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LECTURE NOTES For B. Tech (Dairy Technology) Degree

GENERAL MICROBIOLOGY (Course No: DM 111)

By Dr. Velugoti Padmanabha Reddy, Ph.D Professor and University Head Dept of Dairy Microbiology College of Dairy Technology Tirupati-517 502

COLLEGE OF DAIRY TECHNOLGY SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI- 517 502 2006

INTRODUCTION: ORIGIN OF THE LIFE: Evolutionary principles are the foundation upon which all biology is built; and it is essential to learn about this crucial principle. To grasp the nature of microbiology, an understanding of evolutionary principles is paramount. Evolution means change. The science of evolution is the study of the processes of evolutionary changes. Evolution is a description of a process of how living organisms change in response to environmental factors Evolutionary scientists try to document the changes in species through time

Charles Darwin

Evolutionary sciences, which include paleontology, (the study of the remains of fossils), genetics, microbiology, ecology and biochemistry, attempt to relate changes in life-forms to the development of new species over time. Other evolutionists investigate processes like mutagenesis, gene shuffling, gene jumping, gene duplication etc. so as to define the mechanisms and forces that bring about changes in living forms. Still others investigate the environment, both past and present, to determine how environmental conditions determined, which mutations helped a life form survive while other didn't. Scientific estimates place the origin of the Universe at between 10 and 20 billion years ago. The theory currently with the most acceptances is the Big Bang Theory, the idea that all matter in the Universe existed in a cosmic egg (smaller than the size of a modern hydrogen atom) that exploded, forming the Universe. Brief history of life on earth: 4.5 billion years ago 3.5 billion years ago 1.5 billion years ago 0.5 billion years ago : Formation earth : First life-prokaryotic bacteria dominate : Nucleated cells arise--eukaryotic : Cambrian explosion--multicellular eukaryotes arise

Extra-terrestrial: In 1969, a meteorite (left-over bits from the origin of the solar system) landed near Allende, Mexico. The Allende Meteorite (and others of its sort) have been analyzed and found to contain amino acids, the building blocks of proteins, one of the four organic molecule groups basic to all life. The amino acids recovered from meteorites are in a group known as exotics: they do not occur in the chemical systems of living things. The ET theory is now not considered by most scientists to be correct, although the August 1996 discovery of the Martian meteorite and its possible fossils have revived thought of life elsewhere in the Solar System. Organic Chemical Evolution: Until the mid-1800's scientists thought organic chemicals (those with a C-C skeleton) could only form by the actions of living things. A French scientist heated crystals of a mineral (a mineral is by definition inorganic), and discovered that they formed urea (an organic chemical) when they cooled. Russian scientist and academician A.I. Oparin, in 1922, hypothesized that cellular life was preceded by a period of chemical evolution. These chemicals must have arisen spontaneously under conditions existing billions of years ago (and quite unlike current conditions). In 1950, then-graduate student Stanley Miller designed an experimental test for Oparin's hypothesis. Oparin's original hypothesis called for: 1) little or no free oxygen (oxygen not bonded to other elements); and 2) C H O and N in abundance. Studies of modern volcanic eruptions support inference of the existence of such an atmosphere. Miller discharged an electric spark into a mixture thought to resemble the primordial composition of the atmosphere. From the water receptacle, designed to model an ancient ocean, Miller recovered amino acids. Subsequent modifications of the atmosphere have produced representatives or precursors of all four organic macromolecular classes. Miller's (and subsequent) experiments have not proven life originated in this way, only that conditions thought to have existed over 3 billion years ago

were such that the spontaneous (inorganic) formation of organic macromolecules could have taken place. The simple inorganic molecules that Miller placed into his apparatus produced a variety of complex molecules. The interactions of these molecules would have increased as their concentrations increased. Reactions would have led to the building of larger, more complex molecules. A pre-cellular life would have begun with the formation of nucleic acids. Chemicals made by these nucleic acids would have remained in proximity to the nucleic acids. Eventually the pre-cells would have been enclosed in a lipid-protein membrane, which would have resulted in the first cells. Biochemically, living systems are separated from other chemical systems by three things. The capacity for replication from one generation to another-- Most organisms today use DNA as the hereditary material, although recent evidence (ribozymes) suggests that RNA may have been the first nucleic acid system to have formed. The presence of enzymes and other complex molecules essential to the processes needed by living systems. A membrane that separates the internal chemicals from the external chemical environment. This also delimits the cell from not-cell areas. The cell is the basic structural unit of life, regardless of the complexity of an organism. The word cell was first used by an Englishman, Robert Hooke (1635-1703) in his descriptions (1665) of the fine structure of cork and other plant materials. He observed the honeycomb like structure in a thin slice of cork which was due to the cell walls of cells that were once living. The Cell Theory is one of the foundations of modern biology. Its major tenets are: 1) 2) 3) 4) All living things are composed of one or more cells; The chemical reactions of living cells take place within cells; All cells originate from pre-existing cells; and Cells contain hereditary information, which is passed from one generation to another.

The concept of the cell as the structural unit of life ­the so called cell theory is credited to two Germans Mathias Schleiden & Theodor Schwann (1838-39) who described cells as the basic structural and functional units of all organisms. Characteristics common to all biological systems are: 1. The ability to reproduce 2. The ability to ingest or assimilate food substances and metabolize them for energy and growth. 3. The ability to excrete waste products 4. The ability to react to changes in the environment (some times called irritability) 5. Susceptibility to mutation.

HISTORICAL OVERVIEW OF MICROBIOLOGY Early microbiology :

Ancient cultures and civilizations had no idea that microbes existed but they did comprehend some of their important effects. For example: Ancient Egyptians were among the earliest peoples to use fermentation to brew their own beer. The Romans liked to have good sanitation and prized clean drinking water. Ancient Chinese immunized people against smallpox by having them inhale dried, powdered scabs from those suffering from a mild form of the disease. Many traditional cultures have also recognized and used plants as remedies for certain diseases. For example, South Americans recognized the usefulness of extracts Cinchona tree (containing quinine) to treat malaria. Many cultures recognized the communicability of certain diseases. History can be divided into 4 periods: Pre historic period (until 1850) Golden era (1850- 1900) Modern era (1900- 1960) Molecular era (1960-till date) : Period of speculation in nature : Period of fundamental discoveries : Chemotherapy, Antiseptics, Electronic microscope. : Research at molecular level

THEORIES OF SPONTANEOUS GENERATION: Abiogenesis means starting of life from dead matter and Biogenesis means starting of life from the living matter. Abiogenesis: This long held theory advocated that life could arise spontaneously from nonliving or decomposing matter. For example, many believed that flies could develop directly from rotting meat. Aristotle (248 to 322BC) Life is being originated from non-living things spontaneously from the soil, plants, or other unlike animals. In a simple but elegant series of experiments, Francesco Redi (1626-1697) was among the first to question abiogenesis. He placed meat in a jar covered with gauze. Redi provided nonmicroscopic evidence against abiogenesis by showing flies developed from maggots on meat in uncovered jars but not from meat in covered jars. Attracted by the odour of the meat, flies laid eggs on the covering, and from the eggs maggots developed on the gauze. In the mid 1700's, John Needham (1713 to 1781BC), an English clergyman and proponent of abiogenesis, claimed that life arose spontaneously because of a random or chance clumping of "organic" molecules. He observed the appearance of organisms not present at the start of the experiment and concluded that the bacteria originated from the meat. In 1766 the researcher, Spallanzani (1729-1799), tried to refute Needham's work by heating hay broth after it was sealed in a flask preventing microbes from entering by air. Microbes could not grow spontaneously from such reheated broth. His findings were not totally accepted, as many believed that oxygen was vital to sustain life and nothing would grow if there had not been any in flasks.

A key figure in the debate over abiogenesis was Louis Pasteur who conducted his famous experiments using S-shaped flasks in 1859. Air was allowed to enter flasks but the curved necks trapped bacteria and prevented them from contaminating broth that had previously been boiled. Thus, life could not arise spontaneously from broth even in the presence of air. Henry Bastian conducted many experiments that showed microbes could still grow in various broths' that had been boiled for hours. Although his observations were accurate the growth was not due to abiogenesis. The riddle was solved in 1876 when a German botanist, Cohn identified species of Bacillus, common to hay and cheese that contained heat-resistant endospores. Franz Schulze (1815-1873) Passed air through strong acid solutions into boiled infusions and no growth observed (Chemical sterilization of air). Theodor Schwann (1810-1882) Passed air into flasks through red-hot tubes and no growth observed (Heat sterilization of air). H.Schroder and T.Von Dusch (1850) Passed air through cotton into flasks containing broth. Thus the microbes were filtered out of air by the cotton fibers so that growth will not occur (Filter sterilization of air). Early theories of cause of diseases: 1. Theurgical Theory Of Diseases: The life of a man is governed by superstition. Appearance of disease and death are caused by wrath of divine spirits 2. Miasmatic Theory Of Diseases: Disease was due to emanation from the earth, the influence of stars, the moon, the wind, water and season. Hippocrates(400-370BC)- Father of Medicine proposed a theory of disease called Miasmatic Theory Of Diseases. There are 4 elements causing diseases fire, air, water and earth. The 4 qualities are heat, cold, moisture and dryness respectively corresponds with the four Body fluids i.e Blood, phlegm, yellow bile, black bile. 3. Pore Theory: Symmetry of pores causes healthiness, Disproportion of pores causes diseases. Development of the Germ Theory of Disease: Jacob Hencle (1840) has proposed principles of Germ theory of diseases. This theory provided the knowledge of the causal relationship between microbes and disease and was an outgrowth of the work of numerous scientists and medical professionals. Joseph Lister, a British physician, operating in the mid 1800's, developed antiseptic surgery, which included heat-sterilization of instruments and application of phenols to wounds and dressings. The Missouri physician, Joseph Lawrence, named his now famous mouthwash "Listerine." Ignatz Semmelweis, an obstetrician, noted that deaths due to a streptococcal infection ("childbirth fever") were much higher in the physicians ward as compared to deaths in the midwives ward. Semmelweis subsequently instituted strict hand-washing procedures for physicians and could be considered the father of infection control. Despite the fact that his observations made him extremely unpopular with physicians, simple improvements in hygiene reduced transmission of childbirth fever by 2/3. Robert Koch In 1876, Koch provided a critical link between microbes and disease when he used a series of postulates to uncover the cause of anthrax. Koch's postulates are still in use today in order to prove the cause of an infectious disease.

Koch's postulates: To prove that a disease is caused by an infectious agent The specific causative agent must be identified in ALL cases of the disease. This agent must be obtained from the host with the disease, and be isolated in pure culture in the lab. Another healthy member of a susceptible host species must be inoculated with the lab grown pure culture, and that formerly healthy second host must subsequently get the same disease i.e the pure culture will produce the disease when inoculated into susceptible animal. The agent must be reisolated from the second host and be confirmed as exactly the same agent as the original.

Only when all 4 of these steps have been fulfilled can the original agent be identified as the causative agent of the disease.

Further contributions of Koch

Isolated the bacteria that cause cholera and tuberculosis Developed tuberculin, now used in a skin test for TB (originally intended for use as a vaccine against TB) Developed acid-fast staining Identified bacterial endospores With colleagues, the first to grow cultures on solid media Pour plate technique Received noble prize for medicine 1905. The actual progress in the development of microbiology depended on 1. 2. 3. 4. 5. Invention and progressive evolution of microscope. The use of stains and their development for the study of morphology Finding artificial media for studying cultural characteristics. Development of science of immunology Introduction of chemotherapy-Discovery of Antibiotics

Development of microscope: Marcus Vavro (116-270BC)- Grinding of lenses Roger Bacon (1220-1292)-Clarified the Principles of Optics and he was credited with being the `Founder of Optics' Galileo Galilei (1564-1642)- invented Microscope. Antony Van Leeuwenhoek (1632-1723): is considered by many to be the "Father of Microbiology. Leeuwenhoek was not the first to develop a lens or microscope but was the first to describe LIVING microbes, including bacteria. He constructed 247 complete microscopes with magnifying powers of 40-270 X. He described the "very little animalcules" which we recognize as free-living protozoa. He discovered incredible many little animalcules, in a tiny drop of water. Earnest Abbe (1840-1905)-Perfection in microscopy was obtained by his inventing sub-stage condenser. Robert Hooke: using a compound microscope described cork cells as "little boxes" that reminded him of the cells used by monks. He also described fungi but his microscope was unable to resolve bacteria.

Contributions of Louis Pasteur: 1. Air contained necessary spark that reproduces cells and dust particles provide necessary transportation. 2. Explanation of microbial fermentation 3. Anthrax (Deadly disease) caused by Bacillus anthracis. It comes out the body and formed a rigid body called spore. Spore is then converted into vegetative form. 4. Pasteurization: Louis Pasteur invents Heating the milk or liquid to avoid spoiling. 5. He prepared a flask with a long, narrow gooseneck opening, the nutrient solutions were heated in the flask and no microbes appeared in the solution. Thus he disproved abiogenisis (Spontaneous generation) 6. He produced vaccine for rabies or fowl chorera 7. He introduced vaccination for anthrax. 8. He has found anaerobes 9. Diseases of silk worm due to protozoa Further Inventions of Louis Pasteur: Fermentation: in 1857 Napoleon III requested help to search for the cause of wine spoilage. Pasteur was the first to discover specific biochemical reactions of microbes. He observed that yeast fermented sugar to ethanol and that bacteria oxidized alcohol to acetic acid to cause spoilage. Pasteurization: Pasteur developed a heating process used to kill spoilage germs in wine but which preserved flavor. Prior to pasteurization diseases such as TB were also transmitted in milk. In the early 1900's Mycobacterial-contaminated milk was an important source of TB. Pasteurization also eliminates the milk-borne pathogen Brucella. Pasteur also made huge contributions to the field of vaccination and immunity. Vaccination and Immunology A vaccine is a preparation of microbes or their subunits that is designed to produce immunity to a disease. The ancient Chinese were among the earliest cultures to recognize that material from recovering smallpox victims could be used to immunize others. The problem with crude techniques such as these is that they used living organisms for immunizations, which could potentially be virulent. In the late 1700's the British physician, Edward Jenner demonstrated that inoculation with scrapings from cowpox provided immunity to the more virulent smallpox virus. He used an 8 year old boy called James Phipps to test his vaccine. James was inoculated with virulent matter from cowpox lesions on the fingers of a milkmaid called Sarah Neimes. Phipps developed a mild fever and some cowpox lesions. James was protected from the disease. Jenner noted that this immunity was "a change, which endures throughout life." Louis Pasteur also made large contributions with respect to vaccines. Pasteur found that cholera organisms lost their virulence when passaged in culture. Attenuated (weakened) organisms inoculated into chickens could protect them from virulent strains. Pasteur also developed early vaccines against rabies and anthrax. Ironically, during the course of his work, he lost his father and two sisters to typhoid fever. Elie Metchnikoff was a Russian researcher and is considered the father of cellular immunity largely for his observations of phagocytosis in sea star larvae. Metchnikoff shared the Nobel Prize for medicine with the German microbiologist, Paul Ehrlich in 1908.

Antimicrobial Chemotherapy : Some key figures and events in chemotherapy include: Mid 1600's. The British physician, Thomas Sydenham introduces quinine-containing Cinchona bark from South America. Quinine has long been used as an antimalarial 1910: Paul Ehrlich has developed Salvarsan (compound 606) for the treatment of syphilis. Ehrlich worked under Robert Koch. While working on differential staining techniques Ehrlich developed the idea of using a "magic bullet" that would kill disease-causing microbes but spare the host with minimal toxicity. 1928: Alexander Fleming observed that penicillium mold inhibited growth of Staphylococci growing on a petri plate. Penicillin became widely available and known as the "wonder-drug" with the advent of world war II. Fleming shared the Nobel prize for his efforts along with the researchers, Florey and Chain. 1930's: Domagk develops sulfa drugs. These drugs are especially useful in treating urinary tract infections and are used in combination formulas to prevent pneumocystis pneumonia. 1952: Selman Waksman isolates streptomycin. This antibiotic was the first effective drug available to treat infections with gram negative bacteria and tuberculosis. 1987: AZT becomes the first drug licensed for treating HIV disease. The first cases of AIDS in the U.S. were reported in 1981. 1995: First generation of drugs called protease inhibitors are approved to help treat HIV disease. These drugs continue to extend the improvement the quality of life for many recipients.

STRUCTURE OF PROCARYOTE AND EUCARYOTES There are two general classes of cells: prokaryotic and eukaryotic. The evolution of prokaryotic cells preceded that of eukaryotic cells by 2 billion years. Streptococcus pyogenes, the bacterium that causes strep throat, is an example of prokaryotes. Yeast, the organism that makes bread rise and beer ferment, is an example of unicellular eukaryotes. Humans, of course, are an example of multicellular eukaryotes. The major similarities between the two types of cells (prokaryote and eukaryote) are: 1. They both have DNA as their genetic material. 2. They are both membrane bound. 3. They both have ribosomes. 4. They have similar basic metabolism. 5. They are both amazingly diverse in forms ANIMAL CELL STRUCTURE Animal cells are typical of the eukaryotic cell, enclosed by a plasma membrane and containing a membrane-bound nucleus and organelles. Unlike the cells of the two other eukaryotic kingdoms, plants and fungi, animal cells don't have a cell wall. This feature was lost in the distant past by the single-celled organisms that gave rise to the kingdom Animalia. The lack of a rigid cell wall allowed animals to develop a greater diversity of cell types, tissues, and organs. The ability to move about by the use of specialized muscle tissues is the hallmark of the animal world. (Protozoans locomote, but by nonmuscular means, i.e. cilia, flagella, pseudopodia.). The animal kingdom is unique amongst eukaryotic organisms because animal tissues are bound together by a triple helix of protein, called collagen. Plant and fungal cells are bound together in tissues or aggregations by other molecules, such as pectin. The fact that no other organisms utilize collagen in this manner is one of the indications that all animals arose from a common unicellular ancestor Centrioles-Centrioles are self-replicating organelles made up of nine bundles of microtubules and are found only in animal cells. They appear to help in organizing cell division, but aren't essential to the process. Cilia and Flagella - For single-celled eukaryotes, cilia and flagella are essential for the locomotion of individual organisms. In multicellular organisms, cilia function to move fluid or materials past an immobile cell as well as moving a cell or group of cells. Endoplasmic Reticulum - The endoplasmic reticulum is a network of sacs that manufactures, processes, and transports chemical compounds for use inside and outside of the cell. It is

connected to the double-layered nuclear envelope, providing a connection between the nucleus and the cytoplasm. Golgi Apparatus - The Golgi apparatus is the distribution and shipping department for the cell's chemical products. It modifies proteins and fats built in the endoplasmic reticulum and prepares them for export to the outside of the cell. Lysosomes - The main function of these microbodies is digestion. Lysosomes break down cellular waste products and debris from outside the cell into simple compounds, which are transferred to the cytoplasm as new cell-building materials. Microfilaments - Microfilaments are solid rods made of globular proteins called actin. These filaments are primarily structural in function and are an important component of the cytoskeleton. Microtubules - These straight, hollow cylinders, composed of tubulin protein, are found throughout the cytoplasm of all eukaryotic cells and perform a number of functions. Mitochondria - Mitochondria are oblong shaped organelles that are found in the cytoplasm of every eukaryotic cell. In the animal cell, they are the main power generators, converting oxygen and nutrients into energy. Nucleus - The nucleus is a highly specialized organelle that serves as the information and administrative center of the cell. Peroxisomes - Micro bodies are a diverse group of organelles that are found in the cytoplasm, roughly spherical and bound by a single membrane. There are several types of micro bodies but peroxisomes are the most common. Plasma Membrane - All living cells have a plasma membrane that encloses their contents. Eukaryotic animal cells have only the membrane to contain and protect their contents. These membranes also regulate the passage of molecules in and out of the cells. Ribosomes - All living cells contain ribosomes, tiny organelles composed of approximately 60 percent RNA and 40 percent protein. In eukaryotes, ribosomes are made of four strands of RNA..

BACTERIA CELL STRUCTURE Bacteria are prokaryotes, lacking well-defined nuclei and membrane-bound organelles, and with chromosomes composed of a single closed DNA circle. They come in many shapes and sizes, from minute spheres, cylinders and spiral threads, to flagellated rods, and filamentous chains. They are found practically everywhere on Earth and live in some of the most unusual and seemingly inhospitable places Cell Envelope - The cell envelope is made up of two to three layers: the interior cytoplasmic membrane, the cell wall, and -- in some species of bacteria -- an outer capsule. Capsule - Some species of bacteria have a third protective covering, a capsule made up of polysaccharides (complex carbohydrates).. Cell Wall - Each bacterium is enclosed by a rigid cell wall composed of peptidoglycan, a proteinsugar (polysaccharide) molecule. Cytoplasm - The cytoplasm, or protoplasm, of bacterial cells is where the functions for cell growth, metabolism, and replication are carried out. It is a gel-like matrix composed of water, enzymes, nutrients, wastes, and gases and contains cell structures such as ribosomes, a chromosome, and plasmids. Cytoplasmic Membrane - A layer of phospholipids and proteins, called the cytoplasmic membrane, encloses the interior of the bacterium, regulating the flow of materials in and out of the cell. Flagella - Flagella (singular, flagellum) are hairlike structures that provide a means of locomotion for those bacteria that have them. They can be found at either or both ends of a bacterium or all over its surface. Pili - Many species of bacteria have pili (singular, pilus), small hairlike projections emerging from the outside cell surface Nucleoid - The nucleoid is a region of cytoplasm where the chromosomal DNA is located. It is not a membrane bound nucleus, but simply an area of the cytoplasm where the strands of DNA are found. 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. Ribosomes - Ribosomes are microscopic "factories" found in all cells, including bacteria that translate the genetic code from the molecular language of nucleic acid to that of amino acids -the building blocks of proteins.

DIFFERENCES BETWEEN PROKARYOTE AND EUKARYOTE CELLS Microorganisms include bacteria that are Prokaryotes; Algae, protozoa and fungi that are Eukaryotes; and Viruses which are neither prokaryotes nor eukaryotes.

Characteristic

Group Size of Organism Structure of Nucleus Nucleolus Sexuality

Prokaryote

Bacteria

Eukaryote

Algae, fungi and protozoa

1-2 Micro meter 5 Micro meter Not bounded by nuclear Bound by nuclear membrane Membrane. No mitotic divisions . Mitotic nuclear divisions, Absent Nature of zygote is merozygote Present Zygote is diploid Paired linear Nucleus (membrane present) Mitochondria and Chloroplast Present in endoplasmic recticulum 80 s(40S & 60S) but 70 S in organelle Mitochondria Undulating flagella and cilia , and also amoeboid movement Absent Present Absent Absent Present Present Present Absence of peptytoglycon Present in some Above 40

Chromosome Single circular Chromosome Location Nucleoid (no membrane) Extra Chromosomal DNA Plasmid Distributed in cytoplasm Ribosomes 70 s (30S & 50S) Site of Cellular Respiration Cell membrane Locomotion Pili Cytoplasmic streaming Gas vacuoles Mesosomes Mitochondria, Chloroplast Endoplasmic recticulam Membrane bound vacuoles Cell wall contents Pseudopodia DNA base ratio G+C% Rotating flagella and gliding Sex or attachment pili Absent Present Present Absent Absent Absent Peptytoglycon Absent 28 to 73

COMPOSITION AND FUNCTIONS OF BACTERIAL STRUCTURES

The importance of understanding microbe's structure: Bacteria are very small. Yet despite their size they show a surprising degree of complexity in their structures. Knowing the structure of a microbe helps in understanding how a microbe functions. Disease causing bacteria (pathogens) have various properties that enhance their ability to cause illness. One important property is the ability to attach to the intended victim. pili, a proteinaceous surface structure on the bacteria, are critical in this process. Many bacteria are capable of movement in their environment either by flagella or gliding motility. In the case of flagella, the bacteria have a long, flexible, spiral shaped structure, the flagellum that helps to push the microbe through solution. Flagella also help in the detection of favorable or unfavorable conditions and move the bacteria in an appropriate direction. As a microbe grows it has to synthesize more of itself. Knowing what it is made of and how it is put together is critical to gain an understanding of the growth process. Microbes are also capable of exchanging genetic information by mating. This process involves another type of surface structure, the F-pilus. Bacteria will take steps to insure their survival. This can take the form of creating resting structures that allow the microbe to "sleep" during bad times. During abundant times, many microbes will store excess carbon, nitrogen, sulfur or phosphorous in inclusions in the cell.

Structurally, a typical bacterium usually consists of: A cytoplasmic membrane surrounded by a peptidoglycan cell wall and maybe an outer membrane; A fluid cytoplasm containing a nuclear region (nucleoid) and numerous ribosomes; and Often various external structures like a glycocalyx, flagella, and pili. The Cytoplasmic Membrane: The cytoplasmic membrane, also called a cell membrane or plasma membrane, is about 7 nanometers (nm; 1/1,000,000,000 m) thick. It lies internal to the cell wall and encloses the cytoplasm of the bacterium. It is made of a phospholipid bilayer with integral and peripheral proteins embedded. It maintains the selective permeability of the cell, has respiratory enzymes and during cell division, the chromosome is linked to the cell membrane at a site called Mesosome. Composition: Like all biological membranes in nature, the bacterial cytoplasmic membrane is composed of phospholipid and protein molecules. With the exception of the mycoplasmas, the only bacteria that lack a cell wall, prokaryotic membranes lack sterols. (Mycoplasmas, lacking a rigid cell wall, need the sterols in their cytoplasmic membrane to provide the added strength necessary to hold the cell together.) The phospholipid bilayer is arranged so that the polar ends of the molecules (the phosphate and glycerol portion of the phospholipid which is soluble in water) form the outermost and innermost surface of the membrane while the non-polar ends (the fatty acid portions of the phospholipids which are insoluble in water) form the center of the membrane Functions: The cytoplasmic membrane is a selectively permeable membrane, which determines what goes in and out of the organism. All cells must take in and retain all the various chemicals needed for metabolism. Water, dissolved gases such as carbon dioxide and oxygen, and lipid-

soluble molecules simply diffuse across the phospholipid bilayer. Water-soluble ions generally pass through small pores - less than 0.8 nm in diameter - in the membrane. All other molecules require carrier molecules to transport them through the membrane Functions of the cytoplasmic membrane other than selective permeability: A number of other functions are associated with the cytoplasmic membrane. In fact, many of the functions associated with specialized internal membranebound organelles in eukaryotic cells are carried out generically by the cytoplasmic membrane in bacteria. These include: i. The site of energy production (the electron transport system for bacteria with aerobic and anaerobic respiration; photosynthesis for bacteria converting light

energy into chemical energy). ii. The site of peptidoglycan synthesis, both in the growing cell wall and in the transverse septum that divides the bacterium during bacterial division . iii. The site of phospholipid synthesis and some protein synthesis for production of more cytoplasmic membrane. iv. Involved in amitotic division of the nucleoid, the replication and separation of DNA during bacterial division. v. Contains the bases of flagella used in motility. vi. The site of waste removal. vii. involved in formation of endospores Bacteria divide by binary fission wherein one bacterium splits into two. Therefore, bacteria increase their numbers by geometric progression whereby their population doubles every generation time. In general it is thought that during DNA replication each strand of the replicating bacterial DNA attaches to a different site on the cytoplasmic membrane. As the bacterium grows the, newly replicated chromosomes become separated. In the center of the bacterium the cytoplasmic membrane invaginates and synthesizes a peptidoglycan septum to separate the two daughter cells. THE CELL WALL The Bacteria (eubacteria), with the exception of the Chlamydias have a semi rigid cell wall containing peptidoglycan. The Archaea (archaebacteria), which are often found growing in extreme environments, also have a semi rigid cell wall but it is composed of chemicals distinct from peptidoglycan such as protein or pseudomurein. The mycoplasmas are the only bacteria that naturally lack a cell wall. Mycoplasmas maintain a nearly even pressure between the outside environment and the cytoplasm by actively pumping out sodium ions. Their cytoplasmic membranes also contain sterols that most likely provide added strength. Except this all other bacteria have a cell wall. Composition: The Bacteria, or eubacteria, have a cell wall containing a semi rigid, tight knit molecular complex called peptidoglycan .Peptidoglycan, also called murein, consists of rows of 2 alternating amino sugars (N-acetylglucosamine, or NAG, and N-acetylmuramic acid, or NAM). Tetrapeptide cross bridges coming off of the NAMs link the rows of sugars together. This cross bridging between rows of sugars provides rigidity to the cell wall, functioning much like a molecular chain link fence around the bacterium. Function: Peptidoglycan gives the bacterium its shape and prevents osmotic lysis . In order for bacteria to increase their size following binary fission, enzymes called autolysins break the cross links in the peptidoglycan while transpeptidase enzymes add new peptidoglycan monomers and reseal the wall. The type of transpeptidase determines the shape of the bacterium. Most

bacteria can be placed into one of three groups based on their color after specific staining procedures are performed: gram-positive, gram-negative, or acid-fast. Gram-positive: retain the initial dye crystal violet during the Gram stain procedure and microscopically appear purple. Common gram-positive bacteria of food importance include Streptococcus pyogenes, Staphylococcus aureus, Enterococcus faecalis, and Clostridium species. Gram-negative: decolorize during the Gram stain procedure, pick up the counterstain safranin, and appear pink. Common Gramnegative bacteria of food importance include Salmonella species, Shigella species, Escherichia coli, Proteus species, and Pseudomonas sp.. Acid-fast: resist decolorization with an acid-alcohol mixture during the acid-fast stain procedure, retain the initial dye carbolfuchsin and appear bluish red. Common acid-fast bacteria of food importance include Mycobacterium tuberculosis. These staining reactions are due to fundamental differences in their cell wall. THE GRAM-POSITIVE CELL WALL In electron micrographs, the gram-positive cell wall appears as a broad, dense wall (20-80 nm thick) consisting of several interconnecting layers of peptidoglycans. Chemically, 60 to 90% of the gram-positive cell wall is peptidoglycan. Many gram-positive cell walls also contain teichoic acids

Composition: Has a thick peptidoglycan (murein) layer and 2 classes of teichoic acids. Teichoic acids, which extend through and beyond the rest of the cell wall, are composed of polymers of glycerol, phosphates, and the sugar alcohol ribitol. Lipoteichoic acid which is on the surface, embedded in the peptidoglycan layer and is linked to the cytoplasmic membrane. Wall teichoic acid is on the surface and is linked to only the peptidoglycan layer. Teichoic acid is responsible for the antigenic determinant of the organism. Function: The gram-positive cell wall gives the bacterium its shape and prevents osmotic lysis. In addition, both glycopeptides from peptidoglycan and teichoic acids from the gram-positive cell wall are recognized by defense cells of the body and promote inflammation, one of the first steps

in body defense against microbes. In selected bacteria the cell wall may also enable the organism to adhere to surfaces, penetrate host cells, and/or resist phagocytic destruction The Gram-Negative Cell Wall In electron micrographs, the gram-negative cell wall appears multilayered. It consists of: thin, inner peptidoglycans layer, an outer membrane and Periplasm. The Gram negative cell wall has a thin peptidoglycan (murein) layer with an outer membrane attached to the peptidoglycan layer by lipoproteins. The outer membrane is made of protein, phospholipid and lipopolysaccharide. In the lipopolysaccharide, the lipid portion is embedded in the phospholipid and the O antigen polysaccharide is on the surface. The lipid is called Lipid A and it is toxic, but the whole lipopolysaccharide is called an Endotoxin. The cell wall has channels called Porins for the transport of low molecular weight substances. Between the cytoplasmic membrane and the cell wall is a periplasmic space with hydrolytic enzymes, antibiotic inactivating enzymes and transport proteins. i. A thin, inner peptidoglycan layer Composition: a single layer of peptidoglycan (2-3 nm thick). Chemically, only 10 to 20% of the gram-negative cell wall is peptidoglycan. Function: The peptidoglycan gives the bacterium its shape and prevents osmotic lysis. ii. An outer membrane Composition: a lipid bilayer about 7 nm thick composed of phospholipids, lipoproteins, and lipopolysaccharides(LPS). LPS consists of polysaccharides, which extend outward from the bacterium, attached to lipid A and is present in the external side of the outer membrane. Lipoproteins, proteins combined with a lipid, are located in the internal side of the outer membrane and function to attach the outer membrane to the peptidoglycan layer. The outer membrane, like the cytoplasmic membrane discussed next, is semipermeable and acts as a coarse molecular sieve. Many small molecules may pass through due to pores running through the membrane. These pores are composed of proteins called porins. Functions: Because of its semipermeable nature, the outer membrane helps retain certain enzymes and prevents some toxic substances, e.g., penicillin G and lysozyme, from entering. In addition, LPS from the outer membrane of the gram-negative cell wall is recognized by defense cells of the body and promotes inflammation, one of the first steps in body defense against microbes. In selected bacteria, the outer membrane also enables the organism to adhere to surfaces, penetrate host cells, and/or resist phagocytic destruction.

iii. The periplasm The periplasm is the gelatinous material between the outer membrane, the peptidoglycan, and the cytoplasmic membrane. It contains enzymes for nutrient breakdown as well as binding proteins to facilitate the transfer of nutrients across the cytoplasmic membrane. The Acid-Fast Cell Wall : Bacteria belonging to the genus Mycobacterium and the genus Nocardia are said to be acid-fast because during the acid-fast stain procedure, they resist decolorization with an acid-alcohol mixture. Composition: Only relatively small amounts of peptidoglycan are found in the acid-fast cell wall. Instead, a waxy lipid called mycolic acid makes up approximately 60% of the wall. This mycolic acid helps to form a relatively impermeable outer membrane. The acid-fast cell wall also contains a layer of arabinogalactan. Functions: The acid-fast cell wall gives the bacterium its shape and prevents osmotic lysis. The mycolic acid outer membrane also impedes the entry of chemicals causing the organisms to grow slowly and be more resistant to chemical agents than most bacteria Structures and materials found inside the bacterium The cytoplasm Composition: composed of water (about 80%), nucleic acids (DNA and RNA), enzymes and amino acids, carbohydrates, lipids, inorganic ions, and many low molecular weight compounds. Some bacteria also contain reserve food inclusions in their cytoplasm such as volutin (stored phosphate), polysaccharide granules, lipid inclusions, and sulfur granules. Functions: The cytoplasm is the site of most bacterial metabolism. The nucleoid Composition: The term genome refers to the sum of an organism's genetic material. The bacterial genome is composed of chromosomal deoxyribonucleic acid or DNA and represents the bacterium's nucleoid. The nucleoid is one long, single molecule of double stranded, helical, supercoiled DNA which forms a physical and genetic circle . The chromosome is generally around 1000 µm long and frequently contains around 4000 genes . There is a protein-RNA core to keep it folded into a supercoiled mass about 0.2 µm in diameter but the nucleoid has no nuclear membrane or nucleoli. Bacterial enzymes called DNA gyrase and DNA topoisomerases are essential in the unwinding and replication of the bacterial DNA. They enzymes also enable replicated daughter DNA to unlink and become circular and supercoiled. Function: The nucleoid is the genetic material of the bacterium. Genes located along the DNA are transcribed into RNA which, in the case of mRNA, is translated into protein at the ribosomes. In other words, the primary function of DNA is to code for protein synthesis. Plasmids : In addition to the nucleoid, many bacteria often contain small nonchromosomal DNA molecules called plasmids. Plasmids are not essential for bacterial growth and may be lost or gained without harm. They can, however, provide an advantage under certain environmental conditions. Plasmids usually contain between 5 and 100 genes.

Composition: small molecules of double stranded, helical, nonchromosomal DNA forming a physical circle. Function: Plasmids code for synthesis of a few proteins not coded for by the nucleoid. For example, R-factor plasmids found in some gram-negative bacteria often have genes coding for maleness (production of a sex pilus, and multiple antibiotic resistance. Some exotoxins such as the tetanus exotoxin and Escherichia coli enterotoxin are also coded for by plasmids. Transposons Transposons (transposable elements or "jumping genes") are small pieces of DNA which encode enzymes that transpose the transposon, that is, move it from one DNA location to another. Transposons may be found as part of a bacterium's nucleoid or in plasmids and are usually between one and twelve genes long. One of the genes codes for an enzyme called transpoase which catalyzes the cutting and resealing of the DNA during transposition. Other genes, such as those responsible for bacterial antibiotic resistance, are also often carried in transposons. Such transposons are able to cut themselves out of a bacterial nucleoid or a plasmid and insert themselves into another nucleoid or plasmid and contribute in the transmission of antibiotic resistance. Ribosomes Composition: Ribosomes are composed of two subunits with densities of 50S and 30S. ("S" refers to a unit of density called the Svedberg unit.) The two subunits combine during protein synthesis to form a complete 70S ribosome about 25nm in diameter. A typical bacterium may have as many as 15,000 ribosomes. Function: Ribosomes function as a workbench for protein synthesis, that is, they receive and translate genetic instructions for the formation of specific proteins. During protein synthesis, mRNA attaches to the 30s subunit and amino acid-carrying tRNAs attach to the 50s subunit Structures External to the Cell Wall The glycocalyx : Capsule and slime layer: All bacteria secrete some sort of glycocalyx, an outer viscous covering of fibers extending from the bacterium A gelatinous polysaccharide and/or polypeptide outer covering. The glycocalyx can be identified by negative staining techniques. If glycocalyx appears as an extensive, tightly bound accumulation of gelatinous material adhering to the cell wall it is referred to as a capsule and if it appears unorganized and more loosely attached, it is referred to as a slime layer.

Permit bacteria to adhere to cell surfaces and structures such as medical implants, catheters and soon. This is an important first step in colonization and sometimes leads to disease. Virulence factors, protecting bacteria from phagocytosis by immune cells. Pathogens such as Streptococcus pneumoniae can cause pneumonia if protected by a capsule Capsules can be a source of nutrients and energy to microbes. Streptococcus mutans, which colonizes teeth, ferments the sugar in the capsule and acid byproducts contribute to tooth decay. Prevent cell from drying out (desiccation) Polysaccharides from certain capsules can be the targets of protective immune responses and have therefore been included in 'conjugated' (linked to another immune-stimulating molecule) vaccines. Such a vaccine is used against Hemophilus influenzae (a leading cause

of ear infections and meningitis in young children). Purified polysaccharide vaccines also exist for streptococcal pneumonia and menigococcal meningitis. Composition: usually a viscous polysaccharide or polypeptide slime. Actual production of a glycocalyx often depends on environmental conditions. Functions: Although a number of functions have been associated with the glycocalyx (protect against drying, trap nutrients, etc.), for our purposes there are two important functions. The glycocalyx enables certain bacteria to resist phagocytic engulfment by white blood cells in the body or protozoans in soil and water. The glycocalyx also enables some bacteria to adhere to environmental surfaces (rocks, root hairs, teeth, etc.) and resist flushing. Glycocalyces may also bind numbers of bacteria together to produce microcolonies.

Flagella

Bacterial flagella consist of a filament and a hook which pierces the cell wall and attaches to the base of ring-like structures. The flagella rotate and move the bacterium in a fashion similar to a propellorBacterial flagella consist of intertwining chains of a protein called flagellin and are about 1/10 diameter of a eukaryotic flagellum. Flagella may also be identified by special staining techniques. Composition: Made of the protein flagellin and consists of a filament and basal region. The basal region has a hook and a basal body, which has a rod and rings. Gram positive organisms have 2 rings, one in the cell wall and one in the cell membrane. Gram negative organisms have 4 rings, 2 in the cell wall and 2 in the cell membrane. A bacterial flagellum has 3 basic parts. 1) the outermost region or filament, composed of the protein flagellin arranged in helical chains to form a hollow core; 2) a hook; and 3) a basal body, consisting of a rod and a series of rings that anchor the flagellum to the cell wall and cytoplasmic membrane. The flagellum has no internal fibrils and is noncontractile. Flagella are 10-20 µm long and between 0.01 and 0.02 µm in diameter. Flagella have different numbers and arrangements:

Monotrichous

Lopotrichous

Peritrichous

Amphitrichous

flagellar arrangements 1. monotrichous: a single flagellum, usually at one pole 2. amphitrichous: a single flagellum at both ends of the organism 3. lophotrichous: two or more flagella at one or both poles 4. peritrichous: flagella over the entire surface 5. axial filaments (endoflagella): internal flagella found only in the spirochetes. They are located between the peptidoglycan cell wall and the outer membrane of the organism Counterclockwise rotation is for smooth swimming towards an attractant. Clockwise rotation is for backward or reverse movement. With peritrichous flagella clockwise rotation results in tumbling to change direction.

Functions : Primarily function in motility as these are the organelles of locomotion for most of the bacteria that are capable of motility. Around half of all known bacteria are motile. Motility serves to keep bacteria in an optimum environment via taxis . Taxis is a motile response to an environmental stimulus. Motile bacteria show 'taxis.' A positive taxi is movement toward a favorable environment whereas negative taxis is movement away from a repellent. Flagella can help in identifying certain types of bacteria. For example, Proteus species show a rapid 'swarming' type of growth on solid media. Flagellar antigens are used to distinguish different species and strains of bacteria known as serovars. Variations in the flagellar H antigen are used in such typing. During chemotaxis (a motile response to a chemical stimulus), chemoreceptors located in the cytoplasmic membrane or periplasm of the bacterium bind chemical attractants or repellents. This leads to either the methylation or demethylation of methyl-accepting chemotaxis proteins (MCPs), which in turn triggers either a counterclockwise or clockwise rotation of the flagellum. For example, if the concentration of an attractant remains constant or decreases, the MCPs are demethylated and this eventually leads to clockwise rotation of the flagellum. Clockwise rotation results in a tumble and changes the direction of bacterial movement. On the other hand, if the concentration of attractant increases, the MCPs are methylated and this leads to a counterclockwise rotation of the bacterial flagellum. Counterclockwise rotation leads to longer, straight runs without a change in direction. During a run, which lasts about one second, the bacterium moves 10 - 20 times its length before it stops. In the case of a tumble, the movement lasts only about one-tenth of a second and no real forward progress is made. Therefore, an increasing concentration of attractant or decreasing concentration of repellent (both conditions beneficial) causes longer runs and less tumbling; a decreasing concentration of attractant or increasing concentration of repellent (both conditions harmful) causes increased tumbling and a greater chance of reorienting in a "better" direction. As a result, the organism's net movement is toward the optimum environment Pili (Fimbriae) Short, hair-like structures present in many gram negative bacteria and are NOT responsible for motility as are flagella. Fimbriae act as adhesins and allow bacteria to colonize cells and sometimes cause disease. For example, Neisseria gonorrhoea uses its fimbriae to attach to the lining of the genital tract and initiate an Sexually transmitted diseases. Sex pili act to join bacterial cells for transfer of DNA from one cell to another (bacterial conjugation) Fimbriae also act as receptors for viruses than infect bacteria (bacteriophages). Fimbriae and cell walls of Streptococcus pyogenes are coated with M protein. This acts as an important virulence factor by adhering to host cells and resisting phagocytosis Made of the protein pilin and project from the cell surface. There are 2 types: Sex or conjugation Pili for the transfer of extrachromosomal DNA between donor and recipient. Attachment attachment to Pili or Fimbriae. There are many and are used for surfaces. Pili are virulence factors

Composition: protein (pilin). Pili are thin, protein tubes originating from the cytoplasmic membrane and are found in virtually all gram-negative bacteria but not in many Gram-positive bacteria. There are two basic types of pili: 1) short attachment pili, also known as fimbriae, which are usually quite numerous and 2) long conjugation pili, also called "F" or sex pili which are very few in number.

Function: The short attachment pili are organelles of adhesion allowing bacteria to colonize environmental surfaces or cells and resist flushing. The same bacterium may produce different types of pili in order to adhere to different types of cells. Some bacteria can produce a special pilus called a conjugation or sex pilus that enables conjugation. Endospores : Endospores are dormant alternate life forms produced only by the genus Bacillus (an obligate aerobic ), the genus Clostridium (an obligate anaerobe ; often normal flora of the GI tract of animals), and several other less common genera of bacteria including Sporolactobacillus, Oscillospira, and Thermoactinomyces. Function: An endospore is not a reproductive structure but rather a resistant, dormant survival form of the organism. Endospores are quite resistant to high temperatures (including boiling), most disinfectants, low energy radiation, drying, etc. The endospore can survive possibly thousands of years until a variety of environmental stimuli trigger germination, allowing outgrowth of a single vegetative bacterium . Formation of endospores : Under conditions of starvation, especially the lack of carbon and nitrogen sources, a single endospore form within some of the bacteria. The process is called sporulation . First the DNA replicates and a cytoplasmic membrane septum forms at one end of the cell. A second layer of cytoplasmic membrane then forms around one of the DNA molecules (the one that will become part of the endospore) to form a forespore. Both of these membrane layers then synthesize peptidoglycan in the space between them to form the first protective coat, the cortex. Calcium dipocolinate, which along with the eventual dehydrated state probably contribute to the endospore's heat resistance, is also incorporated into the forming endospore. A spore coat composed of a keratin-like protein and impervious to many chemicals then forms around the cortex. Finally, the remainder of the bacterium is degraded and the endospore is released. Sporulation generally takes around 15 hours. The completed endospore consists of multiple layers of resistant coats (including a cortex, a spore coat, and sometimes an exosporium) surrounding a nucleoid, some ribosomes, RNA molecules, and enzymes. Spore structure and arrangements: The figure on the left shows the general structure of a bacterial Endospore. It shows how the shape, location and the relative size of the formed-spore to the remains of the parent cell can be used to describe a bacterial spore-former. These characteristics are genetic. A = oval, terminal; B = rectangular, terminal; C = rectangular, subterminal, D = rectangular, central; E = circular, terminal; F = circular, central; G = terminal, club-shaped. The endospore is resistant to heat, drying, chemicals, radiation, and practically anything else that normally kills vegetative cells. It is totally ametabolic. It can stay in this state of suspended animation indefinitely. when nutritional conditions improve something triggers endospores to reverse this process of Sporulation. heat: withstand boiling for over one hour desiccation: UV radiation chemical disinfectants the resistance of these spores has serious consequences and some very pathogenic bacteria have the ability to produce such spores. endospores are NOT reproductive structures as only one cell gives rise to one spore. 10 genera of endospore-forming gram positive bacilli and cocci are known; many of them are pathogens endospores can be identified with special stains and differentiated from the vegetative cell.

MORPHOLOGY

Morphology of Bacteria: Morphology is a branch of biological science that deals with size, shape, structure, and arrangement of living organisms. The bacterial cell posses a detailed internal structure. Microbial cytology and bacterial anatomy means the study of cell structure Surface to volume ratio: Because of their small size, bacteria have a large surface-to-volume ratio. Ratio = surface area/volume. In bacteria, surface area is more and volume is less and the ratio is greater than 1 (positive) in bacteria. For example, spherical bacteria with a diameter of 2 µm have a surface area of about 12 µm2 and a volume of about 4 µm3. Their surface-to-volume ratio is 12:4, or 3:1. The large surface-tovolume ratio of bacteria means that no internal part of the cell is very far from the surface and that nutrients can easily and quickly reach all parts of the cell. Large surface area bacteria cell has easy respiration and discharge the waste materials very quickly. The large surface to volume ratio seen in bacteria is one reason that prokaryotes are so successful despite their relatively simple morphologies Bacteria range in size from approximately as small as the largest viruses to large enough for single cells to be visible by the naked eye. The size is, from about 0.1 to about 600 µm over a single dimension. Bacteria vary in size as much as in shape. The smallest (e.g., some members of the genus Mycoplasma) are about 100 to 200 nm in diameter, approximately the size of the largest viruses (poxviruses). Escherichia coli, a bacillus of about average size, is 1.1 to 1.5 µm wide by 2.0 to 6.0 µm long. A few become fairly large; some spirochetes occasionally reach 500 µm in length, and the cyanobacterium Oscillatoria is about 7 µm in diameter (the same diameter as a red blood cell). Recently a huge bacterium has been discovered in the intestine of the brown surgeonfish, Acanthurus nigrofuscus. Epulopiscium fishelsoni grows as large as 600 µm by 80 µm, a little smaller than a printed hyphen. It is now clear that a few bacterium are much larger than the average eucaryotic cell. Epulopiscium fishelsoni is an example of a very big prokaryote. Epulopiscium fishelsoni (is) a prokaryote related to the gram-positive genus Clostridium. .In stark contrast to the large size of Epulopiscium fishelsoni, there exists an extremely small (i.e., bacteria size) eukaryote, Nanochlorum eukaryotum. "Nanochlorum eukaryotum is only about 1 to 2 µm in diameter (about the size of E. coli), yet is truly eucaryotic and has a nucleus, a chrloroplast, and a mitochondrion. Shape and Arrangement: 1. a) b) c) d) f) Coccus (plural = cocci, meaning berries): spherical cells. Generally spherical though with some variation from this theme (i.e., elongation or flattening on one side). Spherical is called coccus. Ex: Chlamydia trachomatis Division along the same plane forms chains; 2 cocci together ­ Diplococcus Ex: Neisseria gonorrhoeae, 4 - 20 in chains ­ Streptococcus Ex: Lactococcus spp, Enterococcus spp Division along 2 different planes ­ Tetrads e) Division along 3 planes Staphylococci Bacillus (plural = bacilli, Ex: Micrococcus luteus

Division along 3 planes regularly ­ Sarcinae irregularly ­ meaning small staffs); rod-

2.

shaped cells. Basically, bacilli are longer than they are wide and lack extreme curvature Variations on rod-shaped bacteria: are rod , tapered rod ,staff, cigar, oval, curved. Bacilli typically divide only across their short axis. Ex: Bacillus anthracis Bacillus subtilis Chlostridium botulinum a) b) Rod shape is called Bacillus Two bacilli together - Diplobacilli c) Chains of bacilli are called Streptobacilli Ex: Bacillus megaterium d) figures Palisades-Rods side by side or in X,V, or Y

Coccobacillus: cells that are not perfectly round, as are true cocci, but appear to have blunted ends

3.

Spirillum (plural spirilla): cells with spiral or curved bodies with one or more twists. Cells tend to be rigid and fairly inflexible. Spirilla are often motile by means of flagella. Spiral shape that is rigid is called Spirillium Spirochetes are also spiral shaped but are more flexible and undulating than spirilla and move by an internal flagellum known as an axial filament Vibrio: comma shaped cells. The word Vibrio also refers to a bacterial genus Other shapes: Square shape is called Archaebacteria. Monomorphic is a trait of a bacterium that tends to display the same shape regardless of physiological or environmental conditions. Pleomorphic [polymorphic, pleiomorphic is a trait of a bacterium that can display different shapes under different physiological or environmental conditions, or even in the same culture. Examples: Corynebacterium diphtheriae , Mycoplasma pneumoniae , Rickettsia rickettsiia

CLASSIFICATION OF MICROBES Identification of an organism is made possible by following the classification and nomenclature guidelines and by various scientific approaches. This allows us to place an organism within its correct position in the classification scheme. Taxonomy: Taxonomy is the science of classification, identification, and nomenclature. Taxonomy is one aspect of classification. Organisms are ordered into groups (taxa) and ranked in a hierarchy according to established procedures and guidelines. In this manner, organisms are placed into taxa of different organizational levels and the inter-relationships and boundaries between groups are established. For classification purposes, organisms are usually organized into subspecies, species, genera, families, and higher orders Classification Classification is the orderly arrangement of bacteria into groups. There is nothing inherently scientific about classification, and different groups of scientists may classify the same organisms differently. Bacteria are classified and identified to distinguish among strains and to group them by criteria of interest to microbiologists and other scientists Identification Identification is the practical use of classification criteria to distinguish certain organisms from others, to verify the authenticity or utility of a strain or a particular reaction, or to isolate and identify the organism that causes a disease. Nomenclature Nomenclature (naming) is the means by which the characteristics of a species are defined and communicated among microbiologists. A species name should mean the same thing to all microbiologists. Bacteria are named so that investigators can define and discuss them without the necessity of listing their characteristics. Nomenclature is another aspect of taxonomy. Names are assigned to organisms in a systematic manner. Species A bacterial species is a distinct organism with certain characteristic features, or a group of organisms that resemble one another closely in the most important features of their organization. In the past, unfortunately, there was little agreement about these criteria or about the number of features necessary to distinguish a species. Species were often defined solely by such criteria as host range, pathogenicity, or ability to produce gas during the fermentation of a given sugar. Without a universal consensus, criteria reflected the interests of the investigators who described a particular species. For example, bacteria that caused plant diseases were often defined by the plant from which they were isolated; also, each new Salmonella serotype that was discovered was given species status. These practices have been replaced by generally accepted genetic criteria that can be used to define species in all groups of bacteria. Species, groups of similar organisms within a genus, are designated by biochemical and other phenotypic criteria and by DNA relatedness, which groups strains on the basis of their overall genetic similarity Diagnostic Identification Bacteria are identified routinely by morphological and biochemical tests, supplemented as needed by specialized tests such as serotyping and antibiotic inhibition patterns. Newer molecular techniques permit species to be identified by their genetic sequences, sometimes directly from the clinical specimen.

Subtyping Because of differences in pathogenicity or the necessity to characterize a disease outbreak, strains of medical interest are often classified below the species level by serotyping, enzyme typing, identification of toxins or other virulence factors, or characterization of plasmids, protein patterns, or nucleic acids. The genetic variability of microbes is further subdivided into subspecies or types: A strain is equivalent to a clone and represents a population of genetically identical organisms that have arisen from a single cell. Some strains of a bacterial species may be virulent, whereas others are not. Serovars are antigenically distinct organisms. For example, over 2,000 serovars of Salmonella have been identified which are typed according to their flagellar (H) and somatic (O) antigens. Biovars are organisms which can differ physiologically. For example, they may possess differing forms of enzymes Before scientists had a clear understanding of the nature of microbes the biological world was classified into two kingdoms: plant and animal. Bacteria were placed into plant kingdom. Clearly, this scheme was inadequate. In 1968 Whittaker proposed his famous 5 Kingdom system of living organisms. Bacteria were classified under Kingdom Prokaryotae (aka Monera). Prokaryotes were defined as "cells in which nuclear material is not surrounded by a nuclear membrane." The Linnean system of Binomial Nomenclature The Swedish naturalist, Carolus Linneaus developed a scientific system of naming organisms. The names used by Linneaus in the Species Plantarum (1753) and the Systema Naturae (1758) are the basis of the system for plants and animals, respectively. He assigned two latinized names to each organism: A genus consists of a group of similar species. Similar genera are grouped into a family. The species name or "specific epithet" is unique to the new species. The genus name is indicated by a capital letter whereas the species name starts with a lower case letter. By convention both names are italicized (or underlined). Example: Streptococcus pyogenes . Once a scientific name has been used in entirety it can subsequently be abbreviated as follows: S. pyogenes Scientific names should be unique, unchanging and descriptive. For example, the name may reflect the name of the person describing the organism, the habitat of the organism, the appearance of the organism, Some names may reflect a disease or infectious process caused by an organism (e.g., 'pyogenes' describes the ability to produce pus) Two alternative approaches to microbial taxonomy Phenetic system: groups organisms based on similarity of shared phenotypic characteristics. For example, we could place anaerobes in one group and aerobes in another. This may not always reflect the correct evolutionary groupings of the organisms. Bergey's manual is an example of a phenetic system. Microbes are organized into groups based on both morphological (staining reactions, cell shape and arrangement, pigment production, appearance on media) and physiological (growth requirements, biochemical tests, type of metabolism). This system can be useful for identifying an unknown organism Numerical Taxonomy Calculates the percentage of characteristics that two organisms or groups have in common A large range of traits (morphology, motility, biochemistry) are considered. The result of this classification is a similarity coefficient (the percentage of the total number of characters

measured that are common to two organisms Phylogenetic system groups organisms based on their shared evolutionary heritage and descent. Unlike, a phenetic system, organisms do not have to be phenotypically similar in order to belong to the same phylogenetic group. For example, based on genetic and molecular evidence, Pneumocystis carinii is now considered to be more closely related to the fungi and is no longer believed to be a protozoan (although it resembles a protozoan in many respects). Molecular methods used to type and identify microbes Two main approaches: Comparing DNA or RNA sequences in one or more ways and comparing amino acid sequences of a protein or proteins Molecular taxonomy : Uses some key assumptions in order to establish a time-line of evolutionary relatedness Genetic mutations are random, once a mutation occurs, all descendants of that cell will carry the mutation, organisms that differ only slightly at the genetic level have diverged more recently over the course of evolution than organisms that differ significantly 16S ribosomal RNA sequences: 16S rRNA (about 1500 nucleotides long) is found in the 30S ribosomal subunit of bacterial ribosomes. This rRNA is also found in the ribosomes of chloroplasts and mitochondria and is therefore present in animals and plants. As the ribosome plays a critical role in protein synthesis most mutations in rRNA are harmful and tend to occur very infrequently. Therefore,16S rRNA is a very useful molecule for comparing relatedness of organisms over the course of evolution. As the 16SrRNA is so highly conserved organisms are classified as separate species if their sequences show less than 98% homology and are classified as different genera if their sequences show less than 93% identity. Specific base sequences in the rRNA known as signature sequences were commonly found in particular groups of organisms. Dr. Carl Woese and colleagues at the University of Illinois examined the 16S rRNA sequences of hundreds of organisms and divided the organisms into three domains based on their signature sequences and their related properties. The domain is now the highest level of organization in the biological world. The Domains are termed: Eukarya , Eubacteria , Archaea DNA base composition: Indicates relatedness of organisms. Base composition is usually expressed as GC content. If the GC content differs by a small percentage the organisms are not closely related. The GC content itself does not always mean that organisms are related. For example, humans and Bacillus have similar GC contents but are very different organisms DNA fingerprinting Comparison of the cleavage pattern (fingerprint) of the DNA from two organisms (one known, the other unknown) can determine if they are related. Each organism has a unique restriction digest profile Hybridization of DNA probes : The most widely used molecular method used to determine relatedness or organisms. ssDNA is separated from double stranded DNA on a filter .The DNA of one organism is radiolabelled and mixed at low concentrations with the nonradioactive denatured DNA of the other organism. The more related the organisms, the higher the degree of complementary base pairing which can be detected by a higher reading of radioactivity. PCR : Amplification and sequencing of DNA is also a powerful tool for establishing the identity of microbes and represents the diagnostic tool of the future.

Other methods for identifying bacteria Direct observation: microscopic examination without culturing organism, wet mounts, stains, many microbes can not be distinguished from each other on the basis of appearance alone Detect biochemical products of metabolism

Serological tests Identify microbes by reactivity with specific antibodies. Serotyping developed by Rebecca Lancefield. Designed A through O system to identify variants (serovars) of Streptococci Enzyme-linked immunosorbent assay (ELISA) Immunofluorescent antibody testing (IFAT) The Bergey's Manual Trust In 1934, the society transferred to Bergey all its rights, title & interests in the manual in order to allow Bergey to create an independent, non-profit trust; In 1974, the manual began a truly international cooperative effort. Authorities from all over the World were invited to prepare the descriptions of various genera and the 8th edition of the Manual released in 1974,contained contributions from 135 authors. In 1984, the scope of the Manual was greatly broadened to bring information dealing with ecology, environment, isolation, perversion & characterization of bacteria all of which concerned with bacterial classification and identification. The new breath of coverage was reflected by the new name `Bergey's Manual of Systematic Bacteriology'. As the knowledge about bacteria continues to increase, so will Bergey's manual continue to change with it and to act as a "Mirror" for the World of bacteria. Bergey's manual is the international standard for bacterial taxonomy and nomenclature. Bergey's manual of systematic bacteriology places all bacteria in the kingdom "Procaryotae" which in turn is divided into 4 divisions. Division:1 Gracilicutes: Bacteria with a thin cell wall. Gram ­ve bacteria are included in this division Division:2 Firmicutes : Bacteria with a thick cell wall structure.( characteristic ofGram +ve bacteria) Division:3 Tenericutes :Bacteria with tender and soft cell wall.Mycoplasmas are included in this division. Division:4 Mendosicutes :Procaryotes that show evidence of an earlier phylogenetic origin than those bacteria included in Div.1 and Div.2. They have faulty (mendose=faulty) cell walls. They do not have typical peptidoglycon, instead they have proteins. Also they live in unusual habitats like hot springs.

BASIC CONCEPTS IN MICROSCOPY The microscope is an important tool used by biologists to magnify small objects to make fine details visible and is important tool for studying cellular structures. There are many different types of microscopes used in studying biology. These include the light microscopes (dissecting, compound bright field, and compound phase-contrast), electron microscopes (transmission and scanning), and atomic force microscope. Magnification is the ability of the microscope to produce enlarged images of the specimen, which is a function of its optical system. It is the ratio of enlargement (or eduction) between the specimen and its image (either printed photograph or the virtual image seen through the eyepiece). The image of an object can be magnified when viewed through a simple lens. By combining a number of lenses in the correct manner, a microscope can be produced that will yield very high magnification values. Normally, resolution is determined by the first lens (i.e. objective) while it, in addition to subsequent lens, enlarge the image to allow resolved detail to be easily visible. Magnification is obtained by a series of two lense systems. The lense system nearest the specimen, called objective, magnifies the specimen and produces a real image. The ocular or eyepiece lens system magnifies the real image , yielding a virtual image that is seen by the eye. The total magnification is equal to the product of the ocular magnification and objective magnification i.e.to calculate magnification we multiply the power of each lens through which the light from the specimen passes. For example: if the light passes through two lenses, an objective lens(4x) and an ocular lens(10x) multiply the 10X ocular value by the value of the objective lens ( 4X): 10 X 4=40, or 40X magnification. Microscopes are generally equipped with three types of objective lenses: Objective lens Focal length Magnification Low power objective lens 16mm 10X High dry objective lens 4mm 44X Oil immersion objective lens 1.8mm 95X Magnification Type of objective lens Eye piece Objective Total L P O (Diameters) 10X 10X 100X H D O (Diameters) 10X 44X 440X O I O (Diameters) 10X 95X 950X Magnification depends on: A. Working distance B. Focal length C. Magnification of objective lens D. Draw tube Resolution The resolution of a microscope is its ability to distinguish two objects, as separate, that are close together. Resolution is mainly a function of the wavelength of the source with which objects are radiated. The closer the two objects are, the easier it is to distinguish and recognize the distance between them. The shorter the wavelength the higher the resolution. The instrument design as well as the contrast properties of the specimen also contribute to set the resolution. Microscopes bring small objects "closer" to the observer by increasing the magnification of the sample. Since the sample is the same distance from the viewer, a "virtual image" is formed as the light (or electron beam) passes through the magnifying lenses. This characteristic of a microscope is a function of wavelength used and a characteristic of the lens system known as its numerical aperture (NA). Numerical aperture: It is the function of the diameter of the objective in relation to its focal length and the light bending power or refractive index of the medium between the specimen and the objective.

Resolving power = Wave length/ numerical aperture. The shorter the wavelength of light used, the smaller the structure visible. Working distance is the distance between the specimen and the magnifying lens. Effective Resolution of Optical Systems for Biological Specimens System Unaided Human Eye Light Microscope Transmission Electron Microscope Incident Wavelength m)( Visible (blue) light (0.4 ) Visible (blue) light (0.4 ) Electron beam (5 x 10-6) Resolution m)( 200 0.2 1 x 10-3 Fold-increase in Resolution 1 1 x 103 2 x 105

Depth of field is a measure of the amount of a specimen that can be in focus. Measurements in microscopy are usually expressed in the metric system. The Greek letter micron (µ) is applied to small measurements (thoud\sandths of a millimeter), producing the micrometer (symbolized as µm). General units in continuing biology careers include micrometer (µm, 10-6m), nanometer (nm, 10-9m), and angstrom (Å, 10-10m). Light microscopes were the first to be developed, and still the most commonly used ones. The best resolution of light microscopes (LM) is 0.2 µm. Magnification of Light microscopes is generally limited by the properties of the glass used to make microscope lenses and the physical properties of their light sources. The generally accepted maximum magnifications in biological uses are between 1000X and 1250X. The optical plan of a transmission microscope, either light or electron, is shown in the adjacent figure. Radiation from a source is focused (i.e. bent) by the condenser lens at or very near the specimen plane. The specimen is placed on a stage that may be moved perpendicularly to the radiation. Radiation passing through the specimen is focused by the objective lens to form an initial image and is refocused by a group of lenses (the projector in the electron microscope or the ocular in the light microscope) to form an image that will be finally placed on a receiver (i.e, photographic emulsion, digital imaging system).

Contrast To see an image of a specimen, contrast or variation of intensity within the image must result from the interaction between incident radiation and specimen. Contrast is both a function of the chemical composition of the specimen and of the microscope design. Illumination: One of the most critical aspects in optical microscopy is to ensure the specimen is illuminated with light that is bright, glare-free, and evenly dispersed in the field of view The easily available source of illumination is ordinary day light but usually artificial light is used. The light from illumination source is reflected into the sub stage condenser via the mirror located just below the

sub stage condenser. The flat surface of the mirror is used in microscopes equipped with a condenser and the concave side with microscopes not equipped with a condenser. Size of the cone of light passing into the microscope differs in each objective. With increasing magnification a larger cone of light must enter the objective. For this the iris diaphragm is opened fully. When low power objectives are used the Iris diaphragm is not opened fully since at these magnification details are more clear when the light is not too intense. When HPO are used the condenser should be in its top position where as when LPO are used this should be in its low position. Microscopes are of two types 1. SIMPLE MICROSCOPE --employs convex lenses 2. COMPOUND MICROSCOPE--employs two separate lense systems i.e. ocular and objective lenses for greater magnification Microscopes are of two types based on magnification 1. Light or optical. 2. Electron Anatomy of a compound microscope is shown in the adjacent figure Dark field microscope: Max. Useful Magnification is 1000-2000X. Appearance of specimen is generally unstained; appears bright or lighted in an otherwise dark field. Useful application of this is for microorganism's morphological features in living state and in fluid suspension The effect produced by the dark field technique is that of a dark back ground against with the objects are brilliantly illuminated. This is accomplished by equipping the light microscope with a special kind of condenser that transmits a hallow cone of light prom the source of illumination. For this the central portion of beam of light that comes from the lens is blocked with small strip of black paper. The peripheral rays while passing through the object is reflected out. Most of the light directed through the condenser does not enter the objective, so the field is essentially dark. However, some of the light rays will be scattered if the transparent medium contains objects such as microbial cells. This scattered or diffracted light will enter the objective and will reach the eyes. Thus the object or microbial cells will appear bright in dark microscopic field. Dark field microscopy is particularly valuable for the examination of unstained microorganisms suspended in fluid-wet mount and hanging ­drop preparations. Fluroscence Microscopy: Max. Useful Magnification is 1000-2000X . Appearance of specimen is Bright and coloured; colour fluorescent dye Useful application of this is for Diagnostic techniques where fluorescent dye fixed to organisms reveals the identity of those organisms. Many chemical substances absorb light. After absorbing light of a particular wavelength and energy, some substances will then emit light of a longer wave length and a lesser energy content. Such substances are called fluorescent and the phenomenon is termed fluorescence. This phenomenon is basis of the fluorescence microscopy. The microorganisms are stained with a fluorescent dye and then illuminated with blue light. Blue light is absorbed and green light is emitted by the dye. The function of the exciter filter is to remove all and allow the blue light only. The barrier filters blocks out blue light and allows green light to pass through and reach the eye. The barrier filters are selected based on dye used.

Phase- Contrast Microscope: Max. Useful Magnification is 1000-2000X. Appearance of specimen is Varying degrees of darkness. Useful application of this is for Examination of cellular structures in the living cells of larger micro organisms Eg.Yeasts,algae, protozoa,and some Bacteria. It is valuable for studying living unstained cells and is widely used in applied and theoretical biological studies. It uses a conventional light microscopy fitted with a phase- contrast objective and a phase contrast condenser. This special optical system makes it possible to distinguish unstained structures within a cell, which differ slightly in their refractive indices or thickness. In principle this technique is based on the fact that light passing through one material into another material of a slightly different refractive index and/ or thickness will undergo a change in phase. These differences in phase or wave front irregularities are translated into variations in brightness of the structures and hence are detectable by the eye. It is opposite to dark field microscopy. Here the back ground will be highly illuminated in which the objects depending on the density there illumination will be seen. Electron Microscope: Appearance of specimen is Viewed on fluorescent screen or photo frame with the help of camera . Useful application is for the examination of viruses and ultrastructure of microbial cells. The electron microscope provides tremendous useful magnification, because of the much higher resolution obtainable due to the extremely short wavelength of the electron beam used to magnify the specimen. The electron microscope uses electron beams and magnetic fields to produce the image, where as the light microscope uses the light waves and glass lenses. The resolving power of the electron microscope is more than 100 times that of the light microscope and it produces useful magnification up to X 4,00,000. Transmission electron microscope: For electron microscope the specimen to be examined is prepared as an extremely thin dry field on small screens & is introduced into the instrument at a point between the magnetic condenser and the magnetic objective. This point is comparable to the stage of the light microscope. The magnetic image may be viewed on a fluorescent screen through an airtight window or recorded on a photographic plate by a camera built into the instrument. Scanning electron microscope: The specimen is subjected to a narrow electron beam, which rapidly moves over (scans) the surface of the specimen. This causes the release at a shower of secondary electrons and other types of radiations from the specimen surface. The intensity of these secondary electrons depends on the shape and the chemical composition of the irradiated object. The secondary electrons are collected by a detector, which generates an electronic signal. These signals are then, scanned in the manner of a television system to produce an image on a cathode ray tube. The scanning electron microscope lacks the resolving power obtainable with transmission electron microscope but has the advantage of revealing a striking three-dimensional picture. The surface topography of a specimen can be revealed with a clarity and a depth of the field most possible by any other method. The great advantage of Electron Microscopy is tremendous resolution and magnification. LIMITATIONS OF ELECTRON MICROSCOPE: 1. Cells cannot be examined under living conditions as vacuum is created in the chamber. 2. Drying process may alter some of the morphology characteristics 3. Low penetration of electron beams necessitates use of thin sections for studying internal structures. 4. Considerable experience in microscope is required before a researcher can correctly interpret the results. 5. Very high initial cost

Microscope type

Major characteristics

Main applications, advantages and limitat

A light microscope Brightfield Uses visible light for illumination; specimen appears against a light background. Total magnification between 1,000-2,000 x Optimal resolving power: 0.2 um Magnification and resolution comparable to light microscope. Exploits differences in refractive indexes. Direct light passes through a ring-shaped diaphragm and the condenser, focusing light on the specimen. A plate in the objective lens captures diffracted light from the specimen. Both the direct and diffracted light rays are brought together at the eye Comparable to phase-contrast in terms of using differences in refractive indexes but gives greater resolution. Uses two beams of light that are split by prisms adding contrasting colors to specimen. Light waves are out of phase when they recombine and give a 3D appearance to specimen Uses condenser with an opaque disc that blocks out light in the center of the beam. Only light reflected by specimen enters objective lens. Specimen is bright against a dark background Uses ultraviolet light (short wavelength) that causes specimen to fluoresce Most commonly used microscope for cell study and cell counts. Specimens must typically be killed, fixed and stained; can lead to distortion. Small bacteria and viruses are not resolved Living cells and internal components are contrasted against the background giving greater definition and detail of cell structure. Cells need not be fixed or stained

Phase Contrast

Differential interference contrast (DIC)

Living specimens can be examined with a 3-D appearance and do not require staining

Dark-field

Fluorescence

Study of microbes that are not visible by bright field microscopy, or are distorted by staining. Major use in microbiology is the detection of living spirochetes such as Borrelia and Treponema spp. Visualization of cells or structures that fluoresce, either naturally or by the addition of special dyes called fluorochromes. Often used to identify cells or structures that react with specific antibodies; a technique known as immunofluorescence

An electron microscope Transmission Electron Uses electron beam (short wave length) instead of visible light. Total magnification: 10,000-100,000X. Resolution ~3 nm Magnets focus electron beams instead of condenser. Image formed on electronsensitive screen Detailed examination of cell ultrastructure and viruses Internal features may be observed through freeze fracture of cells. Specimens must be killed, dried and fixed; often leads to distortion and artifacts (see mesosome)

Scanning Electron

Similar properties to TEM. Electron bean scans surface of specimen. Total magnification: 1,000-10,000X. Resolution: 1-10 nm Uses laser to light one plane of a specimen at a time. Specimens stained with fluorochromes Uses small, pinhole aperture to eliminate blurring of image and improved resolution Metal probe scans surface "landscape" of specimen (rather like reading brail). Greater resolution than e.m. Metal and diamond probe moves along surface of specimen

Study of viruses and surfaces of cells

confocal microscope

Scanning Tunneling microscope Atomic microscope

Usually used in conjunction with a computer to produce 3D images and sections of cells and components. Images can be viewed in different orientations. Detailed views of computer chips and macromolecules such as DNA .No special preparation of specimen needed Detailed 3d images of biological molecules No special preparation of specimen needed.

PREPARATION FOR LIGHT MICROSCOPIC EXAMINATION General techniques Suspend organisms in liquid (The wet mount or hanging drop Method) Drying, fixing and staining of smears or films of the specimen The wet mount or hanging drop Method : Wet preparations permit examination of organisms in a normal living condition. A wet mount is made by placing a drop of fluid containing the organisms into a glass slide and covering the drop with a cover slip. To prevent evaporation and exclude the effect of air currents, the drop may be ringed with petroleum jelly or a similar material to provide a seal between the slide and the cover slip. A special slide with a circular concave depression is used for hanging drop method. A suspension of microbial specimen is placed on a cover slip, and then inverted over the conclave depression to produce a 'Hanging drop' of the specimen. Uses: The morphology of spiral bacteria is greatly distorted when these bacteria are dried and stained, so they should be examined in living condition. E.g. Spirochete that causes syphilis. To determine the motility of organisms. To observe changes during cell division and rate To observe spore formation and germination . To observe cell inclusion bodies like vacuoles, lipid material etc., Stained smears: bacteria. Used for the observation of the morphological characteristics of

Advantages: The cells are made more clearly visible after they are coloured. Differences between cells of different species and within the same species can be demonstrated by use of appropriate staining solution. This technique involves Preparation of the film or smear, Fixation, .Application of one or more staining solutions. Microbiological stains: Dyes are coloured organic compounds .Classification based on chemical behaviour. Acid dyes- Anionic- is one in which the charge on the dye ion is negative. Acid dyes generally stains basic cell components. Basic dyes- cationic- is one in which the charge carried by the dye is positive. It generally stains acidic cell components. e.g. Methylene blue MB+Cl Neutral dye- is a complex salt of a dye acid with a dye base. e.g. Eosinate of methylene blue. The ionic exchange which takes place during staining can be represented by the following equation, in which the MB+ cat ion replaces the Na+ cat ions in the cell. ( Bacterial cell-) (Na+) + (MB+ ) (Cl-) ( Bact.cell-) (Cl-) + (Na+Cl-) Mordant:- is a substance that increases the affinity or attraction between the ell and the dye; that is, it helps to fix the dye in the cell in some way. e.g.:- Acids, bases, metallic salts and iodine. It is difficult to wash out the stain after application of a mordant. Decolourising agent:- a substance that removes the dye from a stained cell. Counter stain:- is a basic dye of a different colour from the initial one. The purpose is to give the decolourised cells a colour different from that of the initial stain.

Simple staining:- The coloration of bacteria by applying a single solution of stain to a fixed smear is termed simple staining. The fixed smear is flooded with a dye solution for a specified period of time, after which this solution is washed off with water and the slide blotted dry. the cells usually stain uniformly. Differential staining:- staining procedures that make visible the differences between bacterial cells or parts of bacterial cell are termed "differential staining" technique. e.g. Grams staining. Preparing Slides for Staining When preparing a slide of a broth media simply put a couple of loopfuls on the slide and let it dry. If preparing a slide of culture from solid media, put a drop of sterile water on the slide, then add a small loopful of culture to the drop of water. Smear it around. The stain will be too thick if the culture very much, and it is difficult to distinguish the details. Allow the slide to air dry. and heat-fix the slide. To do this, hold the slide with a slide holder and pass through a flame 3 or 4 times.

The purpose of doing this is to: Kill the microbes on the slide;Coagulate the protoplasm of the cell to preserve the cell like it was when it was living,; And cause the microbes to adhere to the slide - other wise they wash off during the staining procedure. Donot heat-fix a capsule stain because it would melt the capsule! Grams Staining: One of the most important and widely used differential staining technique. Introduced by Christian Gram in 1884. It has greatest use in characterizing bacteria. This staining is not generally applicable for other groups of microorganisms such as protozoa and fungi; however yeasts consistently stain Gram+ve .The gram stain requires four different solutions: a base dye, a mordant, a decolorizing agent and a counter stain. Those organisms not readily decolorized --- retains the colour of initial basic stain Those organisms readily decolorized --- take the colour of the counter stain The cells that retain the basic dye following decolourization are called Gram +ve and those that are decolorized are Gram ­ve Principle The cell walls of Gram +ve bacteria because of their difference in composition (lower lipid content) become dehydrated during treatment with alcohol. The pore size decreases, permeability is reduced and the CV-I Complex cannot be extracted, so cells remain purple violet. The CV-I. Complex is trapped in the wall following ethanol treatment, which presumably causes a diminution in the diameter of the pores in the cell wall peptidoglycan In Gram negative cells the alcohol treatment extracts the lipids which results in increased porosity or permeability of the cell wall. Thus the crystal violet Iodine (CV-I) complex can be extracted and the Gram-ve bacteria is

decolorized Walls of Gram -ve bacteria have a very much smaller amount of peptidoglycan, which is less extensively cross-linked than that in the walls of Gram +ve bacteria. The pores in the peptidoglycon of Gram-ve remain sufficiently large enough after ethanol treatment to allow the CV-I Complex to be extracted. Procedure: Gram staining is a four-part procedure, which uses certain dyes to make a bacterial cell stand out against its background. The specimen should be mounted and fixed on a slide before proceeding to stain it. The reagents needed to successfully perform this operation are: Crystal Violet (the Primary Stain) ,Iodine Solution (the Mordant), Decolorizer (ethanol is a good choice), Safranin (the Counter-stain) and Water (preferably in a squirt bottle). Different staining procedures and appearance of the cells Staining Appearance Application Technique The Gram stain differentiates bacteria on the basis of structure Gram and composition of the layers of the cell wall. Upon completing Staining the stain, Gram positive bacteria appear purple and Gram negative bacteria appear pink Acid-fast This stain identifies acid-fast bacteria (bacteria that resist staining decolorizing by acid solution). The acid-fast bacteria are magenta pink when the stain is complete. All other bacteria will retain the counter-stain methylene blue. Distinguishes acid-fast bacteria such as Mycobacterium tuberculosis. Spore Staining Capsule Staining Demonstration of spore structure in bacteria as well as free spores. Some species of bacteria that form endospores are Clostridium and Bacillus Capsules are usually polysaccharide material (carbohydrate, or sugary stuff) that keeps bacteria from drying out and the capsule may help bacteria attach to a surface. It Demonstrates presence of capsules surrounding cells

Demonstrates presence of flagella surrounding cells. Several arrangements of flagella for bacterial cells: amphitrichous = two flagella, one at each end of the cell. lophotrichous = two or more flagella at one or each end of the cell. peritrichous = flagella over the entire cell. monotrichous = one flagella at one end of the cell. Cytoplasmic inclusion Staining Identifies intracellular deposits of starch, glycogen polyphosphates hydroxybutyrates and other structures Giemsa stain Particularly applicable for staining rickettsias and some protozoa Flagella Staining (Leiffson's)

BACTERIAL GROWTH Most bacteria divide by a process known as binary fission. Prokaryotes lack a mitotic apparatus. DNA is replicated bidirectionally and then is allocated into two new, genetically identical daughter cells. Cells may remain attached forming characteristic packets, chains or filaments. During the cell division mother cell divides into 2 daughters. Generation time is the average amount of time it takes for a species to mature and divide (double) under optimal conditions. Bacterial generation times vary considerably - from 10 min to 2 or 3 days. Average is 30 mins Increase in the population of cells is called a Culture. Growth of culture goes through 4 phases with time, when plotted on a graph They are: 1. Lag phase 2. Log phase 3. Stationary phase 4. Death phase Lag Phase : Lag is lack in cell division but not lack in synthesis of new protoplasm. Period where cells don't increase in number but are metabolically active and increasing in size- synthesizing structural components DNA, ribosome and enzymes to breakdown nutrients, replicating the chromosome, taking in and storing nutrients to be used for growth, expelling wastes, etc. Cytochemistry reveals an increase an increase in protein content per cell but not in proportion to nucleic acids. RNA synthesis is accelerated and varies with the length of generation time. DNA synthesis is not affected and DNA per cell is constant. Cell size may increase to 2 to 3 times and even more. Although part of the increase is due to higher water content. The increase in size is more apparent in all rod shaped bacteria than cocci. Bacterium may be busy but the growth curve is flat and all this activity is invisible. Organisms are adjusting to the environment and the length of time varies by species and conditions- from an hour or 2 to several days. Log or Logarithmic phase : Log Phase- is also called as period of exponential growth. During this phase one cell divides into 2, those 2 into 4, and these 4 into 16 etc. Log phase requires optimal conditions meaning whatever the bacteria need for nutrients must be available and the temperature must be right. Division is at a constant rate (generation time) but varies with species, temperature and media. Cells are most susceptible to inhibitors. The number of bacteria in a population in log phase can be calculated, if the starting number and the generation time is known. The formula is: Nf = Ni x 2n , where n = the number of generations, Nf = the final number in a population, and Ni = the initial number in a population Nutrients get used up and as they diminish growth slows. Also this heavy metabolism creates wastes, which are toxic or poisonous, and these toxins will build up. If replenishing the nutrients and getting rid of the wastes is there, then log could be continued indefinitely.

C. Stationary phase In a culture where nutrients are not renewed exponential growth continues only for few generations and growth rate starts to decline as a consequence of a) Approaching exhaustion of nutrients b) Accumulation of toxic metabolic products The population remains constant for certain period of time as during this phase dying and dividing organisms are at equilibrium meaning that the number of cells dividing is equal to the number dying and it looks like no growth on the curve. Cells are smaller and have fewer ribosomes. In some cases cells do not die but they are not multiplying. The nutrients continue to diminish, the wastes to build., in addition to pH changes, toxic waste and reduced oxygen. D. Death or Decline phase The population is dying in a geometric fashion so there are more deaths than new cells are produced, if indeed some cells are reproducing. Deaths are due to the factors in stationary phase i.e due to the depletion of essential nutrients and accumulation of inhibitory products in addition to lytic enzymes that are released when bacteria lyse. Most systems that grow bacteria, even nature, are closed systems meaning nutrients are not inexhaustible and wastes do build up. Grow slows until they reach Stationary phase- - Where the number of bacteria in a population dividing is progressively less than the number dying.

REQUIREMENTS FOR MICROBIAL GROWTH Food: "Food" is a very general and vague term with respect to microbial growth requirements. We will consider two broad meanings to the word "food." Chemical requirements Culture media used to grow microbes under laboratory conditions The majority of living cells need over 20 chemical elements in order to function. Six elements in particular are critical for building biological macromolecules and maintaining their structure and function. All life has the same basic nutritional requirements, which include: A Source Of energy. This may be light (the sun or lamps) or inorganic substances like sulfur, carbon monoxide or ammonia, or preformed organic matter like sugar, protein, fats etc. Without energy life cannot exist and quickly dies or becomes inactive. Hydrogen occurs in organic hydrocarbon molecules and in inorganic molecules such as water. A major role of hydrogen in biological systems is to maintain structure and conformation of macromolecules. Hydrogen bonds contribute to base-pairing between complementary DNA strands and to folding of proteins. A source of nitrogen. This may be nitrogen gas, ammonia, nitrate/nitrite, or a nitrogenous organic compound like protein or nucleic acid. Nitrogen is used in protein/ amino acid synthesis and nucleic acid synthesis. Nitrogen-containing bases help spell out information contained within the genetic code. A nitrogenous base ( adenine) is a building block of the energy-rich molecule, ATP. Peptidoglycan is also a nitrogen-rich substance. A source of carbon. This can be carbon dioxide or monoxide, methane, carbon monoxide, or complex organic material . Carbon is needed to build all organic compounds. All organisms require a carbon source for this purpose and many microbes also use some form of carbon as their energy source A source of oxygen. All cells use oxygen in a bound form and many require gaseous oxygen (air), but oxygen is lethal to many microbes. A source of phosphorous, sulfur, magnesium, potassium & sodium. Phosphorus is essential for nucleic acid synthesis and formation of phospholipids that contribute tto the integrity of cell membranes. Sulfur occurs in certain amino acids such as cysteine and methionine . Disulfide bonds help maintain tertiary and quaternary levels of protein structure and are crucial in maintaining the function of many proteins. A source of minerals like iron, zinc, cobalt etc. These are called TRACE metals that are required by some enzymes to function A source of calcium. Most cells require calcium in significant quantities, but some seem to only need it in trace amounts. A source of water. All life requires liquid water in order to grow and reproduce; Some resting stages of cells, like bacterial spores, can exist for long periods without free water, but they do not grow or metabolize. The sources of these various requirements define an organism, so a description of every organism should include this information. Many bacteria can synthesize every complex molecule they need from the basic minerals, but others, said to be fastidious, require preformed organic molecules like vitamins, amino acids, nucleic acids, carbohydrates; Culture Media : A culture medium is any material prepared for growth of an organism in a laboratory condition. It was not until the era of Robert Koch and his coworkers that Agar was introduced as a a common medium for bacterial growth. Agar is a complex polysaccharide derived from a marine red algae. Few bacteria possess enzymes capable of digesting agar and therefore it is useful as a solidifying agent and for isolating microbes in pure culture. Prior to the

advent of agar, gelatin was used as a growth medium. Unfortunately, many bacteria possess enzymes that liquify gelatin and therefore this medium is not useful for isolating pure cultures.. Pure Culture: A pure culture represents a single species (clonal in nature) of microorganisms.A clone is a genetically identical population of microbes that have descended from a single parent cell. Colonies are visible clones that have grown on solid media and represent millions of bacterial cells Distinctive characteristics of colonies should be noted such as: Pigmentation, Odor, Elevation, Margin (border of the colony), Consistency, such as mucoid, irridescence, filamentous, etc. Media vary in their chemical composition. In turn, the composition of the media determines microbial growth and the type of microbes that will grow. In Chemically defined media the exact chemical composition is known. Such media is often commercially prepared. Selective media : Contain chemicals which encourage growth of certain types of microbes but inhibits the growth of others. Differential media: allows different microbes to be distinguished on the basis of various biochemical reactions. Fermentation reactions involving the catabolism of various sugars are particularly useful biochemical tests Many media are both selective and differential, such as MacConkey agar and Mannitol Salt agar. Enrichment media: contains a rich supply of nutrients to encourage the encourage growth of microorganisms. A commonly used enrichment medium is blood agar. This medium is also differential and it permits detection of differnt patterns of hemolysis. Acidity (pH Requirements): The pH scale is a measure of the concentration of hydrogen ions (H+) in a solution. The more the hydrogen ions the more acidic a solution is and vice versa. pH values below pH 7 are considered acidic. pH values above pH 7 are considered alkaline. The majority of bacteria (including pathogens) grow best at neutral pH which is typically between pH 6.8 to pH 7.4. Extreme shifts in pH can damage cells by denaturing proteins and enzymes and by interfering with transport of ions across cell membranes. Foods that are acidic such as vinegars and pickles generaly are not favorable for microbial growth. Fungi such as yeasts and molds prefer slightly more acidic conditions and grow best between pH 5 to pH 6. Certain normal microbiota such as Lactobacilli (an acidophile) help establish pH conditions that are not favorable for the growth of pathogens. Some acidophiles such as those that metabolize sulfur can tolerate extremes as low as pH 1. On the other extreme, bacteria that prefer alkaline (basic) conditions) are known as alkaliphiles. Examples are Vibrio cholerae (prefers pH 9) and the soil bacterium Agrobacterium grows in soil with a pH of 12! When bacteria are cultured in a laboratory most will produce acidic metabolic wastes that would eventually build up and interfere with their own growth. For this reason, many culture media contain physiological buffers to stabilize the pH (a commonly used buffer is phosphatebuffered saline or PBS). Many culture media also contain pH indicators. Often these chemicals are dyes that change color when a shift in pH occurs. For example, phenol red is used in mannitol salt agar. This dye appears red above pH 6.8 but as microbes ferment the sugar in the medium and produce acids the dye turns yellow. Thus, shifts in pH can help determine the products of a biochemical reaction and/or determine if media is contaminated.

Temperature: Temperature is the most important environmental factor. The variation in organisms to grow and multiply at a temperature is due to the capability to synthesize cell constituents at specific temperature. At below minimum temperature the growth does not occur and above maximum temperature the growth ceases and still above organism dies. The effect of the temperature is due to the correlation between maximum growth and heat stability of ribosomes. Guanine and cytosine content and rRNA increase as maximum growth temperature increases. The fine structure of rRNA-protein interaction is the basis for the stability of ribosomes to heat. At higher temperature RNA gets denatured. All microbes have an optimum temperature range for growth Group Temperature Optima Psychrophiles Cold-loving organisms grow best at a temperature of 15 0C but some others can grow between 0 and 200C . Mesophiles 20 C and 400C. The optimum growth temperature is 370C (heat-loving) Capable of growing between between 45 and 600C . The optmum growth temperature is 55oC Organism that are capable of withstanding the pasteurization temperature i.e 63oC for 30 minutes. The optimum growth temperature is 370C Example(s) Most live in cold water and soil. Some can cause food spoilage as, they are capable of growing at refrigerator temperature (40C ). Ex. Pseudomonas sp. Most pathogens grow at or near human body temp of 370C. Mesophiles are responsible for most food spoilage Ex; Bacillus stearothermophilus

Thermophiles

Thermodurics

Micrococcus sp.,

In thermophiles the heat resistance is due to their ability to synthesize heat stable cell constituents in increased amount than other organisms. Ex. For the enzymes are Maleate dehydrogenase, Inorganic pyrophosphotase. Gaseous Requirements: There is great diversity among microbes with respect to their gaseous requirements. Obligate organisms absolutely require an environmental factor for their metabolism Facultative organisms are adaptable and can switch their metabolism depending on their environment. Obligate aerobes, grow only at the top often forming a film (scum) or pellicle, that floats on the top of the liquid while Obligate anaerobes grow at the bottom of a tube of liquid medium.. Facultative aerobes have the best of both as they are able to grow under both aerobic and anaerobic conditions. Microaerophilic bacteria require a little bit of oxygen, but too much is toxic and show growth in sub surface

Group Strict/Obligate Aerobe

Requirements Molecular oxygen required (requiring 21% ) oxygen for aerobic respiration. Glucose is completely oxidized to CO2 and H2O,. Microaerophiles are organisms that require a little oxygen but not as high an oxygen tension as found in the atmosphere. The metabolic process is similar to obligate aerobes, but requires 1-15 % oxygen. Also known as "Capnophilic" or carbon-dioxide loving. Adaptable organisms that use oxygen when present but can switch to anaerobic pathways in its absence. In the presence of O2, glucose is completely oxidized to CO2 and H2O as in obligate aerobes. In the absence of O2, glucose undergoes glycolysis to pyruvic acid, and then fermentation takes place. Can not grow in the presence of Oxygen as molecular oxygen is toxic to these organisms. Lack enzymes catalase and superoxide dismutase (SOD) to neutralize hydrogen peroxide and free radicals, respectively Glucose undergoes glycolysis to pyruvic acid, then fermentation or anaerobic respiration in which oxygen is not the final electron acceptor. Some organisms use nitrate, sulfate or carbonate

Example(s) Ex. Bacillus sp.,

Microaerophilic

Campylobacter jejuni

Facultative anaerobes

Escherichia coli and the large family of gram negative enteric rods

Strict or obligate Anaerobes

Clostridium sp.,

Moisture: All living cells, including bacteria, require an aqueous environment. However certain structures formed by microbes are environmentally resistant and can withstand desiccation for long periods. These include bacterial endospores. Osmosis is the tendency for water to move in and out of the cell to equalize it- cytoplasm more concentrated than pure water so water wants to move in if cell is suspended in pure water. A hypertonic environment has a high solute concentration with respect to the inside of a cell.

Consequently there is more water inside the cell with respect to the exterior and water tends to diffuse out of the cell. The result is the cell shrinks because of plasmolysis. Salting can be used to preserve foods and takes advantage of this important principle. The higher the water content of a food the more it is said to be "potentially hazardous." That is the high moisture content favors bacterial growth and is the food is prone to spoilage. Archaebacteria known as halophiles are salt-loving bacteria found in oceans and salt marshes that actively pump salt into their cells. Halophiles require at least 15% salt concentration (0.9%is intracellular concentration of most cells), prefer 25% and will tolerate a 30% concentration. Examples: Great Salt Lake, pickle spoilers Functions of water: Water serves as medium in which food for the organisms is dissolved for entering into the cell for metabolism Carries away waste products of metabolism from the cell Water is essential in metabolic reactions such as the hydrolysis of proteins, fats etc., It is the final end product of Oxidation of hydrogen from organic compounds to yield energy Water activity is a measure of unbound or free water available for biological and chemical reactions. Water activity is defined as the ratio of partial vapour pressure of water in food (P) to the vapour pressure of pure water (Po) at a given temperature. The term is designated by Scot (1953). aw = ERH/100 Relative humidity is the ratio of partial vapour pressure of water in air to vapour pressure of pure water at a given temperature Most bacteria do not grow below 0.90 aw, Most yeasts do not grow below 0.80 aw, Most molds do not grow below 0.70 aw, No growth of microbes and no spoilage of food below 0.65-0.70 aw aw Importance 0.90 Lower limit for bacterial growth 0.85 Many yeasts inhibited 0.80 Lower limit for the growth of yeasts 0.75 Lower limit for halophilic bacteria 0.70 Lower limit for xerophilic fungi 0.65 Maximum velocity of Maillard reaction 0.60 Lower limit for osmophilic and xerophilic yeasts and molds 0.55 Lower limit for life 0.40 Minimum oxidative velocity Minimum water activity for food borne pathogens Bcateria E. coli Staphylococcus aureus Salmonella sp., Bacillus subtilis Bacillus cereus Psedudomonas fluorescens Clsotridium sp., Min. water activity for growth 0.950 0.860 0.950 0.900 0.920 0.957 0.950

CONTROL OF MICROBIAL GROWTH Terminology related to control of microorganisms Cide means to kill and cidal means an agent that kills the organism e.g. Bactericidal. Germicide/Biocide A chemical agent that demonstrates killing power against various microbes Static or stasis Static Processes or chemical agents that inhibit bacterial growth but do not necessarily kill microbes.. Sepsis is the breakdown of living tissue by organisms and is accompanied by inflammation and pus formation. Antisepsis refers to the killing or removal of microbes on living tissues. Antiseptic is an agent applied externally on living tissues to kill or inhibit the growth of organisms. Antiseptics are milder than disinfectants. Disinfection Refers to the killing of microbes on inanimate objects or materials. Disinfectants are used on inanimate objects, not living tissues, to destroy harmful pathogens in their vegetative state. Disinfections can be achieved by physical and chemical antimicrobial procedures. Most are not sporicidal. Sanitation is the removal of organisms from a location by cleaning but does not imply sterilization. Usually used by the food industry. Reduces microbes on eating utensils to safe, acceptable levels for public health Sterilization Kills or removes all forms of life, including bacterial endospores Sterility indicates free from viable organisms. There are no degrees of sterility. Sterilization is the killing or removal of viable organisms Pasteurization A heating process that reduces the number of spoilage germs and eliminates pathogens in milk and other heat sensitive foods Chemical Methods of Microbial Control: Chemical agents are called disinfectants and germicides. a. Phenol and Phenolics - 5% aqueous solutions kill vegetative forms. Some of their derivatives are Lysol, Dettol, Cresol etc.. They alter the selective permeability of the cytoplasmic membrane of organisms. b. Alcohols - They are bactericidal and fungicidal but not sporicidal. They denature proteins and are solvents of lipids. 60 - 90% is the range for this activity. Water is required for the lethal effect. c. Halogens - Cause oxidation and direct halogenation of proteins thus inhibiting the activity of proteins e.g. hypochlorite, iodophores etc.. d. Surfactants - Surface active agents, good wetting and solubilizing agents e.g. Soap, detergent etc.. They have both hydrophilic and hydrophobic groups (ambivalent) to dissolve compounds. e. Alkylating agents - They substitute alkyl groups for hydrogen of reactive groups in nucleic acids and proteins, causing disruption of metabolic pathways e.g. Formaldehyde, glutaraldehyde etc..

Properties of an ideal antimicrobial agent

Fast-acting Acts against many microbes without harming tissues (selective toxicity) Penetrating power (improves if dirt and debris are first removed) Inexpensive Easy to prepare Chemically stable Inoffensive odor Not harmful to the environment

Any one agent is unlikely to possess all of the above qualities but it is helpful to assess an agent for as many of these characteristics as possible. Germicides are commonly used in hospitals, homes and elsewhere. Germicidal agents are also regularly used as preservatives of foods, cosmetics, vaccines and medical supplies. Many antiseptics and disinfectants act at multiple sites and targets on microbial cells and therefore do not show a great deal of selective toxicity as do many antibiotics. There is also broad spectrum in susceptibility to germicides among different microbes: A Summary of Mechanisms of Inactivation by Biocides Microbial Targets Vegetative bacterium: Cell wall Chemical(s)

Formaldehyde ; CRAs * ; Mercury Phenols Cytoplasmic coagulation Chlorhexidine ;Glutaraldehyde ;Hexachlorophene Mercurial compounds ;Silver salts ;QACS* Cell membrane: membrane potent Hexachlorophene ;Phenols ;Parabens electron transport Weak acids used as food preservatives such as be sorbic and proprionic acids Leakage of cell components Phenols;Chlorhexidine ; Alcohols ; QACs Nucleic acids Alkylating agents such as ethylene oxide gas Bacterial endospores: Spore core Glutaraldehyde; Formaldehyde Spore cortex CRAs ;Glutaraldehyde ;Nitrous acid Nitrates/nitrates act as food preservatives by preve germination of endospores Virus Envelopes Alcohols ;CRAs ;QACs ;Chlorhexidine Viral nucleic acid CRAs Capsid Glutaraldehyde;QACS ;CRAs ;Iodine Phenols ;Alcohols Fungus Cell membrane Chlorhexidine ;Alcohols ;QACS Cell wall Glutaraldehyde Nucleic acid Acridine dyes *CRAs = Chlorine releasing agents QACs = Quaternary ammonium compounds

Physical Methods of microbial control Heat Filtration Radiation Refrigeration Desiccation Heat Sterilization Heating is the most frequently used means to destroy microbes, being both economical and easily controlled. Successful heat sterilization must consider the degree of heat resistance demonstrated by a microorganism. Death from heating is an exponential function and occurs more rapidly as temperature increases. The nature of heat is also important: moist heat penetrates better than dry heat. One should know the heat susceptibility of an organism to know what heat treatment to apply. Thermal death time - The shortest time to kill a suspension of organisms at a specific temperature. Thermal death point - The lowest temperature to kill a suspension of organisms in a given time. Decimal reduction time (D Time) - Time to kill 90 % or 1 log unit of a population at a given temperature/ at a particular temperature. Moist heat : Boiling will kill most vegetative bacteria and viruses within 10 minutes. Bacterial endospores can survive boiling temperatures. Certain bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Moist heat acts by denaturation and coagulation of proteins. To kill vegetative bacteria, yeast and mold -- 80º C for 5 -10 minutes; Mold spores -- 80º C for 30 minutes; Bacterial spores -- 121º C, 15 lbs/sq. in. pressure for at least 15 minutes. Autoclaving: Uses steam heat under pressure to penetrate and kill microorganisms Steam under pressure - 121º C is required to kill spores. To achieve this temperature steam must be placed under pressure of at least 15 lbs/sq. in. in an autoclave. Steam produced at 15 psi heats to 121 C and will kill endospores after 15 minutes. There should be indicators to check for sterility. e.g. Tapes and culture test.. Denser materials or large objects will need to be autoclaved for longer periods. Boiling - Vegetative forms are killed in minutes. However, it is unreliable for killing spores.

Steam at atmospheric pressure - Steam has latent heat and at 100º C has 540 calories. Latent heat is released when steam condenses on a cold surface causing proteins to coagulate Pasteurization : Pasteurization is named for a process developed by Louis Pasteur as he looked for ways to prevent wine spoilage. It is important to note that Pasteurization is not synonymous with sterilization. This process employs heat to destroy pathogens and reduce the number of spoilage microbes in foods. Before this process was developed, milk was a common source of diseases such as tuberculosis, typhoid fever and brucellosis. Today, pasteurization is primarily used to prolong the shelf-life of various foods. Pasteurization employs the concept of equivalent treatments. As temperature increases less time is needed to kill a certain number of microbes that would take more time to kill at a lower temperature. Classical (bulk) pasteurization heated foods at 63 C for 30 minutes. Today, flash pasteurization or high temperature, short-time (HTST) methods are favored as they kill heat-resistant organisms more effectively and are less likely to alter the flavor of foods. The HTST methods involve continuos passage of foods past a heat exchanger. Pasteurization methods include: 72 C for 15 sec (HTST), 140 C for 15 sec (Ultra-High Temp), 149 C for 0.5 sec (UHT) Dry heat sterilization : Dry heat takes more time to kill microbes as it does not penetrate as well in the absence of steam.. Common uses of dry heat sterilization are flaming of inoculating loops and the sterilization of glassware in hot air drying ovens. Causes oxidation of cells. It is slow and requires a higher temperature for sterilization than moist heat. Filtration Filter sterilization is commonly employed for substances that can not tolerate heat. Membrane filters with pore sizes between 0.2-0.45 um are commonly used to remove particles from solutions that can't be autoclaved. Membrane filtration of beer eliminates spoilage germs and pasteurization is no longer needed. Filtered beer is permitted to be sold as "draft beer." Submicron filters are also being marketed for removal of protozoan cysts from drinking water. Radiation One of the most controversial areas of microbial control involves the use of radiation. The controversy largely results from a lack of understanding of the different types and uses of radiation. The effects of types of radiation depend on three important factors: Time (of exposure), Distance (from the source) , Shielding (how penetrating is the radiation) Irradiation of various food has been used in the U.S since the 1960's and has been used to sterilize foods such as herbs and spices. Nonionizing radiation : Includes microwaves and ultra violet radiation. Microwaves are not particularly antimicrobial in and of themselves, rather the killing effect of microwaves are largely due to the heat that they generate. Microwaves are not recommended for cooking large volumes or thick cuts of meat as the heat may not penetrate the foodstuffs sufficiently. UV radiation is of short wavelength, between 220 and 300 nm and is not very penetrating. UV can be stopped by glass, a sheet of paper, or the top layers of your skin! UV rays can kill exposed microbes by causing damage to their DNA. UV radiation is useful for the disinfection of exposed surfaces such as laboratory hoods. However, the usefulness of UV radiation is limited by the fact that certain microbes possess DNA repair mechanisms and can recover after exposure to this kind of radiation. In addition, UV light does not penetrate organisms well that are protected in mucus or debris. Ionizing radiation : Includes gamma rays and X rays which are highly penetrating to cells and tissues and have potent antimicrobial effects. After colliding with a target, ionizing radiation

generates ions and other reactive species from molecules including hydroxyl (free) OH- radicals. These free radicals can cause irreversible breaks in DNA, proteins and enzymes. Radiation is currently used for sterilization by the medical supply and food industries. Irradiation is the only effective means known to eliminate E. coli 0157 from meat. This process can also eliminate the food pathogens, Listeria , Campylobacter and Salmonella Refrigeration Refrigeration will slow down and inhibit the growth of most microbes but it will not kill them! Note: some spoilage germs and psychrophiles can continue to replicate at cooler temperatures. Organisms can be maintained viable at -80 C if suspended in glycerol or DMSO. Desiccation Desiccation of microbes is a very useful means of food preservation and to control the growth of spoilage germs and pathogens. Foods that have a high water activity are most subject to spoilage and typically must be refrigerated or frozen. Numerous foods are preserved by adding salt or sugar to decrease the water activity of the foods. This process creates hypertonic conditions and causes water to leave bacterial cells (plasmolyze). Salting of foods does not protect against all potential pathogens. Many fungi are halophilic as is Staphylococcus aureus, a common source of bacterial food poisoning.

MICROBIAL METABOLISM

Metabolism is defined as the sum of all chemical reactions occurring within a living organism. Catabolic reactions are energy-releasing (exergonic) reactions, which break down more complex molecules, usually by hydrolysis, into simpler components. The chemical processes of digestion typically occur by this route. Anabolic reactions are energy-requiring (endergonic) and build more complex molecules, usually by condensation, from subunit components. The energy for anabolic reactions is provided by catabolic reactions; they are always linked. Energy is ultimately stored in the form of the energy -rich molecule, ATP. Prokaryotes can be organized into groups based upon their nutritional and metabolic needs which are extremely diverse. Traditionally, these groupings have been based on two main criteria: * The nature of the energy source * The nature of the carbon source used for building organic, biological macromolecules Carbon source: from organic compounds made by other organisms Chemoheterotrophs Energy source: from oxidation of organic compounds Examples: most bacteria, protozoa, all fungi and animals Carbon source: CO2 Energy source: oxidize inorganic compounds which are used to fix CO Chemoautotrophs Examples: nitrifying, hydrogen, sulfur and iron-utilizing bacteria. Archaea which live among hydrothermal ocean vents Carbon source: from organic compounds made by other organisms Photoheterotrophs Energy source: light Examples: green and purple nonsulfur bacteria Carbon source: CO2 Photoautotrophs Energy source: light Examples: cyanobacteria, green and purple sulfur bacteria, algae, plan The four main families of small organic molecules in cells. They form the monomeric building blocks, or subunits, for most of the macromolecules and other assemblies of the cell. Some, like the sugars and the fatty acids, are also energy sources.

Metabolic pathways used by bacteria to generate their energy requirements and to illustrate this follow the fate of the energy in glucose as it is catabolized by: Glycolysis (a preparatory, anaerobic process) The Krebs cycle (generation of energy-carriers) The electron transport system (to produce ATP either by aerobic or anaerobic conditions) Fermentation pathways (following glycolysis)

Before following these pathways in more detail we need to review the major steps involved in energy production: Bacteria use three main mechanisms of PHOSPHORYLATION to produce ATP Energy released from certain metabolic reactions can be trapped to form ATP from ADP and a phosphate group by a process called phosphorylation. An enzyme called ATP synthetase catalyzes this chemical reaction. Substrate level phosphorylation: occurs when ATP is formed directly by the addition of a phosphate to ADP. Occurs both in glycolysis and the Krebs cycle. Oxidative phosphorylation: energy in the form of electrons is released stepwise from oxidized organic compounds (e.g. glucose) to electron carriers (usually NAD+ or FADH). Electron carriers enter a membrane-associated electron transport system (ETS). The ETS and ATP synthetase occur on the inner mitochondrial membrane of eukaryotes and on the plasma membrane of prokaryotes. Electrons are ultimately donated to final electron acceptors to form ATP. The electron acceptors include: Molecular oxygen (aerobic respiration), An inorganic molecule other than oxygen (anaerobic respiration)

Photophosphorylation: occurs only in photosynthetic organisms which trap light energy by photosynthetic pigments and convert it to the chemical energy of ATP. This process involves an electron transport system. Photosynthetic bacteria include cyanobacteria and the green and purple sulfur bacteria. Cyanobacteria lack chloroplasts but possess photosynthetic membranes called thylakoids. The thylakoids possess the photosynthetic pigments chlorophyll a, carotenoids and phycobiliproteins. The green and purple sulfur bacteria possess bacteriochlorophyll. Oxidation and Reduction Oxidation and reduction reactions (also known as REDOX reactions) are always coupled in biological systems. Oxidation reactions release energy and Compounds that contain the greatest amount of stored chemical energy are hydrocarbons such as fats and lipids. In biological systems, oxidation typically involves: the loss of hydrogen atoms (equivalent to protons) from a substrate loss of hydrogen atoms are known as dehydrogenation reactions electrons are typically lost together with hydrogen atoms the addition of oxygen is also termed oxidation an "oxidized" molecule has given up energy. For example, the energy carrier molecule NAD+, is an energy-deficient form of an electron carrier as it has given up a hydrogen and two electrons. Reduction reactions harness chemical energy and Reduction involves: the gain of electrons and hydrogen atoms by a substrate.: the loss of oxygen is also termed reduction a "reduced" molecule is energy rich. For example, NADH picks up 2 energetic electrons and a hydrogen atom. These reactions are always coupled, as the electrons lost from an oxidized molecule have to be transferred to another. A source of electrons, or electron donor, is referred to as a reducing agent, while the electron acceptor is the oxidizing agent as it oxidizes some other molecule and becomes reduced in so doing. GLYCOLYSIS Glycolysis literally means "sugar splitting." It is the most common pathway for the breakdown or oxidation of glucose and is typically the first stage in carbohydrate catabolism. Glycolysis (Embden-Meyerhof Pathway) is used by eukaryotic cells, many anaerobic and facultatively anaerobic bacteria. Involves 10 distinct chemical reactions, each catalyzed by different enzyme. Glucose is phosphorylated, cleaved and rearranged into two - 3 carbon compounds that ultimately form 2 pyruvic acids, is accompanied by ATP formation through substrate level phosphorylations. and in the process yields 2 ATP and 2 NADH

During a preparatory stage, glucose becomes energized by the addition of two phosphate groups. This step also alters the conformation of glucose and renders it incapable of leaving the cell. Two molecules of ATP are used to energize glucose. The resulting fructose 1,6 diphosphate is subsequently split into two, three-carbon compounds. Substrate level phosphorylation yields 2 molecules of ATP directly from ADP. As two phosphorylated 3-carbon molecules are formed from each molecule of glucose 4 molecules of ATP are actually formed at this point. There is a net yield of 2 ATP per glucose entering glycolysis. For each three carbon compound a molecule of NAD+ is reduced to a molecule of NADH. The energy rich molecules of NADH can enter the electron transport system so that more ATP can be generated by oxidative phosphorylation. Two molecules of pyruvate result which. Pyruvate may be fermented or enter the Krebs cycle depending on the bacterium.

THE KREBS CYCLE The purpose of the Krebs cycle is to harness useful energy in the form of electrons, carried by the reduced coenzymes, NADH and FADH2. Key events of the Krebs cycle: The reaction of acetyl CoA with oxaloacetate starts the cycle by producing citrate (citric acid). In each turn of the cycle, two molecules of CO2 are produced as waste products, plus three molecules of NADH, one molecule of GTP, and one molecule of FADH2.

Each molecule of pyruvate from glycolysis is decarboxylated (loss of CO2) to become a 2 carbon compound (acetyl group) which attaches to coenzyme A (acetyl CoA). The 2 carbon acetyl groups enter the cycle and pass through a series of reactions whereby chemical energy is released stepwise.

The

carbons that enter the Krebs cycle are oxidized to CO2 and are eliminated as waste For each turn of the Krebs cycle four pairs of electrons are transferred to coenzymes; 3 pairs reduce NAD+ to form three molecules of NADH and 1 pair of electrons reduce FADH to FADH2. Energy is extracted from these coenzymes as they enter the electron transport system. For each turn of the Krebs cycle (two turns), substrate level phosphorylation generates the equivalent of one ATP.

Fermentation Fermentation An additional step to glycolysis where pyruvic acid accepts electrons from NADH to form lactic acid, ethanol, butanol etc. ( depending on the organism), while generating NAD+ for glycolysis. Fermentation produces no more ATP than the 2 ATP formed in glycolysis. Fermentation can have a variety of meanings, ranging from informal to more scientific definitions. The various meanings of fermentation are Any spoilage of food by microbes. For example, the spoilage of wine to vinegar. This is a very general usage of fermentation Any process that produces alcoholic beverages or acidic dairy products (again general use) Any large scale microbial process occurring with or without air (industrial use) Any energy-releasing process that occurs only under anaerobic conditions (more scientific) Any metabolic process that releases energy from a sugar or other organic molecule, does not need oxygen or an electron transport system, and uses an organic molecule as the final electron acceptor.

Fermentation pa End products Lactic acid (HomoLactic acid (2 molecules) Heterolactic Alcohol Lactic acid, ethanol and CO2 Ethanol and CO2

Examples Lactobacillus, Enterococcus, Streptococcus spp. Pathway can result in food spoilage Leuconostoc Used in fermented milk production Saccharomyces (yeast) Important in production of alcoholic beverages, bread and gasohol Proprionibacterium freudenreichii gives flavor to and produces holes in Swiss cheese

Proprionic acid

proprionic acid and CO2

Butyric acid

Butyric acid, butanol, acetone, Clostridium spp. produce butyric acid that isopropyl alcohol and CO2 causes butter and cheese spoilage Butanediol and CO2 Butanediol produced by Enterobacter, Serratia, Erwinia and Klebsiella. The intermediate, acetoin, is detected by the VP test used together with the MR test often to distinguish Enterobacter from Escherichia coli (VP-).

Butanediol

Mixed acid

Ethanol, acetic acid, lactic Variety of acid products. Typically carried acid, succinic acid, formic acid out by members of the Enterobacteriaceae and CO2 including E. coli, Salmonella and Shigella pathogens. Products detected by reaction with methyl red pH indicator. Certain Archaea.

Methanogenesis Methane and CO2 Some other key points that to keep in mind are:

A complete fermentation pathway begins with a substrate, includes glycolysis and results in various end-products. The different fermentation pathways typically are named for the end products that are formed. As far as an energy is concerned, fermentation does not generate ATP directly but recycles a limited amount of NAD+ back into glycolysis to keep glycolysis going. Recall that each pass through glycolysis generates 2 ATP molecules by substrate level phosphorylation. All fermentation pathways are anaerobic. Cells that are capable of both respiration and fermentation will typically use respiration when possible. Respiration yields more energy from a lot less substrate

AEROBIC RESPIRATION In prokaryotes it takes place in the cytoplasmic membrane, while in eukaryotes it occurs in the inner mitochondrial membrane. Pyruvic acid loses a carbon as CO2 and becomes acetyl. At the same time NAD+ is reduced to NADH and is transferred to the electron transport chain. Acetyl combines with Co enzyme A to form Acetyl CoA . Acetyl enters the Krebs cycle to yield 2 CO2, 3 NADH, 1 FADH2 and 1 GTP. NADH and FADH2 are transferred to the electron transport chain, where each NADH produces 3 ATP and each FADH2 produces 2 ATP.

Energy Profile of Aerobic Respiration SOURCE 2 NADH ( glycolysis) Substrate phosphorylation (glycolysis) 2NADH (oxidation of pyruvic acid) 6 NADH (Krebs cycle) 2 FADH2 (Krebs cycle) 2 GTP (Krebs cycle)

AMOUNT OF ATP 6 2 6 18 4 2

Total

38

A total of 38 atp is produced by aerobic respiration in prokaryotes ELECTRON TRANSPORT CHAIN

The carriers of the electron transport system occur on the plasma membrane of bacteria. Energy carrying molecules of NADH and FADH2 donate their electrons to a series of coenzymes. The electrons are ultimately donated to a terminal electron acceptor, which in the case of aerobic respiration, is oxygen. The electrons are then are reunited with protons to form water. Each carrier is reduced as it picks up electrons and oxidized as it passes them on (gives up energy) Most ATP generated by aerobic respiration is by oxidative phosphorylation: occurs at 3 sites at ETS so ADP is phosphorylated to ATP FAD enters lower down the ETS and only 2 sites are utilized for this coenzyme

CHEMIOSMOSIS

This theory was formulated by the British biochemist, Peter Mitchell, who won the Nobel prize 1978. This theory proposes the mechanism whereby the electron transport system siphons off electrons from the Krebs cycle and uses the energy to produce ATP by oxidative phosphorylation. A molecule of NADH entering the ETS donates a pair of electrons to the first coenzyme in the pathway. For each molecule of NADH, three pairs of hydrogen ions (protons) are pumped across the inner mitochondrial membrane into the intermembrane space (of eukaryotes). This generates a concentration and electrical gradient between the membrane space and mitochondrial matrix. Protons diffuse back down this concentration gradient through channels in the inner membrane into the mitochondrial matrix. ATP synthetase, associated with the channels, phosphorylates ADP to ATP. Each pair of hydrogens drives formation of a molecule of ATP. A rule of thumb is that for each molecule of NADH entering this system, three molecules of ATP are produced. FADH2 enters the ETS further down the pathway at coenzyme Q and donates two pairs of electrons. Consequently, each molecule of FADH2 only gives rise to 2 ATP molecules

BACTERIAL GENETICS Genetics is the study of the inheritance (Heredity) and the variability of the characteristics of an organism. Inheritance concerns the exact transmission of genetic information form parents to their progeny. Variability of the inherited characteristics can be accounted for by a change either in the genetic makeup of a cell or in environmental conditions The study of Microbial Genetics has contributed much to the genetics of all organisms. Bacteria became the principal experimental tools for unraveling the basic knowledge of genetics at the molecular level. The advantages in the use of bacteria for genetic experiments: 1. Bacterial cultures contain millions of individual cells. So by appropriate selection techniques, rare genetic events can be discovered. 2. Bacteria contains a single chromosomes thus a change in the genetic material results in an immediate observable change in characteristics 3. The rapid growth rate of microbes or ease of growing bacteria in the lab is advantageous. The inheritance of characteristics and variability: A characteristic of all forms of life, is the general stability or "likeliness" in the characteristics of progeny & parent. However in addition to the inheritance of characteristics there is variability or change expressed in the progeny. These changes are associated with two fundamental properties of the cell or organism namely a) The genotype b) The phenotype Phenotype is the expression of the genotype in observable properties or characteristics of the cell or organism. The genotype refers to the genetic constitution of the cell. Genotype of a cell remains relatively constant during the growth. However it can change by mutation. This change can result in an alteration in the observable properties or phenotypes of the cells. So the genotype represents the heritable total potential characteristics of a cell where phenotype represents the characteristics expressed. Chromosome: This consists of the entire double Helix DNA macromolecule occurring in the nucleus as a fine thread to which the genes are attached. In bacteria the chromosomes is a single largely circular DNA molecule. In some viruses the chromosome is composed of RNA. The molecular weight of the chromosomes of the E.coli are 2.5X109 daltons and contain about 3800 genes. In bacteria, the chromosomes are always haploid i.e., they are unpaired. In large animals, they are Diploid (46 chromosomes). They have paired homologous chromosomes. Structure of D N A: D N A from any cell is a long rope like molecule composed of two stands, each wound around the other to form a double helix. James Watson and Francis crick first proposed the model for this structure in 1953. They received noble prize in 1962. Each stand of the DNA helix is made up of nucleotides linked together to from a chain i.e., polynucleotide. DNA is long polymers of only 4 nucleotides, adenine, guanine, cytosine and thymine (or uracil for RNA). Nucleic acids are polymers of nucleotide residues joined together by sugar phosphate esterifications. The nucleotide structure can be broken down into 2 parts. The sugar-phosphate backbone and the base. All nucleotides share the sugar-phosphate backbone. Nucleotide polymers are formed by linking the monomer units together using oxygen on the phosphate, and a hydroxyl group on the sugar.

DNA is a polymer of nucleotides. They have three components. 1. 2. 3. A cyclic five carbon sugar: DNA has 2 deoxy ribose. Nitrogen base: They are two types; Purines and pyrimidines. They are attached to 1' carbon atom of the sugar by N-glycosidic bond. Purines are Adenine and Guanine. The Pyrimidines are Cytocin, Thymine. DNA contain A,G and C T . Phosphoric acid: Phosphate group is attached to 5' carbon of the sugar by phosphoester linkage.

Nitrogen base with sugar molecule is known as Nucleoside. Nitrogen base with sugar molecule and phosphate group is known as Nucleotide. A, T, G and C are capable of being covalently linked together to form a long chain. The 3'hydroxyl group on the ribose unit, reacts with the 5'-phosphate group (phophodiester bond) on it's neighbor to form a chain. The ratio of A: T or G: C in double stranded DNA is always 1:1. The amount of purine is equal to the amount of Pyrimidine. The DNA of each species shows a characteristic composition that is not effected by age; growth; conditions; environmental changes etc. The molar ratio of A+T/G+C indicates a characteristic composition of DNA of each species. Human 1.52 E.coli 0.93 Sheep 1.36 Staphylococcus aureus 1.5 Wheat 1.19 Clostridium perfringens 2.7 Micrococcus luteus 0.35 Base Pairing Adenine is capable of forming hydrogen bonds with thymine and cytosine can base pair with guanine. Adenine forms two hydrogen bonds with thymine, cytosine forms 3 with guanine.

DNA Structure

The prime features of the structure are:

The length of each pair is about the same and helix can fit into a smooth cylinder Two strands of DNA wrap around each other The sugar-phosphate backbone is on the outside The bases are perpendicular to the axis of symmetry The bases are in the middle Two bases in each base pair lie in the same plane. The strand polarities are opposite each other It is a right-handed helix i.e each strand appears to follow a clockwise path moving away from a viewer looking down the helix axis. Plane of each pair is perpendicular to helix axis The base pairs are rotated with respect to each to produce 10 pairs per helical turn There is a 2-fold axis of symmetry There is a wide (major) and a narrow (minor) groove between the backbones on opposite strands Distance between two bases on one of strand is 3.4 A0. One Helical turn is equal to 10 bases. Therefore the distance of Helical turn is 34 A0 The two strands are antiparallel in DNA i.e. 3' OH terminus of one strand is adjacent to 5' ­ p terminus of the other. Thus in a linear double helix there is one 3'-OH and 5' -p terminus at each end of helix

RNA: RNA is similar in structure to DNA, except that uracil (U) takes the place of thymine in the molecule and the ribose unit on each sugar contains a hydroxyl group. RNA serves 3 functions in ribosomes. These functions center around translating the genetic information in DNA into protein. Types of RNA: Messenger RNA (mRNA): This helps in transcription. The sequence of one strand of the chromosomal DNA is enzymatically transcribed in the form of a single strand of mRNA. Now mRNA reaches ribosome where it serves as a template for the sequential ordering of amino acids during biosynthesis of proteins. Messenger or mRNA is a copy of the information carried by a gene on the DNA. The role of mRNA is to move the information contained in DNA to the translation machinery.

Transfer RNA (tRNA): Acts as carriers of specific individual amino acids during synthesis of proteins on ribosomes. Ribosomal RNA (rRNA): constitutes about 65% mass of ribosomes Differences between DNA and RNA

RNA usually forms intramolecular base pairs The information carried by RNA is not redundant because of these intramolecular base pairs. the major and minor grooves are less pronounced The structural, informational adaptor and information transfer roles of RNA are all involved in decoding the information carried by DNA

N bases: The nitrogen bases of DNA are Adenine, Guanine Cytosine, Thymine while in RNA they are Adenine, Guanine Cytosine, Uracil Sugar: DNA has 2-deoxy ribose while RNA has D-Ribose Monomers: DNA is made up of Deoxy ribo nucleotides. RNA is made up of ribo nucleotides Location: DNA is found in nucleus (entirely or almost entirely). Extra chromosomal bodies do occur (Plasmids) RNA is found in nucleus and in cytoplasm but mostly in cytoplasm Structure: DNA is mostly double stranded except in few viruses where it is single stranded and RNA is mostly single stranded except in few viruses Function: DNA is the genetic material of all animals, plants and microbes and some viruses while RNA is the genetic material in most viruses. RNA plays important role in synthesis of biomolecules. Types: DNA exists as chromosome or plasmid There are basically three types of RNA i.e. Messenger RNA (mRNA), Ribosomal RNA (rRNA) Transfer RNA (tRNA) All these occur in multiple molecular forms. Cell contains 2 to 8 times as much RNA as DNA MUTATIONS: A Gene is a functional unit of inheritance. It specifies the formation of a particular `polypeptide' as well as various types of RNA. Each gene consists of hundreds of nucleotide pairs. For instance, if polypeptide chain contains 300 amino acids, then the gene coding for this polypeptide must contain 900 base pairs (three pairs for each coding amino acid). It has been estimated that the bacterial chromosome has the capability to code for approximately 3,500 different proteins. Any gene is capable of changing or mutating to a different form so that it specifies formation of an altered or new protein which may in turn change the characteristics of the cell Mutant: An organism with a changed or new gene. Mutagen: Any agent that increases the mutation rate is called a mutagen. Mutation: Bacterial multiplication in which a gene becomes altered or lost so that daughter cell and its progeny in a genetic constitution is different to that of a cell is called mutation or a stable change of a gene such that the changed condition is inherited by offspring cells. In the normal process of reproduction it is usual for the genes to be exactly replicated. In this way the daughter cells of each bacterial generation maintain the properties of the parent cell. The factors in the bacterial environment may be responsible for a mutation occurring in which case the activity of the gene may be modified by extrinsic cause. These mutants can be detected if they survive in the environment in competition with parent cell. Often this may not occur and mutant strain will the die out. Some time they may survive and grow into easily detectable properties.

Max. Delbruck and Salvador Luria believed that bacteria have stable hereditary mechanism. In 1943, they performed an elegant experiment that proved the point for which they received noble prize. E.g. Bacteriophages that are capable of killing bacteria. When susceptible bacteria are exposed to a phage, some of the bacterial cells survive and they and their descendants or progeny are resistant to the phage. This is due to the result of mutation and these scientists proved statistically by their fluctuation tests. Types of Mutations: Spontaneous mutation: DNA is relatively more stable but occasionally a gene become altered either in its chemical structure or translocated with respect to other genes in the bacterium. In either event the activity of the gene will be modified by an intrinsic cause resulting in the daughter cell becoming mutants. This occurs during normal growth conditions. The rate is 10-6 to 10-10 per generation. Ex. Development of resistance or sensitivity to toxic agents Increased virulence of pathogens. Induced mutation: Mutations obtained by the use of mutagen. Mutagenic agents exert (directly or indirectly) their effect by reacting with DNA. Ex. X rays, UV rays, Carcinogenic chemicals. The rate is 10 to one lakh times more frequent than spontaneous Point mutations: They occur as result of the substitution of one nucleotide for another in the specific nucleotide sequence of a gene. i) ii) iii) iv) v) Transition type of point mutation: The substitution of one purine for another purine or one pyrimidine for another pyrimidine is called transition type of mutation. Tran version: It is the replacement of purine by a pyrimidine or vice-versa. This base pair substitution may result in one of three kinds of mutations affecting the translation process. Mis-sense mutation: The altered gene triplet produces a codon in the mRNA which specifies on Amino acids different from the one present in the normal protein. This mutation is called Missence mutation.E.g. Sickle cell anaemia in Human Non-sense mutation: The altered gene triplet produces a chain terminating codon in mRNA, resulting in premature termination of protein formation during translation. This leads to formation of incomplete polypeptide which is non-functional. Neutral mutation: The altered gene triplet produces a mRNA codon, which specifies the same amino acid for all amino acids, because the codon resulting from mutation is a synonym for the original codon.

Frame shift mutations: These mutations resulting from an addition or loss of one or more number of nucleotides in a gene and are termed insertion or deletion mutations, respectively. This results in the shift of the reading frame and proteins thus produced are non functional. Phenotypes of bacterial mutants: 1. Exhibit an increased tolerance to inhibitory agents, particularly antibiotics. 2. Demonstrate an altered fermentation ability i.e increased or decreased ability to produce the end products. 3. Nutritional requirements i.e., some may require no complex medium for growth (Auxotrophic mutants). 4. Exhibit changes in colonial form and ability to produce pigments. 5. Shows a change in the surface structure and composition of the microbial cell. (Antigenic mutants). 6. Resistant to be action of bacteriophages.

7. Shows changes in morphological features like spore formation, capsule or flagella formation etc. 8. Looses a particular function but retains the intracellular enzymatic activities to catalyze the reactions of the function . E.g. Loss of permease (Cryptic mutants) 9. Mutants yield a wild type phenotype under one set of conditions and a mutant phenotype under another.(Conditionally expressed mutants). BACTERIAL RECOMBINATON: Genetic recombination is the formation of a new genotype by rearrangement or reassortment of genes following an exchange of genetic material between two different chromosomes, which have similar genes at corresponding sites. These are called Homologous chromosomes and are from different individuals. Progeny from the recombination have combinations of genes different from those that are present in the parents. Different types of gene transfer in bacteria: Conjugation Transduction Transformation protoplast fusion and electrification. In bacterial recombination, the cells do not fuse and usually only a portion of the chromosome from the donor cell (Male) is transferred to the recipient cell (Female). The recipient cell thus becomes a 'merozygoate' a zygoate that is a partial diploid. Once merozygoate transformation has occurred, recombination can takes place. Mechanism : Inside the recipient cell the donor DNA fragment is positioned alongside the recipient DNA in such a way that homologous genes are adjacent. Enzymes act on the recipient DNA, causing nicks and excision of a fragment. Then the donor DNA is integrated into the recipient chromosome in place of the excised DNA. The recipient cell then becomes the recombinant cell because its chromosome contains DNA of both the donor and recipient cell Bacterial Conjugation: Luria and Delbruck had demonstrated in 1943 that bacteria have a stable hereditary system. Lederberg and Tatum (1946) achieved the first demonstration of recombination in bacteria in E.coli. They combined two different strains of E.coli and gave them an opportunity to mate. Strain 'A' grow only in the presence of Tryptophan, whereas strain 'B' grow in the presence of Histidine only. When both are combined the recombinant has grown in the absence of both the amino acids in the medium. The cell to cell contact is required for bacterial conjugation. It involves the transfer of some DNA from one cell to another followed by separation of the mating pair of cells. It is possible that large segment of the chromosome or in special cases the entire chromosome is transferred but in other recombination only a fragment is transferred. Sex factor: In E.coli male cells contain a small circular piece of DNA, which is in the cytoplasm, and not a part of chromosomes is called the sex factors or F factor (F Fertility). These cells are referred to as F+ and are donors in mating. The

female cells lack this factor and are labelled F -. They are recipient cells. High frequency recombination strains or Hfr strain: Hfr strains arise from F + cells in which F factor becomes integrated into the bacterial chromosome. They differ from F + cells in that the F factor of the Hfr strain is rarely transferred during recombination. Thus in Hfr X F- cross, the frequency of combination is high and the transfer of F factor is low; in F+ X F- cross the frequency of recombination is low and the transfer of F factor is high. Transduction: Transduction is the transfer of a portion of DNA from the bacterium i.e., donor to another i.e., recipient by a bacteriophage serving as a `vector'. Discovered by Zinder and Lederberg in 1952. Most bacteriophage undergoes a rapid lytic growth cycle in their host cells. They inject their nucleic acid usually DNA into the bacterium wherein it replicates rapidly and directs the synthesis of new phage proteins. With in 10 to 20 minutes depending on the phage the new DNA combines with new proteins to make whole phage particles, which are released by the destruction of cell wall and lysis of cell. Some phages are ordinarily don't lyse the cell "Temperate phage" carrying DNA that can behave as a kind of episome. Now if the viral DNA gets integrated into bacterial chromosome it is known as "Prophage" and bacteria carrying these prophages are called lysogenic bacteria. Prophages start replicating under the influence of UV light and other agents and through lytic growth cycle results in lysis of cell with the releases of new phage particles. Thus bacterial transduction is the transfer by the bacteriophage serving as a vector of a portion of DNA from one bacterium to another. Usually only one gene at a time can be carried from one bacterial cell to another by a temperate phage. Sometimes however when the genes are situated very close together on the donor chromosome both of them are included within one phage particle and are transmitted together as a unit. There are two types of transductions: i. Generalized Transduction by virulent phages A lytic virus uses the bacterial machinery to make viruses. The bacterium lyse and viral parts are released. The bacterial chromosome is degraded into fragments and one piece is accidentally packaged into a phage head in place of the phage DNA. If this phage infects another bacterium, the bacterial DNA in the phage head can be integrated into the second bacterial DNA, if their sequences are homologous. Any bacterial gene can be transferred by a generalized transducing phage. Such a phage cannot lyse another bacterium it invades because the DNA in the phage head does not have the genetic information to produce phage genome and proteins. This is a defective phage. It is like a transformation only because only a small fragments of bacterial DNA are transferred. Any gene on a chromosome can be transferred. However their sizes are much more uniform and are only limited by the capacity of phage coat. ii. Restricted or Specialized Transduction When a bacterium with an integrated prophage is induced to be lytic, the phage DNA is excised. Sometimes a piece of bacterial DNA adjacent to the phage DNA remains attached to the excised

phage DNA. The phage is released from the lysed cell and when it infects another bacterium, both phage and the adjacent bacterial DNA are integrated into a new bacterial chromosome. Only genes adjacent to the phage can be transduced The phage transduces only those bacterial genes adjacent to the inserted prophage in the chromosome of bacteria transduced as a result of excision of prophage DNA. This phenomenon is generated only on induction of prophage (the previous stage of lysogenic phage) and not in lytic infection. Transformation: It is observed by Griffith in 1928. Transformation is simply the process where bacteria manage to "uptake" or bring in a piece of external DNA. So the uptake of free or naked DNA by a cell resulting in acquisition of a phenotypic trait is called transformation. It is the process where by cell free or naked DNA containing a limited amount of genetic information is transferred from one bacterial cell to another. Donor cells lyses, a fragment of DNA is released and passed into a recipient cell. Enzyme dissolves one strand of the fragment, and the other strand displaces a homologous segment of the recipient's DNA. The recipient then has a recombinant DNA. The displaced fragment is dissolved by an enzyme. Only competent cells can be transformed and DNA of both organisms must be similar. Competence is the ability to take up DNA from the environment Conditions suitable for uptake of donor only during a short interval during growth i.e; Only during a short interval during the late log phase. During this period, the transformable bacteria are said to take up and incorporate donor DNA. Competence results from alterations in the cell wall making it permeable to large molecules (or) governed by the synthesis of specific receptor sites on the bacterial cell surface. Protoplast fusion: Protoplasts have been produced by digesting the cell wall with lysozyme , mutanolysin or a combination of amylase and lysozyme. The advantage of fusion studies is that plasmid DNA enters the cell as intact plasmid and does not generate deletions as in other plasmid transformation systems. PEG-polyethylene glycol has been used o fuse protoplasts together. Transfer of both plasmid and chromosomal genes between two strains is achieved. The process is non specific and involves total genome contact rather unidirectional transfer of pieces of DNA. But it is observed that only certain strains are capable of being transformed or capable of generating cell walls at frequencies needed to detect transformation. Electroporation: In this process high voltage electric field pulses on bacterial membrane are given. These in a very narrow range of intensity and duration result in a reversible, experimentally controllable increase in cell membrane permeability which leads to transient exchange of matter across the membrane and cause membrane-membrane fusion when two membranes are in close contact.

IMMUNOLOGY Immunology is the study of the body's defenses against infection. The disinfectants and antibiotics kill microbes, but the most important way to stay healthy and keep pathogens from making us sick is not through drugs or microbial control mechanisms, but through our own body's defenses against microbial infection. Three things required for infection: adherence, colonization, and invasion. The human body has an elaborate system for stopping a potentially pathogenic microbe from accomplishing any or all of those three things. Mechanisms used by the body to defend against and eliminate foreign agents include blood, lymph system, phagocytic system and tissue factors. Blood - Composed of White Blood cells (WBC), Red Blood Cells (RBC), Platelets and Plasma. WBC: i. Granulocytes : Neutrophils - They are phagocytic and one of the first to attack a foreign cell. ; Eosinophils - Important in allergic reactions ; Basophils ­ Have receptors for IgE. ii. Agranulocytes : Lymphocytes - B and T Lymphocytes. ; Monocytes - They are immature macrophages and are phagocytic. RBC: They are not part of the immune system. Platelets: Produced from Megakaryocytes. They are important in clot formation and have mediators for allergic reactions. Plasma: Contains dissolved substances and gases. The principal proteins are albumin, fibrinogen (clot formation) and globulins (antibody and complement). The order in which theses defenses work is called "lines of defense". Barrier protection.(1st line of defense) Barrier defenses are natural physical or chemical ways of keeping microbes from getting to, or removing them from a potential site, in or on the body, where the microbe might be able to start the process of infection. Physical: skin, cilia in the respiratory tract ,nasal hair, coughing, sneezing, blinking, tearing, sweating ,vomiting ,urination ,defecation Chemical: lysozyme - a natural substance found in tears that is bacteriocidal by dissolving peptidoglycan ; pH -is low on skin and helps keep fungi from colonizing (fungi hate acid ph), salt in sweat ; osmotic pressure- helps keep microbes from colonizing skin Nonspecific defenses (2nd line) Nonspecific 2nd line defenses become important after barrier protection has failed. They are called nonspecific because the same defense mechanism is used for any microbe. The response is not specific to a particular microbe. Phagocytosis- literally "eating" the microbe- Phagocytic cells are found in the blood and tissues. Main tissue phagocyte is the macrophage. The main blood phagocytes are neutrophils and monocytes. They circulate in the blood and eat bacteria and foreign invaders. Mechanism of phagocytic cells to know what's foreign ( Self vs. nonself) : There are markers or receptors on the surface of cells (usually proteins) known as antigens. During fetal development the blood cells learn to recognize which markers or antigens are on their own cells (called tolerance) learning to tolerate our own antigens but not a foreign antigen. Phagocytic cells phagocytize microbes with foreign antigens on their surface. Some bacteria and other microbes have mechanisms to help them resist phagocytosis. Those mechanisms are virulence factors. Ex. Capsules, acid fast cell wall, viral envelopes, etc.

Specific Immune Defenses (3rd line defenses) Lymphocytes are a group of white blood cells that are made in the bone marrow as stem cells. They mature and differentiate into either T lymphocytes or B lymphocytes. These 2 main categories of lymphocytes are both involved in specific immune defenses but they have different functions. T cells are involved in cell-mediated immunity and B cells are involved in humoral immunity. Humoral immunity- B cells are Responsible for the production of antibodies (also called immunoglobulins). Antibodies are proteins, which recognize and bind to specific antigens. The Blymphocytes have a specific receptor on their surface they will recognize only 1 foreign antigen. The B cells don't make antibodies directly, but they circulate in the blood as surveillance cells. Most B lymphocytes will ignore a microbial invader when they meet it, but the one B lymph that has the receptor that matches that foreign antigen will become activated when it meets its match. This is why lymphocytes is of specific immunity. To respond to a foreign invader, there must be specific recognition between 1 particular foreign antigen and that specific lymphocyte. B cell activation : When the B cell meets its foreign antigen, it makes more and more copies of itself (clonal expansion). Then some of those copies turn themselves into plasma cells. Plasma cells are activated B-lymphocytes that produce antibodies (immunoglobulins). There are 5 different kinds of immunoglobulins: IgG ,IgM ,IgA ,IgD and IgE. These antibodies are produced by B Lymphocytes and have two functions. i. To recognize an antigen and initiate clones to secrete antibodies specific to the antigen. ii. ii. To eliminate the remaining antigen. IgG and IgM are the most important. Both bind to the specific foreign antigens and do several different things when they make antigen-antibody complexes. Properties of Immunoglobulins: IgG - makes up 75 - 80% of all immunoglobulins in serum. It is the only Ig that crosses the placenta to protect the fetus. It functions in the activation of the classical pathway of complement. The Fc region of Ig attaches to phagocytes allowing phagocytosis of Ag-Ab complex. IgM - The largest immunoglobulin and accounts for 7 - 10% of all immunoglobulins in serum. It is the first that is synthesized in life, and the first synthesized in response to an antigen. It can occur as a monomer on B lymphocytes or as a pentamer in serum. It functions in the activation of complement (classical). IgM as a pentamer, can bind many Ags and cause agglutination. IgA - Occurs in serum as a monomer, but on epithelial surfaces and in secretions (Breast milk, mucus, saliva etc.)it is a dimer (sIgA) which resists enzymatic digestion. IgA inhibits organisms from adhering to tissues and aggregates of IgA can activate the alternate pathway of complement. IgD - Small amounts are in serum, but along with IgM it serves as membrane receptors on B cells for antigenic determinants. IgE - It is associated with hypersensitive reactions. IgE binds to mast cells and basophils by the Fc portion and produces sensitization. Antibodies fight microbial Invaders by Neutralization, Opsonization, Agglutination, Complement fixation. All of these mechanisms act to signal the body to remove this tagged complex from the body after making it nonfunctional. These antibodies don't kill or remove the invader directly and

antibodies require help from: macrophages, Complement, phagocytes, etc. tags the invader so that the nonspecific systems can take over. Primary vs. Secondary response. Primary Response The first time the B lymphocytes meet the antigen they match, it takes awhile for clonal expansion and differentiation into plasma cells and production of antibodies. 7- 10 days are required for the primary response. In this first meeting of antigen with B cell, IgM is the first immunoglobulin made after the B cells differentiate into plasma cells. Later IgG will be made. IgG is the most efficient at getting rid of invader. That is the primary response. Secondary Response The second time the body meets the same foreign invader with the same antigens, IgG is made in larger quantities and much more quickly. In the secondary response only 2-3 days are required for a significant quantity of IgG to be produced because Memory cells are lymphocytes that remain in the circulation for years, and remember that foreign antigen. They are primed so that if the invader with that specific antigen shows up again the response is quicker and more efficient. We get mostly IgG produced much faster than in the first meeting. That is Humoral Immunity. But that is only half of the specific defenses. The other half of the specific immune response is called: Cell medicated Immunity - By T lymphocytes.

Cell medicated immunity is called so because this type can kill some foreign invaders directly, and this type also mediates or controls the function of humoral immunity (B lymphocytes). T Lymphocytes - Stem cells in the bone marrow produce precursor T cells (Prothymocytes) that migrate to the thymus where they differentiate into 4 subsets. Two Regulators - Helper (TH) and Suppressor (TS) and two Effectors - Cytotoxic (TC) and Delayed type hypersensitive (TD). From the thymus they migrate to peripheral lymphoid organs. They circulate in the blood with a specific receptor that matches a specific foreign antigen. When they meet that foreign antigen they are activated. They make more of themselves (clonal expansion) and differentiate into one of several different subgroups of T lymphocytes viz., cytotoxic Ts (killer Ts), helper Ts, suppressor Ts, delayed type hypersensitivity Ts, Functions of T Lymphocytes: Cytotoxic- only cytotoxic Ts kill cells directly. They destroy antigen binding target cells. TC must get a signal from TH before it activates and proliferates, then binds and destroys the target. Cytotoxic Ts can recognize viral infected cells. Viruses usually leave tell tale viral antigens on the outside of the cell they infect. This is 3rd line specific immune defense so most cytotoxic T's that circulate through the blood will ignore that infected cell, but there is at least one T lymphocyte with a matching specific receptor for that foreign antigen. When that T cell meets it's specific antigen they bind. This binding causes release of cellular poisons into the infected cell that kills that cell. Cytotoxic T lymphocytes kill viral infected cells and then are able to go off to seek out and kill any others like them that are around.

Helper - They recognize antigen early in an infection and induce antigen specific B cells and effector T cells to proliferate and differentiate.Antigens that require B cell response are of 2 types: i. Thymus dependent antigens that require Helper T cells to participate in the response. ii. Thymus independent antigens activate B cells without the assistance of Helper T cells. They require large amounts of the antigen to produce enough non-specific binding to trigger B cell activation. e.g. Bacterial flagellin and lipopolysaccharide. Helper T's are the main mediators of all the specific immune defenses( all the other lymphocyte functions). Helper Ts start helping the B lymphocytes and the cytotoxic T's to activate. When the fight with the foreign invader is over, they also stimulate suppressor Ts to stop all this proliferation and action. Helper T lymphocytes are the mediators of the specific immune response. Suppressor - They interact with TH to prevent an immune response or to suppress an ongoing response. TS can also regulate Effector T cells. Delayed Type Hypersensitive - They do not destroy an antigen. They attract macrophage, neutrophils and other cells to destroy and dispose of the antigen. Allergies and hypersensitivity. Memory T lymphocytes are also produced in initial meeting with foreign antigen. Memory T's circulate for years primed and ready to make the response to any additional meeting with the same foreign invader much swifter and more intense. Types of immunity- Immunity is the resistance to infection by a particular microbe or invader. I. Innate (genetic) - comes from genetically determined characteristics. It is species specific. The individual is not susceptible to certain antigens because of inherited characteristics Example: host range is determined by species immunity. Only certain groups of potential hosts share a receptor that allows adherance of a virus, so that virus can only cause disease in that group. Genes determine what receptors we have on our cells, so we are immune to some infections because of the absence of some genes. A whole species shares many genes so that is innate species or genetic immunity. II. Acquired- not hereditary or based on what genes we have or don't have. It can be artificially acquired by vaccines (antigen) or serum (antibody), or it can be naturally acquired (infection). The acquired immunity can be active if the individual's body did produce the antibody, or it can be passive if the individual's body did not produce the antibody, but it was injected as antigen or immunoglobulin from another source. A. Naturally acquired: Immunity that is acquired through some process that occurs naturally (without intentional intervention) 1. Active- long lasting immunity that is acquied through being naturally exposed to an infectious microbe. Once body gets the infection and body's immune defenses take care of it. After recovery body has circulating memory T's and B lymphocytes for years. Sometimes forever so the next time the same microbe tries to adhere, colonize and invade the response to get rid of it is so swift, that there will be no sickness. 2. Passive- short-term immunity that is acquired through a natural process by being given preformed antibodies that the own plasma cells didn't make. Example: mother to fetal transmission of antibodies made by the mother through breast milk or transmission through the placenta. IgG crosses the placenta since smaller but IgM can't. IgA primarily in breast milk.

B. Artificially acquired:Another type of acquired immunity that results not from a natural process, but through some human purposeful intervention. 1. Passive- short-term resistance to infection through being given injections of preformed antibody. Example: immune sera (gamma globulins) which is pooled IgG antibodies taken from thousands of donors. Immune sera to prevent Hepatitis virus infection. 2. Active- long lasting immunity to disease through being given an immunization or a vaccination. This is not a natural exposure to an infectious microbe but a purposeful, controlled exposure to a specially prepared form of the microbe's antigens. Exposure to the microbe, if done carefully, can result in the specific immune defenses (lymphocytes) making both antibodies and memory cells, so that if the body ever comes in contact naturally with the infectious microbe, it can quickly wipe it out before it causes disease. SEROLOGY Reactions between antigen and antibody, but one must be known to determine the other. The test can be qualitative or quantitative. i. Neutralization - Antigen and antibody neutralize each other. This is used for identification of toxins, antitoxins, viruses, antibodies etc. ii. Precipitation - Antigen and antibody cross link at multiple sites to form a lattice that precipitates. iii. Agglutination - Antigen and antibody react on the surface of objects or cells causing clumping. iv. Immuno- diffusion - Antigen and antibody diffuse and react to form lines of precipitate. 2 antigens with the same antigenic determinants show a continuous line which indicates common identity. A spur indicates partial identity. One antigenic determinant (a) is common to both antigens, but precipitate from Ag bAb b forms a spur. Antigen is not present in the well the spur is directed towards. Cross lines indicate non-identity and the antigenic determinants are different.

MICROBIOLOGY OF WATER Water cycle or Hydrolytic cycle: The earth's moisture is in continuous circulation, a process known as water cycle or hydrolytic cycle. The continental air moves over ocean to become moist with conversion to maritime air with precipitation over the oceans. Total evaporation per year Oceans : 80,000 cubic miles of water Lakes & land surface : 15,000 -doTotal : 95,000 -doThe total evaporation is equaled by the total precipitation of which about 24,000 cubic miles fall on the land surfaces Importance of microbiology of water o They may affect the health of humans and other animal life. o They may occupy a key position in the food chain by providing rich nutrients for the higher level of aquatic life. o They are instrumental in the chain of biochemical reactions, which accomplish recycling of elements E.g. in mineralization There are three types of natural water Atmospheric water Surface water Ground water Atmospheric water: The moisture contained in clouds and precipitated as snow, sleet, hail and rain constitutes atmospheric water. The air contributes the microflora of this water. The air is washed by atmospheric water, which carries with it the particles of dust to which microorganisms are attached. Surface water: Bodies of water such as lakes, streams, rivers and oceans represent surface water. These waters are susceptible to contamination with microorganisms from atmospheric water (precipitation), the surface run off from soil, and any wastes dumped into them.The microbial number vary in number and kind with The source of water Composition of water in terms of microbial nutrients. Geographical, biological and climatic conditions. Ground water: Ground water is subterranean water that occurs where all pores in the soil or rock containing materials are saturated Bacteria as well as suspended particles are removed by filtration, in varying degrees. Wells and springs that are properly located produce water of very good bacteriological quality. Lakes And Ponds Littoral Zone: Along the shore, has considerable roofed vegetation and includes regions where light penetrates to the bottom. Limnetic Zone: in open areas, the light compensation level i.e., the depth of effective light penetration determines this zone. Profundal Zone: Photosynthetic activity decreases progressively in the regions of the open water. Benthic Zone: It is composed of soft mud or ooze at the bottom. The profundal zone and the benthic zone is largely populated by Heterotrophic organisms.

Bloom: Lakes and ponds of the temperate region exhibit interesting seasonal changes in their microbial populations due to stratification of the water as a result of temperature difference. In summer, the top layers tend to be warmer than the lower regions, but in winter, ice, which is less dense, collects on the top; there fore a reversal of temperature and mixing occurs in the springs and in the fall, often resulting in massive growth of algae i.e. called Bloom. The Aquatic Environment Temperature- The temperature of surface waters varies near 00 C in polar regions to 30-40 0 C in equatorial regions. Marine environment--Below 50 C --favorable for psychrophilic organisms Natural hot springs -- 75-800 C -- Thermus aquaticus a common bacteria inhabitant of hot springs. Hydrostatic pressure -- Barophilic microorganisms grow under pressure ( >100 atms) Light-- Photosynthetic activity is confined to the upper 50-125 m. CO2 is available largely from HCO-3 Salinity-- Zero to saturation in salt lakes. Seawater has high salt--33-37/kg of water. pH --pH of sea is 7.5 --8.5 Turbidity -- near shore rivers are often turbid. Limnology: The study of the flora and conditions for life in lakes, ponds, streams is called limnology. Microbiology of domestic water and waste water: The drinking water of most communities and municipalities is obtained from surface sources like rivers, streams and lakes. They are likely to be polluted with domestic and industrial waste i.e., the used water of a community (waste water). Municipal water purification systems have been very effective in protecting the inhabitants against polluted water. As a potential carrier of pathogenic microbes, water can endanger health and life. The common pathogens are can endanger health and life. The common pathogens are Typhoid and paratyphoid bacteria Dysentery (bacillary and amoebic) Cholera bacteria Enteric viruses Potable Water: water that is free of disease-producing microbes and chemicals deleterious to health. Polluted or Non-Potable Water: Water contaminated with either domestic or industrial wastes. WATERBORNE DISEASES: Salmonellosis: Salmonella are gram negative facultative rods, motile with peritrichous flagella and placed in serological groups based on O(somatic) and H (flagella) antigens. Transmission is by ingestion of food and water contaminated by feces. Gastroenteritis is caused by S. typhimurium, S. enteriditis and S. newport. Organisms multiply in the intestine causing abdominal pain and diarrhea. Typhoid fever is caused by S. typhi,. Organisms bind to the intestinal wall penetrate into Peyer's patch where they invade macrophages and multiply. Intracellular survival is associated with inhibition of oxidative metabolic burst and protection from antibodies. Organisms spread from Peyer's patch to the liver, spleen and blood. In the blood organisms release endotoxins. Immune response is humoral and cell mediated. The disease is characterized by High fever and headache.

Shigellosis The agents are Sh. sonnei, Sh. dysenteriae, and Sh. flexnerii.. They are gram negative nonmotile rods facultative anaerobes placed in groups based on their O antigen and biochemical reactions. Organisms are transferred by fecal contamination of food and water. They are acid resistant and therefore survives in the stomach to reach the small and large intestine where they replicate and remain, causing ulcer and abscess. They rarely enter parenteral tissues. Shigellosis is self limiting and requires the replacement of fluid and electrolytes. The disease is characterized by Abdominal pain, fever, cramps and diarrhea.

Cholera Caused by Vibrio cholera that are gram negative rods, aerobic, oxidase positive, motile with polar flagellum and are found in marine and fresh water. Organisms can grow in alkaline pH. Sero type O1 is the agent for cholera and is characterized based on biochemical reactions, enterotoxin and O antigen. Transmission is by ingestion of contaminated water and uncooked food, especially fish. The disease is characterized by Vomiting, diarrhea and abdominal pain. Organisms do not spread beyond the GI tract. Cholera is self limiting with the replacement of fluids and electrolytes. The drinking water is not always required for transmission of some water borne infections. E.g. Leptospirosis - Non-intestinal disease characterized by bacteremia and kidney damage. It can be acquired merely by coming into contact with water contaminated with the urine from infected domestic or wild animals9 E.g. By swimming in a pond frequently contaminated by infected cattle or by working in a rat infested sewers). The Leptospirosis can penetrate the conjunctivas of the eye, abrasions in the skin, or mucous membranes of the nose and mouth. Municipal Water Purification: The principle operations are: Sedimentation Filtration Disinfection Sedimentation occurs in large reservoirs where water remains for a holding period. Large particular matter settles to the bottom. Sedimentation is enhanced by the addition of Alum (Aluminum sulfate) at the treatment plant, which produces a sticky flocculent precipitate. Any microorganisms as well as finely suspended matter, are removed as this is passed through sand filter beds, a process which removes 99% of bacteria. Subsequently, the water is disinfected to ensure it potability. Many employ chlorination for disinfection. The dosage must be sufficient to leave a residual of 0.2 to 2 mg per liter, free chlorine. Other disinfection methods are ozonation and irradiation with ultraviolet light. Purification includes procedures for Removal of hardness Adjusting the pH, Removing undesired colour and taste and Adding fluoride for the prevention of dental caries.

Microbiology of Soil Directly or indirectly the wastes of humans and other animals, their bodies and the tissues of plants are dumped onto or buried in the soil. Somehow they all disappear, transformed into the substances that makeup the soil. It is the microorganisms that make these changes-the conversion of organic matter into simple inorganic substances that provide the nutrient material for the plant world. Thus microorganisms play an important role in maintaining life on earth, as we know it. Soil : Soil has been defined as that region on the earth's crust where geology and biology meet. From a functional point, the soil may be considered as the surface of earth, which provides the substratum for plant and animal life. Soil Profile Horizon A: organic debris in various stages of decomposition and minerals Horizon B: Fine particles and minerals Horizon C: weathered mineral material excluding bedrock. Horizon D: Unearthed rock, bedrock. Dominate minerals: Silicon, Aluminium, Iron and in lesser amounts of Calcium, magnesium, Potassium, titanium, manganese, sodium, nitrogen, phosphorus and sulfur Humus: The plant and animal remains deposited on or in the soil contribute organic substances. In the last stages of decomposition, such substances composed of residual organic matter not readily decomposed by microorganisms. Micro flora of soil: fertile soil is inhabited by The roots of higher plants Many animal forms( eg. Rodents, insects and worms) Tremendous number of microorganisms. Factors influencing the growth of microorganisms in soil: Amount and type of nutrients Available moisture Degree of aeration Temperature PH Practice and occurrences which contribute large number of organisms to soil e.g. Floods or addition of manure. The activity of microorganisms on the earth is often rather grandly referred to as their "geochemical" activities. All this means is that they do their chemistry on a grand scale and perform a large variety of fascinating chemical processes enrich us in many cases. However, they are, quite simply, going about their daily lives. All life transforms chemicals through their biochemical activities. For example, photosynthetic organisms convert carbon dioxide, water and light to organic matter, but somewhere down the line that chemistry is reversed and the polymers made in the synthetic process are converted back into the original chemicals (CO2 and H2O) from whence they came. This is where the concept cycle comes in. All the nutrients of life endlessly turnover in a cyclic way and each nutrient has a cycle involving a group of microorganisms that are responsible for carrying out this process.. The Cycles of Matter All living organisms carry out catabolism which results in degradation and all living organism carry out anabolic processes which expend energy to construct macromolecules or to move etc.

The Carbon Cycle: This is an easy one. Carbon dioxide + water + energy + plant/microbes = organic molecules in live organisms = organic molecules in dead organisms + oxygen + microbes = energy + carbon dioxide + water; However, some carbon stays around for a really long time in the form of coal, methane, oil and CaCO3 deposits. One important component of the carbon cycle is methane (CH4) production and metabolism (oxidation). Methane is a major byproduct of many anaerobic biochemical processes, including those that occur in our very own intestines. Methane is much more effective than carbon dioxide in absorbing and radiating energy (heat) back to the earth; i.e., methane is a major greenhouse gas. Methane-producing microbes produce about 400 million metric tons of methane each year, but only under anaerobic conditions (inside rumens, insects, wetlands, rice paddies, sewage digesters, landfills, biogas generators. The increase in the greenhouse gas methane that is attributed to man is ~1%/year. Conversely, methane is an important carbon and energy source for those microbes that are able to metabolize (oxidize) it into carbon dioxide and water. That is, methane is a major component of the carbon-cycle. The Nitrogen Cycle: Although nitrogen gas (N2) constitutes 80% of the earth's atmosphere, N2 can not be used by most forms of life. However, nitrogen is a major component of protein and nucleic acids and so is a major required nutrient.

The majority of organisms can only use nitrogen if it is in the form of ammonia, nitrates or nitrites. But these are scarce chemicals in nature and nitrogen deficiencies often limit crop yields. Fortunately, there are a number of microbes that are able to convert the N2 in the air to ammonium nitrogen. These organisms, (and this will surprise you) are called nitrogen-fixing bacteria. The only other natural source of usable nitrogen for plants is from lightening. The nitrogen cycle starts with N2-fixing bacteria. The nitrogen-fixing bacteria are able to take N2 gas + a lot of energy + a lot of electrons and convert it to ammonia (NH3) which they use to make the many nitrogen-containing organic molecules they required to grow and make offspring. When they, and any other living organism die, much of their organic-bound nitrogen is released by microbes decomposing the dead as ammonia (some of it, however is used directly by the degrading organisms); which you can smell when you turn over a compost pile. The ammonia is oxidized to nitrite (NO2) by a group of specialized microorganisms to extract the energy trapped in it. Another group of bacteria subsequently oxidize the nitrite to nitrate (NO3) and squeeze out some more energy. Ammonia, nitrite and nitrate can be assimilated by plants and a lot of microbes and converted into nitrogen-containing organic molecules.

The final phase to the nitrogen cycle occurs under anaerobic conditions where still another group of bacteria use nitrite/nitrate as electron acceptors (substituting for oxygen) and in the process reduce the nitrogen back into the atmosphere as N2 gas, thus completing the cycle. In summary nitrogen is continuously cycled from N2 gas into living organisms and back into N2 gas. Other Nutrient Cycles: There are two other cycles that are often presented in this section. They are the sulfur and phosphorous cycles. Microbial population in a fertile agricultural soil Type 1.Bacteria Direct count Dilution plate 2. Actinomycetes 3. Fungi 4. Algae 5. Protozoa No. per gram 2,500,000,000 15,000,000 700,000 400,000 50,000 30,000

Types of soil bacteria; 1. Autotrophs & heterotrophs 2. Mesophiles 3. Thermophiles 4. Psychrophiles 5. Aerobes & Anaerobes 6.Cellulose digesters & sulfur oxidizers 7. Nitrogen fixtures and protein digesters. Actinomycetes: Present in dry warm soil. These organisms are responsible for the characteristic musty or earthy odor of a freshly plowed field. They are capable of degrading many complex organic substances and consequently play an important role in building of soil fertility. The actinomycetes are also noted for their ability to synthesize and excrete antibiotics. Fungi: Hundreds of different species of fungi inhibit the soil. They are abundant near the surface, where an aerobic condition is likely to prevail. They exist in both the mycelia and spore stage. Fungi are active in decomposing the major constituents of plant tissues, namely cellulose, lignin and pectin. The crumble structure and the binding together of the fine soil particles to form water stable aggregates is important in agriculture. Algae: Green algae and diatoms are present. Predominant on the surface or just below the surface layer of soil because of its photosynthetic nature. On barren and eroded lands , they may initiate the accumulation of organic matter because of their ability to carry out photosynthesis and other metabolic activities. Protozoa: Maintain equilibrium of microorganisms in soil by their dominant mode of nutrition involves ingestion of bacteria.

MICROBIOLOGY OF AIR Air is not a natural environment for the growth and reproduction of microorganisms because It does not contain the necessary amount of moisture required by the microbes It does not contain nutrients in available form So air does not have a flora yet the microorganisms are found in air and their presence is of considerable importance because of economic and public health point of view. Microflora of air is transient and variable. Air is not a medium in which microbes can grow but `is a carrier' of particulate matter, dust and droplets which may be laden with microbes. Microbes are introduced in to by various sources. The principle source is dust particles containing dry vegetative cells and spores. Most important organisms are saprophytes i.e. from that live on dead organic matter and are important in food and dairy establishments, in biological laboratories. Micro organisms exist in three forms in the air As passengers on the solid particles of dust or skin or hair With in the droplets form by the atomization of liquids by sneezing, stirring or their activities. Aerosols--droplets remain suspended in air for some time known as droplet nuclei As isolated organisms resulting from the evaporation of water from droplets(or) in case of mold spores as result of their natural method of propagation.

Species vary depending on location: The fate of air borne organisms is governed by a complex set of circumstances including atmospheric conditions. Ex: temperature , humidity, sunlight, size of particle bearing microbe, nature of organism i.e.; ability to form spores ,susceptible / resistant to new environment 1. Yeast &Molds are not most commonly found in air and in some localities even out number bacteria. Important organisms are cladosporium spp , Penicillium sp. and Asperigillus sp. 2. Aerobic spore formers- especially from soil are found quite frequently in the air. Best-known form is B. subtilis (Hay bacillus, found in soil and on vegetation) 3. Other Nonspore formers: Sarcina sp, Staphylococcus sp, Micrococcus sp, cornybacterium sp, Alcaligenes sp, Chromobacterium sp. Factors controlling number & kind of organisms: Type of soil Amount of dust stirred up Activity in the environment

Outdoor: Rich, fertile, cultivated silo show a higher viable count then sandy or clay or uncultivated land. Air above bare land contains more organisms than land covered with vegetation i.e. the organisms can be blown more easily into air because earth is not protected from air currents. Marine air contains less number of organisms than terrestrial air. Damp or humid air contains fewer organisms than dry one owing to the fact that organisms are carried down by the droplets of moisture. Air in summer months contains many more organisms than in wet winter months. Refrigerator air is usually free and organisms. Organisms in free state are heavier than air and settle out slowly in quiet atmosphere. However, a gentle air current is capable of keeping than in suspension almost indefinitely.

Organisms attached to dust particles or in droplets of moisture settle out at much faster rate . Indoor: - Degree of microbial contamination depends on Ventilation rates Nature and degree of activity Crowding of individuals Droplets expelled from respiratory tract vary in their dimensions from micrometers to millimeters. Low micrometer range can remain air borne for long period of time routine laboratory techniques produce aero sole- fine sprays producing droplets that remain suspended in for a time ­ of microbes. Ex: Loop & culture Opening of lyophilized culture Streaking on agar Centrifugation Sources of aerial contamination in food factories: Soil: Raw materials received in the factory may bring with them dust & microbes Persons: workers , officials &visitors, Floor drains: microbes are aerolised by water flowing into the drains. Wash basin serves minor but potential source. Water from dairy fluid products that drains on the floor or flow with sufficient turbulence in open equipments will foam minute droplets and contribute to air flora. Faulty constructed rooms requiring repairs. insect like flies & rodent like mice effect air quality < 200/ m3 2000/m3 < 100/m3 1000/m3

Standards for air quality: Total air count: good Poor Yeast &molds: good Poor

ENUMARETION OF BACTERIA IN AIR Most organisms in air are spores since vegetative cells can not resist conditions with exceptions of staphylococci & mycobacteria which resist drying in better way. Sedimentation:- Air borne organisms may be collected by exposing sterile agar plates for definite periods of time . Then the plates are incubated after replacing the covers. This is not very useful for quantitative analysis &gives only relative results. Results are influenced by air movement and directions of air current .Usually plates are exposed for 15 mts. Medium used are: SPC medium with incubation period of 2 days at 370c for bacteria and PD agar with incubation period of 5 days at 20--250c for fungi are used. Results are expressed as the rate of fall of organisms i.e Number of organisms/SQ ft/min. Improved method to use as quantitative comprises of using devices such as sieve or slit type .known as "impaction on solid surface". Air is drawn through the small holes even spaced in a metal cover under which is located Petridis containing the medium. Measured volume of air is drawn.

Particulate matter impinges on agar. Slit type: - Air is drawn at a high speed through narrow slit on to agar plate. Petri dish is rotated at a uniform speed. One complete revolution is made during the sampling operation. Uniform spreading of organisms over agar surface is possible. Speed of rotation can be adjusted according to the density of microbial population. Slow rotation if fewer organisms are expected. It is more efficient in recovering air borne organisms. Liquid Impingement Method:It is a quantitative method . The air is drawn through small holes on the bulb of the tube and bubbled through the sterile liquid. Air borne particles are wetted and entrapped in the collecting medium. Due to bubbling fragmentation of bacterial clumps occurs. Air is drawn at the rate of 25--30lts per min. The collecting medium is subjected to serial dilutions before proceeding as normal plate count technique. Not suitable if few organisms are found in air. Some organisms are destroyed by high impingement velocity but dehydration loss of organisms is minimized. Filtration Polypore or Millipore membranes are used for removal of organisms from air .The diameters of the collected particles are larger than the pores of membranes and are retained on or within a few microns of filter surface .membranes are then agitated in a suitable liquid to disperse the particles and then used for plating (OR) Membranes can be directly placed on agar surface clumps of bacteria are broken during agitation in liquid method and so the counts usually are higher. Centrifugation It involves a technique employing centrifugal force for propulsion of air particles to the collecting surfaces. When air moves in a circular direction at high-speed, the suspended aerosols are impacted on collecting surfaces by a force proportional to the particle velocity and mass. Two types are there and in one type, sampler remains stationary while the aerosol travels in a circular path, the larger particle being collecting at the bottom. In second type, the collecting vessel and aerosol rotate at high velocity resulting in the impaction of particles on the walls. The culture medium in fluid state is poured into the sample tube the tube is inserted in the centrifuge and current is switched on .The in coming air is mixed with the agar and the medium is spread as a thin film on the wall of the sample tube by the centrifugal force of rotation .The organisms are deposited in the film and after incubation may be counted. Electrostatic Precipitation Samplers using this method collect particles by drawing air over an electrically charged surface. A sampler holds two Petri dishes in separate units with removable caps. Units are two and each unit has two electrodes i.e. one lower electrode and one top electrode. The two electrodes are +ve and -ve in each unit . The arrangement is reverse in another unit. Both -ve and +ve charged organisms exit in the air simultaneously. Those having +ve charge are collected on the Petri dish placed over -ve electrode and those -ve charged are collected on Petri dish over the +ve electrode. Airborne Bacterial Diseases Streptococcal Diseases: Streptococcus are gram positive chains, nonmotile and are either alpha, beta or gamma hemolytic. They are in serological groups A to T based on their cell wall carbohydrate, but the main pathogens are A,B,C,D,F and G. Most human diseases are caused by Group A beta hemolytic streptococcus, also called S. pyogenes. The diseases that are caused include pharyngitis, scarlet fever, rheumatic fever and impetigo.

Diphtheria The causative organism is Corynebacterium diphtheria which is a gram positive rod, pleomorphic and produces catalase. Transmission is by respiratory or cutaneous discharge.Symptoms are Sore throat, fever and pharyngitis or tonsillitis. Immunized with diphtheria toxoid (DTP), Whooping cough (Pertussis) Bordetella pertussis is the agent for whooping cough (Pertussis). It is a gram negative, aerobic, non motile coccobacilli, with pili and is very fastidious. It is pathogenic especially to the young. Humans are the only reservoir and transmission is by respiratory droplets. There are three symptomatic stages. A. Catarrhal - Conjunctivitis, watery eyes, sneezing, cough and a low fever. B. Paroxysmal - The most contagious stage with severe coughing, mucoid secretion, typical whooping and fever. C. Convalescence - Mild cough periodically. Whooping cough is self limiting but drugs lessen its communicability. Immunize with DTP. Tuberculosis It is caused by Mycobacterium tuberculosis and M. bovis that are acid fast rods, require 5 - 10% CO2 and divide every 18 - 24 hours. Transmission is via droplet nuclei, contaminated milk or dust particles. Lesions develop in the alveoli and lymph nodes. TB is associated with malnutrition, alcoholism, diabetes and old age. It is characterized by chronic fever, weight loss, night sweat, and productive coughing. For the Mantoux test purified tuberculin protein antigen is injected intracutaneously. There is an inflammatory reaction involving macrophages and lymphocytes resulting in a reddened, raised and hard area (induration). Symptoms - Fever, malaise, fatigue and weight loss. Bacillus of Calmette and Guerin (BCG) isolated from M. bovis is used as a vaccine. Meningococcal meningitis The agent is Neisseria meningitidis that is gram negative diplococci, aerobic, fastidious and produces cytochrome oxidase. Transmission is by direct contact or by respiratory droplets. Bacterial meningitis can be caused by other organisms. (H. influenzae, & S. pneumoniae ) but only N. meningitidis causes septic meningitis in epidemic form. Symptoms- Fever, headache, stiffness (neck, back and shoulders), nausea and vomiting.

Hemophilus Influenzae Diseases Hemophilus are gram negative, aerobic coccobacilli. Some are capsulated and some are not. Invasive influenza infection are caused by encapsuled strains causing meningitis, epiglottitis, sellulitis and pneumonia. The non-capsulated H. influenzae are non-invasive and cause infections such as otitis media, sinusitis, bronchitis, etc. Treatment - ampicillin, chloramphenicol and cephalosporin. There are many drugs resistant strains so drugs are given in combinations.

AIR SANITATION The pre requisite measures for a successful air sanitation

Proper ventilation systems. Installation of proper drainage system. Minimum number of worker operating in the food processing. Correct design and construction of plants with smooth ceiling &walls & floors . Preventing insects and rodents by self closing doors, fumigation etc

Ventilation: Good ventilation will remove the moisture released during processing. It will protect the finish of the building and equipment and prevent condensation and subsequent mould growth on surfaces. Humidity in processing rooms should not exceed 95% R.H. ventilation with out air filtration increases the probability of contamination considerably. The actual number of microbes in the filtered ventilating air depending not only on the efficiency of air filtration media but also on the level of microbial contamination in the air outside the factory. If the microorganisms in the fresh outside air are more and the air filter media efficiency is relatively low serious problems may arise in the processing rooms. Air Filtration In this process air is forced through a network of fibrous materials able to retain microbes. Efficiency of air filters depend on: Flow of air. Size of the particle. Depth of the particle. Types of Filters:A. Rough filters--- Made of viscous coated fibers of metal, glass wool, hair or loosely packed cotton, glass. B. Medium efficiency filters---- Used by compressed glass fibers or paper fibers. A & B are used to remove large particles. C. High efficiency filters ------ Used to obtain 90% efficiency. They are made by small diameter fiber and with decreasing porosity. D. Ultra high efficiency filters made up of Cellulose acetate material (97-99%) Borosilicate filters (99-99.9%) Polytetra fluoro ethylene (100%) Germicidal Chemicals: -- Used as mists and aerosols. Hypochlorite in a final concentration of 1:2 million is sufficient to produce 90-99% reduction of bacteria. The effectiveness depends on moisture content. The organisms are rapidly killed between R.H 70 & 90. Propylene glycol atomized in 1:4million produces an immediate sterilization. opt .temp: 270 c R.H :45-70% Glycols exert hygroscopicity. When glycol reaches certain concentration in the moisture surrounding the organisms, moisture is drawn out of the cell resulting in its death. Chlorine: 0.61 gr of available chlorine/1000cubic ft of air. Calcium hypochlorite :0.48ppm available chlorine results in 99.99% inactivation

Ultraviolet radiations: Germicidal range of UV rays is between 2000 and 2967 Ao but maximum is at 2650 Ao UV rays exert pronounced destructive action on bacteria, viruses and other organisms suspended in air. Radiation efficiency in terms of disinfection of air depends on Quantity of radiation Strength of ray Length of ray Total exposure Uniformity of exposure

Type of contamination State of suspension of bacteria in air Humidity of atmosphere Volume of space of air Installation of UV lamps on the side wall to irradiate the floor and air below 30 inches level or from ceiling of a room to irradiate the air above 7 ft level. In between these levels it is harmful to humans. Drawbacks : Lower penetration power-does not inactivate the microbes beneath surface layers Efficiency of radiation falls with square distance from the source of irradiation Mutagens to humans.

BACTERIOPHAGES Bacteriophages are the viruses that infect bacteria. Viruses are not plants, animals, or bacteria, but they are the quintessential parasites of the living kingdoms. `Phage' literally means devouring just like phagocyte (to swallow or eat up greedily). Widely distributed in nature & most abundant in intestinal contents of animals and first is invented by Twort in 1915. Typical bacteriophage has a structure like tadpole. Head ­ enclosing nucleic acids in protein sheath. Tail ­ a hollow tube of proteins & bearing tail plate & tail fibers. All viruses contain nucleic acid, either DNA or RNA (but not both), and a protein coat, which encases the nucleic acid. Some viruses are also enclosed by an envelope of fat and protein molecules. Without a host cell, viruses cannot carry out their lifesustaining functions or reproduce. They cannot synthesize proteins, because they lack ribosomes and must use the ribosomes of their host cells to translate viral messenger RNA into viral proteins. Viruses cannot generate or store energy in the form of adenosine triphosphate (ATP), but have to derive their energy, and all other metabolic functions, from the host cell. They also parasitize the cell for basic building materials, such as amino acids, nucleotides, and lipids (fats). The virus has a "tail" which it attaches to the bacterium surface by means of proteinaceous "pins." The tail contracts and the tail plug penetrates the cell wall and underlying membrane, injecting the viral nucleic acids into the cell. Viruses are further classified into families and genera based on three structural considerations: 1) the type and size of their nucleic acid, 2) the size and shape of the capsid, and 3) whether they have a lipid envelope surrounding the nucleocapsid (the capsid enclosed nucleic acid). They are significant from processing point of view. Phages attack Lactic acid bacteria & lyses them by multiplying inside the host cell leading to the release of many phages which can reinfect the fresh cells in the culture. This results in the failure of starters to act & bring about the changes during preparation of fermented products. Bacteriophages are highly host specific, a rotation of starter cultures help to control the problem to some extent. Ca ion deficient medium for maintaining starter cultures prevent phage attack as the ion helps in phage adsorption to host cell. Genetic manipulations to construct phage resistant strains. Typical Lactococcus lactis ssp. lactis phage might have 40-90 microns head & 100-120 microns tail.Bacteriophages can persist for a long time in dried up whey & on utensils. Phages attach themselves to living cells & as they divide phages also multiply. The infective material is injected (nucleic acid) through hollow tail of the phage particle into the bacterial cell. The rate of phage proliferation is greater than that of bacterium. Consequently, a point is reached at which lysis begins. Lysis is preceded by swelling of the bacteria to 6-16 times their normal size. Phage action may cause a number of variations in the properties of bacteria.Irreversible adsorption of phage to the cell triggers a no. of steps which lead to the transport of viral nucleic acids into the cytoplasm & nucleic acid gets metabolically active transforming cellular function to one of synthesizing viral components. As the phage assembly

completed the lysis of the host cell occur. All this occur approx. in one generation time of the host phage multiplying to hundred in one generation & in two generations to 10000 & so on. Capsid - The capsid is the protein shell that encloses the nucleic acid; with its enclosed nucleic acid, it is called the nucleocapsid. This shell is composed of protein organized in subunits known as capsomers. They are closely associated with the nucleic acid and reflect its configuration, either a rod-shaped helix or a polygon-shaped sphere. The capsid has three functions: 1) it protects the nucleic acid from digestion by enzymes, 2) contains special sites on its surface that allow the virion to attach to a host cell, and 3) provides proteins that enable the virion to penetrate the host cell membrane and, in some cases, to inject the infectious nucleic acid into the cell's cytoplasm. Under the right conditions, viral RNA in a liquid suspension of protein molecules will selfassemble a capsid to become a functional and infectious virus. Envelope - Many types of virus have a glycoprotein envelope surrounding the nucleocapsid. The envelope is composed of two lipid layers interspersed with protein molecules (lipoprotein bilayer) and may contain material from the membrane of a host cell as well as that of viral origin. The virus obtains the lipid molecules from the cell membrane during the viral budding process. However, the virus replaces the proteins in the cell membrane with its own proteins, creating a hybrid structure of cell-derived lipids and virus-derived proteins. Many viruses also develop spikes made of glycoprotein on their envelopes that help them to attach to specific cell surfaces. Nucleic Acid - Just as in cells, the nucleic acid of each virus encodes the genetic information for the synthesis of all proteins. While the double-stranded DNA is responsible for this in prokaryotic and eukaryotic cells, only a few groups of viruses use DNA. Most viruses maintain all their genetic information with the single-stranded RNA.

GENERAL PROPERTIES OF FUNGI The fungi are more evolutionarily advanced forms of microorganisms, as compared to the prokaryotes (prions, viruses, bacteria). They are classified as eukaryotes, i.e., they have a diploid number of chromosomes and a nuclear membrane and have sterols in their plasma membrane. Genetic complexity allows morphologic complexity and thus these organisms have complex structural features that are used in speciation. They reproduce atleast in part by means of spores They lack chlorophyll. Because of this they can not prepare their own food They get food from other organisms and organic matter. They are in general decomposers They are classified under plant kingdom because they have cell wall around their cells. Fungi are heterotrophic organisms that require organic compounds for nutrition. Fungi can be divided into two basic morphological forms, yeasts and molds ( hyphal form). Yeasts are unicellular fungi which reproduce asexually by blastoconidia formation (budding) or fission. Molds are filamentous, multi-cellular fungi, which reproduce asexually and/or sexually. Dimorphism is the condition where by a fungus can exhibit either the yeast form or the hyphal form, depending on growth conditions. Very few fungi exhibit dimorphism Fungi Division: Mycota Subdivision: Mycotina Class: Mycetes Sub class: Mycetidae Order: ales Family: aceae The classification is mainly based on the characteristics of sexual spores and fruiting bodies present during their life cycles. But many produce bodies only in certain environmental conditions and thus the complete life cycles of many are unknown (Imperfecti) or form class Deuteomycetes. The fungi with known sexual cycles/stages form class ascomycetes and class basidiomycetes. MOLDS: Molds are complex forms. They are aerobic and grow over a wide range of temperature pH, osmotic pressure. Molds produce cotton or wooly colonies that may be pigmented and white, cream, green, black, or brown in colour. Most molds occur in the hyphae form as branching, threadlike tubular filaments. A mass of hyphal elements is termed the mycelium. These filamentous structures either lack cross walls (coenocytic) or have cross walls (septate) depending on the species. The cross walls divide it into a number of uninucleate cells. The portion of hyphal structure of mold involved in securing nutrient from food stuffs and attaching to solid substrate are termed as vegetative hyphae. Septa have pores that allow the movement of cytoplasm from one cell to another. Hyphae are composed of an outer tube like wall surrounding a cavity, the lumen which is lined by protoplasm. Between the protoplasm and the wall is plasmalemma, a double layered membrane which surrounds the protoplasm. In some cases septate hyphae develop clamp connections at the septa, which connect the hyphal elements. Aerial hyphae often produce asexual reproduction propagules termed conidia (synonymous with spores). Relatively large and complex conidia are termed macroconidia while the smaller and more simple conidia are termed microconidia. When the conidia are enclosed in a sac (the sporangium), they are called endospores

Asexual reproduction, via conidia formation, does not involve genetic recombination between two sexual types whereas sexual reproduction does involve genetic recombination between two sexual types. A. Yea st cell s repr odu cing by blas toco nidi a for mati on; B. Yea st divi ding by fission; C. Pseudohyphal development; D.Coenocytic hyphae; E.Septate hyphae;F.Septate hyphae with clamp connections Some important molds are Aspergillus, Penicillium , Geotrichum, Trichophyton, Microsporum, and Rhizopus. Penicillium;Penicillia have septate vegetative mycelia which penetrate the substrate and then produce aerial hyphae on which conidiophores develop. Conidiophores may be branched and have brush like heads bearing spores. Clusters of sterigmata are usually in one place and from each is formed a chain of conidia. The important species of dairy importance are P. Roquefort, P. camembert, P. casei Aperigillus : Septate branching mycelia with vegetative portions submerged in nutrient. Conidiophore or fertile hyphae arise from thickened foot cells which may also be sumerged. At the apex conidiophore inflates to form a vesicle which gives rise to sterigma which may be single layered or double layered. Conidia arise from sterigmaata and borne in chains. Conidia are of various colors and are quite characteristic of the species.Conidiophores may be septate or Non-septate. Rhizophus : Bread mold. Non-septate, sporangiophores form at the nodes where rhizoids are formed. sporongia are quite large and black. It has stolenferous type of spread. Ex. Rhizophus stolenifer

Asexual fruiting structure of Aspergillus sp. Asexual fruiting structure of Rhizopus sp. illustrating septate hyphae, conidiophore, illustrating sporangium, sporangiophore, sporangiospores, coenocytic hyphae and vesicle, phialides and conidiospores . rhizoids.

Yeasts: Yeats are unicellular and ovoid/elliptical in shape. They are Gram positive, non-motile,larger than bacteria with width of 1-5 microns and length of 10-15 microns. They reproduce by budding. They grow over a temperature range of 25°-40°C. they are acid tolerant. They are strongly fermentative/oxidative in the metabolism of carbohydrates or organic acids. Generally non proteolytic in nature with few exceptions. May produce "ascospores" . True yeasts consists of ascomycetes and others that do not sexual spores are false yeasts belonging to `fungi imperfecti' (Deuteromycetes. Some important yeasts are : Saccharomyces sp. Kluyveromyces sp. Candida p. Torulopsis sp.

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