – can survive with or without atmospheric oxygen.