Introduction to General Microbiology PDF

Microbiology is the study of microorganisms, which are tiny organisms that live us inside our body. An organism is a living thing that ingests and breaks down food for energy and nutrients, excretes undigested food as waste, and is capable of reproduction. A microorganism is simply very, very small...

Microbiology is the study of microorganisms, which are tiny organisms that live us inside our body. An organism is a living thing that ingests and breaks down food for energy and nutrients, excretes undigested food as waste, and is capable of reproduction. A microorganism is simply very, very small organism that you cannot see with your naked eye, but with a microscope. **I.1TYPES OF MICROORGANISMS** Pathogenic microorganism An infection is caused by the infiltration of disease causing microorganisms infect humans, other animals and plant. **Example:** *Yersinia pestis* is the microorganism that caused the Black Plague and killed more than 25 million Europeans. You might say that *Yersinia pestis* first infected fleas that were carried into populated areas on the black of rats. Rodents traveled on ships and then over land in search of food. Fleas jumped from rodents and bit people, transmitting *Yersinia pestis* into person's blood stream. 1.2Non-pathogenic microorganism Not all microorganisms are pathogens. In fact many microorganisms help to maintain homeostasis in our bodies and used in the production of food and other commercial products. For example, flora are microorganisms found in our intestine that assist in the digestion of food and critical role in the formation of vitamins such as vitamin B and K. they help by breaking down large molecules into smaller ones. **I.2**-definitions **What is a microorganism?** Microbiology is the subject of microbiology, which is the branch of science that studies microorganisms. A microorganism can be on cell or cluster of cells that can be seen only by using a microscope. Microbiology is classified or organized into five fields of study: bacteriology, virology, mycology, phycology, and protozoology. We will also study some other smaller groups such as prions and viroid's. The one property that links these groups together is their very small size! **I.2.1Branches of microbiology** **I.2.1Bacteriology** Bacteriology is the study of bacteria. Bacteria are prokaryotic organisms. A prokaryotic organism is one-celled organism that does not have a true nucleus. Many bacteria absorb nutrients from their environment and some make their own nutrients by photosynthesis or other synthetic processes. Some bacteria can move freely in their environment while others are stationary. Bacteria occupy space on land and can live in aquatic environment and in decaying matter. They can even cause disease. *Bacillus anthracis* is a good example. It is bacterium that causes anthrax. *Bacteriologists-* study bacteria; there are medical, agricultural, biotechnological specializations. **I.2.2Virology** Virology is the study of viruses. A virus is a submicroscopic, parasitic entity composed of nucleic acid core surrounded by a protein coat. Parasitic means that a virus receives food and shelter from another organism and is not divided into cells. An example of virus is the varicella-zoster virus, which is the virus that causes chickenpox in humans. Virologists -- study viruses, there are medical, agricultural, biotechnological specializations. **I.2.3Mycology** Mycology is the study of fungi. A fungus is eukaryotic organism, often microscopic, that absorbs nutrients from its external environment. Fungi are not photosynthetic. A eukaryotic microorganism is a microorganism whose cells have a nucleus, cytoplasm and organelles. These include yeast and some molds. *Tinea pedis* better known as athlete's foot is caused by fungus. *Mycologists* -- study fungi, there are medical, agricultural, biotechnological specializations. **I.2.4 Phycology** Phycology is the study of algae. Algae are eukaryotic photosynthetic microorganisms that transform sunlight into nutrients using photosynthesis. A eukaryotic photosynthetic microorganism has cells containing a nucleus, nuclear envelope, cytoplasm and organelles and is able to carry out photosynthesis. **I.2.5Phycologists -- study algae**. Protozoology Protozoology is the study of protozoa, animal-like single-cell microorganisms that can be found in aquatic and terrestrial environment. Many obtain their food by engulfing or smaller organism. An example is *Amoeba proteus.* Scope of microbiology *Why do we Study Microbiology?* 1. Impact on Animal and Human Health 2. Balance of Nature -- food source, play a role in decomposition, help other animals digest grass (cattle, sheep, termites). 3. Environmental -- provide safe drinking water; development of biodegradable products; use bacteria to clean up oil spills, etc. -- called **bioremediation.** 4. Industrial -- foodstuff (beer, wine, cheese, bread), antibiotic, insulin, genetic engineering. 5. Agricultural -- research has led to healthier livestock and disease-free crops. **I.3 HISTORY OF MICROBIOLOGY** -- The microscope **I.3.1 Zacharias Janssen** In 1590, Zacharias Janssen developed the first compound microscope in Middleburg, Holland. Janssen's microscope consisted of three tubes. One tube served as the outer casing and contained the other two tubes. At either ends of the inner tubes were lenses used for magnification. Janssen's design enabled scientists to enlarge the image of specimen three and nine times the specimen's actual size. **I.3.2. Robert Hooke (1635 -- 1703)** In 1635, Robert Hooke, an English scientist, popularized the use of compound microscope when he placed lenses over slices of cork and viewed little boxes that he called cells. It was this discovery that led to the development of cell theory in the nineteenth century by Mathias Schleiden. Theoder Schwann, and Rudolf Virchow, cell theory state that all living things are composed of cells. **I.3.3 Antony van Leeuwenhoek (1632 -- 1723)** Hooke's experiments with a crude microscope inspired Antony van Leeuwenhoek to further explore the micro world. Van Leeuwenhoek, an amateur lens grinder, improved Hooke's microscope by grinding lenses to achieve magnification. His microscope required one lens. With his improvement, van Leeuwenhoek became the first person to view a living microorganism, which he called Animalcules. This discovery took place during the 1600s, when scientists believed that organisms generated spontaneously and did not come from other organisms sound preposterous today; however, back then scientists were just learning that a cell was the basic component of an organism. Origin if Organisms **I.3.4 Francesco Redi** In 1668, Italian physician Francesco Redi developed an experiment that demonstrated that an organism did not spontaneously appear. He filled jars with rotting meat. Some jars he sealed and other he left opened. Those that were open eventually contained maggots, which is the larval stage of the fly. The other jars did not contain maggots because flies could not enter the jar to lay eggs on rotting meat. His critics stated that air was the ingredient required for spontaneous generation of an organism. Air was absent from the sealed jar and therefore no spontaneous generation could occur, they said Redi repeated the experiment except this time he placed a screen over the opened jars. This presented flies from entering the jar. There were not any maggots on the rotting eat. Until that time scientists did not have a clue about how to fight disease. However, Redi's discovery gave scientists an idea. They used Redi's finding to conclude that killing the microorganisms that caused a disease could prevent the disease from occurring. New microorganisms could only be generated by other microorganisms when it underwent a reproductive process. Kill that microorganism and you will not have new microorganisms, the theory went -- you could stop the spread of the disease. Scientists called this the Theory of Biogenesis. The Theory of Biogenesis state that a living cell generated from another living cell. **I.3.5 Louis Pasteur (1822 -- 1895)** Although the Theory of Biogenesis disproved spontaneous generation, spontaneous generation was hotly debated among the scientific community until (1861) when Louis Pasteur, a French scientific, resolved the issue once and for all. Pasteur showed that microorganisms were in the air. He proved that sterilized medical instruments became contaminated once they were exposed to the air. Pasteur came to this conclusion by boiling beef broth in several short-necked flasks. Some flasks were left open to cool. Other flasks were sealed after boiling. The opened flasks became contaminated with microorganisms while no microorganisms appeared in the closed flasks. Pasteur concluded that airborne microorganism had contaminated the opened flasks. In a follow-up experiment, Pasteur placed beef broth in an open long-necked flask. The neck wasp bent into an S-shape. Again, he boiled the beef and let it cool. The S-shaped trapped the airborne microorganisms The beef broth remained uncontaminated even after months of being exposed to the air. The very same flasks containing the original beef broth exist today in Pasteur Institute in Paris and still show no sign of contamination. Pasteur's experiments validated that microorganisms are not spontaneously generated. Based on Pasteur's finding, concerned effort was launched to improve sterilization techniques to prevent microorganisms from reproducing. Pasteurization, one of the best-known sterilization techniques, was developed and named for Pasteur. Pasteurization kills harmful microorganisms in milk, alcoholic beverages, and other foods and drink by heating it enough to kill most bacteria that cause spoilage. **I.3.6 John Tyndall and Ferdinand Cohn** The work of John Tyndall and Ferdinand Cohn in the late 1800s led to one of the most important discoveries in sterilization. They learned that some microorganisms are resistant to certain sterilization techniques. Until their discovery, scientists had assumed that no microorganisms could survive boiling water, which became a widely accepted method of sterilization. This was wrong. Some thermophiles resisted heat and could survive a bath in boiling water. This means that there was not one magic bullet that killed all harmful microorganisms. **I.3.7** **Robert Koch (1843 -- 1910)** **Germ theory** Until the late 1700s, not much was really known about diseases except their impact. It seemed that anyone who came in contact with an infected person contracted the disease. A disease that is spread by being exposed to infection is called a contagious disease. The unknown agent that causes the disease is called a contagion. Today we have known that a contagion is a microorganism, but in the 1700s many found it hard to believe something so small could cause such devastation state the germ theory. Koch made some observations on the disease caused *Bacillus anthracis* called anthrax. Based on his finding, Koch developed the Germ Theory. The Germ **Theory states that disease-causing microorganisms should be present in animals infected by the disease and not in healthy animals**. The microorganism can be cultivated away from the animal and use to inoculate a healthy animal. The healthy animal should then come down with the disease. Samples of a microorganism taken from several infected animals are the same as the original microorganism from the first infected animals. **Four steps used by Koch to study microorganisms are referred to as Koch's Postulates.** **I.3.8 Koch's Postulates state**: 1. The microorganism must be present in the disease animals and not presence in the healthy animal. 2. Cultivate the microorganism away from the animal in pure culture. 3. Symptoms of disease should appear in the healthy animal after the healthy animal is inoculated with the culture of the microorganisms. 4. Isolate the microorganism from the newly infected animal and culture it in the laboratory. The new culture should be the same as the microorganism that was cultivated from the original diseased animal. Koch's work with anthrax also developed techniques for growing a culture of microorganisms. He eventually used a gelatin surface to cultivate microorganisms. Gelatin inhibited the movement of microorganisms. As microorganisms reproduced, they remained together, forming a colony that made them visible without a microscope. The reproduction of microorganisms is called colonizing. The gelatin was replaced with **agar** that is derived from seaweed and still used today. **Richard Petri** improved on Koch's cultivating technique by placing the agar in a specially designed disk that was later called the Petri dish, which is still used today. **I.3.9 Edward Jenner** Edward Jenner, an English physician, discovered something very interesting about both smallpox and cowpox in 1796. Those who survived smallpox never contracted smallpox again; even when they were later exposed to someone was infected with smallpox. Milkmaids who contracted cowpox never caught smallpox even though they were exposed to smallpox. Jenner had an idea. He took scrapings from a cowpox blister found on milkmaid and, using a needle scratched the scraping into the arm of James Phipps, an 8-year-old. Phipps became slightly ill when the scratch turned bumpy. Phipps recovered and was then exposed to smallpox. He did not contract smallpox because his immune system developed antibodies that could fight off variola. Jenner's experiment discovered how to use body's own defense mechanism to prevent disease by inoculating a healthy person with a tiny amount of the disease-causing microorganism. Jenner called this vaccination, which is an extension of the Latin word vacca (cow). The person who received the vaccination became immune to the disease-causing microorganism. **I.3.10 Elie Metchnikoff** Elie Metchnikoff, a ninetieth-century Russian zoologist, was interested by Jenner's work with vaccinations. Metchnikoff wanted to learn how our bodies react to vaccination by exploring our body's immune system. He discovered that white blood cells (leukocytes) engulf and digest microorganisms that invade the body. He called these cells phagocytes, which means, "Cell eating". "Metchnikoff was one of the first scientists to study the new area of biology called immunology, the study of the immune system. **1.3.11 Ignaz Semmelweis** Great studies were made during the late 1800s in the development of antiseptic techniques. It began with a report by Hungarian physician Ignaz Semmelweis on a dramatic decline in childbirth fever when physicians used antiseptic techniques when delivering babies. Infections become preventable through the use of antiseptic techniques. **I3.12 Joseph Lister** Joseph Lister, an English surgeon, developed one of the most notable antiseptic techniques. During surgery, he sprayed carbolic acid over the patient and then bandaged the patient's wound with carbolic acid-soaked bandages. Infection following surgery dramatically dropped when compared with surgery performed without spraying carbolic acid. Carbolic acid, also known as phenol was one of the first surgical antiseptic. **I.3.13 Paul Ehrlich (1854 -- 1955)** Antiseptic prevented microorganisms from infecting a person, but scientist still needed a way to kill microorganisms after they infected the body. Scientists needed a magic bullet that cured diseases. At the end of the ninetieth century, Paul Ehrlich, a German chemist, discovered the 'magic bullet' against infectious disease called syphilis. Ehrlich blended chemical elements into a convocation that, when inserted into area, killed microorganism without affecting the patient. Today we call Ehrlich's concoction a drug. Ehrlich's innovation has led to chemotherapy using drugs that are produced by chemical synthesis. **I.3.13 Alexander Fleming (1881 -- 1955)** Scientists from all over set out to use Ehrlich's findings to find drugs that could make infected patients well again. One of the most striking breakthroughs came in 1929 when Alexander Fleming discovered *Penicillium notatum,* the organism that synthesizes penicillin. *Penicillium notatum* is a fungus that kills the *Staphyloccus aureus* microorganism and similar microorganisms. Fleming grew cultures of *Staphyloccus aureus,* a bacterium, in laboratory. He was also conducting experiments with the *Penicillium notatum,* a mold. By accident, the *Staphyloccus aureus* was contaminated with *Penicillium notatum,* causing the *Staphylococcus* to stop reproducing and die. *Penicillium notatum* became one of the first antibiotics. An antibiotic is a substance that kills bacteria. **1.2 Taxonomy** The classification of living organisms is refered to as taxonomy and it aims to classify living organisms by differentiating the them and establishing relationships between groups of organisms. The kingdomof living organismswas termed as domain **(Naming and classifying microorganisms)** Taxonomy is the science of the classification of organisms. Taxonomy is a system of orderly classification of organism into categories called taxons. Carl Linnaeus developed the system for naming organisms in 1735. This system is referred to as binominal nomenclature. Each organism is assigned two Latinized named because Latin or Greek was the traditional language used by scholars. The first name is called the genus. The second name is called specific epithet, which is the name of the species, and it is not capitalized. The genius and the epithet appear italicized. Sometimes an organism in named after a researcher, as in the case with *Escherichia coli,* better known as *(E. coli)* the genus is Escherichia, which is named after Theodor Escherichia, a leading microbiologist. The epithet or species is coli, which implies that the bacterium lives in a colon. The formal binomial naming method created by Linnaeus is still used, but the modern classification rational is based on evolutionary relatedness. All living cellular things (biological entities other than viruses and prions) have a species and genus designation, and organisms are placed into groupings that reflect their evolutionary relationships. For us *Homo sapiens,* our genus is Homo and species is sapiens. Note the correct form used, genus name is underlined and capitalized; species name is not capitalized and underlined. Alternatively the two words can be separately underlined or can both be entirely capitalized (through this rarely done). These look like really fussy, picky rules, but this is essential, serious misunderstandings can occur if this convention is not followed. *Escherichia coli* are incorrect (not in bold or underlined or italicized). *Escherichia Coli* is incorrect (species epithet "coli" first letter must not be capitalized). *escherichia coli* are incorrect, the term is italicized but the genus must begin with a capital letter. It is acceptable and usual the first letter of the genus of a species PROVIDING that in any section of text the entire taxonomic name has FIRST been given in full, thus once the name *Escherichia coli* has been given in a written work it can then be referred to as *E. coli.* Organisms were classified into either the animal kingdom or the plant kingdom before the scientific community discovered microorganisms in the seventeenth century. it was at that time when scientist realized that this classification system was no longer valid. **Carl Woese** Developed a new classification system that arranged organisms according to their molecular characteristics and then cellular characteristics. However, it wasn't until 1978 when scientists could agree on the new system for classifying organism, and it took 12years after this arrangement before the new system was published. **Woese** devised three classification groups called domain. A domain is larger than a kingdom. These are: **Domain** **The domain bacteria includes all of the pathogenic and nonpathogenic prokaryotes. They have cell wall composed of peptidoglycan and muramic acid. They also have membrane lipids with ester --linked straight chained fatty acids that resembles eukaryotic membrane lipids** - ***Eubacteria:*** Bacteria that have peptidoglycan cell walls. (Peptidoglycan is the molecular structure of the cell walls of eubacteria which consists of Nacetyglucosamine, N-acetylmuramic acid, Tetrapeptide, side chain and murein). ***THE TAXONOMIC CLASSIFICATION SCHEME FOR PROKARYOTES IS ELABORATED IN BERGEYS MANUAL OF SYSTEMATIC BACTERIOLOGY WHICH DESCRIBES THE CLASSIFICATION OFPROKARYOTES INTO TWO DOMAINS*** - ***Archaea:*** Prokaryotes that do have peptidoglycan cell walls. - ***Eukarya:*** Organisms from the following kingdoms: ANIMALS PLANTS; FUNGI PROTIST AND HAVE DEFINED NUCLEUS AND MEMBRANE BOUND ORGANELLES Kingdoms -------- - ***Protista:*** Examples -- Algae, protozoa, slime, molds. - ***Fungi:*** Examples -- one-celled yeasts, multicellular molds and mushrooms. - ***Plantae:*** Examples -- moss, conifers, ferns, flowering plant, algae. - ***Animalia:*** Examples -- insects, worms, sponges (ocean invertebrate animals) and vertebrates. **FURTHER CLASSIFICATION OF DOMAIN IS AS FOLLOWS** **DOMAIN, KINGDOM, PHYLA, CLASS, ORDER, FAMILY, GENUS, SPECIES** **THE BINOMIAL NOMENCLATURE ASSIGNS EACH MICROBE 2 names -- genus -- noun always capitalised and specie-both names are italicised or underlined.** N/B: Prokaryotes belong to the kingdom ***Monera*** Thus, the five Kingdom Classification System includes Animalia, Fungi, Plantae, Protista, and Monera. Within each kingdom each organism is nested into a hierarchical of taxons in the order-kingdom, Phylum-Division, Class, Order, Family, Genus, and Species. The order of this list is important (which accounts for use of the term hierarchical), each taxon holds progressively more numbers of taxonomically different organisms as one moves up the list from species level, thus a genus contains a number of species, a family contains a number of genera an thus contains more species than a single genus in that classification since each family contains a number of genera each with their own species. ***Taxonomists --*** those who study the classification of organisms can be a HIGHLY argumentative bunch, put two of them together ad you may get three opinions as to how an organism should be classified. ***THERE IS DIFFICULTY IN DEFINING SPECIES IS THAT EVEN THOUGH ALL ALL SPECIES ARE KINDS OF ORGANISMS , ALLKINDS OF ORGANISMS ARE NOT SPECIES.*** THIS IS BECAUSE MOST WIDELY ACCEPTED DEFINITION OF A SPECIE IS A GROUP OF ORGANISMS WITH SIMILAR MORPHOLOGICAL AND PHYSIOLOGICAL FEATURES THAT ARE ABLE TO BREED TOGETHER AND PRODUCE FERTILE OFF SPRINGS **I.2.3 The Prokaryotes (kingdom Monera) are GENERAL CHARACTERISTICS** 1ALL ARE PRIMITIVE PROKARYOTIC ORGANISMS 2THEY ARE GENERALLY UNICELLULAR 3A MEMBRANE BOUND NUCLEUSE IS ABSNT 4LIVES IN AEROBIC OR UNDER ANEAROBIC CONDITIONS :ORGANISMS BELONGING TO THIS KNGDOM DO NOT CONTAIN A TRUE NUCLEUS. THESE ARE THE OLDEST KNOWN MICROORGANISMS ON EARTH. THEIR DNA IS NOT ENCLoSED WITHIN THE NUCLEUS EXAMPLES ARE BLUE GREEN ALGAE, CYANOBACTERIA, BACILLUS SHAPED BACTERIA E.COLI VIBROCHOLERAE SPIRAL SHAPED BACTERIAETC 1. The ***Eubacteria*** (so called "true" bacteria -- in future lectures, when I use the word bacteria -- it refers to the eubacteria unless I state otherwise). 2. Some of the eubacteria cause human disease and this is why the eubacteria are those bacteria that are of main interest to medical microbiologists. This is one of the possible classification "trees", there are others, and as usual in taxonomy, this is a contentious issue, but the five kingdom system is consistent with numerous sources of evidence. 3. The ***Cyanobacteria*** (referred to as blue-green algae) -- common photosynthesizing bacteria often noted as the green scum on ponds in summer months. 4. The ***Purple photosynthetic bacteria,*** these perform photosynthesis but they do not use chlorophyll, they use special purple pigments instead, they are found in brine ponds for instance. 5. The ***Archaebacteria,*** a group of evolutionarily ancient bacteria which are adapted to living in extreme environments such as high salt, intense cold, high temperature, high acidity etc. ALL the other kingdoms contain eukaryotic organisms: **I.2.4The kingdom Protista.** These are set of organisms ***Algae --*** fresh and saltwater, single cells to simple multicellular photosynthesizing organisms. Autotrophs most have cell walls. ***Slime molds --*** they are NOT fungi, look like fungi but can also be animal or even plant-like in morphology. Heterotrophs. ***Protozoa --*** a diverse group of single celled creatures that look and act like animals often, but are not, includes amoeba, paramecia etc. They are heterotrophs (some have autotrophic algae as symbiotic partners). Some protozoa cause human disease. Some protists have plant and animal-like characteristics and are hard to classify -- the Euglenoid Protista are Heterotrophic and Autotrophic. **VI.4The kingdom Fungi.** Non photosynthesizing single celled to multicellular organisms most of which have cell walls. Heterotrophs. Includes yeasts (which are unicellular) and mycelial (filamentous) organisms such as bread mold and the mushroom forming fungi. Some fungi cause human disease, some are important in food preparation. **The kingdom Plantae --** autotrophs[.] Multicellular photosynthesizing organisms with cell walls. Trees, shrubs, grasses, moss, ferns etc. **The kingdom Animalia.** -- Heterotrophs. Complex multicellular organisms of diverse types, no cell walls, showing characteristic irritability and movement. Some cause human disease (parasitic infections, worms etc. Or act as vector for other disease causing organisms). - **Viruses** Viruses are not classified by using Bimodal naming system and do not belong to any of the five kingdoms. Viruses are not cellular and are dependent on host cells for their replication. Viruses are classified in two different ways: 1. According to their structure -- genetic (DNA or RNA?) and physical (shape etc.) -- this scheme is favored by scientists doing fundamental work 2. According to type of disease they cause, this scheme is favored by medical workers who need to correlate given viruses with given diseases. Size of microorganism --------------------- Microorganisms are measured using the metric system. In order to give you some idea of the size of a microorganism, let us compare a microorganism to things that are familiar to you. A human red cell -- 100 micrometers (µm); A typical bacterium cell -- micrometers (µm); A virus -- 10 nanometers (nm); an atom -- 0.1 nanometers (nm). **ENDOSYMBIOTIC THEORY** In the prokaryotic cells mitochondria and chloroplasts have 70s ribosomes, where as eukaryotic cells have 80s ribosomes, this provides the support for the endosymbiotic theory which states that the mitochondria and chloroplasts in eukaryotic cells were once aerobic bacteria (prokayotes) that were ingested by a large anaerobic bacteria (prokaryotes) **Section 2: General Structures and morphologies of bacteria** #### II.1 The cell All living beings are cellular (most biologists do not regard viruses as being "alive"). The broadest definition of the structure of a cell is that it is a bag made of lipid enclosing a thick water based soup of life's chemicals and processes. This is an absurdly inadequate definition though, which fails to impress on you haw extremely complex and dynamic a cell is. The cell is bounded by a plasma membrane which is made of special phospholipids and is studded with many complex protein pores, channels, gates, receptors, recognition proteins etc. within is the cytoplasm which contains water, and many chemicals and special structures, as well as the genome, the sum total of cells genetic information -- in the form of genes -- linear DNA sequences, or in the case of bacteria -- a single circular DNA macromolecule. Cells are exceedingly complex -- they possess thousands of different but interrelated reactions, simultaneously, at enormous speed and under exquisite control. All cell membranes are based on what is known as the lipid bilayer structure, and are "studded" with very large numbers and many types of proteins, some of these span the membrane and are transport proteins, some are receptor or recognition proteins. There is a high capacity for these protein molecules to move around in the cell membrane, as can lipid molecules of the membrane to some extent, so that the modern understanding of the cell membrane is referred to as the fluid-mosaic model. The prokaryotic and eukaryotic cell membranes perform similar functions but differ in the chemical nature of their lipids, carbohydrate, and protein components. Cell membranes function in protection, transport, cell-to-cell recognition, and especially in bacterial cells also participate in the biochemical reactions of metabolism. Surface area to volume ratio (SA/V).the ratio of a cells surface area to its volume places critical limits on its lifestyle -- its activities, and its size. As a cells increases in size (let us assume that the cell is a sphere), its volume increases far more than its surface area (in other words, as the radius of the cell increases by amount proportional to X3). This place a limit on how large a cell can grow, because the huge increase in volume relative to the increase in surface area means that there is now too little membrane surface area to cope with the increased import and export processes across the cell membrane required by the hugely increase cell volume. Eukaryotic cells have evolved some means to cope with this, principally by means of membrane bound organelles, but particularly by means of an extensive proliferation of internal membrane surface area called the endoplasmic reticulum, but the SA/V problem ratio does place fundamental limits on how large a cell can become. Bacteria do not "solve" this SA/V problem, they remain small -- this has its advantages, a high surface area to volume area allows rapid growth for instance, and when this is coupled with the fact that their DNA is much more subject to mutation than is the case for eukaryotic cells -- the result is rapidly proliferating growth and many mutation derived variants in the bacterial population that can quickly adapt to changing ecological circumstances. There are two basic types of cell: prokaryotic and eukaryotic. #### II.2 Characteristics of Prokaryotic and Eukaryotic cell All living beings are cellular (most biologists do not regard viruses as being "alive", nevertheless, they consider them to be biological entities of course). Hence, do not make the common mistake of thinking of viruses as being cells. They are not! The broadest definition of structure of a cell is that it is a bag made of lipid enclosing a thick water based soup of life's chemicals and processes. This is an absurdly inadequate definition though, which fails to impress on you how extremely complex and dynamic a cell is. #### The life processes of a living thing include - Metabolism. Breakdown nutrients for energy or extract from the environment. - Responsiveness. React to internal and external environment changes. - Movement. Whether it is the entire organism relocating within its environment, cells within that organism or the organelles inside those cells. - Growing. Increase the size or number of cells. - Differentiation. The process whereby cells that are unspecialized become specialized. (An example would be a single fertilized human egg, developing into an individual). Prokaryotic cells do not differentiate. - Reproduction. Form new cells to create a new individual. All cells have: 1. Cell wall or plasma membrane (separate the cell from the outer environment) 2. Genetic material (DNA) 3. Cytoplasm. Two basic types of cell are known: prokaryotic and eukaryotic. The membranes of these cells perform similar functions but differ in the chemical nature of their lipids, carbohydrate, and protein components. A. **Prokaryotic** ("true nucleus") -- a cell lacking a membrane-bound nucleus and membrane-bound organelles ([ex. Bacteria]); these cells do have some organelles, but they are not membrane-bound; all prokaryotic cells have cell wall, its primary component being peptidoglycan; prokaryotic cells are much smaller than eukaryotic cells (about 10 times smaller); their small size allows them to grow faster and multiply more rapidly than eukaryotic cell (they can easily meet their modest nutritional needs and grow rapidly). This group includes all bacteria. B. **Eukaryotic** ("true nucleus") -- a cell having a membrane-bound nucleus and membrane-bound organelles ("little organs" -- specialized structures that perform specific functions within the cell); evolved about 2 million years after the prokaryotes; cell walls are sometimes present, but they are composed of cellulose or chitin; organisms with eukaryotic cells include fungi, algae, protozoa, plant and animals. It is important to know the differences between prokaryotic and eukaryotic cells; allows us to control disease-causing bacteria without harming our own cells. In summary, a tropical prokaryotic cell, there is nucleus, but a region where the DNA exists called a nucleoid, a cell wall, and a cytoplasm with no membrane bound organelles **II.3 General comparison between prokaryotic cells and eukaryotic cells:** +-----------------------------------+-----------------------------------+ | Prokaryotes | Eukaryotes | | | | | ('pre-nucleus') | ('true nucleus') | +===================================+===================================+ | - Cells typically 0.2-2.0 µm | Cells typically 5-100 µm diameter | | diameter | | +-----------------------------------+-----------------------------------+ | - No nuclear membrane around | Nucleus with double nuclear | | genetic material | membrane houses genetic material | | | separate from cytoplasm | +-----------------------------------+-----------------------------------+ | - DNA = one circular chromosome | DNA = multiple linear chromosomes | +-----------------------------------+-----------------------------------+ | - DNA not associates with | DNA would around histone proteins | | histones | | +-----------------------------------+-----------------------------------+ | - Lack membrane-enclosed | Have membrane-enclosed | | organelles | organelles: Mitochondria, | | | Endoplasmic Reticulum, Golgi | | | Complex, Lysosomes, Chloroplasts, | | | etc. | +-----------------------------------+-----------------------------------+ | - Chemical complex cell walls | Chemical simple cell walls (if | | e.g. peptidoglycan | present) e.g. chitin, cellulose | +-----------------------------------+-----------------------------------+ | - Simple flagella: two protein | Complex flagella or cilia | | building blocks, no cilia | composed of microtubules with | | | membrane | +-----------------------------------+-----------------------------------+ | - Capsule or slime layer | Glycocalyx in cells that lack a | | glycocalyx (if present) | wall | +-----------------------------------+-----------------------------------+ | - No carbohydrates or sterols | Plasma membrane contains sterols | | in plasma membrane | and carbohydrates | +-----------------------------------+-----------------------------------+ | - Cytoplasm lacks cytoskeleton | Cytoskeleton and cytoplasmic | | and cytoplasmic streaming | streaming present in cytoplasm | +-----------------------------------+-----------------------------------+ | - Small 70s ribosomes | Large 80s ribosomes | +-----------------------------------+-----------------------------------+ | - Cell division by binary | Cell division by mitosis | | fission | | +-----------------------------------+-----------------------------------+ | - Genetic recombination | Genetic recombination involves | | involves DNA fragment | meiosis | | exchange | | +-----------------------------------+-----------------------------------+ | - Bacteria and Archaea | Algae, Protozoa, Fungi, Plants, | | | Animals | +-----------------------------------+-----------------------------------+ **II.4 Parts of Prokaryotic Cells** **General structure and morphology Microbial physiology**  **Glycocalyx** Glycocalyx is a strictly, sugary envelope composed of polysaccharides and/or polypeptides that surround the cell. Glycocalyx is found in one or two state. It can be firmly attached to the cells surface, called capsule, or loosely attaché, called slime layer. A slime layer is water-soluble and is used by the prokaryotic to adhere to surfaces external to the cell. Glycocalyx is used by prokaryotic cell to protect it against attack from the body's immune system. #### Flagella Flagella mad of protein and appear "whip-like". They are used by the prokaryotic cell for mobility. Flagella propel the microorganism away from harm and food in a movement known as taxis. Movement caused by a light stimulus is referred to as phototaxis and chemical stimulus causes a chemotaxis movement to occur. **Flagella can exist the following form** ========================================= ***Monotrichous:*** one Flagellum Lophotrichus: two or more Flagella that are at one end of the call. ***Amphitrichus:*** Flagella at two ends of the cell. ***Endoflagellum:*** A type of amphitrichous flagellum that is tightly wrapped around spirochetes. A spirochete is a spiral-shaped bacterium that moves in a corkscrew motion. *Borrelia burgdorferi,* which is the bacterium that causes Lyme disease, exhibits an endoflagellum. C:\\Users\\dell\\Desktop\\dvm 305\\stock-vector-schematic-illustration-of-bacterial-flagella-types-1734245219.jpg Structure of different forms of flagella set up in bacteria FIND OUT AN EXAMPLE FOR EACH OF THEM ###### Fimbriae ###### Fimbriae are proteinaceous, sticky, bristle-like projections used by cells to attach to each other and to objects around them. *Neisseria gonorrhoeae,* the bacterium that causes gonorrhea uses fimbriae to adhere to the body and to cluster cells of bacteria. **Pili** Pili are tubules that used to transfer DNA from one cell to another cell similar to tubes used to fuels aircraft in flight. Some are also used to attach one cell to another cell. The tubules are made of protein and are shorter in length than flagella and longer than fimbriae. ###### Cell Wall The prokaryotic cell's wall is located outside the plasma membrane and gives the cell its shape, providing rigid structural support for the cell. The cell wall also protects the cell from its environment. Pressure within the cells builds as fluid-containing nutrients enters the cells. It is the job of the cell wall to resist the pressure the same way that the walls of balloon resist the build-up pressure when it is inflated. If pressure inside the cell becomes too great, the cell wall bursts, which is referred to lysis. The cell wall of many bacteria is composed of peptidoglycan, which covers the entire surface of the cell. Peptidoglycan is made up of a combination of peptides bonds and carbohydrates, either N-actyImuramic acid, commonly referred to as NAM, or Nacetylglycosamine, which is known as NAG the wall of bacterium is classified in two ways: ***Gram-positive:*** A gram-positive cell wall has thick exposed layer of Peptidoglycan that retain crystal violet dye when the cell is stained. This gives the cell a purple color when seen under a microscope. Gram-positive walls also contain a unique substance called teichoic acid. ***Gram-negative:*** A gram-negative cell wall is thin with an extra outer lipid membrane covering a thin layer of Peptidoglycan. The Inside made of Peptidoglycan. The cell wall does not retain the crystal violet dye when the cell is stained. The cell appear pink when view with a microscope. The outer membrane is composed of phospholipids and lipopolysaccharide (LPS) also known as endotoxin. The term "Gram stain" refers to a dye staining procedure involving sequential treatment of bacteria on slide with crystal violet dye, iodine, alcohol and safranin dye. We will look at this in detail in later tutorial/practical session.  C:\\Users\\dell\\Desktop\\dvm 305\\g-pos-g-neg-cell-wall-structure-final1566305996142.jpg **Cross sectional structure of gram positive and gram negative bacteria cells** **Cytoplasmic membrane** The prokaryotic cell has membrane called the cytoplasmic membrane that forms the outer structure of the cell and separates the cell's internal structure from the environment and the cell's internal structures. **The function of the Cytoplasmic Membrane** The function of the Cytoplasmic membrane regulates the flow of molecules (such as nutrients) into the cell removes waste from the cell by opening and closing passages called channels. In Photosynthesis prokaryotes, the cytoplasmic membrane functions in energy production by collecting energy in the form of light. The cytoplasmic membrane is selectively permeable because it permits the transport some substances and inhibits the transport of other substances. Two types of transport mechanisms are used to move substances through the cytoplasmic membrane. These are passive transport and active transport. **Cytosol and Cytoplasm** The cytosol is the intracellular fluid of prokaryotic cell that contains proteins, lipids, enzymes, ions, waste and small molecules dissolved in water, commonly referred to as semifluid. Substances dissolved in cytosol are involved in cell metabolism. The cytosol also contains a region called the nucleoid, which is where the DNA of the cell is located. Unlike human cells, a prokaryotic microorganism has a single chromosome that is not contained within a nuclear membrane or envelop. Cytosol is located in cytoplasm of the cell. Cytoplasm also contains the cytoskeleton, and inclusions. **Ribosome** A ribosome is an organelle within the cell that synthesizes polypeptide. There are thousands of ribosomes in the cell. A ribosome is comprised of subunits consisting of protein and ribosomal RNA, which is referred to as RNA. Ribosomes and their subunits are identified by their sedimentation rate. Sedimentation rate is the rate is expressed in Sydberg (s) units. The sedimentation rate reflects mass, size and shape of ribosome ad its subunits. **Inclusions bodies** An inclusion is a storage area that serves as a reserve for lipids, nitrogen, phosphate, starch and sulfur within the cytoplasm. Scientists use inclusion to identify types of bacteria inclusions are usually classified as granules. \****Endocytosis*** occurs when a small fragment of cell membrane captures food material from the exterior, pinches off to form a sphere and enters the cell. ***Exocytosis*** is the reserve process where small vacuoles from the cytoplasm travel to and then merge with the cell membrane and release material to the cell exterior, such as waste molecules. ONLY eukaryotic cells undertake these processes, and not all eukaryotes do it. **SECTION III: BACTEROLOGY** **III.1 Classification of Bacteria** The artificial scheme of classification in *Bergey's Manual of Systematic Bacteriology* is widely used. *Bergey's Manual* disregards evolutionary relationships they often group bacteria into assemblages that cannot be easily identified by standard laboratory procedures. Instead, the manual takes a strictly practical approach so that it can be used as a comprehensive and quick reference when accuracy and speed are important, as is often the case in diagnostic labs. *Bergey's Manual* divides bacteria into 4 divisions on the basis of their cell wall \[G (+) or G (-)\], their lack of a cell wall (mycoplasmas), and walls lacking peptidoglycan (archaeobacteria). Bacteria species in each division are assigned to one or two sections; sections have no taxonomic standing; they are simply groups of bacteria, which share certain easily identifiable properties. **How do we identify bacteria?** 1. We begin with morphological characteristics (shape, arrangement, etc.). 2. Reply primarily on physiological characteristics (ability to grow on a selective medium, metabolic end-products, etc.). 3. Knowing the source of the bacterium is also important. 4. Can also use DNA probes. **THE FOLLOWING IS A LIST OF THE MEDICALLY IMPORTANT MEMBERS OF SELECTED SECTIONS DEFINED IN *BERGEY'S MANUAL OF SYSTEMATIC BACTERIOLOGY.*** 1. **GRAM-NEGATIVE BACTERIA** (eubacteria) -- have an outer membrane, a periplasmic space &a thin peptidoglycan cell wall. A. **Section 1 -- Spirochetes --** Distinguished by their corkscrew shape; possess axial filaments (bundled flagella contained within the periplasm). That enables them to move through viscous environments (mud, mucous). Some live harmlessly in our mouths. Ex. Of pathogenic species: *Treponema pallidum --* syphilis, *borrrelia burgdorferi --* Lyme disease (carried by ticks); *leptospira --* leptospirosis. B. **Section 2 -- Aerobic/Microaerophilic, Motile, Helical/Vibrioid Bacteria --** Helical members are corkscrew shaped, but flagella are ordinary; vibrioid members are comma-shaped. Ex. of species: *Campylobacter jejuni --* major cause of diarrhea. *Helicobacter pylori --* cause gastric ulcers in humans. C. **Section 4 -- Aerobic Rods & Cocci --** large & diverse group. Ex. of species: D. **Section 5 -- Facultatively Anaerobic Rods --** Grouped into 3 Families; many can be distinguished by their characteristic firmament reactions; includes the enterics; Examples: *Salmonella typhi --*typhoid fever; other species cause food poisoning; *Shigella spp. --* shigellosis, a form of dysentery; *Yersinia pestis --* bubonic plague; *Vibrio cholera --* cholera; *Escherichia coli --* some species cause diarrhea & dysentery; *Enterobacter cloacae --* opportunistic infections; *Proteus vulgaris; Haemophilus influenza --* upper respiratory infections (epiglottitis, sinusitis, ear infections), pneumonia, & meningitis; *Klebsiella pneumonia --* pneumonia. E. **Section 6 -- Aerobatic Straight, Curved & Helical Rods --** most abundant microbes in mouth & intestinal tract; Example: *Bacterioides gingivalis --* causes gingivitis &periodontal disease. Other species cause digestive& respiratory infections, urinary tract infections, infections of wounds. F. **Section 9 -- The Rickettsias &Chlamydias --** Once thought to be viruses because of small size. Most species are [obligate intracellular parasites] & can't be cultivated outside a living host cell. In general, rickettsial pathogen is transmitted by arthropods (ticks, lice, mites, fleas); chlamydiae are spread directly from one infected human or animal to another. Chlamidiae alternate between 2 cell types, [elementary bodies] and [vegetative cells]. Elementary bodies are tiny, round structures released when an infected host cell lyses. When phagocytized, they differentiate into rod-shaped vegetative cells that multiply within the host cell \[This is different from other bacteria, which do not invade the host cell!\]. They then differentiate into elementary bodies again before the host cell lyses. Examples: *Rickettsia spp. --*typhus (transmitted by body lice & rat fleas), Rocky Mt. Spotted Fever (transmitted by ticks); *Coxiella --* Q fever; *Chlamydia trachomatis --*trachoma, sexually transmissible nongonococcal urethritis or NGU; *Chlamydia psittaci --* ornithosis (parrot fever) a respiratory disease. 2. **MYCOPLASMAS** (eubacteria) -- **Section 10 --** All lack a rigid cell wall. To maintain turgor pressure: 1. Their cell membrane contain sterols to add strength (sterols are also found in eukaryotic cell membranes), 2. They maintain their cytoplasm at the same pressure as their external environment by actively pumping sodium ion out of the cell. All are parasites of humans, animals, or plants. Almost all are obligate fermenters (they ferment even in the presence of oxygen). Their colonies have a distinctive fried egg appearance. They have various shapes, but when growth conditions are suboptimal, they become distorted, forming long strands that resemble fungi (thus accounting for the name *myco,* which means "fungus"). Their wall-less structure allows them to squeeze through even the tiny pores in filters used to sterilize liquids. *Mycoplasma pneumonia-*common cold & primary atypical pneumonia (walking pneumonia). 3. **GRAM-POSITIVE BACTERIA** (eubacteria) -- lack an outer membrane & a periplasmic space; have a thick peptidoglycan cell wall. A. **Section 12 -- Cocci --** large group. Some examples: B. **Section 13 -- Endospore-Forming Rods & Cocci --** These bacteria are the most heat-resistant living things; they are used as an index of fertilization; location of endospore can be used to distinguish species. Some examples: (i) *Clostridium spp. --* all strict anaerobes, inhabiting soil & mud; *C. tetani* causes tetanus (fatal rigid paralysis); *C. perfringens* causes gas gangrene & food poisoning; *C. chauvoei* causes [black quarter disease in cattle]*. botulin*causes botulism (food poisoning); some species are harmless. (ii) *Bacillus* spp. - aerobes, some facultative anaerobes; *B. anthracis* -- causes anthrax; *B. cereus* causes food poisoning. C. **Section 14 -- Non-sporing Rods --** *Listeria monocytogenes* -- food poisoning (listeriosis); in young, old & immunocompromised patients it can cause form of meningitis. D. **Section 15 -- Irregular Non-sporing Rods-** Members have irregular shapes (branched, club-shaped, etc.); shapes can change with growth phase of culture. E.g. *Propionibacterium acnes --* causes acne; *Corynebacterium diphtheriae* -- cause diphtheria. 4. **MYCOBACTERIUM (Section 16) --** have a waxy outer layer composed of polysaccharides & mycolic acid; protects against hostile environments & affects staining; identified by the acid-fast stain procedure; examples: *Mycobacterium tuberculosis --* causes tuberculosis; *Mycobacterium leprae --* causes leprosy. 5. **OTHER SECTIONS** A. B. **III General structure and morphology Microbial physiology** (  **\[In this figure\]** The bacteria shape and morphology. **Cocci** Cocci are usually round shape but can be oval or elongated. The name of the cocci is based on how they reproduce and what they look like after dividing. For example, cocci that remain in pairs after dividing are called **diplococci** and those that divide and remain attached together in a chain are called **streptococci**. Those that divided in two planes and remain in groups of four are called **tetrads**. Those that divide in three planes and remain in groups of eight are called **sarcinae**. Those that divided in multiple planes and remain in a cluster like a grape are called staphylococci. These shape characteristics are very useful for the identification of cocci. **Bacilli** Bacilli divide only across their short axis, so there are fewer groups of bacilli than cocci. Most bacilli are single rods. Those that remain in pairs after dividing are called **dipobacilli**. Similarly, those that remain together in a chainlike shape after dividing called **streptobacilli**. Some bacilli are oval and look like cocci, so they called **coccobacilli**. **Spiral bacteria** Spiral bacteria have one or more twists. **Vibrios** are bacteria that look like curved rods. **Spirilla** are bacteria that have a helical shape, like a corkscrew and rigid bodies. Another type of spiral bacteria called **spirochetes,** which are also helical but with flexible bodies. Spirilla use flagella, a whiplike external appendage, to move while spirochetes use axial filaments to twist. Most of the bacteria maintain in one single shape. However, there are also some bacteria that can change their shape based on the environment condition. This will make identification more difficult.   First, a reminder from general high school biology: Organisms can be categorized based on how they obtain nutrition: ***Autotrophs --*** form their own organic molecules from simple inorganic compounds, there are two types of autotrophs (this word literally means self-feeder): ***Photoautotrophs --*** use light energy to form organic compounds from simple inorganic materials such as carbon dioxide, water and salts (plants, algae, cyanobacteria). ***Chemoautotrophs --***use energy obtained by breaking down simple inorganic molecules (some bacteria). ***Heterotrophs --*** most use pre-formed organic compounds (such as glucose) that were formed the autotrophs. Fungi, animals, many bacteria, protozoa are heterotrophs. **III.1 Bacteria growth** The term **microbial growth** refers to the growth of a population (or an increase in the number of cells), not to an increase in the size of the individual cell. Cell division leads to the growth of cells in the population. **Two Types of Asexual Reproduction in Microbes:** 1. **Binary Fission --** Bacterial reproduction occurs through **fission**, a primitive form of cell division that does not employ a **spindle fiber apparatus**. \[A spindle fiber apparatus made of protein filaments is responsible for moving the chromosomes around during cell division (mitosis & meiosis) in most eukaryotic cells. Bacteria do not have these structures.\] The bacterial cell doubles in size and replicates its chromosome. Following DNA replication, the two chromosomes attach to separate sites on the plasma membrane, and the cell wall is laid down between them, producing two **daughter cells**. 2. **Building -A** few bacteria and some eukaryotes (including yeasts) may also replicate by **building**, forming a bubble-like growth that enlarges and separates from the parent cell. A typical cell of the bacterium *E. coli* can reproduces in as little as twenty minutes under good conditions (lots of nutrient and a warm temperature -- 37°C). This is much faster than the average eukaryotic cell (Baker's yeast for instance, might manage to reproduce asexually by a process called budding once an hour, at best). If you do the math, this means that ONE bacterial cell becomes millions within a day, this has great significance, potentially, for your health and safety. Bacterial cells attain a very limited cell size and they reproduce frequently, so that bacterial growth is defined as the orderly increase in numbers of bacteria over time. Bacterial cells do not have multiple linear chromosomes as our eukaryotic cells do, so they do not undergo mitosis or meiosis. If a single bacterium the size of *E. coli* has a generation time of 20 minutes, and nothing impedes the division of successive progeny, within 48 hours a mass of bacteria will be formed that is 4000 times as great as the mass of the earth! Obviously, there are factors which limit this growth! -- Principally availability of resources, presence of competitors, buildup of toxic metabolites etc. C:\\Users\\dell\\Desktop\\dvm 305\\image841.png Bacterial cells undergo a replication of their single circular chromosome and then the cell "splits in two" in a process called binary fission. Many bacteria also contain much smaller circular DNA molecules, these are classified as extrachromosomal DNA structures, and they are called plasmids, they commonly hold genes for antibiotic resistance or toxin production, and they also replicate as the bacterium undergoes reproduction, the plasmids are key concern in the pathogenesis and virulence of many bacteria that cause human disease. Extrachromosomal DNA is also introduced into bacteria by some infecting viruses, this is called transduction, these viruses can errantly include bacterial genes in their capsids as new viruses form, and then they introduce these pieces of DNA into the bacterium they infect. So, bacteriophage can transfer bacterial genes from one bacterial species to another; in some cases genes transferred by transduction can increase the virulence of disease caused by the receiving bacterium. A rod shaped bacterium (technically, a rod shape is described as a bacillus) will grow in length while binary fission is underway, and then produce a "cross wall" called a septum which results in two individual bacteria. The name "binary fission" was given by researchers because this is what was observed (a literal translation of binary fission into simple English is "splitting in two), binary fission is a complex vent mechanically and chemically but it looks like a simple splitting in two under a microscope. Imagine that you have inoculated a flask of culture fluid (containing nutrients and agitated to mix oxygen into the fluid) with a small loop full of bacteria (it will hold millions of bacteria even though there does not appear to be anything on the loop). One can take samples regularly and count the number of bacteria present. The typical results of bacterial growth when plotted in a graph with numbers of bacteria expressed in logarithmic form will be what you see below. A. ***Phases of Growth --*** A microbial lab culture typically passes through 4 distinct, sequential phases of growth that form the [standard bacterial growth curve]: (Not all growth phases occur in all cultures). **See graph; be able to draw & label.**  ***Different shapes of bacteria colonies during bacteria multiplication*** 1. **Lag Phase --** In the lag phase, the number of cells does not increase. However, considerable metabolic activity is occurring as the cells prepare to grow. (This phase may occur, if the cells used to inoculate a new culture are in a log phase & provided conditions are the same). 2. **Log Phase** (logarithmic or exponential phase) -- cell numbers increase exponentially; during each generation time, the number of cells in the population increases by a factor of two. The number of microbes in an exponentially increasing population increases slowly at first, then extremely rapidly. A limited time, as nutrients are used up, metabolic wastes accumulate, and microbes suffer from oxygen depletion. 3. **Stationary Phase --** The number of cells does not increase, but changes in cells occur: cells become smaller and synthesize components to help them survive longer periods without growing (some may even produce endospores); the signal to enter this phase may have to do with overcrowding (accumulation of metabolic byproducts, depletion of nutrients, etc.). 4. **Death Phase -** In this phase, cell begin to die out. Death occurs exponentially, but at a low rate. Death occurs because cells have depleted intracellular ATP reserves. Not all cells necessarily die during this phase! In some bacterial species (notably the Gram-positive genera *Bacillus* and *Clostridium)*, endospores may be formed as the culture nears death phase, these are tough dry spherical bodies that can endure long periods (often many years) in harsh chemical and climatic conditions, and will germinate to produce a new bacterial cell when conditions are suitable. **III.2 Continuous Culture of Microbes** In the lab, cultures usually pass through all growth phase -- not in nature. In nature, nutrients continuously enter the cell's environment at low concentrations, and populations grow continually at a low but steady rate. The growth rate is set by the concentration of the scarcest or limiting nutrient, not by the accumulation of metabolic byproducts -- in nature there is always some other microbe that can use these metabolic byproducts for their own metabolism. In the lab, we have to continually replace the media. ***Measuring Numbers of Microbes*** **i). Indirect Measurements** (measure a property of the mass of cells and then [ESTIMATE] the number of microbes) **III.3. Metabolic Activity -- 3 ways:** 1. **Direct Counts -** Coulter Counter -- electronic counter; rapid & accurate only if bacterial cells are the only particles present in the solution. \[Gives a total count -- live & dead cells\]. 2. **Plate Count --** Bacterial colonies are viewed through the magnifying glass against a colony-counting grid; called a Quebec colony counter (we have this in the lab). \[Gives a viable count\] 3. **Filtration --** A known volume of liquid or air is drawn through a membrane filter by vacuum. The pores in the filter are too small for microbial cells to pass through. Then the filter is placed on an appropriate solid medium and incubated. The number of colonies that develop is the number of viable microbial cell in the volume of liquid that was filtered. This technique is great for concentrating a sample, ex. a swimming pool, where small population's may go undetected using some other methods. \[Gives a viable count\] **III.4 Growth Factors** **III.4.2 Chemical factors** i. **Oxygen Requirements** 1. **Strict or obligate anaerobes** -- oxygen kills the bacteria; ex. *Clostridium tetani* 2. **Strict or obligate aerobes** -- lack of oxygen kills the bacteria; ex. *Pserdomonas* 3. **Facultative anaerobes** -- can shift their metabolism (anaerobic if oxygen is absent or aerobic if oxygen is present); ex. *E. coli, Staphylococcus* 4. **Aerotolerant** -- the bacteria don't use oxygen, but oxygen doesn't harm them; ex. *Lactobacillus* 5. **Microaerophiles** -- like low oxygen concentrations and higher carbon dioxide concentrations; ex. *Campylobacter* **ii). Nutritional (Biochemical) Factors --** Nutrients needed by microorganisms includes: i. **Carbon --** carbon containing compounds are needed as an energy source (ex. glucose) and for building blocks. ii. iii. iv. v. vi. **VI Microscopy and staining** **V.1 Microscopy principles** Microscopy is the technology of making very small things visible to the human eye. Most microbes are also small that they are measured in micrometers or nanometers. A typical bacterial cell is about 1 *u*m while a virus is more in range of 10 -- 100 nm. Resolution is the ability to see two objects as separate, discreet entities... kind of like the ability to see railroad tracks as being separate tracks... GOOD resolution is being able to distinguish the two tracks as separates... once the two tracks merge into one, the resolution is poor!!! Refraction is the bending of light as it passes from one medium to another of different density. Immersion oil, which has the same index of refraction as glass, is used to replace air and to prevent refraction at a glass-air interface. An example would be when one looks at objects just below the surface of water in pond or other body of water... the objects become refracted or "distorted" from the true image. The total magnification of a light microscope is calculated by multiplying the magnifying power of the objective lens by the magnifying power of ocular lens. Increased magnification is of no value unless good resolution can also be maintained. Scanning (3X) x (10X) = 10X total Low power (10X) x (10X) = 100X total High "dry" (40X) x (10X) = 400X total Oil immersion (100X) x (10X) = 100X total Most microscopes are designed so that when the microscopist increases or decreases the magnification by changing from one objective lens to another, the specimen will remain very nearly in focus. Such microscopes are said to be *parfocal* (par means equal). **V.2 Types of microscope:** - Compound Light -- (this is what we will use... known the parts and functions... we will spend more time on this in the lab. - Dark-Field; Phase-Contrast; Fluorescence; Transmission; Scanning Electron. **V.3 Preparation of specimens** Wet mounts are used to view living organisms. The hanging drop technique is a special type of wet mount, often used to determine whether organisms are motile. Smears of appropriate thickness are allowed to air-dry completely and are then passed through to the slide and more readily accept stains. 1. **Principles of Staining** A stain, or dye, is a molecule that can bind to a structure and give it color. Most microbial stains are cationic (positively charged), or basic dyes, such as methylene blue, crystal violet, or safranin. Somme are anionic dyes (negatively charged), or acidic dyes, such as nigrosin or India ink. MOST bacterial surfaces are negatively charged so they will attract the basic dyes. Simple stains use one dye and reveal basic cell shapes and arrangements. Differential stains use two or more dyes and distinguish various properties of organisms. [The Gram stain], spore stain, and acid-fast stain are examples. Negative stains color the background around cells and their parts, which resist taking up the stain (acidic dyes will "stick" to the glass slide since glass has a + charge). Imagine a magnet when thinking of basic an acidic dyes... basic dyes (+) will attract to bacteria due to their (-) parts but will be repelled by the glass because of its (+) charge!! Acidic dyes, on the other hand, will attract to the (+) glass but be repelled by the (-) bacterial parts! **Quick Guide for Staining Techniques** +-----------------+-----------------+-----------------+-----------------+ | type | Number of Dyes | Observations | Examples | | | used | | | +=================+=================+=================+=================+ | Simple stains | Use a single | Size, shape and | - Methylene | | | dye | arrangement of | blue | | | | cells | | | | | | - Safranin | | | | | | | | | | - Cristal | | | | | violet | +-----------------+-----------------+-----------------+-----------------+ | Differential | Use a two or | - Distinguish | - Gram stain | | stains | more dyes to | es | | | | distinguish | gram-positi | - Zehl-Nielse | | | different types | ve | n | | | or different | or | acid-fast | | | structures of | gram-negati | stain | | | bacteria | ve. | | | | | | | | | | - Distinguish | | | | | es | | | | | the member | | | | | of | | | | | mycobacteri | | | | | a | | | | | and | | | | | nocardia | | | | | from other | | | | | bacteria | | +-----------------+-----------------+-----------------+-----------------+ | Special stains | These stains | - Exhibits | Schaeffer-Fulto | | | identify | the | n | | | specialized | presence of | spore staining | | | structures | flagella. | | | | | | | | | | - Exhibits | | | | | endospores | | +-----------------+-----------------+-----------------+-----------------+ Will also be covering these in detail in laboratory. **Types of stains** There are two types of Stains: simple and differential. **Simple Stain** A simple stain has a simple basic dye that is used to show shapes of cells and the structures within a cell. Methylene blue, safranin, carbolfluchsin and crystal violet are common simple stains that are found in most microbiology laboratories. **Differential Stain** A differential stain consists of two or more dyes and is used in the procedure to identify bacteria. Two of the most commonly used differential stains are the Gram stain and the Ziehl-Neelsen acid-fast stain. In 1884 Hans Christian Gram, a Danish physician, developed the Gram-stain. Gram-stain is a method for the differential staining of bacteria. Gram-positive microorganisms stain purple. Gram-negative microorganisms stain pink. *Staphylococcus aureus*, a common bacterium that causes food poisoning, is gram-positive, *Escherichia coli* is gram negative. The Ziehl-Neelsen acid-fast stain, developed by Franz Ziehl and Fredrick Neelsen, a red dye that attaches to the waxy material in the cell walls of bacteria such as *Mycobacterium tuberculosis*, which is the bacterium that causes tuberculosis, and *Mycobacterium leprae*, which is the bacterium causes leprosy. Microorganisms that retain the Ziehl-Neelsen acid-fast stain are called acid-fast. Those that do not retain it turn blue because the microorganism does not absorb the Ziehl-Neelsen acid-fast stain. ***How to Gram-stain a specimen.*** (Observing Microorganisms) 1. Prepare the specimen using the heat fixation process. 2. Place a drop of crystal violet stain on the specimen. 3. Apply iodine on the specimen using an eyedropper. The iodine helps the crystal violet stain adheres to the specimen. Iodine is a mordant, which is a chemical that fixes stain to the specimen. 4. Wash the specimen with ethanol or alcohol-acetone solution, then wash whit water. 5. Wash the specimen to remove excess iodine. The specimen appears purple in color. 6. Apply the safranin stain to the specimen using an eyedropper. 7. Wash specimen. 8. Use a paper towel and blot the specimen is dry. 9. The specimen is ready to be viewed under the microscope. Gram-positive bacteria appear purple and gram-negative bacteria appear pink. ***Here is how to apply the Ziehl-Neelsen acid-fast stain to a specimen.*** 1. Prepare the specimen 2. Apply the red dye carbon-fuchsin stain generously using an eyedropper. 3. Let the specimen sit for a few minutes. 4. Warm the specimen over steaming water. The heat will cause the stain to penetrate the cell wall. 5. Wash the specimen with an alcohol-acid or acid-alcohol decolorizing solution consisting of 3% hydrochloric acid and 93% ethanol. The hydrochloric acid will remove the color from non-acid-fast cells and the background. Acid-fast cells will stay red because the acid cannot penetrate the cell wall. **Special Stains** Special stains are paired to dye specific structure of microorganisms such as endospores, flagella and gelatinous capsules. One stain in the pair is used as negative stain. A negative stain is used to stain the background of microorganism causing the microorganisms to appear clear. A second stain is used to colonize specific structures within the microorganism. For example, nigrisin and India ink are used as a negative stain and methylene blue is used as positive stain. The Schaeffer-Futon endoscope stain is a special stain that is used to colorize the endospore. The endospore is a dormant part of bacteria cell that protects the bacteria from the environment outside the cell. **Here is how to apply the Schaeffer-Fulton endospore stain.** 1. Prepare the specimen. 2. Heat the malachite green stain over a Bunsen burner until it becomes fluid. 3. Apply the malachite green to the specimen using an eyedropper. 4. Wash the specimen for 30 seconds. 5. Apply the safranin stain using an eyedropper to the specimen to stain parts of the cell other than the endospore. 6. Observe the specimen under the microscope. **VI CULTURING BACTERIA** **VI.1 Pure culture technique** a. ***Clonal populations*:** i. *Pure culture technique* is a method of culturing microorganisms which all of the individuals in a culture have descended from a single individual. ii. This is done so as to: a. Inhibit evolutionary change within culture b. Allow the characterization of type's microorganisms without the confounding presence of other, different types of microorganisms **b. *Colony isolation:*** i\. The basis of *pure culture technique* is the isolation, in colonies, of individual cells, and their descendants, from other colonies of individuals. ii\. This is usually done by culturing methods employing petri dishes such as: 1. **Streaking** 2. **Pouring** 3. **Spreading** c. ***Isolation from the wild:*** i. When isolating microorganisms from complex mixtures it is always a good idea to repeat the isolation procedure at least once (e.g. re-streak an isolated colony) to make sure that an isolated colony is truly derived from only a single cell (i.e. closely overlapping colonies can be indistinguishable from colonies founded from single cells). ii. Following their isolation from the wild, microorganisms may be characterized by inoculation into differential medium to determine what type nutrients they require or can use, and what types of by-products they produce. This aids in identification. d. ***Pouring a plate*** a. A *pour plate* is a method of melted agar inoculation followed by petri dish incubation. b. Steps include: i. Culture are inoculated into melted agar that has been cooled to 45°C ii. The liquid medium is well mixed then poured into a petri dish (or *vice versa*) iii. Colonies from within the agar matrix rather than on top as they do when streaking a plate c. Pour plate are useful for quantifying microorganisms that grow in solid medium. d. Because the "pour plate" embeds colonies in agar it can supply a sufficiently oxygen deficient environment that it can allow the growth and quantification of microaerophiles. e. ***Spreading a plate*** a. Quantification technique : - *Spreading a plate* is an additional method of quantifying microorganisms on solid medium. - Instead of embedding microorganisms into agar, as is done with the pour plate method, liquid cultures are *spread* on the agar surface using a devise that looks more or less like a hockey stick. b. An advantage of spreading a plate over the pour plate method is that cultures are never exposed to 45°C melted agar temperatures. **VI. 2 CULTURE MEDIA** A. ***Types of Media*** 1. **Synthetic medium --** prepared in the lab from materials of precise or reasonably well-defined composition. 2. B. ***Selective & Differential Media*** (we will learn about these in detail in lab!) 1. **Selective --** one that encourages the growth of some bacteria but suppresses the growth of others. 2. **Differential --** has an ingredient that causes an observable change in the medium when a particular biochemical reaction occurs (ex. a color pH change). C. **Controlling Oxygen Content of Media** 1. **Candle jars -** the inoculated tube or plate is placed in a jar; a candle is lit before the jar is sealed to it; when the carbon dioxide extinguishes the flame, condition are optimum for the growth of microorganisms that require small amounts of carbon dioxide (ex. *Neisseria gonorrhoeae*) 2. **Thiolglycollate medium --** oxygen-binding agent added to the medium to prevent oxygen from exerting toxic effects on anaerobes; media is usually dispensed in sealed screw-cap tubes. 3. **Anaerobic Chamber (Brewer Jar) --** a catalyst is added to a reservoir in the lid of jar. Water is added to the gas-pack Water is converted into hydrogen gas and carbon dioxide. The hydrogen gas can then bind with any oxygen in the jar to from water. A methylene blue test strip is included in the jar to ensure that anaerobic conditions are reached. When oxidized (oxygen is present) the strip is blue; when reduced (no oxygen), the strip is clear. **Preservation of cultures** Culture Preservation is important for the following reasons: i. Scientific reasons ii. Identification iii. Vaccine production iv. Industrial use Methods of preserving cultures include: i. Refrigeration ii. Stabs iii. Slants iv. Lyophylisation v. Freezing i. **Refrigeration**(at 4°C) This is effective short term preservation. All following can be refrigerated: - Broth cultures - Stabs - Slants - Streaks ii. **Stabbing** In this method, Cultures are usually stabbed deeply into agar using in inoculating needle in bijou bottles, test tubes and other holding wares. The stabs are incubated until visible cultures are formed; they are then sealed and stored at room or lower temperature. iii. **Slant method** Slant method involves streaking the organism of interest into the surface of a solid medium in a slant tube. The slant is then inoculated until visible culture formation. The slant is then sealed and stored at room or lower temperature. iv. **Lyophilisation** Lyophilisation is the freeze-drying of cultures. Cultures are first frozen and then dried under high vacuum. To revive cultures they are rehydrated by broth. Lyophilisation can be an effective long term method of storage. v. **Freezing** Broth cultures are mixed whit various ingredients (e.g. glycerol) to limit damage upon freezing and the frozen to temperatures ranging from -50°C to 95°C. To revive cultures, they are thawed, pelleted, and resuspended into broth. Freezing can be an effective long-term of storage. **VII Disinfection and Sterilization** **VII.1 Natural disinfection** Although the deliberate application of disinfectant techniques is of great importance in medicine, appreciable disinfection occurs spontaneously under natural conditions -- pathogenic organisms which have become adapted to a parasitic role in the animal body as a rule surviving poorly outside it. The more important factors operating under such conditions and leading to the death of bacteria are deprivation of nutriment and the disinfectant action of light and desiccation. In certain natural environments other special factors come into play; thus in water bacteria are ingested by algae and protozoa, while in the soil they are exposed to the action of antibiotic substances produced by various soil organisms. **VII.2 Physical methods of sterilization** ***Mechanical trauma*** Bacteria can be killed by simple mechanical trauma, e.g. by grinding, by shaking with particles, by ultrasonic irradiation and by repeated freezing and thawing. Mechanical methods, however, have no practical application in disinfection -- although they are of considerable value as research procedures for the rupture of bacteria, so as to release intracellular constituents. ***Heat*** The application of heat is most important of all the methods of disinfection and provided the heat used is adequate it is the most certain and rapid; it is also easily controlled and unlike chemical disinfection leaves no potentially harmful residue. As with all other types of disinfection the sterilization of a bacteria population by heat is a gradual process. Throughout most of the process the rate of disinfection is approximately a logarithmic one, i.e. if the logarithm of the number of survivors is plotted against time the resultant curve is a straight line. The time required for sterilization is inversely related to the temperature of exposure -- the higher the temperature the shorter the time required. Thus a culture which requires one hour for fertilization at 60°C will be sterilized only a few minutes at 80°C; at 100°C sterilization will be virtually instantaneous. **Dry heat** Dry heat is the preferred method of sterilization for glassware, e.g. glass syringes, and for materials such as oils, jellies and powders which are impervious to steam. It is unsuitable for material, e.g. fabrics, which may be damaged by heat. As compared with stream sterilization it has the disadvantages that a longer time and a higher temperature are required. The most widely used type of dry heat sterilizer is the hot air oven, which is usually electrically heated. In the simpler gravity convection ovens the air circulates by convection. This form of oven is unsatisfactory since it is difficult to ensure that there has been adequate air circulation. The mechanical convection type of oven in which the air is circulated by a fans is much more efficient. Suitable sterilizing times in the hot air oven are three hours at 140°C, one hour at 160°C and 20 to 30 minutes at 180°C. **Moist heat** A previously mentioned most vegetative bacteria can be fairly rapidly killed by temperatures in the range of 60 to 65°C. The most important applications of temperatures in this range are in the pasteurization of milk and in the preparation of bacterial vaccines. Boiling is frequently used for the sterilization of glass syringes, surgical instruments and small pieces of apparatus. Since, however, many spores will withstand boiling for a considerable time boiling must be regarded as inadequate for the sterilization of such materials. ***Steam sterilization*** Exposure to steam is most widely used and the most effective technique of moist heat sterilization. Steam is a more efficient sterilizing agent than hot air at the same temperature, for the following reasons. First, bacteria are intrinsically more susceptible to moist than to dry heat. Second, steam is a more rapid sterilizing agent than hot air because on condensation it gives up its latent heat of vaporization rapidly, steam has a much greater power of penetrating porous material. This is due to the fact that on condensation a partial vacuum is created which has the effect of sucking in more steam from outside until the material becomes thoroughly permeated. Steam may be employed in three ways. *Low temperature steam.* This method has recently been advocated for the sterilization of materials such as blankets and polythene tubing, which would be damaged by steam at high temperatures. Saturated steam at temperatures of from 70 to 90°C may be generated I sterilizers operating at pressures of 30 to 65 kPa (10 to 20 in Hg). Under these conditions steam has been found to be a much more effective sporicidal agent than water at the same temperature. The efficiency of the process can be significantly increased by the injection of a small amount of formalin along with the steam. *Steam at temperatures greater than 100°C*. This is achieved by the use of the autoclave. The use of such high temperatures necessitated by the high thermal resistance of bacterial spores. Essentially an autoclave is a very strong boiler or cylinder with a door which can be hermetically sealed and constructed sufficiently strongly to withstand the high pressure is maintained at the appropriate level during sterilization by means of a safety valve set to blow off at a predetermined pressure. In construction, autoclaves vary enormously from the simpler laboratory types -- I which the steam is generated in the autoclave itself, and which are essentially little more than glorified pressure cookers -- to the highly sophisticated hospital sterilizers with virtually automatic control of the sterilizing cycle. ***Cold*** Bacteria may also be killed by exposure to cold. Two different circumstances must be distinguished. **Cold shock** This is sudden reduction in temperature without actual freezing. Some species are highly susceptible to cold shock. Thus a 95% drop in the number of variable *Escherichia coli* has been reported to occur following sudden chilling from 45 to 15°C. The mechanism of this effect is unknown but it is possibly due to a difference in the rate of contraction on cooling of different intracellular constituents, with a resultant disorganization of cellular structure. **Freezing** It was at one time thought that the lethal effect of freezing was due to physical damage to the cell membrane and/or cell wall by ice crystals. It is now generally accepted that such a mechanism can play only a mirror role, if any, in the death of frozen organisms. There are probably two major factors involved: 1. The formation of ice crystal outside the cell by the withdrawal of water from the cell interior increases the intracellular salt concentration. This would in turn be capable of causing considerable damage by protein denaturation and is probably a major cause of death when bacteria are frozen to temperatures of not lower than -- 30 to -- 35°C. 2. Formation of ice inside the cell. There is evidence that this can only occur if the bacteria are frozen to temperatures lower than -35°C and, even then, only under conditions of rapid cooling. The lethal effect of intracellular ice does not appear to be due to a direct action of the ice on the cytoplasm but only occurs during defreezing. It appears to be maximal when the bacteria are heated slowly and minimal when they are heated rapidly. ***Ultra-violet radiation*** Light has considerable disinfectant properties and plays a very important part in the spontaneous sterilization which occurs under natural conditions. Its disinfectant action is due mainly to the ultra-violet rays, most of which are screened out by glass and also to a considerable extent by smoky and foggy atmospheres. The most active of ultra-violet rays are those with wave-lengths in the region 240 to 280 nm. Radiation in this region is, however, present to only a small extent in the solar radiation which succeeds in penetrating the earth's atmosphere, the latter containing little radiation with wave--length bellow 290nm. Ultra-violet radiation can be produced artificially by special lamps. Those most commonly employed are of the low pressure mercury vapor type. With these, over 95% of the radiation emitted is of wave-length 253.7 nm. Germicidal UV lamps must be used whit care since the radiation has a damaging effect on the eyes; they must consequently be sited in such a way that the eyes are not exposed directly to the radiation. **VII.3 Chemical disinfection** Chemical disinfection agents exhibit two distinct types of antibacterial effect: a *bactericidal* or killing effect and a *bacteriostatic* or growth-inhibiting effect. Bactericidal activity may be demonstrated by mixing a suspension of bacteria with the disinfectant and then after a period of contact under defined conditions the mixture is subculture to a nutrient medium. If the compound has a bactericidal action no growth of the organism will occur on subculture. To demonstrate bacteriostatic activity the disinfectant is incorporated in a suitable nutrient medium, e.g. broth which is then inoculated with the organism and inoculated. A bacteriostatic effect is shown by the absence of growth after incubation. Most chemical compounds which can kill bacteria exhibit a bacteriostatic effect in concentrations lower than those required to kill. The relationship between bactericidal and bacteriostatic concentrations is not, however, the same for all disinfectants. Some compounds, e.g. the mercurial and the quaternary ammonium compounds, have very marked bacteriostatic properties inhibiting bacterial growth in dilutions very much higher than those required to kill. Others, e.g. the halogens, which of the commonly used disinfectants are unique in this respect, appear to be exclusively or almost exclusively bactericidal. On the whole we know relatively little of the precise ways in which disinfectants kill bacteria; it is probable that in most cases killing occurs through some form of enzyme inactivation either by protein denaturation, oxidation or by a combination of the antibacterial agent with specific groups of enzyme proteins. Some of the compounds used as disinfectants, e.g. the quaternary ammonium compounds, are capable of damaging the bacterial cell membrane and thus permitting leakage of essential intracellular compounds from the cell into its environment. This type of effect is believed to be of primary importance in the killing action of such compounds. ***Kinetics of disinfection*** When a bacterial population is exposed to a disinfectant there is a progressive reduction with time in the number of surviving organisms. Disinfection is therefore a gradual process. In this connection it is important to remember that when we study the killing of bacteria by a disinfectant the observable effect is the effect on bacterial cell. It follows therefore that the larger the bacterial population exposed the longer is the time required for its sterilization. It is generally accepted as a working hypothesis that the rate at which disinfection occurs is uniform in the sense that during each unit of time a constant proportion of the organisms present at the beginning of the time is killed. ***Acid and alkalis*** The disinfectant of strong acids and alkalis depends in general on their acid or basic strength. Many weak organic acids have, however, a higher activity than would be expected from their dissociation constants. This activity is due to the toxicity of the organic anion. The tubercle bacillus is appreciably more resistant than other organisms to disinfection by acid alkali. Consequently material from which it is proposed to culture tubercle bacilli may be treated with acid or alkali to free it from other bacteria present. ***Salts*** All salts have some degree of toxicity for bacteria. The most toxic are those of the heavy metals, mercury and silver, and the least toxic the salts of sodium and potassium. Of the heavy metals the most frequently used is mercury. Two types of compound are employed -- the *organic salts* and the *organic mercurial* in which mercury is combined with an organic radical. Both types of compound have very marked bacteriostatic properties but cannot be regarded as good disinfectants since their bactericidal activity is relatively slight. Mercury compounds appear to act on bacteria by combining with sulphydryl (SH) groups of bacterial proteins and other essential intracellular compounds. With these they form a relatively loose combination from which they can be dissociated by the addition of SH-containing variety of such compounds they antagonize the action of the mercurial. This constitutes a further serious limitation to their medical utility. ***Halogens*** *Chlorine* and *iodine* are the only halogens which have any practical application as disinfectants. For certain purposes -- chlorine as a water disinfectant and iodine as a skin disinfectant -- they are unequalled. Their great value as disinfectants is due to the following: 1. They are bactericidal in very high dilution. They are in fact unique amongst the disinfectants in that their activity is practically exclusively bactericidal. 2. Their action is very rapid. 3. They possess considerable activity against sporing organisms. In addition to chlorine itself three types of chlorine compound -- the hypochlorites, the inorganic chloramines and the organic chloramines -- are available. The disinfectant action of all chlorine compounds is due to their capacity to liberate free chlorine. In solution the liberated chlorine forms hypochlorus acid is least dissociated at acid pH values chlorine disinfectants are in general most active under acid conditions. ***Alcohols*** Ethyl alcohol is non-specific in action, being active against both Gram-positive and Gram-negative organisms. It possesses in addition appreciable activity against tubercle bacilli. It has no significant sporicidal activity; in fact the recovery of anthrax spores after 20 year's exposure to ethyl alcohol has been recorded. Ethyl alcohol almost certainly acts by protein denaturation and exhibits a bactericidal effect only in the presence of water. Appreciable disinfection activity only occurs in concentrations of from 40 to 95% - the optimum range being from 50 to 70%. In general the disinfectant activity of the alcohol increase in chain length. Above ten carbon atoms, however the solubility of the compound becomes too low for practical use. Apart from ethyl alcohol the only alcohols which have so far had any significant application have been isopropyl alcohol and dihydric alcohols ethylene and propylene glycol. Isopropyl alcohol, which is appreciably more active and less volatile than ethyl alcohol, has been recommended for the sterilization of clinical thermometers. ***Dyes*** Two groups of dyes, the *aniline* dyes and the *acardines* have been considerably used as skin and wound antiseptics. Both groups are bacteriostatic in high dilution but are of low bactericidal activity. Of the aniline dyes brilliant green, malachite green and crystal-violet (an impure form of the latter is known as gentian violet) have been mainly used. All are amino derivatives of triphenylmethane. The aniline dyes are highly selective being much more active against Gram-positive than against Gram-negative organisms **VIII THE NORMAL BODY FLORA** **Flora** is defined as a group of microbes that reside in different anatomical sites of the human body. Flora is commonly divided into normal flora and transient flora. **Normal flora** includes the microbes that permanently reside in a specific body site and is also known as **resident flora**. In contrast, **transient flora** is only temporarily present in the superficial regions of the body. Normal flora of the body maintains a positive host-microbe relationship and helps the host in maintaining good health. However, some normal flora are **opportunistic pathogens** that can cause diseases when the person is seriously ill or demonstrates low immunity. Disruption of the normal flora can also lead to colonization of pathogenic bacteria that can cause diseases. *Staphylococci* is the common resident flora of the skin and *Streptococci* is the common resident flora of the mouth, or oral cavity. Unlike transient flora that can be easily removed from the surface of the hand, normal or resident flora are difficult to clean off. Normal flora demonstrates several protective functions of our body until the ecosystem is disrupted At birth the skin and mucous membranes are, as might be excepted, sterile. It is in fact possible by the adoption of stringent isolation procedures to maintain laboratory animals in a germ-free state. Such animals have been of considerable value for research, particularly in connection with the development of immunity mechanisms. In the normal animal, however, the surfaces of the body which are in contact with the environment, namely the skin and mucous membranes, rapidly become colonized by organisms present in the environment. The organisms that can establish themselves in this way differ in the various parts of the body, which consequently show variations in their normal flora. Such differences are due to the operation of local and environmental factors which favor and tend to select certain species. We know relatively little of precise nature of these environmental factors and of the way in which they tend to favor particular bacteria. We do know, however, that changes in environmental conditions may result in marked changes in local flora. The organisms of this normal flora are known as *commensals* and obtain their nutriment from the secretions and waste products of the body. Occasionally species of relatively high pathogenicity may appear in the normal flora without causing disease. When this happens the individual in whose body the pathogen is found is known as a carrier. The carrier state is, however, unusual -- the highly pathogenic organisms either initiating an infection or being rapidly eliminated from the body. A knowledge of the types of bacteria found different areas is great importance in diagnostic bacteriology and these will be briefly considered. The skin is constantly receiving bacteria from the air or from object with which it has come in contact, but the majority of these do not grow on it because of the absence of suitable growth conditions. Few are capable even of surviving on the skin for more than a very short time because of substances which are bactericidal for them. The most constant of the normal flora of the skin are anaerobic diphtheroids -- which belong to the genus *Propionibacterium* -- and non-pathogenic staphylococci. These organisms are found mostly in the sebaceous glands; their numbers are little affected by washing. The organisms most frequently and most constantly found in the nose are non-pathogenic coryne bacteria. The nose is also the natural home of *Staph. aureus* and this organism can be isolated from about 50% of nasal swabs taken from normal persons. The occasional *Staph. aureus* on the skin is usually secondary to nasal carriage. About the throat, viridians streptococci, *N. catarrhalis* and staphylococci commonly occur while virulent strains of pneumococci are not infrequent. The deeper portions of the respiratory tract, the finer bronchioles and the alveoli of the lung are normally sterile. The mouth has a very mixed bacterial flora, the abundant moisture and the constant presence of small food particles providing an ideal environment for bacterial growth. Both aerobic and anaerobic types are found. The most important of the aerobic organisms are non-haemolytic streptococci and neisseriae, but miscellaneous fungi and actinomycetes are frequently present. Some of the last are believed to be of importance in the formation of dental tartar. Prior to the eruption of the teeth and in the edentulous adult, the flora of the mouth consists almost exclusively of aerobic organisms. Following the eruption of the teeth