WEEK-1-2-INTRO-TO-MICROBIO-PARASITOLOGY PDF

BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) 1. Nature and Scope of Microbiology and Parasitology History & Development of Microbiology Science is the study of nature that proceeds by posing questions a...

BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) 1. Nature and Scope of Microbiology and Parasitology History & Development of Microbiology Science is the study of nature that proceeds by posing questions about observations. Many early written records show that people have always asked questions. For example, the Greek physician Hippocrates 460-377 B.C.) wondered whether there is a link between environment and disease, and the Greek historian Thucydides (460–404 B.C.) questioned why he and other survivors of the plague could have close contact with victims and not fall ill again. Aristotle (384–322 BC) was one of the earliest recorded scholars to articulate the theory of spontaneous generation, the notion that life can arise from nonliving matter. Aristotle proposed that life arose from nonliving material if the material contained pneuma (“vital heat”). For many centuries, the answers to these and other fundamental questions about the nature of life remained largely unanswered. But about 350 years ago, the invention of the microscope began to provide some clues. The discovery of the microbial world is much attributed to Antonie van Leeuwenhoek (1677) who observed and described single-celled organisms, which he originally referred to as animalcules with his handcrafted microscopes. However, the different activities and functions of these organisms were identified after approximately 200 years later while performing fermentations, understanding diseases in humans and animals and in agriculture. Leeuwenhoek first reported the existence of most types of microorganisms in the late 1600s. Though Leeuwenhoek is regarded as the Father of Microbiology, microbiology did not develop significantly as a field of study for almost two centuries. There were a number of reasons for this delay. First, Leeuwenhoek was a suspicious and secretive man. Though he built over 400 microscopes, he never trained an apprentice, and he never sold or gave away a microscope. In fact, he never let anyone—not his family or such distinguished visitors as the czar of Russia—so much as peek through his very best instruments. When Leeuwenhoek died, the secret of creating superior microscopes was lost. It took almost 100 years for scientists to make microscopes of equivalent quality. Another reason that microbiology was slow to develop as a science is that scientists in the 1700s considered microbes to be curiosities of nature and insignificant to human affairs. But in the late 1800s, scientists began to adopt a new philosophy, one that demanded experimental evidence rather than mere acceptance of traditional knowledge. This fresh philosophical foundation, accompanied by improved microscopes, new laboratory techniques, and a drive to answer a series of pivotal questions, propelled microbiology to the forefront as a scientific discipline. Incidentally the term microbe was given by Prof. Charles E. Sedillot (1804– 1833) who is one of the pioneers of modern medicine, surgery, anesthesiology, histopathology and etiology. Sedillot understood the existence and action of microorganisms which he termed as microbes while studying the development of post-operative infections. Louis Pasteur demonstrated that there are specific activities of yeasts and bacteria, which are responsible for specific fermentations which he published in papers between 1857 and 1860. He was able to demonstrate the development of wine diseases and role of pasteurization to preserve wine storage. Martinus W. Beijerinck was one of the great general microbiologists who made fundamental contributions to microbial ecology by highlighting microbial association with plants for fixing the atmospheric nitrogen. He isolated the aerobic nitrogen fixing microorganism Azotobacter as well as root nodule organism Rhizobium. The first microbe (prokaryote) evolved around 3.6 billion years ago and since then has undergone a process of evolution by exploiting a vast range of energy sources and thriving in different habitats which existed during the course of evolution. All the basic biochemical processes of the life evolved and developed from their microbial ancestors. Some Contributors in Microbiology & Parasitology Antonie Philips van Leeuwenhoek (16761) He was the first scientist who observed bacteria and other microorganisms, using a single-lens microscope constructed by him and he named diose small organisms as “animalcules”. 1 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) Animalcule is an old term for microscopic organisms that included bacteria, protozoans, and very small animals. Edward Jenner 1796, developed the first vaccine of the world, the smallpox vaccine. He used the cowpox virus (Variolae vaccinae) to immunize children against smallpox from which the term 'vaccine' has been derived. Microbiology developed as a scientific discipline from the era of Louis Pasteur (1822- 1895). He is also known as Father of Modern Microbiology and Father of Bacteriology (together with Koch). His contributions in microbiology are as follows: o He had proposed die principles of fermentation for preservation of food. o He introduced the sterilization techniques and developed steam sterilizer, hot air oven and autoclave. o He described the method of pasteurization of milk. o He had also contributed for the vaccine development against several diseases, such as anthrax, fowl cholera and rabies. o He disproved the theory of spontaneous generation of disease and postulated the “germ theory of disease”, stating that disease cannot be caused by bad air vapor but it is produced by the microorganisms present in it. Joseph Lister (1867) is considered to be the father of antiseptic surgery. Infections were greatly reduced by using disinfectants during surgery, sterilizing instruments and cleaning the wounds. Robert Koch provided remarkable contributions in the field of microbiology. His contributions are as follows: o He introduced solid media for culture of bacteria, o He also introduced methods for isolation of bacteria in pure culture. o He discovered bacteria such as die anthrax bacilli, tubercle bacilli and cholera bacilli. o He introduced staining techniques by using aniline dye. o Koch's phenomenon: Robert Koch observed that guinea pigs already infected with tubercle bacillus developed a hypersensitivity reaction when injected with tubercle bacilli or its protein. Since then, this observation was called as Koch's phenomenon. o Koch's postulates: Robert Koch had postulated that a microorganism can be accepted as the causative agent of an infectious disease only in certain conditions (This will be discussed under “infections” during the finals.) Paul Ehrlich (1854- 1915) was a German scientist and is also known as father of chemotherapy. His contributions are as follows: o He was the first to report the acid-fast nature of tubercle bacillus. o He developed techniques of stain tissues and blood cells. o He proposed a toxin-antitoxin interaction called Ehrlich phenomenon and also introduced methods of standardizing toxin and antitoxin. o The bacteria' Escherichia' was named after him. Chemotherapy may involve drugs that target cancerous cells or tissues, or it may involve antimicrobial drugs that target infectious microorganisms. Antimicrobial drugs typically work by destroying or interfering with microbial structures and enzymes, either killing microbial cells or inhibiting their growth. Hans Christian Gram (in 1884): He developed a method of staining bacteria which was named as 'Gram slain' to make them more visible and differentiable under a microscope. Charles Chamberland: He is one of Pasteur's associates, constructed a porcelain bacterial filler in 1884 by which the discovery of viruses and their role in disease was made possible. Ernst Ruska: He was the founder of electron microscope (1931). Alexander Fleming (in 1929): He discovered the most commonly used antibiotic substance of the last century, i.e. penicillin. Elie Melchnikoff: He described phagocytosis and termed phagocytes. Waller Gilbert and Frederick Sanger were the first to develop (1977) the method of DNA sequencing. Karry B Mullis: Discovered polymerase chain reaction (PCR) and was awarded Noble prize in 1993. 2 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) Microbes Microbes or microorganisms are too small to be seen by the naked eye. They are considered the common ancestors of all organisms, which not only grow everywhere but are present in abundance. Microorganisms correspond to the richest collection of molecular and chemical diversity. They drive the ecosystem processes by maintaining the nutrient cycles as well as maintain elegant relationships between themselves and higher organisms. Microbial diversity is a great resource of biotechnological exploration of novel microorganisms and their products for exploitation in different processes. There is a wide range of microbes present in our biosphere depending on their physical and other characteristics. Microbes fall into two groups, prokaryotes and eukaryotes, depending upon whether they have nucleus or not. Prokaryotes lack this membrane around their genetic material, and this group includes viruses, bacteria, and related archaea. The other category of microbes includes algae, fungi, protists, and other microscopic animals, having cell nucleus. Microbiology is the study of all living organisms that are too small to be visible with the naked eye. Microbiology as the study of microbes can be differentiated as follows: o Immunology: the study of the immune system o Bacteriology: the study of bacteria o Virology: the study of viruses o Mycology: the study of fungi o Parasitology: the study of parasites; it has two sub branches ✔ Protozoology: the study of protozoa (single-celled eukaryotes) ✔ Helminthology: the study of helminths (worms, multi-celled eukaryotes) Realm of Microbial Existence Microbial diversity from different environments has been studied using culture-dependent and culture-independent methods. 1. Terrestrial Communities A community of microbes and their environment that occurs on the landmasses of continents and islands form a terrestrial microenvironment. Terrestrial microenvironment is distinguished from the aquatic microenvironment ecosystems by the lower availability of water and the consequent importance of water as a limiting factor. Rain forests are the most diverse and productive terrestrial microenvironment, but their soil is nutrient deficient due to extensive leaching by rainwater. 2. Aquatic Communities Aquatic microenvironments occupy more than 70 % of the earth’s surface including mostly ocean but also others such as estuaries, harbors, river, lakes, wetlands, streams, springs, aquifers, etc. The microbiota, living in aquatic environment, are the primary producers (responsible for approximately half of all primary production on earth) and primary consumers as well. Aquatic microenvironment is further classified into three microenvironments, occupied by microbes living in freshwater, brackish water, and marine water. 3. Extremophile Communities The organisms living in physically or geochemically extreme conditions that are detrimental to most life on earth are termed as extremophiles. Most of the extremophiles are microbes and belong to the domain Archaea. They thrive in extreme hot niches, high pressure, ice, and salt solutions, as well as 3 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) acid and alkaline conditions; some may grow in toxic waste, organic solvents, heavy metals, or in several other habitats that were previously considered inhospitable for life. NOTE: The atmosphere is a habitat for micro-organisms, and not purely a conduit for terrestrial and aquatic life. However, air can be considered one of the least hospitable environments for microbes because it holds fewer nutrients and thus supports relatively fewer organisms. Associations of Microorganisms with other Organisms 1. Neutral Associations Neutralism or neutral association between microbes refers to the occupation of two different species of microbes in the same environment without affecting each other. This type of association is generally transitory in nature, meaning, as conditions in the environment change, like nutrient availability, their might be a change in relationship. However, it has been suggested that true neutralism is probably rare in nature. Example: Some bacteria may exhibit neutralism as it has been reported that some species of Lactobacillus and Streptococcus can coexist without affecting each other positively or negatively. 2. Positive Associations Positive associations comprise of mutualism, syntrophism, proto-cooperation and commensalism. Mutualism is essentially a relationship in which each organism is benefitted from the association. Syntrophism is a mutualistic association which involves the exchange of nutrition between two species. Commensalism refers to a relationship between organisms in which one species of a pair benefits whereas the other is not affected. Examples: Mutualism: In a mutualism, both partners benefit from the relationship. Many coral reefs have "cleaning stations" where some species of fish remove parasites from other fish. The cleaner fish get nutrition from the consumed parasites while the cleaned fish enjoy freedom from their parasites. Syntropism: Methanogenic bacteria have a syntrophic relationship with protozoans living in the guts of termites. The protozoans break down cellulose, releasing H2, which is then used in methanogenesis. 3. Negative Associations The negative associations comprise of antagonism, competition, parasitism, ammensalism and predation. Antagonistic relationship between microorganisms generally results in inhibition or adversely affects the growth and survival of other species. This is generally mediated by signal molecules which induce the inhibition or adverse effects and have been referred to as antibiotics, and this phenomenon is known as antibiosis. This interaction has a great importance in the discovery and development of a variety of antimicrobial drugs. Competition refers to the interaction between microorganisms for limited nutrients and space. In this interaction, a microorganism which out competes or eliminates other for the limited resource in lesser time is found to predominate. Microorganisms exhibiting adaptability and faster growth rate are better competitors. A relationship in which one organism lives in or on other organism is referred to as parasitism. The parasite lives in intimate physical contact with the host and forms metabolic association with the host. Examples: Parasitism: A parasite is physiologically dependent upon its host for nutrition. While the host is negatively affected by the loss of nutrients to the parasite, parasitism rarely leads directly to the 4 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) host's death. Unfortunately, humans are hosts to any number of parasites, including liver flukes, tapeworms, lice, pinworms, giardia, and many others. Amensalism: Amensalism takes place when one individual is negatively affected by interaction with another individual who is not affected by the relationship. Many molds, including Penicillium, secrete chemicals that kill bacteria in their vicinity. The phenomenon of parasitism is studied by a special branch under microbiology, called parasitology. Parasites are pathogens that lives on or in a host organism and gets its food from or at the expense of its host. While the diseases caused by viruses and bacteria are called infection, the diseases caused by animals, protozoans and fungi are called invasional or parasitogenic. There are four kinds of microorganisms that can cause disease namely, bacteria, fungi, parasites and viruses. What Gives Rise to Microorganisms? Many philosophers and scientists of past ages thought that living things arose via three processes: through asexual reproduction, through sexual reproduction, or from nonliving matter. The appearance of shrimp and toads in the mud of what so recently was a dry lakebed was seen as an example of the third process, which came to be known as abiogenesis, or spontaneous generation. The theory of spontaneous generation as promulgated by Aristotle (384–322 BC) was widely accepted for over 2000 years because it seemed to explain a variety of commonly observed phenomena, such as the appearance of maggots on spoiling meat. However, the validity of the theory came under challenge in the 17th century. 1. Redi’s Experiments In the late 1600s, the Italian physician Francesco Redi (1626– 1697) demonstrated by a series of experiments that when decaying meat was kept isolated from flies, maggots never developed, whereas meat exposed to flies was soon infested with maggots. As a result of experiments such as these, scientists began to doubt Aristotle’s theory and adopt the view that animals come only from other animals. 2. Needham’s Experiments The debate over spontaneous generation was rekindled when Leeuwenhoek discovered microbes and showed that they appeared after a few days in freshly collected rainwater. Though scientists agreed that larger animals could not arise spontaneously, they disagreed about Leeuwenhoek’s “wee animalcules”; surely, they did not have parents, did they? They must arise spontaneously. The proponents of spontaneous generation pointed to the careful demonstrations of British investigator John T. Needham (1713–1781). He boiled beef gravy and infusions of plant material in vials, which he then tightly sealed with corks. Some days later, Needham observed that the 5 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) vials were cloudy, and examination revealed an abundance of “microscopical animals of most dimensions.” As he explained it, there must be a “life force” that causes inanimate matter to spontaneously come to life because he had heated the vials sufficiently to kill everything. Needham’s experiments so impressed the Royal Society that they elected him a member. 3. Spallanzani’s Experiments Then, in 1799, the Italian Catholic priest and scientist Lazzaro Spallanzani (1729–1799) reported results that contradicted Needham’s findings. Spallanzani boiled infusions for almost an hour and sealed the vials by melting their slender necks closed. His infusions remained clear unless he broke the seal and exposed the infusion to air, after which they became cloudy with microorganisms. He concluded three things: o Needham either had failed to heat his vials sufficiently to kill all microbes or had not sealed them tightly enough. o Microorganisms exist in the air and can contaminate experiments. o Spontaneous generation of microorganisms does not occur; all living things arise from other living things. Although Spallanzani’s experiments would appear to have settled the controversy once and for all, it proved difficult to dethrone a theory that had held sway for 2000 years, especially when so notable a man as Aristotle had propounded it. One of the criticisms of Spallanzani’s work was that his sealed vials did not allow enough air for organisms to thrive; another objection was that his prolonged heating destroyed the “life force.” The debate continued until the French chemist Louis Pasteur conducted experiments that finally laid the theory of spontaneous generation to rest. 4. Pasteur’s Experiments Louis Pasteur (1822–1895) was an indefatigable worker who pushed himself as hard as he pushed others. Pasteur’s determination and hard work are apparent in his investigations of spontaneous generation. Like Spallanzani, he boiled infusions long enough to kill everything. But instead of sealing the flasks, he bent their necks into an S-shape, which allowed air to enter while preventing the introduction of dust and microbes into the broth. 6 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) Crowded for space and lacking funds, he improvised an incubator in the opening under a staircase. Day after day, he crawled on hands and knees into this incommodious space and examined his flasks for the cloudiness that would indicate the presence of living organisms. In 1861, he reported that his “swan- necked flasks” remained free of microbes even 18 months later. Because the flasks contained all the nutrients (including air) known to be required by living things, he concluded, “Never will spontaneous generation recover from the mortal blow of this simple experiment.” Pasteur followed this experiment with demonstrations that microbes in the air were the “parents” of Needham’s microorganisms. He broke the necks off some flasks, exposing the liquid in them directly to the air, and he carefully tilted others so that the liquid touched the dust that had accumulated in their necks. The next day, all of these flasks were cloudy with microbes. He concluded that the microbes in the liquid were the progeny of microbes that had been on the dust particles in the air. Common Laboratory Tools, Reagents and Procedures Used in Microbiology and Parasitology Tools 1. An analytical balance is a type of balance that is commonly used for the measurement of mass in the sub-milligram range. 7 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) 2. An autoclave is a pressurized chamber used for the process of sterilization and disinfection by combining three factors: time, pressure, and steam. 3. Bunsen burner is a standard tool used in laboratories, named after Robert Bunsen. It is a gas-fueled single open flame. 4. A centrifuge is a device that allows the rotation of an object about a single axis, where an outward force is applied perpendicularly to the axis. A laboratory centrifuge is motor-based and allows the rotation of a liquid sample resulting in the separation of the components of the mixture. 5. A colony counter is used to estimate the density of a liquid culture by counting the number of CFU (colony forming units) on an agar or culture plates. 6. Deep freezers are based on the principle that under extremely low temperatures, there is minimum microbial growth which allows for the protection and preservation of different substances. Based on this principle, we can even preserve cultures over a long period of time without any change in the concentration of the microorganism. 8 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) 7. Homogenizer is a device used in laboratories for the mixing of various liquids and materials like tissue, plant, food, soil, and many others. 8. A hot plate is a stand-alone appliance used in microbiology laboratories as a tabletop heating system. In a laboratory, hot plates are used to heat glassware and its components. They are used over water baths as water baths might be hazardous in case of any spills or overheating. 9. A hot air oven is an electrical device that is used for sterilization of medical equipment or samples using dry heat. A hot air oven can be used to sterilize materials like glassware, metal equipment, powders, etc. It allows for the destruction of microorganisms as well as bacterial spores. 10. Magnetic Stirrer is a device commonly used in microbiology laboratories for the purpose of mixing liquids. It is usually used for mixing various liquid components in a mixture in a chemical or microbiology laboratory. This device is used in place of other stirrers as it is noise-free and because the size of the stir bar is so tiny, there is less chance of contamination. 11. Microscopes are devices that allow the observer to have an exceedingly close view of minute particles. 12. Glass slides and cover slips are where specimen are mounted and secured for viewing under the microscope. 9 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) 13. Petri dish is a shallow transparent lidded dish that biologists use to hold growth medium in which cells can be cultured like bacteria and fungus. 14. A pH meter is a device used in laboratories that measure the H-ion concentration in water-based solutions to determine the acidity or alkalinity of the solution. A pH meter is often termed a “potentiometric pH meter” as it measures the difference in electric potential between the reference and a pH electrode. 15. The spectrophotometer is an optical instrument for measuring the intensity of light in relation to wavelength. In a microbiology laboratory, a spectrophotometer is applied for the measurement of the substance concentration of protein, nucleic acids, bacterial growth, and enzymatic reactions. 16. Water Bath is a conventional device that is used for chemical reactions that required a controlled environment at a constant temperature. Water baths are primarily used for heating samples under a controlled temperature. These are suitable for heating chemicals that might be flammable under direct ignition. Procedures and Techniques Used in Microbiology Microorganisms are small. This may seem an obvious statement, but it is one that should not be taken for granted. Exactly how small are they? How can we measure the width and length of microbes? Typically, a unit of measurement is smaller than the object being measured. For example, we measure a person’s height in feet or inches, not in miles. So that they can work with units that are simpler and in standard use the world over, scientists use metric units of measurement. Unlike the English system, the metric system is a decimal system, so each unit is one-tenth the size of the next largest unit. Even extremely small metric units are much easier to use than the fractions involved in the English system. The unit of length in the metric system is the meter (m), which is slightly longer than a yard. One-tenth of a meter is a decimeter (dm), and one-hundredth of a meter is a 10 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) centimeter (cm), which is equivalent to about a third of an inch. One-tenth of a centimeter is a millimeter (mm), which is the thickness of a dime. A millimeter is still too large to measure the size of most microorganisms, but in the metric system we continue to divide by multiples of 10 until we have a unit appropriate for use. Thus, one-thousandth of a millimeter is a micrometer (mm), which is small enough to be useful in measuring the size of cells. One thousandth of a micrometer is a nanometer (nm), a unit used to measure the smallest cellular organelles and viruses. 1. Microscopy Microscopy refers to the use of light or electrons to magnify objects. The science of microbiology began when Antoni van Leeuwenhoek (lā′ven-hŭk, 1632–1723) used primitive microscopes to observe and report the existence of microorganisms. Since that time, scientists and engineers have developed a variety of light and electron microscopes. General Principles in Microscopy Both light and electron microscopy include the wavelength of radiation, the magnification of an image, the resolving power of the instrument, and contrast in the specimen. a. Wavelength of Radiation Visible light is one part of a spectrum of electromagnetic radiation that includes X rays, microwaves, and radio waves. Note that beams of radiation may be referred to as either rays or waves. These various forms of radiation differ in wavelength—the distance between two corresponding parts of a wave. The human eye discriminates among different wavelengths of visible light and sends patterns of nerve impulses to the brain, which interprets the impulses as different colors. For example, we see wavelengths of 400 nm as violet and of 650 nm as red. White light, composed of many colors (wavelengths), has an average wavelength of 550 nm. Electrons are negatively charged particles that orbit the nuclei of atoms. Besides being particulate, moving electrons also act as waves, with wavelengths dependent on the voltage of an electron beam. For example, the wavelength of electrons at 10,000 volts (V) is 0.01 nm; that of electrons at 1,000,000 V is 0.001 nm. As we will see, using radiation of smaller wavelengths results in enhanced microscopy. b. Magnification Magnification is an apparent increase in the size of an object. It is indicated by a number and “*, which is read “times.” For example, 16,000* is 16,000 times. Magnification results when a beam of radiation refracts (bends) as it passes through a lens. Curved glass lenses refract light, and magnetic fields act as lenses to refract electron beams. Let us consider the magnifying power of a glass lens that is convex on both sides. A lens refracts light because the lens is optically dense compared to the surrounding medium (such as air); that is, light travels more slowly through the lens than through air. The leading edge of a light beam slows as it enters glass, and the beam bends (FIGURE 4.2a). Light also bends as it leaves the glass and reenters the air. Because of its curvature, a lens refracts light rays that pass through its periphery more than light rays that pass 11 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) through its center, so that the lens focuses light rays on a focal point. Importantly for the purpose of microscopy, light rays spread apart as they travel past the focal point and produce an enlarged, inverted image. The degree to which the image is enlarged depends on the thickness of the lens, its curvature, and the speed of light through its substance. Microscopists could combine lenses to obtain an image magnified millions of times, but the image would be so faint and blurry that it would be useless. Such magnification is said to be empty magnification. The properties that determine the clarity of an image, which in turn determines the useful magnification of a microscope, are resolution and contrast. Total Magnification To figure the total magnification of an image that you are viewing through the microscope is really quite simple. To get the total magnification take the power of the objective lens (e.g. 10x, 40x, 100x) and multiply by the power of the eyepiece or ocular lens (e.g. 5x, 10x, 15x). 12 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) c. Resolution Resolution, also called resolving power, is the ability to distinguish two points that are close together. An optometrist’s eye chart is a test of resolution at a distance of 20 feet (6.1 m). Leeuwenhoek’s microscopes had a resolving power of about 1 mm; that is, he could distinguish objects if they were more than about 1 mm apart, whereas objects closer together than 1 mm appeared as a single object. The better the resolution, the better the ability to distinguish two objects that are close to one another. Modern microscopes have fivefold better resolution than Leeuwenhoek’s; they can distinguish objects as close together as 0.2 mm. FIGURE 4.3 illustrates the size of various objects that can be resolved by the unaided human eye and by various types of microscopes. Why do modern microscopes have better resolution than Leeuwenhoek’s microscopes? A principle of microscopy is that resolution is dependent on (1) the wavelength of the electromagnetic radiation and (2) the numerical aperture of the lens, which refers to the ability of a lens to gather light. Resolution may be calculated using the following formula: The resolution of today’s microscopes is greater than that of Leeuwenhoek’s microscopes because modern microscopes use shorter wavelength radiation, such as blue light or electron beams, and because they have lenses with larger numerical apertures. d. Contrast Contrast refers to differences in intensity between two objects or between an object and its background. Contrast is important in determining resolution. For example, although you can easily distinguish two golf balls lying side by side on a putting green 15 m away, at that distance it is much more difficult to distinguish them if they are lying on a white towel. Most microorganisms are colorless and have very little contrast whether one uses light or electrons. One way to increase the contrast between microorganisms and their background is to stain them. Stains and staining techniques are covered later in the chapter. As we will see, the use of light that is in phase—that is, in which all of the waves’ crests and troughs are aligned—can also enhance contrast. 13 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) 2. Slide Preparation Techniques The main methods of placing samples onto microscope slides are wet mount, dry mount, smear, squash and staining. a. Mounting A ‘mount' is just the way during which a specimen is placed on the slide. It also involves securing the specimen on the slide prior to focusing it under the microscope. Mounting is a process of suspending a specimen in a medium for observation or preservation of specimen. Water, PBS, glycerin/ DPX etc. are common mounting media. It is useful for both temporary and permanent slide preparations. 1) Dry Mount The dry mount is the most basic technique: simply position a thinly sliced section on the center of the slide and place a cover slip over the sample. Dry mounts are ideal for observing hair, feathers, airborne particles such as pollens and dust as well as dead matter such as insect and aphid legs or antennae. Opaque specimens require very fine slices for adequate illumination. Since they are used for primarily inorganic and dead matter, dry mounts can theoretically last indefinitely. 14 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) 2) Wet Mount Used for aquatic samples, living organisms and natural observations, wet mounts suspend specimens in fluids such as water, brine, glycerin and immersion oil. A wet mount requires a liquid, tweezers, pipette and paper towels. Although wet mounts can be used to prepare a significantly wide range of microscope slides, they provide a transitory window as the liquid will dehydrate and living specimens will die. Organisms such as protozoa may only live 30 minutes under a wet mount slide; applying petroleum jelly to the outer edges of the cover slip creates a seal that may extend the life of the slide up to a few days. In addition, larger protozoa such as paramecium may be too large and/or move too quickly under the wet mount. In these circumstances, adding ground pieces of cover glass to the water before the slip layer will create added space and chemicals or strands of cotton can be added to slow the movement of paramecium, amoeba and ciliates. or 15 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) Techniques: Smear Slides Smear slides require two or more flat, plain slides, cover slips, pipette and tissue paper. Smear slides are generally used for analysis of blood cells, seminal fluids, tissue culture, throat swabs and differential staining of bacteria. Squash Slides: 16 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) Squash preparations are prepared by placing a tiny (1-2mm) fragment of tissue onto a glass slide, placing another glass slide over it, pressing the slides together, squashing the tissue between them, then sliding the 2 slides past each other, dragging squashed tissue across each slide. Squash preparations are widely used to study cell division, polyteny or polyploidy. Both plant and animal tissues are used such as root tips, blood cells, grasshopper testes and salivary glands of Drosophila larva. In Experiment 4 and 5 squashes of onion root tips and buds will be prepared to study the stages of mitosis and meiosis, respectively. b. Staining Most microorganisms are colorless and difficult to view with bright-field microscopes. Microscopists use stains to make microorganisms and their parts more visible because stains increase contrast between structures and between a specimen and its background. Electron microscopy requires that specimens be treated with stains or coatings to enhance contrast. Staining solutions such as iodine, methylene blue and crystal violet can be added to 17 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) Stains are especially useful in the fields of histology, virology and pathology, allowing researchers to study and diagnose diseases, identify gram positive and negative bacteria as well as examine detailed attributes of a variety of cells. 3. Aseptic Techniques Aseptic technique is a fundamental and important laboratory skill in the field of microbiology. Aseptic technique is a set of routine measures that are taken to prevent cultures, sterile media stocks, and other solutions from being contaminated by unwanted microorganisms (i.e., sepsis). While such actions are sometimes called “sterile technique,” that terminology is appropriate only in reference to preventing the introduction of any organisms to the laboratory or medical equipment and reagents. Since the goal of a biologist is to grow microorganisms or eukaryotic cells without the introduction of extraneous organisms, aseptic techniques are crucial for accurate and meaningful experimentation. One should always remember that a completely sterile working environment does not exist. However, there are a number of simple, common sense procedures that will reduce the risk of culture contaminations. The aseptic techniques control the opportunities for contamination of cultures by microorganisms from the environment, or contamination of the environment by the microorganisms being handled. Examples of aseptic technique are: 1. Cleaning and disinfecting lab surfaces prior to use. 2. Limiting the duration that cultures or media are uncapped and exposed to the air. 3. Keeping petri dishes closed whenever possible. 4. Effectively sterilizing inoculating loops and other equipment that comes into contact with cultures or media, and 5. Avoiding breathing on cultures or sterile instruments. 18 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph  BSEd (Sci) SESE 116: Microbiology and Parasitology Course Content (Lecture Notes) There are some general rules to follow for any aseptic technique. 1. Close windows and doors to reduce drying and avoid contamination from air-borne microorganisms. 2. Make transfers over a disinfected surface. Ethanol disinfection is recommended because of its rapid action. If the bench surface is difficult to clean, cover the bench with a sheet of tough material, which is more easily disinfected. 3. Start the operations only when all apparatus and materials are within immediate reach. 4. Complete all operations as quickly as possible, but without any hurry. 5. Vessels must be open for the minimum amount of time possible. 6. While vessels are open, all work must be done close to a Bunsen burner flame where air currents are drawn upwards. 7. On opening a test tube or bottle, the neck must be immediately warmed by flaming (see below) with the vessel held as near to horizontal as possible and so that any movement of air is outwards from the vessel. 8. During manipulations involving a Petri dish, limit exposure of the sterile inner surfaces to contamination from the air. 9. The parts of sterile pipettes which will be put into cultures or sterile vessels must not be touched or allowed to come into contact with other non-sterile surfaces, such as clothing, the surface of the working area, or the outside of bottles/ test tubes. 10. All items which come into contact with microorganisms must be sterilized before and after each such exposure. This could be either by the technical team preparing for and clearing up after a piece of practical work (for example, in the case of glassware to be used), or by the worker during the course of the practical (for example, in flaming a wire loop). 2 Worksheets/Laboratory or Learning Activities 1 SUMMATIVE TEST End of Week 1 - 3 19 |Prepared by MMANALANG mmanalang@dmmmsu.edu.ph