General Microbiology PDF

DMCB 221 GENERAL MICROBIOLOGY Introduction Microbiology is the study of microscopic organisms, such as bacteria, fungi, and protists. It also includes the study of viruses. Microbiology encompasses all aspects of these microorganisms such as their behavior, evolution, ecology, beneficial roles, bioc...

DMCB 221 GENERAL MICROBIOLOGY Introduction Microbiology is the study of microscopic organisms, such as bacteria, fungi, and protists. It also includes the study of viruses. Microbiology encompasses all aspects of these microorganisms such as their behavior, evolution, ecology, beneficial roles, biochemistry, and physiology, along with the pathology, of diseases that they cause. Branches of microbiology In general, the field of microbiology can be divided into the more fundamental branch (pure microbiology) and the applied microbiology (biotechnology). Pure microbiology refers to where scientists from different angles study microbes for the main purpose of comprehending them. Applied microbiology refers to the fields where the micro-organisms are applied in certain processes such as brewing or fermentation. The organisms itself are often not studied as such, but applied to sustain certain processes. Pure microbiology Bacteriology: Bacteriology is the branch of microbiology concerned with the study of bacteria. Mycology: Mycology deals with the study of fungi which are eukaryotic in nature, the different types of fungi (mold and yeast) can be highly beneficial or harmful. Protozoology: this is one of the newer branches of microbiology based on taxonomy. It is the sub-discipline that deals with the study of protozoa. Like fungi, these are eukaryotic organisms that include such groups as amoeboids, ciliates, sporozans, and flagellates. Phycology/algology: deals with the study of different types of algae that can be found in different types of environment. Parasitology: this is a wide field of microbiology that deals with the study of parasites. Those who study parasitology are known as parasitologists. Immunology: Immunology is the sub-discipline that deals with the study of the immune system. A person who studies immunology is known as an immunologist. Virology: Virology is the branch of microbiology that is concerned with the study of viruses. Nematology: Nematology is the sub-disciplines that deal with the study of multicellular nematodes. Applied microbiology Medical microbiology: the study of the pathogenic microbes and the role of microbes in human illness. Includes the study of microbial pathogenesis and epidemiology and is related to the study of disease pathology and immunology. It is related to a number of other fields including virology, bacteriology and immunology. Pharmaceutical microbiology: the study of microorganisms that are related to the production of antibiotics, enzymes, vitamins, vaccines, and other pharmaceutical products and that cause pharmaceutical contamination and spoilage. Industrial microbiology: the exploitation of microbes for use in industrial processes. Examples include industrial fermentation and wastewater treatment. This branch of microbiology is concerned with the use of given microorganisms for industrial production. Microbial biotechnology: the manipulation of microorganisms at the genetic and molecular level to generate useful products. THE HISTORY OF SCIENCE OF MICROBIOLOGY The term Microbiology was given by French chemist Louis Pasteur (1822-95). Microbiology had its roots in the great expansion and development of the biological sciences that took place after 1850. The term microbe was first used by Sedillot (1878) TRANSITION PERIOD When microorganisms were known to exist, most scientists believed that such simple life forms could surely arise through spontaneous generation. That is to say life was thought to spring spontaneously from mud and lakes or anywhere with sufficient nutrients. This concept was so compelling that it persisted until late into the 19th century. The main aspects were to solve the controversy over a spontaneous generation which includes experimentations mainly of Francesco Redi, John Needham, Lazzaro Spallanzani, and Nicolas Appert, etc, and to know the disease transmission which mainly includes the work of Ignaz Semmelweis and John Snow.  · Francesco Redi (1626-1697) The ancient belief in spontaneous generation was first of all challenged by Redi, an Italian physician, who carried out a series of experiments on decaying meat and its ability to produce maggots spontaneously. · John Needham (1713-1781) He was probably the greatest supporter of the theory of spontaneous generation. He proposed that tiny organisms the animalcules arose spontaneously on his mutton gravy. He covered the flasks with cork as done by Redi and even heated some flasks. Still the microbes appeared on mutton broth. · Lazzaro Spallanzani (1729-1799) He was an Italian Naturalist who attempted to refute Needham’s experiment. He boiled beef broth for longer period, removed the air from the flask and then sealed the container. Followed incubation no growth was observed by him in these flasks. He showed that the heated nutrients could still grow animalcules when exposed to air by simply making a small crack in the neck. Thus, Spallanzani disproved the doctrine of spontaneous generation. · Nicolas Appert He followed the idea of Spallanzani’s work. He was a French wine maker who showed that soups and liquids can be preserved by heating them extensively in thick champagine bottles. Ignaz Semmelweis and John Snow These were the two persons who showed a growing awareness of the mode of disease transmission. Thereafter two German scholars Schulze (1815-1873) and Theodor Schwan (1810-1882) viewed that air was the source of microbes and sought to prove this by passing air through hot glass tubes or strong chemicals into boiled infusions in flasks. The infusion in both the cases remained free from the microbes. · George Schroeder and Theodor Von Dusch (1854) These were the first to introduce the idea of using cotton plugs for plugging microbial culture tubes. · Darwin (1859) in his book, ‘Origin of the Species’ showed that the human body could be conceived as a creature susceptible to the laws of nature. He was of the opinion that disease may be a biological phenomenon, rather than any magic. THE GOLDEN AGE The golden age of microbiology began with the work of Louis Pasteur and Robert Koch who had their own research institute. More important there was an acceptance of their work by the scientific community throughout the world and a willingness to continue and expand the work. During this period, we see the real beginning of microbiology as a discipline of biology. The concept of spontaneous generation was finally put to rest by the French chemist Louis Pasteur in an inspired set of experiments involving a goosenecked flask. When he boiled broth in a flask with a straight neck and left it exposed to air, organisms grew. When he did this with his goose-necked flask, nothing grew. The S-shape of this second flask trapped dust particles from the air, preventing them from reaching the broth. By showing that he could allow air to get into the flask but not the particles in the air, Pasteur proved that it was the organisms in the dust that were growing in the broth. Pasteur, thus in 1858 finally resolved the controversy of spontaneous generation versus biogenesis and proved that microorganisms are not spontaneously generated from inanimate matter but arise from other microorganisms. Pasteur, also found that fermentation of fruits and grains, resulting in alcohol, was brought about by microbes and also determined that bacteria were responsible for the spoilage of wine during fermentation. Pasteur in 1862 suggested that mild heating at 62.8°C (145°F) for 30 minutes rather than boiling was enough to destroy the undesirable organisms without ruining the taste of the product, the process was called Pasteurization. Pasteurization was introduced into the United States on a commercial basis in 1892. His work led to the development of the germ theory of disease. · Louis Pasteur is known as the “Father of Modern Microbiology / Father of Bacteriology. Summary of important contributions of Louis Pasteur. 1. He has proposed the principles of fermentation for the preservation of food. 2. He introduced sterilization techniques and developed steam sterilizers, hot air oven, and autoclave. 3. He described the method of pasteurization of milk. 4. He had also contributed for designing the vaccines against several diseases such as anthrax, fowl cholera, and rabies 5. He disproved the theory of spontaneous generation of disease and postulated the ‘germ theory of disease’. He stated that disease cannot be caused by bad air or vapor, but it is produced by the microorganisms present in the air. 6. Liquid media concept- He used nutrient broth to grow microorganisms. 7. He was the founder of the Pasteur Institute, Paris. Louis Pasteur proposed the Principles of fermentation, Pasteurization of milk, sterilization techniques and the germ theory of disease John Tyndall (1820 – 1893) An English physicist, deal a final blow to spontaneous generation in 1877. He conducted experiments in an aseptically designed box to prove that dust indeed carried the germs. He demonstrated that if no dust was present, sterile broth remained free of microbial growth for indefinite period even if it was directly exposed to air. He discovered highly resistant bacterial structure, later known as endospore, in the infusion of hay. Prolonged boiling or intermittent heating was necessary to kill these spores, to make the infusion completely sterilized, a process known as Tyndallisation. Around the same time that Pasteur was doing his experiments, a doctor named Robert Koch was working on finding the causes of some very nasty animal diseases (first anthrax, and then tuberculosis). He gave the first direct demonstration of the role of bacteria in causing disease. He was a german physician who first of all isolated anthrax bacillus (Bacillus anthracis, the cause of anthrax) in 1876. He perfected the technique of isolating bacteria in pure culture. He also introduced the use of solid culture media in 1881 by using gelatin as a solidifying agent. In 1882 he discovered Mycobacterium tuberculosis. He proposed Koch postulate which were published in 1884 and are the corner stone of the germ theory of diseases and are still in use today to prove the etiology (specific cause) of an infectious disease. Koch’s four postulates are: · The organism causing the disease can be found in sick individuals but not in healthy ones. · The organism can be isolated and grown in pure culture. · The organism must cause the disease when it is introduced into a healthy animal. · The organism must be recovered from the infected animal and shown to be the same as the organism that was introduced. Germ theory of disease The combined efforts of many scientists and most importantly Louis Pasteur and Robert Koch established the Germ theory of disease. The idea that invisible microorganisms are the cause of disease is called germ theory. This was another of the important contributions of Pasteur to microbiology. It emerged not only from his experiments disproving spontaneous generation but also from his search for the infectious organism (typhoid) which caused the deaths of three of his daughters. · Fanne Eilshemius Hesse (1850 – 1934) He was one of Koch’s assistant first proposed the use of agar in culture media. Agar was superior to gelatin because of its higher melting (i.e. 96°C) and solidifying (i.e. 40-45°C) points than gelatin and was not attacked by most bacteria. One other Koch’s assistant Richard Petri in 1887 developed the Petri dish (plate), a container used for solid culture media. Thus contribution of Robert Koch, Fannie Hesse and Richard Petri made possible the isolation of pure cultures of microorganisms and directly stimulated progress in all areas of microbiology. HISTORY OF MICROBIOLOGY RELATING TO MEDICAL PRACTICE · Once scientists knew that microbes caused disease, it was only a matter of time before medical practices improved dramatically. Surgery used to be as dangerous as not doing anything at all, but once aseptic (sterile) technique was introduced, recovery rates improved dramatically. Hand washing and quarantine of infected patients reduced the spread of disease and made hospitals into a place to get treatment instead of a place to die. · Lord Joseph Lister (1827-1912) A famous English surgeon is known for his notable contribution to the antiseptic treatment for the prevention and cure of wound infections. Lister concluded that wound infections too were due to microorganisms. In 1867, he developed a system of antiseptic surgery designed to prevent microorganisms from entering wounds by the application of phenol on surgical dressings and at times it was sprayed over the surgical areas. He also devised a method to destroy microorganisms in the operation theatre by spraying a fine mist of carbolic acid into the air, thus producing an antiseptic environment. Thus Joseph Lister was the first to introduce aseptic techniques for control of microbes by the use of physical and chemical agents which are still in use today. Because of this notable contribution, Joseph Lister is known as the Father of Antiseptic surgery. · Vaccination was discovered before germ theory, but it wasn’t fully understood until the time of Pasteur. In the late 18th century, milkmaids who contracted the nonlethal cowpox sickness from the cows they were milking were spared in deadly smallpox outbreaks that ravaged England periodically. The physician Edward Jenner used pus from cowpox scabs to vaccinate people against smallpox. · Edward Jenner (1749-1823) He was an English physician was the first to prevent small pox. He was impressed by the observation that countryside milk maid who contacted cowpox (Cowpox is a milder disease  caused by a virus closely related to small pox) while milking were subsequently immune to th small pox. On May 14 , 1796 he proved that inoculating people with pus from cowpox lesions provided protection against small pox. Jenner in 1798, published his results on 23 successful vaccinators. Eventually this process was known as vaccination, based on the latin word ‘Vacca’ meaning cow. Thus the use of cow pox virus to protect small pox disease in humans became popular replacing the risky technique of immunizing with actual small pox material. · The significance of Jenner’s experiment was realized by Pasteur who next applied this principle to the prevention of anthrax and it worked. He called the attenuated cultures vaccines (Vacca = cow) and the process as vaccination. Encouraged by the successful prevention of anthrax by vaccination, Pasteur marched ahead towards the service of humanity by making a vaccine for hydrophobia or rabies (a disease transmitted to people by bites of dogs and other animals). As with Jenner’s vaccination for small pox, principle of the preventive treatment of rabies also worked fully which laid the foundation of modern immunization programme against many dreaded diseases like diphtheria, tetanus, pertussis, polio and measles etc. · Elie Metchnikoff (1845-1916) He proposed the phagocytic theory of immunity in 1883. He discovered that some blood leukocytes, white blood cells (WBC) protect against disease by engulfing disease causing bacteria. These cells were called phagocytes and the process phagocytosis. Thus human blood cells also confer immunity, referred to as cellular immunity. HISTORY OF MICROBIOLOGY IN THE DEVELOPMENT OF ANTIBIOTICS AND CHEMOTHERAPEUTIC AGENTS · Emile Roux (1853-1933) and Alexandre Yersin, These two notable French bacteriologists demonstrated the production of toxin in filtrates of broth cultures of the diphtheria organism. Emil von Behring (1854 -1917) and Shibasaburo Kitasato (1852-1931) both colleagues of Robert Koch, in 1890 discovered tetanus (lock jaw) antitoxin. Only about a week after the announcement of the discovery of tetanus antitoxin, Von Behring in 1890 reported on immunization against diphtheria by diphtheria antitoxin. The discovery of toxin-antitoxin relationship was very important to the development of science of immunology. · Paul Ehrlich (1854-1915) Erlish in 1904 found that the dye Trypan Red was active against the trypanosome that causes African sleeping sickness and could be used therapeutically. This dye with antimicrobial activity was referred to as a ‘magic bullet’. Subsequently in 1910, Ehrlich in collaboration with Sakahiro Hata, a japanese physician, introduced the drug Salvarsan (arsenobenzol) as a treatment for syphilis caused by Treponema pallidum. Ehrlich’s work had laid important foundations for many of the developments to come and the use of Salvarsen marked the beginning of the eni of chemotherapy and the use of chemicals that selectively inhibit or kill pathogens without causing damage to the patient. Summary of Paul Erlich contribution 1. He was the first to report the acid-fast nature of tubercle bacillus. 2. He developed techniques to stain tissues and blood cells. 3. He proposed a toxin-antitoxin interaction called an Ehrlich phenomenon and also introduced methods of standardizing toxin and antitoxin 4. He proposed the ‘side-chain theory for antibody production’. 5. He discovered ‘salvarsan’, an arsenical compound (magic bullet) for treatment of syphilis, hence Paul Ehrlich is known as the father of chemotherapy. 6. The bacteria ‘Ehrlichia’was named after him. Discovery of the wonder drugs - antibiotics Sir Alexander Fleming · The credit for the discovery of this first ‘wonder drug’ penicillin in 1929 goes to Sir Alexander Fleming of England, a Scottish physician and bacteriologist. Fleming had been actually interested in searching something that would kill pathogens ever since working on wound infections during the first world war (1914-1918). · Fleming left a solid culture in a Petri dish (called a plate) of bacteria was left to sit around longer than usual. As will happen with any food source left sitting around, it became moldy, growing a patch of fuzzy fungus. The colonies in the area around the fungal colony were smaller in size and seemed to be growing poorly compared to the bacteria on the rest of the plate. The compound found to be responsible for this antibacterial action was named penicillin. The first antibiotic, penicillin was later used to treat people suffering from a variety of bacterial infections and to prevent bacterial infection in burn victims, among many other applications. · Waksman at the Rutgers University, USA., discovered another antibiotic, streptomycin produced by strains of actinomycete, Streptomyces griseus in 1944. Waksman received the  noble prize in 1952 for his discovery of Streptomycin used in the treatment of tuberculosis, a bacterial disease caused by Mycobacterium tuberculosis that had been discovered by Robert Koch in 1882 · A dramatic turn in microbiology research was signaled by the death of Robert Koch in 1910 and advent of World war I. The Pasteur Institute was closed, and the German laboratories converted for production of blood components used to treat war infections. Thus came to an end what many have called the Golden Age of Microbiology. · THE ERA OF MOLECULAR BIOLOGY · By the end of 1900, science of microbiology grew up to the adolescence stage and had come to its own as a branch of the more inclusive field of biology. · In the later years the microorganism were picked up as ideal tools to study various life processes and thus an independent discipline of microbiology, molecular biology was born. · The relative simplicity of the microorganism, their short life span and the genetic homogeneity provided an authentic simulated model to understand the physiological, biochemical and genetical intricacies of the living organisms. · The field of molecular biology made great strides in understanding the genetic code, how DNA is regulated, and how RNA is translated into proteins. Until this point, research was focused mainly on plant and animal cells, which are much more complex than bacterial cells. When researchers switched to studying these processes in bacteria, many of the secrets of genes and enzymes started to reveal themselves. Molecular Koch’s postulates: It was a modification of Koch’s postulates (by Stanley Falkow). He stated that the gene (coding for virulence) of a microorganism should satisfy all the criteria of Koch’s postulates rather than the microorganism itself. Other important contributors in Microbiology 1. Antonie Philips van Leeuwenhoek: Discovered single-lens microscope and named organisms as ‘Little animalcules’. 2. Edward Jenner: Developed the first vaccine of the world, the smallpox vaccine by using the cowpox virus. 3. Joseph Lister: Joseph Lister is considered to be the father of antiseptic surgery. He used carbolic acid during surgery. 4. Hans Christian Gram: He developed a ‘Gram stain’. 5.Ernst Ruska: He was the founder of the electron microscope. 6.Alexander Fleming: He discovered the antibiotic penicillin. 7.Elie Metchnikoff: He described phagocytosis and termed phagocytes. 8.Kleinberger: He described the existence of L forms of bacteria. 9.Barbara McClintock: She described transposons. 10. Walter Gilbert and Frederick Sanger: were the first to develop (1977) the method of DNA sequencing. 11. Karry B Mullis: Discovered polymerase chain reaction (PCR). 12. Robert Hooke, a 17th-century English scientist, was the first to use a lens to observe the smallest unit of tissues he called “cells.” Soon after, the Dutch amateur biologist Anton van Leeuwenhoek observed what he called “animalcules” with the use of his homemade microscopes. PROKARYOTES and EUKARYOTES These are the two fundamental cells, the prokaryote cell and eukaryote cell. Prokaryotes are relatively small cells surrounded by the plasma membrane, with a characteristic cell wall that may differ in composition depending on the particular organism. Prokaryotes lack a nucleus (although they do have circular or linear DNA) and other membrane-bound organelles (though they do contain ribosomes). Bacteria and Archaea are the two domains of prokaryotes. Eukaryotes: Eukaryotes are the first of complex cells, which were labeled proto-eukaryotes. Over a period of time these cells acquired a mitochondrial symbiont and later developed a nucleus. This amongst other changes, have posed as the significant difference between the two. Animals fungi, and protoctista. Prokaryotic Cell The term “prokaryote” is derived from the Greek word “pro “, (meaning: before) and “karyon” (meaning: kernel). It translates to “before nuclei. “ Prokaryotes are one of the most ancient groups of living organisms on earth, with fossil records dating back to almost 3.5 billion years ago. These cells are comparatively smaller and much simpler than eukaryotic cells. The other defining characteristic of prokaryotic cells is that it does not possess membrane-bound cell organelles such as a nucleus. Reproduction happens through the process of binary fission. Structurally, prokaryotes have a capsule enveloping their entire body, and it functions as a protective coat. This is crucial for preventing the process of phagocytosis (where the bacteria gets engulfed by other eukaryotic cells, such as macrophages) The pilus is a hair-like appendage found on the external surface of most prokaryotes and it helps the organism to attach itself to various environments. The pilus essentially resists being flushed, hence, it is also called attachment pili. It is commonly observed in bacteria. Certain pili, known as conjugation pili, unite prokaryotic cells to one another and permit the passage of DNA between the cells. The term fimbriae is often used for the attachment pili. Right below the protective coating lies the cell wall, which provides strength and rigidity to the cell. Further down lies the cytoplasm that helps in cellular growth, and this is contained within the plasma membrane, which separates the interior contents of the cell from the outside environment. Within the cytoplasm, ribosomes exist and it plays an important role in protein synthesis. It is also one of the smallest components within the cell. Some prokaryotic cells contain special structures called mesosomes which assist in cellular respiration. Most prokaryotes also contain plasmids, which contain small, circular pieces of DNA. To help with locomotion, flagella are present, (Flagella are long, ultrathin structures, many times the length of the cell. Common examples of Prokaryotic organisms are bacteria and archaea. Also, all members of Kingdom Monera are prokaryotes The Eukaryotic Cell The term “Eukaryotes” is derived from the Greek word “eu“, (meaning: good) and “karyon” (meaning: kernel), therefore, translating to “good or true nuclei.” Eukaryotes are more complex and much larger than prokaryotes. They include almost all the major kingdoms except kingdom monera. Structurally, eukaryotes possess a cell wall, which supports and protects the plasma membrane. The cell is surrounded by the plasma membrane and it controls the entry and exit of certain substances. The nucleus contains DNA, which is responsible for storing all genetic information. The nucleus is surrounded by the nuclear membrane. Within the nucleus exists the nucleolus, and it plays a crucial role in synthesising proteins. Eukaryotic cells also contain mitochondria, which are responsible for the creation of energy, which is then utilized by the cell as well as many other cell organelles which performs many different functions in the cell. Examples of eukaryotes include almost every unicellular organism with a nucleus and all multicellular organisms. SOME DIFFERENCES BETWEEN PROKARYOTES AND EUKARYOTES FEATURES PROKARYOTES EUKARYOTES Organism Bacteria, archea Fungi Protist, plants and animals All are unicellular organisms Unicellular and multicellular Cell Size A diameter (0.5 - 10Nm) 10 – 100nm, 1000-10000 µm sometimes Cell division Mainly by binary fusion Mitosis, Meiosis or both Genetic material DNA is circular lies free in the DNA is linear contained in the cytoplasm nucleus Protein synthesis 7OS ribosomes (smaller) no 8OS ribosomes which may be endoplasmic reticulum present attached to E. R` Organelles Few to none is surrounded by Many organelles enveloped by envelope single/double membrane Cell wall Rigid contain polysaccharides, Cell wall of plants fungi contain amino acids, murein as polysaccharide, cellulose is the strengthening agent. strengthening agent Plasmids Present Very rarely found in eukaryotes Endoplasmic reticulum Present Absent Flagella Simple, lacking microtubules Complex Respiration Mesosomes in bacteria Mitochondria for aerobic respiration Lysosome Lysosomes and centrosomes are Lysosomes and centrosomes are absent present Cell division Through binary fission Through mitosis Flagella The flagella are smaller in size The flagella are larger in size Reproduction Asexual Both asexual and sexual Classification of prokaryotes into archaea and eubacteria. DNA sequence comparisons together with structural and biochemical comparisons consistently categorize all living organisms into 3 primary domains: Bacteria, Archaea, and Eukarya (also called Eukaryotes). All three domains of life share a single common ancestor. Both Bacteria and Archaea are prokaryotes, single-celled microorganisms with no nuclei. Both belong to the kingdom Monera plus Gram negative bacteria Archaea and Bacteria Archaea and Bacteria generally have a single circular chromosome: a piece of circular, double- stranded DNA located in an area of the cell called the nucleoid. They both reproduce asexually through binary fission, a process where an individual cell reproduces its single chromosome and splits in two; eukaryotes reproduce asexually through mitosis, Archaea and Bacteria can share genomic information between individual cells which result in a phenomenon called horizontal gene transfer, also called lateral gene transfer. (Horizontal gene transfer is in contrast to vertical gene transfer, which is where offspring inherit genetic information from a parent as a result of reproduction). Horizontal gene transfer occurs when a cell acquires DNA from an external source, and it results in shared DNA between individuals that may not be genetically related. Horizontal gene transfer is the primary way that antibiotic resistance spreads through microbial populations. Almost all prokaryotes have a cell wall, a protective structure that allows them to survive in extreme conditions, which is located outside of their plasma membrane. The composition of the cell wall differs significantly between the three domains of life. a. Bacterial cell walls are composed of peptidoglycan, a complex of protein and sugars. b. Archaeal cell walls are composed of polysaccharides (sugars). c. Eukaryotic cell walls found in plants are composed of cellulose, and the cell walls of fungi are composed of chitin. Some differences between archae and eubacteria: -Cell Wall Archae: Cell wall is mainly composed of pseudo peptidoglycans. Eubacteria: The cell wall is mainly composed of peptidoglycans with muramic acid -Growth and Reproduction Archae: Asexual reproduction- binary fission, fragmentation, and budding. Eubacteria: In addition to other asexual methods they also produce spores in order to remain dormant during unfavorable conditions. Archae are usually found in extreme conditions whereas eubacteria are found everywhere on the surface of Earth. DISINFECTION AND STERILIZATION i. Sterilization Sterilization is the process of killing all microorganisms (bacterial, viral, and fungal) with the use of either physical or chemical agents. A disinfectant is a chemical substance that kills microorganisms on inanimate objects, such as exam tables and surgical instruments. Skin can never be completely sterile. Sterilization in the microbiological laboratory denotes sterilization process implemented in preparation of culture media, reagents and equipment where the work warrants maintaining sterile condition. Sterilization in microbiology laboratory is done by following methods Physical method i.e., use of heat, filters, radiation Chemical method i.e., by use of chemicals. Types of Sterilization methods a. DRY HEAT STERILIZATION: Inoculation loops or needle are sterilized by heating to 'red' in Bunsen burner or spirit lamp flame. Sterilization in hot air oven is performed at a temperature of 160°C and maintained or holding for one hour. Spores are killed at this temperature and this is the most common method of sterilization of glassware, swab sticks, pestle and mortar, mineral oil etc. Dry heat sterilization causes protein denaturation, Oxidative damage, toxic effect of elevated electrolyte in absence of water. b. WET HEAT OR MOIST HEAT STERILIZATION Moist heat sterilization is accomplished by (1). Boiling at 100°C for 30 minutes is done in a water bath. Syringes, rubber goods and surgical instruments may be sterilized by this method. Almost all bacteria and certain spores are killed in this method. (2). Steaming at 100°C for 20 to 30 minutes under normal atmospheric pressure are more effective than dry heat at the same temperature because bacteria are more susceptible to moist heat, Steam has more penetrating power and sterilizing power as more heat is given up during condensation. Suitable for sterilizing media which may be damaged at a temperature higher than 100°C. 3).Tyndallization (Fractional Sterilization) is the steaming process performed at 100°C is done in steam sterilizer for 20 minutes followed by incubation at 37°C overnight and this cycle is repeated for successive 2 days. Spores, if any, germinate to vegetative bacteria during incubation and are destroyed during steaming on second and third day. Heat labile media containing sugar, milk, gelatin can be sterilized using this method. 4). Autoclaving is done by steam under pressure. Steaming at temperature higher than 100°C is used in autoclaving. This is achieved by employing a higher pressure. The autoclave is closed and made air-tight for pressure development and at 15 lbs per sq. inch pressure, 121°C temperatures will be reached and this temperature is given as sterilizing holding time for further 15 minutes. This process kills spores and this works like a pressure cooker and one of the most common methods of sterilization. 5). Pasteurization is another one method of moist heat sterilization which works below 100°C heat. This process is used in heating of milk and other liquid food. The product is held at temperature and for a period of time to kill pathogenic bacteria that may be present in the product. This process does not destroy complete organism including spores. All these moist heat sterilization causes denaturation and coagulation of protein, breakage of DNA strands, and loss of functional integrity of cell membrane. c). FILTRATION: This method of sterilization is used for media particularly heat labile in nature (e.g., sera an media containing proteins or labile metabolites. If the study warrants bacteria- free filtrates it can be obtained through 0.45micron sized filter membranes and if the study requires viral particle free solution, then 0.22micron sized filter membranes are use. In earlier days absorptive filters of asbestos or diatomaceous earth were replaced by unglazed porcelain or sintered glass are used. Nowadays these are replaced by nitrocellulose membrane filters of graded porosity, PVDF etc. d). ULTRAVIOLET RADIATION: at wavelength between 330nm and 400nm causes sterilizing effect. This method is used in surface sterilization of laminar airflow, biosafety cabinet and in certain cases in laboratory. In microbiology laboratory autoclaving, hot air oven sterilization, filtration and UV radiation are commonly used. ii. DISINFECTION The term disinfection refers to the use of a physical process or the use of a chemical agent to destroy vegetative microbes and viruses Disinfectants are applied to inanimate surfaces, medical equipment, and other man-made objects whereas antiseptics are used to disinfect skin.. This does not include bacterial endospores. The ideal disinfectant would result in complete sterilization without harming other forms of life. Unfortunately, ideal disinfectants as such do not exist and most of them only partially sterilize. In addition to the most resistant pathogens, endospores, other bacteria, and viruses are also highly resistant to many disinfectants. Substances that kill bacteria are bactericidal and those that interfere with cell growth and reproduction are bacteriostatic. Disinfectants and antiseptics are bactericidal and bacteriostatic depending on the concentration applied. All disinfectants are by their nature potentially harmful, even toxic, to humans and animals. They should be handled with appropriate care to avoid harm to the handler or recipient. The type of disinfectant to be used depends on the surface or material to be disinfected. iii. SANITIZATION Several applications in everyday life and medicine do not require sterilization, disinfection, or antisepsis but need to reduce microorganisms in order to control possible infections or spoilage of substances. Sanitization achieves this by using any cleansing technique that mechanically removes microorganisms and other debris to reduce contamination to safe levels. Often the sanitizer used is a compound such as soap or detergent. Restaurants, dairies, breweries, and other food industries handle soiled utensils on a daily basis and must take appropriate measures to sanitize them for prevention of infection, spoilage, and contamination. This includes controlling microbes to a minimal level during preparation and processing. Degermation This is the process by which the numbers of microbes on the human skin are reduced by scrubbing, immersion in chemicals, or both. Some examples of degermation include the process of pre-surgical scrubbing of the hands with sterile brushes and germicidal soap before putting on sterile surgical gloves, the application of alcohol wipes to the skin, and the cleansing of a wound with germicidal soap and water. ECONOMIC IMPORTANCE OF MICROORGANISMS Bacteria play very important role in the continuous sustenance of life. They are man’s best friends as well as enemy. There are many useful as well as harmful bacteria around us. Economic importance of bacteria are studied under two headings: Beneficial activities and Harmful activities. The following are some of the important beneficial activities of microorganisms. A) Agricultural Importance: The activities of bacteria are very important in agriculture in the following aspects: (a) Decaying of organic substance: Most of the bacteria are very useful in bringing about decomposition of dead organic matter of plants and animals by the secretion of enzymes. The enzymes convert the fats, carbohydrates and nitrogenous compounds into simpler forms, such as, CO2 water, ammonia, hydrogen sulphide, phosphates, nitrates etc that are used as raw material by the green plants. Thus, these bacteria not only decompose the organic compounds but also remove the harmful waste from the earth and thus function as nature's scavengers. saprophytes {b) Fertility of the soil: Some bacteria maintain and others increase the fertility of the soil. They bring about physical and chemical changes in the soil by converting insoluble materials into soluble ones. These bacteria are the ammonifying, nitrifying and the nitrogen fixing Bacteria. (i) Ammonifying Bacteria: The decay bacteria decompose the proteinous compounds into amino acids, which are reduced to ammonia by ammonifying bacteria. The free ammonia combines in the soil to form ammonium salts. This conversion is known as ammonification. Examples are Bacillus ramosus, B. vulgaris etc. (ii) Nitrifying Bacteria: These bacteria convert ammonium salts into nitrates, which are absorbed by the plants. The nitrifying bacteria are the Nitrobacter and Nitrosomonas. The Nitrosomonas oxidize the ammonium salts into nitrous acid, which forms nitrites in the soil. The Nitrobacter then converts the nitrites into nitrates. This conversion of ammonium salts into available nitrates is called nitrification. (iii) Nitrogen Fixing Bacteria: These bacteria take up nitrogen from the atmosphere and convert it into organic nitrogen compounds. It is known as nitrogen fixation. The nitrogen-fixing bacteria are of two types. One type includes Azotobacter and Clostridium, which live freely in the soil and fix nitrogen of the air in their bodies in the form of nitrogenous organic compounds. The other types of bacteria are the nodule bacteria, the Bacillus radicicola. Rhizobium lives as symbiont in the roots of leguminous plants and forms nodules. The leguminous plants thus enrich the fertility of the soil. They are grown for green manuring and rotation of crops. (2) Role in Industry Bacteria play a very important role in various industries. The products obtained as a result of bacterial activities cannot be chemically prepared. Their activities are involved in the following industries: (a) Preparation of Alcohols: Ethyl alcohol and butyl alcohol are manufactured by the bacterial activities in the sugar solution, e.g., Clostridium acetobutylicum. (b) Preparation of Vinegar: Vinegar is prepared by the activities of Acetobacter aceti in the sugarcane juice. (c) Preparation of Butter, Cheese etc.: The preparation of butter, cheese etc. is done by bacteria. The Lactobacillus lactis is responsible for souring of milk resulting in curd (dahi) preparation. Bacterial activities also impart the typical flavours. (d) Preparation of Tea, Coffee etc.: Bacteria are very useful in preparation and flavouring of tea, coffee, cocoa etc. e.g., Micrococcus. (e) Preparation of Tobacco: Tobacco leaves are cured and flavoured by the bacteria. Typical types of bacteria are cultured for this purpose, e.g., Micrococcus. (f) Preparation of Hemp fibres: Fibres from the hemp are isolated after rotting the stems by activity of bacteria (e.g., Clostridium butyricum). The bacteria eat up the protoplasmic tissues but leave the sclerenchyma fibres. (g) Preparation of Leather and Tanning: The hairs and fats are removed from the skin by the action of bacteria in the leather industry. h) Food Industry: A good number of food products are manufactured due to bacterial activities e.g. Production of fermented food products such as yoghurt, butter, Cheese etc. C) IMPORTANCE IN HUMAN MEDICINE Antibiotics Antibiotics are compounds produced by microorganisms which either kill the target pathogen or inhibit its growth. The first antibiotic was discovered by Alexander Fleming in 1929. He accidentally discovered that Pencillium notatum inhibited the growth of gram-positive bacterium staphylococcus. Antibiotics are secondary metabolites, mostly produced by streptomyces, a bacterial genus belonging to Actinomycetes. One of the most common antibiotics produced by bacteria is streptomycin. In addition to antibacterial antibiotics, bacteria also produce antifungal drugs like Nystatin and amphotericin B. Nystatin, an antibiotic produced by Streptomyces is used in treatment of superficial mycoses caused by fungi. Nystatin is active against infections of skin, vagina or alimentary tract caused by Candida. Vaccines A vaccine is a preparation of either the altered whole microorganism or its specific components that is used to induce immunity in host. Bacteria can be thus used for preparation of vaccines which evoke hormonal and cell mediated responses to ward off pathogens during active infection. Vaccines are of different types with their corresponding advantages and disadvantages. Attenuated bacterial vaccines include vaccine against tuberculosis (Mycobacterium tuberculosis) and typhoid (Salmonella spp) bacteria. Inactivated whole agent bacterial vaccines have been formulated for Vibrio cholera, Pneumococcus, Bordetella and Salmonella. Bacterial vaccines prepared from bacterium Clostridium tetani and Corynebacterium diptheriae are the toxoid vaccines that is they contain the inactivated toxins of these bacteria. Steroid Biotransformation Steroids are used as drugs in the treatment of number of diseases like asthma, rheumatoid arthritis, reproductive and other hormonal disorders. Animal steroids are produced in limited quantity and cannot possibly be extracted from human sources Plant sterols like stigma sterol, Sapogenins are abundant in nature and can be converted into animal steroids using microorganisms. Corynebacterium simplex is used for production of prednisolone. Insulin synthesis This has been achieved using Recombinant DNA technology by inserting the insulin gene into a suitable vector, the Escherichia coli bacterial cell B. Harmful Activities of Bacteria Bacteria are also harmful to man directly or indirectly. They cause various diseases in plants, human beings or domestic animals. The harmful bacteria are of the following types; (i) Animal pathogenic Bacteria. (ii) Plant pathogenic Bacteria. (iii) Food destroying Bacteria. (iv) Soil fertility destroying Bacteria. (i) Animal Pathogenic Bacteria: There are a large number of parasitic bacteria, which cause various serious diseases in man and domestic animals, sometimes in epidemic form. They are invisible enemies. Some of the common human diseases producing bacteria are Mycobacterium tuberculosis causing tuberculosis, Salmonella typhi causing typhoid fever, Clostridium tetani causing tetanus, Shigella dysenteriae causing dysentery, Heamophilus influenzae causing influenza, Corynebacterium diphtheriae causing diphtheria, Vibrio cholerae causing cholera, Streptococcus causing blood poisoning, Treponema pallidium causing syphilis, Gonococcus causing gonorrhoea, Bacillus pestis causing plague etc. In domestic animals various diseases are caused by bacteria, e.g., Anthrax, Pneumonia, Tuberculosis, Cholera, Glanders etc. (ii) Plant Pathogenic Bacteria: Many parasitic bacteria cause serious diseases in cultivated plants, which cause great harm to the crops. Important diseases are Citrus canker, fire blight of pear, cotton root rot, walnut blight, potato rot, pineapple rot etc. The Canker of Citrus (orange and lemon) is caused by Xanthomonas citri. The rot diseases cause black spots on potato, tomato, cabbage, carrot etc. (iii) Food destroying Bacteria: Some saprophytic bacteria are responsible for the decay of human foodstuffs including meat, milk, vegetables, fruits etc. These bacteria spoil foodstuffs and make them unpalatable and poisonous, e.g., souring of milk, rotting of meat, vegetables, fruits etc. Food poisoning- Some bacteria like Staphylococcus aureus, Clostridium botulinum secrets toxic substance due to which food becomes poisonous resulting even death too, (iv) Soil Fertility destroying Bacteria: These are denitrifying bacteria in the soil, which reduce the nitrates, and the ammonium salts to free nitrogen, which escapes into the atmosphere. This process is known as denitrification, 5) Denitrification and desulphurification of soil-plants can absorb NO3 and SO4 form of nitrogen and sulphur by the process of nitrification and sulphurificarion which decreases the fertility of the soil, e.g., Bacillus denitrificans. These bacteria are often abundant in the poorly drained and heavily manured soil. So, the denitrifying bacteria are the natural enemies of the farmers. Thus, with the study of economic importance of bacteria we conclude that bacteria are our friends due to their beneficial aspects and enemies due to their harmful aspects. But in the bacteria beneficial aspects overweigh their harmful aspects. We can control the harmful activities but their beneficial activities cannot be replaced by artificial processes. So the bacteria are our friends more and enemies less 4) Deterioration of Domestic articles- Wooden articles, fibres, leather deteriorate by action of bacteria e.g. (Spirochaete). Economic Importance of Fungi Fungi belong to one of the five kingdom classification of living organisms. It includes microorganisms such as yeast and molds. They are cosmopolitan and occur in air, water, soil and on animals and plants. Some common economic importance of fungi are i. Fungi is a kingdom of microorganisms which includes yeast and moulds, Yeast, Penicillium, Mushroom, Moulds i.e., Rusts ii. They find various applications in medical industries such as penicillin is made from a fungi penicillium. iii. It is also used in agricultural industries for the enhancement of crop quality and nutrients in the soil. iv. Some specific fungi also help in the improvement of plant growth. v. The most common economical uses of fungi include production of bread, beer, cheese, enzymes and organic acids, and in food such as mushroom. vi. Some are of economic importance in medicine in their ability to cause diseases ranging from superficial to systemic infections vii. The most common fungi yeast is used in the production of bread and beer. viii. A free living fungi named Trichoderma sp present in the root ecosystem act against plant pathogens. ix. Some species of fungi penicillium is used to produce roquefort and camembert cheese. x. Fungi are also used in the commercial production of different enzymes and organic acids. xi. Eaten as a food; There are some edible fungi such as mushrooms which provide different essential nutrients like vitamins, amino acids and lipids. xii. It also plays an economically important role in recycling processes. Specific roles of fungi in medicine Fungi plays an essential role in the production of medicines and is very helpful to cure various health issues. Some of the commonly used fungi include: · The antibiotic penicillin produced from the fungi penicillium. · Micafungin is used as an antifungal agent. · Mycophenolate is a fungi which is used to prevent tissue rejection. · Rosuvastatin is a fungi which has found uses in reducing cholesterol. Aspergillus niger is used for the synthesis of different steroids. Specific roles of fungi in agriculture Fungi also find uses in various agricultural activities and plays an important role in the following: · Fungi has the ability to decompose the organic matter in the soil which leads to the increase in its nutritional quality. · They decrease the amount of nitrogen in the soil by converting it to protein for plants. · Fungi form a symbiotic relationship with plants known as mycorrhizae through which plants obtain phosphorus and other essential minerals. Fungi produce various bioactive metabolites which help in the improvement of plant growth. · Some important fungi in the agriculture industry are Fusarium, Chaetomium, Chytridium, Penicillium, and Aspergillus. Some Economic importance of Viruses i. Viruses are commonly used as vector in recombinant DNA technology because of their ability to insert their content into host cells. A vector is an autonomously replicating DNA fragment into which genes of interest can be integrated for cloning ii. Viruses as biopesticides: The most commonly used microbial biopesticide is produced from the bacterium Bacillus thuringiensis. However, viruses are also used as a pesticide for killing a number of insect species like moths, bollworms, fruitworms etc. The Biopesticidal agents can either prey on pests, be parasitic on them, compete with insects or are insect pathogens. iii. Viruses as causal organism of plant disease Plant diseases caused by viruses lead to massive agronomic losses. Viruses causes a number of diseases iv. Viruses as causal agents of several human/animal diseases v. Vaccine production: Viral vaccines confer immunity against infection with the pathogenic strains of same virus. The conventional vaccines synthesized using live attenuated viruses or killed viruses are easy to produce and economic vi. Gene therapy: The introduction of functional gene into human cells to correct defective genes by replacing them is known as Gene Therapy. Gene therapy is largely used in cancer treatment. vii. Cancer therapy: Viruses can be directly used to prevent cancer by being the source of anti- cancer vaccines, e.g., vaccines against hepatitis B virus (causes hepatic cancer) and human papillomavirus (cervical cancer) are commercially available. viii. Bacteriophage therapy: This is involve the use of bacteriophages for destruction of bacterial pathogens. This therapy has been used to successfully treat staphylococcal and E. coli infections and appears to be very promising. ix. Virus based diagnosis: virus play a pivotal role in diagnostic procedure commonly employed in various biological sciences viz. Microbiology, Molecular Biology, Immunology, Genetic Engineering etc. CULTURE OF MICRO-ORGANISMS. ISOLATION OF MICRO-ORGANISMS. ISOLATION OF BACTERIA To study bacteria and other microorganisms, it is necessary to grow them in controlled conditions. Microbes are grown in substances that provide the nutrients necessary to sustain their metabolic activities and reproduction called "growth media" or simply "media" (singular is "medium"). Growth media can be either liquid or solid. Nutritional requirements of particular microorganisms range from a few simple inorganic compounds to a complex list of specific inorganic and organic chemicals. Access to carbon, the essential component required for molecular life, is obtained in different ways by microorganisms. Autotrophs acquire carbon from carbon dioxide in the atmosphere and heterotrophs obtain their carbon from organic compounds. This diversity is seen in the different types of media needed to ensure the growth of the organism for investigation. Media vary in nutrient content and consistency and can be classified according to their physical state, chemical composition, and functional type. Physical State of Media Liquid media are water-based solutions that do not solidify at temperatures above freezing and flow freely in the containers when tilted. Most commonly, liquid media are supplied in tubes or bottles and are called broths, milks, or infusions. A common laboratory medium is nutrient broth, which contains beef extract and peptone (partially digested protein) dissolved in water. Methylene blue milk and litmus milk are opaque liquids prepared from skim milk powder and dyes. After inoculation, growth occurs throughout the container. Enriched broths are used to grow bacteria that are present in few numbers such as in small specimen samples obtained from patients. Semisolid media contain a limited amount of a solidifying agent such as agar or gelatin, giving the medium a clot like consistency. Semisolid media are often used to determine motility and growth patterns of bacteria. Solid media are dispensed in Petri plates or slanted in tubes or bottles to provide firm and maximal surfaces for growing bacteria or fungi. By far the most widely used and effective of these media is agar, composed of a complex polysaccharide from the red alga Gelidium. Agar is solid at room temperature and liquefies at the boiling temperature of water. Once in liquid form it does not solidify until it cools to 42° C. It then can be inoculated and poured in liquid form at temperatures that will not harm the microbes or the handlers. Agar added to media simply gels them into a solid form. Any medium containing 1% to 5% agar usually has the agar in the name of the specific medium as, for example, nutrient agar, phenylethyl alcohol agar, blood agar, and others. Chemical Classification of Media Depending on their chemical content media can be classified as complex or non-synthetic media and as chemically defined or synthetic media. Chemically defined media or synthetic media are media with a defined, exact chemical composition. They are prepared by means of an exact formula, adding precise amounts of inorganic and/or organic chemicals to distilled water. Some of these media contain minimal amounts of chemicals such as some salts and a source of carbon; others are special media containing a variety of precisely measured substances. Complex media or non-synthetic media contain at least one component that cannot be chemically defined and thus the medium cannot be represented by an exact chemical formula. Complex media contain extracts from animals, plants, or yeast. They may include blood, serum, meat extracts, milk, yeast extracts, soybean digests, and peptone. Functional Types of Media General-purpose media are designed to grow a broad spectrum of microbes that do not have any special growth requirements. Other media are available for special growth conditions of selected organisms. These include enriched, selective, and differential media. Enriched media contain complex organic substances such as blood, serum, hemoglobin, or growth factors for the growth needs of specific species. An example is blood agar, made by adding sterile sheep, horse, or rabbit blood to a sterile agar base. It is widely used to grow certain streptococci and other pathogens. Another enriched medium is chocolate agar. Chocolate agar is enriched with heat-treated blood, which turns brown and gives the medium the color and thus its name. Selective media inhibit the growth of selected organisms while allowing the growth of others. These media are useful in isolating bacteria or fungi from specimens that contain several different organisms. For example, mannitol salt agar contains 7.5% NaCl, inhibitory to most human pathogens with the exception of the genus Staphylococcus, which thrives in mannitol salt agar and consequently its growth can be amplified in mixed samples. Differential media can grow several different organisms that show visible differences. These differences can be variations in colony size or color, a change in medium color, or the formation of gas bubbles and precipitates. Dyes can be used as differential agents because many of them are pH indicators that change color in response to acid or base production by a specific microbe. For example, MacConkey agar contains neutral red, which is a dye that is yellow when neutral and pink or red when acidic. Escherichia coli, a bacterium common to the intestinal tract, produces acid when it metabolizes the lactose in the medium and develops red or pink colonies. In contrast, Salmonella does not give off acid and therefore remains in a natural off-white color. A comparison of general, selective, and differential media is shown below Simple or basal media: It consists of sodium chloride, peptone, meat extracts, and water, for example, Nutrient Broth. Indicator media: It contains an indicator that changes color when a certain bacterium grows on the medium. For example, the addition of sulfite in the Wilson and Blair medium changes color to black when Salmonella typhi colonies grow on the medium. Transport media: It’s a buffer solution containing peptone, carbohydrates, and other nutrients (except growth factors) to maintain the viability of the bacteria during transport without allowing their multiplication. An example is the Stuart medium for gonococci. Anaerobic media: It contains ingredients that support the growth of anaerobic bacteria. An example is Robertson’s cooked meat media. Now, the common culture techniques used in microbiology labs include: (A) tryptic soy agar—a complex medium used as an all-purpose growth medium, and (B) xylose lysine deoxycholate agar—a chemically defined agar that is both selective and differential and used primarily in selecting for and differentiating Pink colonies are probably Gram-negative enteric bacilli, especially Shigella. Yellow indicates the organism is utilizing the carbohydrate xylose and the black colonies indicate the production of hydrogen sulfide (i.e in Salmonella typhi) CULTURING MICROORGANISMS Inoculation In simple terms, inoculation in Microbiology is the process of introducing microbes into a culture media, in the introduced culture, the microbes can reproduce and grow. Mostly, it is used in research and laboratory practices wherein researchers aim to cultivate and study some species of bacteria. Bacteria and other microbes can be inoculated into a range of media where it thrives. It also describes the introduction of vaccines, serum or any antigenic substance substances through the absorptive surface of the skin into the body so as to boost immunity against a particular disease. It also finds its usage in commercial applications such as baking, brewing, wine making, producing antibiotics and more. A good example is the production of blue cheese through the inoculation of ripening cheese with dedicated mounds of bacteria. Isolation of microorganisms: Microorganisms occur in natural environment like soil. They are mixed with several other forms of life. The isolation and growth of suspected microbe in pure culture is essential for the identification of microorganisms. The primary culture from natural source will normally be a mixed culture containing microbes of different kinds. A culture which contains just one species of microorganism is called a pure culture. The process of obtaining a pure culture by separating one species of microbe from a mixture of other species, is known as isolation of the organisms. The methods used to achieve this include Streak Plate Method This is most widely used method of isolation. The technique consists of pouring a suitable sterile medium into sterile petri dishes and allowing the medium to solidify. It is used to obtain completely isolated colonies from a culture or specimen inoculum through the creation of sections of increasing dilution on a single plate. Inoculate the specimens through the use of sterile inoculation loops into the agar media. Spread the specimen gently on a section of the culture media surface Extract loop from the inoculated area and distribute into a second part Extract the loop from the other section and disperse it to the 3rd section. Continue for the 3rd and 4th section. Make sure that sections 1 and 4 are not overlapping. Unload inoculation loop used into suitable containers It is important to note that intermittently between streaking, the needle is then flamed and streaking in done at right angles to and across the first streak. Pour Plate Method The pour plate method is a laboratory technique for isolating and counting viable microorganisms like bacteria and fungi in a liquid sample that is added to a molten agar medium. In general, this technique counts the total number of CFUs (colony-forming units/ml) on the surface of the solid medium. Sometimes it may involve carrying out serial dilutions; Serial dilution – If the sample is a liquid and presumed to have high microbial load, it can be diluted serially with sterile broth or distilled water. If the sample is semisolid or solid, it must first be emulsified before being serially diluted to reduce the microbial load to the permissible limits. In the pour plate method, the sample is either added to the Petri plate or then poured with the molten agar medium, or the sample is mixed with the molten agar medium before pouring. Now the medium is allowed to solidify before being incubated at the appropriate temperature to grow the microbes present in the sample. The number of isolated colonies is counted after incubation. The major difference between the streak and pour plate method is that in the streak plate, the melted agar is added first, followed by a loop of bacteria from a slant, whereas in the pour plate, the bacterial broth is added first, followed by the agar. Agar stab technique It is used in the preparation of stab cultures, from a plate select single colonies. Select a well-isolated colony through aseptic technique using an inoculating stab needle (sterile) and stab it a few times via the agar to the base of the tube Substitute the cap and secure loosely during incubation enabling exchange of gases Incubation of this stabbed plate at the suitable temperature is carried out Spread Plate Method It is used for evenly spreading cells to ensure growth of the isolated separate colonies. Further, it can be used for serial dilutions. The spread plate method is used for enrichment, enumeration and screening and selection of microorganisms. Onto the agar media, with the help of a sterile spreader, inoculate the clinical specimen where we spread the bacteria gently on the whole culture media surface. This is done by rotating the plate while spreading it backwards and forward. Refrain from allowing the spreader to touch the edges of the plate Substitute the lid and ensure the plate is standing in an upright position for drying (10-12 minutes) Now incubate the spread agar plate at the optimum temperature with the lid at the base (inverted) The biggest advantage of a spread plate method is that the morphology of the isolated bacteria can be seen vividly. The only disadvantage is that sometimes fungal colonies may grow. INCUBATION An incubator is a temperature-controlled chamber whose main role is to permit microbial growth macroscopically. Its main role is to control environmental factors like temperature, humidity, and CO2 to create a safe and reliable environment free of contaminants for work with cell and tissue cultures. Microorganisms are grown in incubators in various fields. Types of incubators The most common types of incubators are – BOD incubators Bacteriological incubators CO2 incubators The main difference between these two types of incubators is due to temperature. Bacteriological incubators This type of incubator is used mainly in laboratories usually for the growth of bacteria. A constant temperature set according to the requirement is possible because of having a thermostat that maintains it. BOD incubators (Low-Temperature Incubators) These types of incubators are often called low-temperature incubators used for the growth of fungi i.e., yeast and mold as they require a low temperature to grow. @ Low temperature around 20-25˚C. CO2 Incubators Inside incubators, also known as a gassed incubators, an atmosphere is created that is as natural as possible to develop cell and tissue cultures. INSPECTION: Culture plates are checked to see if it contains pure or, mixed cultures or contaminated. Sequel to incubation of plates, cultures are checked for formation of discrete colonies. Where this is absent/mixed cultures, sub-culturing must be done Sub-culturing Involves the aseptic transfer of microbial colonies from its original growth media to another sterile growth medium. Sub-culturing is done in microbiology for several reasons but most importantly it is done to obtain pure culture. The simplest and commonest technique of sub- culturing is the streaking methods. This method involves the use of a sterile inoculating loop or needle to aseptically transfer microbial colonies by making streaking on the agar plate. The most appropriate streaking method is the quadrant streak method. Identification of microorganisms Maintenance and preservation of cultures Maintenance and preservation of cultures involves periodic sub-culturing technique to ensure the live organisms remains alive although with the risk of contamination and mutation. Culture organisms are preserve for the purpose of generating a stock culture of standard strains which imperatively serves as control or for other studies. There are different ways of maintaining and preserving culture organisms. They include: Culture plate, culture tube, freeze drying (eg; lyophilisation). Serial sub-culturing techniques are employed to keep fast growing microorganisms alive. Some of the useful medium used for preserving bacteria are: Robertson’s cooked meat medium while for Fungi is Sabouraud dextrose medium Maintenance of Pure Culture: After obtaining the pure culture of a particular microbe, it may be needed for further analysis which may not be immediate. This makes it necessary to grow and maintain the pure culture in viable states. This can be achieved in different ways: Maintenance under low temperature in a Refrigerator: This common practice involves growing the culture on suitable medium until it reaches the stationary phase of growth, and then storing in a refrigerator at 0-4C; this slows down the microbes metabolic activities. If they are to be kept alive for a long period, successive transfer to a fresh sterile medium must be done every 2- 3weeks for bacteria and 3-4weeks for fungi. Setbacks to this method include a. Risk of contamination – in time, it is possible for important isolates to be completely overgrown by contaminants b. Loss of viability – if sub culturing is not carried out at the required intervals and the cultures are inadequately stored, sensitive isolates may lose viability and be irrecoverable c. Continued growth at chill temperatures – some organisms, such as Listeria monocytogenes, are capable of slow growth at 0oC or even less d. Labelling mistakes – sub culturing a large number of agar slants many times introduces a significant chance of a culture being wrongly labelled Genetic drift and mutation – every subculture carries a potential for genotypic and phenotypic changes, such as loss of virulence and resistance factors, or reduced motility, to occur 2. Freeze-drying Also known as lyophilisation, freeze-drying is a method that can be used to suspend the metabolism of bacterial and fungal cultures and to stabilize them for long-term storage. A thick suspension of bacterial cells or fungal spores is first prepared in a suitable suspending medium, such as 10% skim milk, or a specific lyophilisation buffer. This suspension is then dispensed into small glass vials and frozen. When drying is complete the vial is sealed and then stored in the dark at 8oC or less. Many bacterial and fungal species will remain stable and viable for at least a year under these conditions and in some cases cultures have been successfully resuscitated many years later. Freeze-drying is the preferred method keeping reference strains and other isolates for long periods and is standard practice for large commercial and national culture collections Set back: Some fastidious or delicate species may be damaged by the process, or may not remain viable. e.g. the cells of Campylobacter spp and moulds 3. Cryogenic Storage: Cryogenic storage involves use of liquid nitrogen. Suspensions of bacterial cells or fungal spores are prepared in a cryoprotectant medium, generally containing 10-15% glycerol to minimize damage during freezing. The suspension is then dispensed into suitable containers, such as small screw-capped vials, which are then immersed in, or suspended above, liquid nitrogen. The temperature of liquid nitrogen is -196oC, well below the temperature at which all metabolic activity is thought to cease. 4. Preservation on sterile soil: This method of maintaining pure culture is most suitable for spore forming species. The microorganisms are grown in pure culture in suitable media. A suspension of microorganisms is then transferred to cotton stoppered tubes of sterilized dry soil. The spores remain viable, though dormant, for long periods of time, in dry soil. The organism can be grown after a desired period, by transferring the soil into a suitable medium and incubating it under suitable temperature. IDENTIFICATION: Cultures are observed for obvious growth characteristics that could be useful in analyzing the specimen contents. This involve macroscopic and microscopic examinations, biochemical tests, genetic characterizations and sometimes immunological testing Colonial characteristics (Macroscopic) The visual properties of a bacterial colony observed on a culture media are known as colony morphology. An essential ability for identifying bacteria in the microbiology lab. The following are the features of the bacterial colony that are typically observed include surface appearance, size Shape, texture or consistency, elevation, edge and colour – (Pigments are produced by certain bacterial species.) LECTURE TITLE: VARIATION IN BACTERIA AND HEREDITY VARIATION IN BACTERIA AND HEREDITY INTRODUCTION In comparison to other groups of organisms, the diversity of form and function among bacteria is unparalleled. This is because they exhibit an enormous capacity to evolve new potentialities due to the possession of these characteristics 1) Short generation time 2) Manifold means of variation 3) Existence of a haploid vegetative phase which allows expression of recessive genes. Variation refers to changes in an organism relative to its parent or former state. From the viewpoint of the bacteriologist/microbiologist not only the expressed change in appearance or function (the phenotype) of a bacterium is of interest because, even the most latent changes in the gene pattern, (the genotype) may have evolutionary significance. Thus, although visual changes at the cellular level in turn reflect changes in the genes or cytoplasm, the absence of expressed differences does not preclude differences in genotype. Variation may be environmental or hereditary depending on whether the change results from interplay of gene and environment, or from the introduction of new genes (by intra-change or inter-change). Since heredity is the transfer of genes, hereditary (gene) variation is transmissible to the progeny. Environmental variation however, does not alter the genes, and is therefore considered non-transmissible Bacterial genetics is the study of how genetic information is transferred, either from a particular bacterium to its offspring or between interbreeding lines of bacteria, how genetic information is expressed, and how the genetic information (genotype) determines the physiology of the bacterium (phenotype). Structurally and functionally, a microbial cell is composed of genes (or nucleus containing genes) and a surrounding cytoplasm. In considering environmental variation, the environment may be pictured as controlling the level of expression within the range permitted by the gene, by reacting directly or indirectly with the gene in a reversible physicochemical reaction. Since the immediate environment of the gene is the cytoplasm, environmental stimuli originating outside the cell may be modified here. Environmental stimuli include response to specific medium constituents and their concentration, pH, temperature, moisture, light, aeration, a change in pH level of the medium, for example, could alter solubility of metabolites, membrane permeability, enzyme activity through dissociation of enzyme-substrate complexes, equilibria, hydrogen bonding, etc. These effects in turn would trigger other reactions or reaction sequences. The effected changes cease when equilibrium is restored in the reaction between gene and environmental reactant. etc. Industrial microbiologists, especially those involved in fermentation have noted that an organism's past environmental history may markedly affect the future course of development of the culture even in the absence of any demonstrable change in its heredity. A plausible hypothesis for these observations includes carryover of mitochondria, microsomes, and enzyme molecules with high turnover conferring a significant higher activity. In all cases, however, the continuation of this pattern depends ultimately on replication and carryover of the genes. The sum total of responses or changes the organism undergoes in approaching a new equilibrium in a new environment is adaptation. Environmental variation is only one facet of adaptation, since the range of ability to adapt, which is gene-controlled, is also subject to variation. Other classic examples of non-heritable or environmental variations include a. Loss of flagella in Salmonella typhi when grown in phenol agar (H-O variation) b. Pleumorphism (variation in shape); the ability of some bacteria to alter their shapes and size in response to environmental conditions in old culture c. Lack of pigment production by Staph aureus under anaerobic conditions. d. S-R (smooth to rough) variation in Salmonella typhi that is characterized by loss of O antigen and change in colony morphology to rough type e. Variation in Salmonella typhi characterised by loss of Vi (a virulence capsular polysaccharide) antigen f. Production of flagella in Listeria monocytogenes at temperature less than 20ͦͦC g. Formation of spheroplasts and protoplast, mostly after lysing/treating cell wall with lysosomes Bacterial genetics is the study of how genetic information is transferred, either from a particular bacterium to its offspring or between interbreeding lines of bacteria, how genetic information is expressed, and how the genetic information (genotype) determines the physiology of the bacterium (phenotype). Structurally and functionally, a microbial cell is composed of genes (or nucleus containing genes) and a surrounding cytoplasm. In considering environmental variation, the environment may be pictured as controlling the level of expression within the range permitted by the gene, by reacting directly or indirectly with the gene in a reversible physicochemical reaction. Since the immediate environment of the gene is the cytoplasm, environmental stimuli originating outside the cell may be modified here. Environmental stimuli include response to specific medium constituents and their concentration, pH, temperature, moisture, light, aeration, a change in pH level of the medium, for example, could alter solubility of metabolites, membrane permeability, enzyme activity through dissociation of enzyme-substrate complexes, equilibria, hydrogen bonding, etc. These effects in turn would trigger other reactions or reaction sequences. The effected changes cease when equilibrium is restored in the reaction between gene and environmental reactant. etc. Industrial microbiologists, especially those involved in fermentation have noted that an organism's past environmental history may markedly affect the future course of development of the culture even in the absence of any demonstrable change in its heredity. A plausible hypothesis for these observations includes carryover of mitochondria, microsomes, and enzyme molecules with high turnover conferring a significant higher activity. In all cases, however, the continuation of this pattern depends ultimately on replication and carryover of the genes. The sum total of responses or changes the organism undergoes in approaching a new equilibrium in a new environment is adaptation. Environmental variation is only one facet of adaptation, since the range of ability to adapt, which is gene-controlled, is also subject to variation. Other classic examples of non-heritable or environmental variations include a. Loss of flagella in Salmonella typhi when grown in phenol agar (H-O variation) b. Pleumorphism (variation in shape); the ability of some bacteria to alter their shapes and size in response to environmental conditions in old culture c. Lack of pigment production by Staph aureus under anaerobic conditions. d. S-R (smooth to rough) variation in Salmonella typhi that is characterized by loss of O antigen and change in colony morphology to rough type e. Variation in Salmonella typhi characterized by loss of Vi (a virulence capsular polysaccharide) antigen f. Production of flagella in Listeria monocytogenes at temperature less than 20ͦͦC g. Formation of spheroplasts and protoplast, mostly after lysing/treating cell wall with lysosomes Unlike environmental variation. in which the reaction of the environmental stimulus with the gene is a reversible one, hereditary variation may be hypothesized to be an irreversible reaction directly involving the gene. Since the self-replicating gene is affected, and is also transferable, this type of variation tends to be perpetuated. Hereditary variation can occur in microorganisms by numerous means. a. MUTATION When a bacterial cell divides, the two daughter cells are generally indistinguishable. Thus, a single bacterial cell can produce a large population of identical cells or clone. On solid medium, a clone is manifested as an easily isolated colony. Occasionally, a spontaneous genetic change occurs in one of the cells. This change (mutation) is heritable and passed on to the progeny of the variant cell to produce a sub clone with characteristics different from the original (wild type) parent. This is termed vertical inheritance. If the change is detrimental to the growth of the cell, the sub clone will quickly be overrun by the healthy, wild type population. However, if the change is beneficial, the sub clone may overtake the wild type population. This is an example of how evolution is directed by natural selection. Spontaneous mutations are of two classes: (1) point mutation, or change of a single nucleotide, and (2) DNA rearrangement, or shuffling of the genetic information to produce insertions, deletions, inversions, or changes in structure. DNA rearrangements can affect a few to several thousand nucleotides. Both types of mutations generally occur at a low frequency (roughly once in 106 to 108 cells for any particular gene) and lead to a continuous, slow evolution of bacterial populations. The effect may be deleterious (inactivation or lower activity) or beneficial (enhanced or new activity The vast majority of mutations are undesirable, in that the organism has must have adapted to its environment over a period of time and sudden changes in the genes would throw the organism out of equilibrium. The cause of spontaneous mutation is not entirely known, however, the location of the gene (its chemical environment) and its chemical nature can be a contributor. Bacterial variation can also occur by horizontal transfer of genetic material from one cell to another. Consider two cells from different populations: bacterium B has features distinct from those of bacterium A. There are three possible mechanisms for transferring a trait from B to A: (1) Transformation The uptake of naked DNA molecules and their stable maintenance in bacteria is called transformation. This phenomenon was discovered in 1928 by Griffith, who was studying the highly virulent pathogen Streptococcus pneumoniae. He showed that injecting into mice a mixture of heat killed virulent (smooth) S. pneumoniae with a live attenuated (rough) strain led to the development of a live virulent strain, which ultimately killed the mouse. Avery, MacCleod, and McCarty purified the transforming substance and identified it as DNA. This experiment was the first to demonstrate that DNA was the genetic material. It was also the first discovery of gene transfer between bacteria. Since then, other bacteria, e.g Bacillus, E. coli, etc have been found to be naturally transformable. (2) Transduction (packaging and transfer of bacterial DNA by viruses) In transduction, phage carries genetic information, apparently single genes, from the cell of one strain to another. The transferred genes become part of the genotype of the receptor cell. The resulting infecting organism is called a bacteriophage. Viruses are molecules of nucleic acid wrapped in a protein coat (and sometimes lipid). They are found outside the cell, but they can only replicate within a living cell. Viruses that infect bacterial cells are called bacteriophages or phages. Bacteriophages represent a highly successful strategy for survival in nature. all bacteriophages have the same basic life cycle. They infect the bacterial cell, subvert the cell's machinery to replicate themselves, then lyse the cell to release hundreds of new bacteriophage particles into the environment. They are medically relevant because they can mediate the efficient transfer of genes between bacteria, including genes for antibiotic resistance and toxins. Some bacteriophages have the ability to convert harmless bacteria into pathogens Conjugation: This is bacterial mating in which cells must be in contact and for the transfer of plasmids between bacterial cells to occur One of the most important properties of plasmids is their ability to transfer to other bacterial cells. There are two categories of conjugative plasmids with respect to transfer: (1) self- transmissible plasmids, which encode all the genes necessary to promote cell-to-cell contact and transfer of DNA, and (2) mobilizable plasmids, which do not promote conjugation, but can be efficiently transferred when present in a cell that contains a self-transmissible plasmid For all three process, the transferred DNA must be stably incorporated into the genetic material of the recipient bacterium. This can occur in two ways: (1) recombination, or integration of the transferred DNA into the bacterial chromosome; or (2) establishment of a plasmid. In this case, the transferred material essentially forms a mini-chromosome capable of autonomous replication. Hereditary variation may occur in the asexually reproductive stage of microorganism in these instances. i. The cytoplasm of two cells of different strains (presumably established by mutation) may fuse. If the nuclei remain distinct in the common cytoplasm, the newly synthesized organism. called a heterokaryon. may be at an advantage over either homokaryotic strain, if the respective nuclei are complementary. ii. A heterokaryon may also be formed by mutation of one or more nuclei in a homokaryotic multinucleate cell. Changes brought about by hereditary variation may include the following i. Changes in the targets for several antibiotics can result in functional proteins that are no longer sensitive to the antibiotic (e.g. certain mutants of DNA gyrase are resistant to quinolones) (Antibiotic resistance). ii. In non-coding regions, point mutations can affect a variety of signals for expression and regulation of a gene. iii. Often a gene from one bacterial species is not expressed after transfer to another bacterial species because of differences in promoters, ribosome binding sites, codon usage, etc. iv. Sometimes, the spontaneous single nucleotide changes can result in the generation of a functional gene. Such a process may account for the relationship of cholera toxin of Vibrio cholerae and enterotoxin of E. coli. The toxin proteins are highly homologous, but the genes are only expressed in the bacterial species from which they were isolated. MICROORGANISMS OF WATER HABITAT; Aquatic microbial Ecology” is the study of the ecology of microbes in water or aquatic environments. Major types of aquatic environment The freshwater ponds, lakes, marshes, rivers, estuaries and canals and the marine environment which are characterized by high salt content and extreme depth. The freshwater makes up a small percentage of the earth’s water but are extremely important as sources of drinking water. Over two third of the earth’s is marine. Factors affecting establishment of microbial community in aquatic environments. Nutrients The colonization and continuous existence (microbial community) of microorganisms in this environment is greatly dependent on mixing and movement of nutrients in the water column Important gases in the aquatic environment Oxygen: Plants and animals need oxygen to survive. A low level of oxygen in the water is a sign that the habitat is stressed or polluted. The obligate aerobes in water require oxygen while the obligate anaerobes are harmed by presence of oxygen Carbon dioxide, also called CO2, is found in water as a dissolved gas. It can dissolve in water more easily than oxygen. Aquatic plants depend on carbon dioxide for life and growth, primary producers/autotrophic microbes and Plants use carbon dioxide during the process of photosynthesis. Other important gases are H2, N2 and CH2. Hydrogen nitrogen and methane. Temperature: Microorganisms can live in a wider range of temperatures than humans, but they grow best in a warm, moist environment. pH: Most bacteria grow best at a pH of around 7, and grow poorly or not at all below a pH of 4. Salinity: Salinity is a major factor that shapes the structure and function of microbial communities in water. Aquatic Microorganisms Microorganisms include members of the plant kingdom, protozoa, bacteria, and fungi. Bacteria display the greatest range in metabolic ability of any group of organisms. There are both autotrophic and heterotrophic bacteria. Heterotrophic bacteria are a crucial link in the decomposition of organic matter and the cycling of nutrients in aquatic systems. Autotrophic bacteria are primary producers in aquatic systems as are true algae Fungi Fungi occur as single cells, and in filaments called hyphae. Most aquatic fungi are microscopic; those known as hyphomycetes are the most abundant and important. Algae and Phytoplankton Several groups of largely autotrophic protists are referred to as algae. Like the term 'microorganisms' it is an informal term, used for convenience to describe microorganisms that carry out photosynthesis; (acts as primary producers and are often positioned in the upper layer of water column to access sunlight) the cyanobacteria are often included as algae. Periphyton and Biofilm Algae, bacteria, fungi, protozoa, and the breakdown products of dying cells form layers on submerged surfaces, including bottom sediment, rocks, submerged leaves and branches, and macrophytes. Sources of microbial pathogens in aquatic ecosystem Microbial pathogens in water come from a variety of sources, including sewage, agriculture, and storm water runoff and microbes present in atmospheric water during mixing action Sources can be point, non-point Point sources: These are easily identifiable sources, such as sewage treatment plants, factories, and landfills. Non-point sources: These are harder to identify, such as agricultural runoff and storm water runoff, overland flow etc. Types of microbial pathogens in water Bacteria: Examples include Escherichia coli, Salmonella, and Vibrio cholerae Viruses: Examples include Hepatitis A, Norwalk-type viruses, rotaviruses, adenoviruses, Enteroviruses, and Reoviruses Protozoa: Examples include Giardia lamblia, Cryptosporidium, and Entamoeba histolytica Parasites: Examples include Schistosoma and Dracunculus medinensis (Guinea worm) Surface water is susceptible to contamination because it covers a large portion of the Earth's surface and is easily affected by human activities due to its large surface area and open nature Microorganisms in the air Environmental microbiology is not limited to microbes in water and soil, but also the air. The air is a microbial habitat though it can be considered one of the least hospitable environments for microbes because it holds fewer nutrients and thus supports relatively fewer organisms Microbes that are suspended in air are usually in small airborne particles called bioaerosols. Pathogenic bioaerosols are dependent upon prevailing physicochemical properties of the atmosphere, such as temperature, humidity, solar radiation, wind, precipitation and air pressure, for transport and survival. Typical Airborne Microbes Mycobacterium tuberculosis (bacteria) The very small size of the bacterium (1-5µm range) allows it to remain suspended in air within bioaerosols when released from the lung, others include Bacillus anthracis, Mycobacterium tuberculosis, Legionella pneumophila Airborne Fungal Spores Fungi are found in air mostly as spores. Spores are generally able to survive harsh environmental conditions for the following reasons: possession of a thicker cell wall; protective small molecules (sugars, amino acids, sugar alcohols and betaine) and expression of heat shock proteins. Fungal genera found in the air include Aspergillus, Penicillium, Rhizopus and Alternaria, along with other fungi, such as Coccidioides imitis and Histoplasma capsulatum Viral airborne diseases Viruses are transported in air through either respiratory droplets or aerosols, in which they remain viable (depending on the prevailing environmental conditions) until they are transmitted to a susceptible host. NOTE that Aerosols cannot be seen with the naked eye (except with the use of specialized equipment), while droplets can be seen as the spray of fluid released during a sneeze. Dairy microbiology This is the study of microorganisms in dairy products, such as milk and cheese. It involves analyzing the microorganisms in dairy products to determine their quality and safety. What microorganisms are found in dairy products? Bacteria Milk naturally contains bacteria from the cow and environment. Bacteria like Lactobacillus and Streptococcus are used to produce fermented dairy products. Yeasts and fungi are also included. What role do microorganisms play in dairy products? Activities of microorganisms in milk Microbes are capable of carrying out the following activities in dairy products a. Fermentation in milk is a process that uses microorganisms to break down milk proteins and lactose. This process produces fermented milk products like yogurt, cheese, and kefir. It is considered the oldest way to preserve milk. Lactic acid bacteria like Lactobacillus, Lactococcus, and Leuconostoc are used to ferment milk. These microorganisms are introduced into the milk under controlled conditions. The fermentation process breaks down milk proteins and lactose, and produces lactic acid, ethyl alcohol, and other flavor substances. Fermentation increases the shelf life of milk products and makes milk easier to digest and can help with lactose intolerance and milk protein allergies. Probiotics are produced during fermentation can improve digestion and may help with digestive disorders like irritable bowel syndrome. In addition, it enhances the taste of milk products. b. Flavor Microorganisms produce enzymes, gases, and organic acids that can change the flavor of milk and dairy products. c. Spoilage: Microorganisms can cause spoilage, which is when a food's texture, color, odor, or flavor deteriorates to the point where it's no longer safe to eat Sources of contamination in dairy foods Dairy foods can be contaminated by biological, chemical, and physical sources. Biological sources i. Toxigenic molds and fungi: Molds like Aspergillus flavus and Aspergillus parasiticus that produce toxic substances ii. Parasites: Parasites that can contaminate dairy foods iii. Viruses: Viruses that can contaminate dairy foods iv. Veterinary drugs: Antibiotics, hormones, and other drugs used to treat diseases, promote growth, and increase milk production Chemical sources i. Pesticides: Pesticides that cattle consume through feed, forages, and water ii. Food additives: Additives used in the processing and packaging of dairy foods iii. Heavy metals: Metals that can contaminate dairy foods iv. Environmental contaminants: Chemicals from packaging materials, soil, and other environmental sources Physical sources Insects and their parts, Glass pieces, Hair or fur Bacterial contaminants of raw milk They include Salmonella, E. coli, Campylobacter, Listeria, Staphylococcus aureus, Coxiella: and Mycobacterium tuberculosis Reducing risk of contamination in milk and dairy foods i. Bedding type and management can prevent mastitis on dairy farms. Bedding should be topped off at least weekly and as needed. ii. Provide sufficient ventilation. Mechanical ventilation may be necessary, particularly during warmer months. iii. Alleys should be kept clean, and manure should be removed regularly to prevent splatter onto the udder. Splatter can lead to contamination with feacal pathogens like E. coli, Shigella, Enterobacter species iv. Stalls should be cleaned daily to remove any soiled bedding as cows can spend 12 to 14 hours per day lying and resting. v. Handle cows in a low stress environment. Stress can prevent milk letdown, and compromise teat integrity. vi. Wear disposable gloves and change them as needed to reduce the spread of pathogens to the teat. vii. Milk handers should not show the symptoms of any communicable disease, should be routinely examined, and must not have open cuts and wounds Mastitis is a bacterial infection or inflammation of the mammary gland in milk-producing animals, such as cows, goats, and sheep. It can be caused by physical injury, stress, or bacteria. Symptoms include redness, swelling, heat, and pain in the udder, Milk that is foul-smelling, contains blood clots, or is reduced in quantity and elevated body temperature in affected cow. It is commonly caused by Str. agalactiae; Str. pyogenes among others