Chapter 1-7 (MicroPara) PDF

CHAPTER The Science 1 of Microbiology LEARNING OBJECTIVES At the end of this chapter, the student should be able to: 1. define Microbiology and give the importance of the study of Microbiology; 2. name important persons wit...

CHAPTER The Science 1 of Microbiology LEARNING OBJECTIVES At the end of this chapter, the student should be able to: 1. define Microbiology and give the importance of the study of Microbiology; 2. name important persons with significant contributions to the field of Microbiology; 3. differentiate the various types of microscopes and their uses; 4. compare the various staining methods used to visualize microorganisms; and 5. classify the different types of culture media based on their physical state, chemical composition, and functional type. Microbiology is derived from the Greek words mikros (“small”), bios (“life”), and logia or logos (“study of”). It is therefore the study of organisms that are so small they cannot be seen with the naked eye. These organisms are called microorganisms or microbes and are categorized into two: (1) cellular, which may either be prokaryotes (bacteria, cyanobacteria, and archeans) or eukaryotes (fungi, protozoa, and algae); and (2) acellular, which includes viruses. Microbiology is further classified into different fields of study, namely: (1) bacteriology, the study of bacteria; (2) virology, the study of viruses; (3) mycology, the study of fungi; (4) parasitology, the study of protozoa and parasitic worms; (5) phycology, the study of algae; and, (6) immunology, the study of the immune system and the immune response. Why study microbiology? The study of microbiology is important for the following reasons: 1. Microbiology has an impact in the daily lives of humans. Microorganisms are everywhere—in the air one breathes, in the environment, and even in one’s body. About a thousand or more organisms inhabit the human body. These are collectively called normal flora or indigenous flora which only produce disease in persons with compromised immune systems 4 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences 2. Some microorganisms are essential in biotechnologyand a wide range of industries which include food and beverage, pharmaceuticals, mining, genetics, and many more. Much of the knowledge available in the study of genetics and biochemistry utilize microorganisms as model organisms. 3. Some microorganisms, especially bacteria and fungi, are important sources of antimicrobial agents. For example, penicillin was derived from the fungus Penicillium. 4. Some microorganisms act as saprophytes or decomposers of waste products and dead organisms, making them essential in maintaining a balanced ecosystem. 5. The study of microorganisms has led to a better understanding of how microorganisms produce disease, paving the way to better disease management and control. This was further improved through the discovery of vaccines that helped prevent sickness from infections diseases. By knowing the sources of disease producing microbes, sanitation practices improved immensely, leading to better mitigation of infectious diseases. 6. Certain diseases which were thought to have been eradicated are now re emerging. Some have the potential as biological warfare agents. At the same time, there are now a number of pathogens that are developing resistance to antibiotics. In this context, the study of microbiology is relevant for better understanding of the negative instances in which science can be used. Evolution of Microbiology Archaeologists and evolutionists have uncovered evidence demonstrating the existence of primitive microorganisms. In Western Australia, as many as eleven different types of fossils of primitive microorganisms have been found in ancient rock formations, dating back to as early as 3.5 billion years ago, long before the existence of animals and humans. Infectious diseases have existed for thousands of years. In 3180 BC, an epidemic known as the “plague” broke out in Egypt. In 1122 BC, an outbreak of a smallpox like disease that originated in China spread worldwide. The exhumed mummified remains of Rameses V showed skin lesions resembling smallpox. In the mid 1600s, the microscope was discovered and with the use of this instrument, Robert Hooke was able to discover the cell—the basic unit of living organisms. His discovery heralded the cell theory that stated living organisms are made up of cells. Then in the 1670s, Anton von Leeuwenhoek, a Dutch merchant, created a single lens microscope that he used to make observations of microorganisms which he then called animalcules. Through his observations, he became known as the “Father of Microbiology” and was the one who first provided accurate descriptions of bacteria, protozoa, and fungi  The Science of Microbiology In the middle and late 1800s, Louis Pasteur performed countless experiments that led to his germ theory of disease. He postulated that microorganisms were in the environment and could cause infectious diseases. He also developed the process of pasteurization, which kills microorganisms in different types of liquids, and which became the basis for aseptic techniques. He also introduced the terms aerobes and anaerobes and developed the fermentation process. Pasteur’s attempts to prove his germ theory of disease were unsuccessful. It took Robert Koch to prove that microorganisms caused certain diseases through a series of scientific steps which led to his formulation of the Koch’s postulates. This led to an increased effort by other scientists to prove and illustrate further the germ theory that was initially formulated by Louis Pasteur. Thus, the late 1800s and the first decade of the 1900s came to be known as the Golden Age ofMicrobiology. Since then, numerous scientists have made significant contributions to the field of Microbiology. Edward Jenner discovered the vaccine for smallpox. Joseph Lister applied the theory to medical procedures paving the way for the development of aseptic surgery. After World War II, antibiotics were introduced to the medical world. Paul Ehrlich discovered Salvarsan for the treatment of syphilis. This drug was heralded the “magic bullet” of chemotherapy, which is treatment of disease by using chemical substances. Alexander Fleming discovered the antibiotic penicillin from the mold Penicillium notatum. With the discovery of antibiotics, the incidence of infectious diseases like tuberculosis, pneumonia, meningitis, and others was significantly reduced. Most of the experiments conducted in the field of microbiology during the early 20th century involved the study of bacteria. During this time scientists were not yet equipped with advanced technology in their study of microorganisms. It was only in the 1930s when the electron microscope was developed that experimentations in microbiology became more complex. It was also during that time when viral culture was introduced paving the way for rapid discoveries on viruses. The vast knowledge gained from the experiments performed by microbiologists together with the discovery of other vaccines in the 1940s and 1950s have led to better prevention and control of numerous potentially fatal infectious diseases. Microscopy Microorganisms are miniscule organisms that cannot be seen with the naked eye. The discovery of the microscope has led to their close observation, allowing microbiologists and other scientists to study them further. A microscope is an optical instrument that can magnify organisms a hundredfold or even a thousand fold. From the time of its initial discovery in the 1600s, the microscope has undergone great revolutionary changes. Making it more advanced and complex throughout time. The following are the different types of microscopes that have evolved from von Leeuwenhoek’s simple prototype. 6 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Compound Microscope The compound microscope is a type of microscope that contains more than one magnifying lens. It can magnify objects approximately a thousand times their original size. Visible light is its main source of illumination. As such, it is also known as the compound light microscope. The compound microscope utilized today consists of two magnifying lens systems. The eyepiece (or ocular) contains what is called the ocular lens that has a magnifying power of 10x. The second lens system is located in the objective that is positioned directly above the organism to be viewed. Eyepiece (ocular lens) Head Diopter adjustment Locking screw Revolving nose piece Arm Objectives Stage Slide holder Coarse focus Condenser Fine focus Iris diaphragm Stage controls Built in light source Brightness adjustment On/off switch Base With built in light source Eyepiece (ocular lens) Body tube Coarse focus Fine focus Revolving nose piece Arm Objectives Stage clips Stage Condenser Iris diaphragm Mirror Base With mirror to direct an external light source Figure 1.1 Two compound light microscopes which differ in their light sourc  The Science of Microbiology 7 Table 1.1 Components of the compound light microscope Component Function Ocular lens or Topmost part of the microscope which is the lens the viewer looks eyepiece through to see the specimen. Revolving nose piece Located above the stage, it holds the objective lenses. Diopter adjustment It is used to change focus on one eyepiece in order to correct any difference in vision between the two eyes. Body tube or head It connects the eyepiece to the objective lenses. Arm It connects the body tube to the base of the microscope. Coarse adjustment It brings the specimen into general focus. Fine adjustment It fine tunes the focus and increases the details of the specimen. Objective lenses This is held in place above the stage by the revolving nosepiece and are the lenses that are closest to the specimen. It contains 3 to 5 objectives ranging in power from 4X to 100X. Stage Located beneath the revolving nosepiece, it is the flat platform on which the specimen is placed. Stage clips Situated above the stage, these are metal clips that hold the slide in place. Stage control Found beneath the stage, these knobs move the stage either left or right or forward and backward. Aperture The hole in the middle of the stage that allows light from the illuminator to reach the slide containing the specimen. On/off switch The switch located at the base of the microscope that turns the illuminator on or off. Illuminator The light source of the microscope. Iris diaphragm Found on the condenser, it is used to adjust the amount of light coming through the condenser. Condenser It is found beneath the stage and contains a lens system that focuses light onto the specimen. It gathers and focuses light onto the specimen. Base It supports the microscope and it is where the illuminator is found. Brightfield Microscope Made up of a series of lenses and utilizing visible light as its source of illumination, the brightfield microscope can magnify an object 1,000 to 1,500 times. This is used to visualize bacteria and fungi. Objects less than or thinner than 0.2 μm cannot be visualized by this type of microscope. The term “brightfield” is derived from the fact that the specimen appears dark against the surrounding bright viewer field of this microscope. However, it has very low contrast and most of the cells need to be stained to be properly viewed 8 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Darkfield Microscope This microscope utilizes reflected light instead of transmitted light, with a special condenser that has an opaque disc that blocks the light, such that only the specimen is illuminated. The specimen to be studied appears bright against a dark background. This type of microscope is ideal for studying specimens that are unstained or transparent and absorb little or no light. It is also useful in examining the external details of the specimen such as its outline or surface. This type of microscope is used to view spirochetes. Phase contrast Microscope Phase contrast microscopy is based on the principle that differences in refractive indices and light waves passing through transparent objects assume different phases. This type of microscopy was first introduced by Frits Zernike, a Dutch physicist, in 1934. The phase contrast microscope has a contrast enhancing optical technique in order to produce high contrast images of specimens that are transparent which include thin tissue slices, living cells in culture, and subcellular particles (such as nuclei and organelles). Image Plane Digital Camera Diffracted System Direct Light Transmitted (Surround) Observation Light Light Biological Microscope Objective Specimen Phase Plate Condenser Condenser Annulus Figure 1.2 Phase contrast microscope and its part  The Science of Microbiology 9 Living Cells in Brightfield and Phase Contrast Figure 1.3 a Appearance of cells under brightfield microscope where cells appear semi transparent. The only visible structures are the highly refractive regions, such as the membrane, nucleus, and unattached cells (rounded or spherical). b Same specimen viewed using phase contrast a b microscope showing significantly more structural detail. Differential Interference Contrast Microscope The differential interference contrast microscope is similar to the phase contrast microscope except that it utilizes two beams of light instead of one and therefore has higher resolution. The resulting contrasting colors of the specimen being studied are due to the prisms that split the light beam. It was developed by Georges Nomarski in 1952 as an improvement to the phase contrast microscope. It is useful in examining living specimens when normal biological processes might be inhibited by standard staining procedures. However, the three dimensional image of the specimen produced may not be accurate since the enhanced areas of light and shadow may distort the appearance of the image. Fluorescence Microscope The fluorescence microscope makes use of ultraviolet light and fluorescent dyes called fluorochromes. The specimen under study fluoresces or appears to shine against a dark background. Fluorescence microscopy is based on the principle that certain materials emit energy that is detectable as visible light when they are irradiated with the light of a given wavelength. It uses a higher intensity of light source and this in turn excites a fluorescent species. The fluorescent species then emits a lower energy light of a longer wavelength which produces the magnified image instead of the original light source. Fluorescence microscopy can be used to visualize structural components of small specimens such as cells and to detect the viability of cell populations. It may also be used to visualize the genetic material of the cell (DNA and RNA) 10 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Confocal Microscope Also known as the confocal laser scanning microscope (CLSM) or laser confocal scanning microscope (LCSM), the confocal microscope uses an optical imaging technique that increases optical resolution and contrast of the micrograph by using a spatial pin hole to block out of focus light in image formation. The specimen is stained with a fluorescent dye to make it emit or return light. The object is scanned with a laser into planes and regions. This is used, together with computers, to produce a three dimensional image. It is also useful in the study of cell physiology. Electron Microscope The electron microscope utilizes a beam of electrons to create an image of the specimen. The electron beams serve as the source of illumination and magnets are used to focus the beam. The first prototype of this microscope was built by the German Engineer Ernst Ruska in 1933, which had a resolution power of up to 50 nm. Modern electron microscopes are capable of magnifying objects up to 2 million times. It is used to visualize viruses and subcellular structures of the cell. There are two types of electron microscopes—transmissionelectron microscope and scanning electron microscope. The transmission electron microscope (TEM) is the original form of the electron microscope. It produces two dimensional, black and white images, and magnifies objects up to 200,000 times. The scanning electron microscope (SEM) relies on interactions at the surface rather than transmission. It can magnify bulk samples with greater depth of view so that the image produced represents the 3 D structure of the sample, but the image is still only black and white. Generally, it can magnify the object 10,000 times. Scanning Probe Microscope The scanning probe microscope was developed in the 1980s by the Swiss scientists Dr. Gerd Binnig and Dr. Heinrich Rohrer. It is used to study the molecular and atomic shapes of organisms on a nanoscale. A physical probe is used to scan back and forth over the surface of a sample. A computer then gathers data that are used to generate an image of the surface. It can also be used to determine the variations in temperature inside the cell as well as its chemical properties. Staining Most microorganisms besides being very tiny are also devoid of any color and are thus difficult to see, even with the use of the microscope. To facilitate visualization, staining procedures have been developed by various scientists. These staining procedures are meant to give color to the organisms, making them easier to see under the microscope  The Science of Microbiology 11 Simple Stains Simple stains make use of a single dye which can either be aqueous (water based) or alcohol based. This method of staining is a quick and easy way to visualize cell shape, size, and arrangement of bacteria. It uses basic dyes such as safranin, methylene blue, or crystal violet. These stains give up or accept hydrogen ion, leaving the stain positively charged. Most bacterial cells and cytoplasm are negatively charged and since the dye is positively charged, it adheres readily to the cell surface enabling the visualization of bacterial cell morphology. a b Figure 1.4 a Cocci in clusters and b Bacilli Differential Stains Differential stains are used to differentiate one group of bacteria from another. There are two types of differential staining procedures commonly used, namely: 1. Gram stain – distinguishes gram positive bacteria from gram negative bacteria. gram positive bacteria stain blue or purple, while gram negative bacteria stain red or pink. As a general rule, all cocci are gram positive except Neisseria, Veilonella, and Branhamella. On the other hand, all bacilli are gram negative except Corynebacterium, Clostridium, Bacillus, and Mycobacterium. Table 1.2 Reagents used in Gram staining and expected results Reagent Function Result if Gram positive Result if Gram negative Crystal violet Primary stain Purple or blue Purple or blue Gram’s iodine Mordant* Purple or blue Purple or blue Acetone or 95% Decolorizer Purple or blue Colorless alcohol Safranin Counterstain or Purple or blue Red or pin secondary stain *A mordant enhances the uptake of the primary stain. 12 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences 2. Acid fast stain – stain used for bacteria with high lipid content in their cell wall, hence cannot be stained using Gram stain. Two methods are used, namely: a. Ziehl Neelsen stain – also known as the “hot method” because it requires steam bathing the prepared smear after addition of the primary dye. This is because the primary stain used is aqueous and will not bind to the cell wall of the organism. Acid fast organisms will appear red on a blue background. b. Kinyoun stain – also known as the “cold method” as it does not utilize heat after addition of the primary stain, which is oil based. The acid fast organisms will appear red on a green background. Table 1.3 Reagents used in acid fast staining and the expected results Reagent Result Function Ziehl Neelsen Kinyoun Acid fast Non acid fast Carbol fuchsin Carbol fuchsin Primary stain Red or pink Red or pink Acid alcohol Acid alcohol Decolorizer Red Colorless Methylene Malachite Counterstain Ziehl Neelsen: Ziehl Neelsen: blue green or secondary red organism/ blue organism/ stain blue background blue background Kinyoun: red organism/ Kinyoun: green organism/ green background green background Special Stains These are used to demonstrate specific structures in a bacterial cell. For instance, metachromatic granules can be visualized using the LAMB (Loeffler Alkaline Methylene Blue) stain. Other special stains include Hiss stain (capsule or slime layer); Dyer stain (cell wall), Fischer Conn stain (flagella), Dorner and Schaeffer Fulton stain (spores), and India ink or nigrosine (capsule of the fungus Cryptococcus neoformans). Capsule Staining Capsules Background Rods Flagella a b Figure 1.5 a Demonstration of the capsule using India ink and b flagella surrounding the bacteria demonstrated using the Leifson method of stainin  The Science of Microbiology 1 Culture Media Staining procedures only give clues as to the probable organism being studied. To identify a specific organism, culture using specific culture media is the most ideal. Media (sing. medium) are used to grow microorganisms. A culture medium is basically an aqueous solution to which all the necessary nutrients essential for the growth of organisms are added. These are classified into three primary levels: physical state, chemical composition, and functional type. According to Physical State 1. Liquid media – commonly called broths, milk, or infusions, these are water based solutions that do not solidify at temperatures above the freezing point. These contain specific amounts of nutrients but do not contain gelling agents such as gelatin or agar. Liquid media are suited for the propagation of a large number of organisms, fermentation studies, and other tests. 2. Semi solid media – exhibit a clot like consistency at ordinary room temperature and contain agar at concentrations of 0.5% or less that allows thickening of the media without producing a firm substance. They have a soft consistency similar to custard and are best suited for culture of microaerophilic bacteria or for the study of bacterial motility. 3. Solid media – contain a solidifying agent such as 1.5%–2% agar, giving them a firm surface on which cells can form discrete colonies. They are used for isolation of bacteria and fungi or for determining the colony characteristics of the organism under study. Solid media come in two forms: (a) liquefiable (or reversible) solid media and (b) non liquefiable (or non reversible) solid media. According to Chemical Composition 1. Synthetic media – contain chemically defined substances which are pure organic and/or inorganic compounds. The precise chemical composition of a synthetic medium is known. They may be simple or complex, depending on what supplement is added to it. 2. Non synthetic media – complex media that contain at least one ingredient that is not chemically defined, which means that it is neither a simple or pure compound. It is not representable by an exact chemical formula. Most are extracts of animals, plants, or yeasts. Non synthetic media can support the growth of more fastidious organisms. 14 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences According to Functional Type 1. General Purpose media – are designed for primary isolation of a broad spectrum of microbes and contain a mixture of nutrients that support the growth of both pathogenic and non pathogenic organisms. Examples are peptone water, nutrient broth, and nutrient agar. 2. Enrichment media – contain complex organic substances such as blood, serum, or special growth factors, and are designed to increase the number of desired microorganisms without stimulating the rest of the bacterial population. These are used to grow fastidious or nutritionally exacting bacteria. There are two commonly used enrichment media, namely: a. Blood agar – contains general nutrients with 5%–10% (by volume) blood added to a blood agar base. Certain gram positive bacteria produce exotoxins that cause hemolysis of red blood cells contained in the blood agar. Their hemolytic reaction is categorized into three, which is useful in the classification of these bacteria. The hemolytic patterns are: i. Beta hemolysis – shows complete lysis of red blood cells resulting in complete clearing around the colonies. ii. Alpha hemolysis – shows incomplete lysis of red blood cells, producing a greenish discoloration of the blood agar around the colonies. iii. Gamma hemolysis – shows no hemolysis, resulting in no change in the medium. a b c Figure 1.6 Three types of hemolytic reactions seen in the culture: a beta hemolysis or complete hemolysis; b alpha hemolysis or incomplete hemolysis; and c gamma hemolysis or no hemolysis b. Chocolate agar – a type of nutrient medium that is used for the culture of fastidious organisms such as Haemophilus sp. Heat is applied to lyse the red blood cells, causing the medium to turn brown  The Science of Microbiology 1 3. Selective media – contain one or more substances that encourage the growth of only a specific target microorganism and inhibit the growth of others. It is designed to prevent the growth of unwanted contaminating bacteria or commensals so only the target bacteria will grow. Examples of approaches that will make the medium selective include changing the pH of the culture medium or adding substances such as antibiotics, dyes, or other chemicals. These are usually agar based solid media that allow isolation of individual bacterial colonies. Examples of this type of culture medium include the following: a. Thayer Martin agar – contains the antibiotics trimethroprim, nystatin, vancomycin, and colistin. It is used for the isolation of Neisseria. b. Mannitol Salt agar – contains 10% NaCl and used for the isolation of Staphylococcus aureus. c. MacConkey’s agar – promotes the growth of gram negative bacteria, primarily those belonging to the family Enterobacteriaceae, and inhibits the growth of gram positive bacteria through the addition of bile salts. It is both selective and differential. d. Löwenstein Jensen medium – a selective medium used to recover Mycobacterium tuberculosis. It is made selective by the incorporation of malachite green. e. Saboraud’s dextrose agar – used for the isolation of fungi. 4. Differential media – allow the growth of several types of microorganisms. These are designed to show visible differences among certain groups of microorganisms. The differences may be in the form of variations in colony size or color, changes in color of culture media, or formation of precipitates or gas bubbles. Differential media allow the growth of more than one target microorganism that demonstrate morphologic variations in colony morphology. Examples include MacConkey’s agar and Triple Sugar Iron agar. 5. Transport media – used for clinical specimens that need to be transported to the laboratory immediately after collection. These media prevent the drying of specimen and inhibit the overgrowth of commensals and contaminating organisms. Charcoal is added to neutralize inhibitory factors. Examples are the Cary Blair transport medium for transport of feces of suspected cholera patients and Pike’s medium which is used to transport throat specimens of patients with streptococcal infection. 6. Anaerobic media – media used specifically for organisms that cannot survive in the presence of oxygen and require reduced oxidation reduction potential and other nutrients. These are supplemented with nutrients such as vitamin K and hemin. They undergo boiling to remove dissolved oxygen. To reduce the oxidation reduction potential, substances such as 1% glucose, 0.1% ascorbic acid, 0.1% thioglycolate, or 0.05% cysteine are added. Methylene blue or resazurin is added as an indicator of the oxidation reduction potential. Examples are chopped cooked meat and thioglycolate broth. 16 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences CHAPTER SUMMARY Microbiology is the study of small, living microorganisms or microbes that cannot be seen with the naked eye. These organisms may be cellular (prokaryotes, eukaryotes, and the like) or acellular such as viruses. Microbiology is divided into several fields that deal with the study of bacteria (bacteriology), viruses (virology), fungi (mycology), protozoa and parasitic worms (parasitology), algae (phycology), and the immune system (immunology). Microorganisms may be beneficial or harmful. Some microorganisms are used in different industries such as in food and beverage. Some microorganisms are sources of antibiotics while some are used in the field of biotechnology and genetic engineering. Microorganisms are also important in maintaining a balanced ecosystem. While some microorganisms are essential and have beneficial uses, there are also numerous microorganisms that produce disease in humans, some of which are potentially fatal. Some microorganisms have the potential to be used as biological warfare agents. Microorganisms are so miniscule that for them to be visualized, they need to be stained and studied using the microscope. Several types of microscopes have been developed for this purpose—from the compound microscope to the more sophisticated electron microscopes. The use of various staining procedures has made visualization of microorganisms easier. These stains may be classified into simple, differential, and special stains. › Simple stains make use of a single water or alcohol based dye that is used to demonstrate the shape and basic structures of the organism. › Differential stains are used to distinguish one group of bacteria from another group. These include the Gram stain and the acid fast stain. › Special stains are mainly used to demonstrate specific bacterial structures such as the spores (Dorner or Schaeffer Fulton), flagella (Fischer & Conn), capsule (Hiss stain), or the metachromatic granules (LAMB stain). Specific culture media are the most ideal in identifying specific organisms. Several classes of culture media have been developed and these culture media can be classified into three primary levels: physical state (liquid, semi solid, solid), chemical composition (synthetic and non synthetic), and functional type (general purpose, enrichment, selective, differential, transport, and anaerobic)  The Science of Microbiology 17 SELF ASSESSMENT QUESTIONS Name: Score: Section: Date Multiple Choice. 1. Which among the following groups of organisms are not considered cells? a. Bacteria c. Viruses b. Fungi d. Algae 2. Which among the following types of microscopes is used together with computers to produce a three dimensional image and is also useful in the study of cell physiology? a. Phase contrast microscope c. Fluorescent microscope b. Scanned probe microscope d. Confocal microscope 3. Which among the following parts of the microscope is used to gather and focus light onto the specimen? a. Coarse adjustment c. Eye piece b. Fine adjustment d. Condenser 4. Who among the following scientists made the initial postulates regarding the germ theory of disease? a. Louis Pasteur c. Edward Jenner b. Alexander Fleming d. Robert Koch 5. You discovered a new organism and you want to study its molecular and atomic properties. Which among the following types of microscopes would be suited for this purpose? a. Electron microscope c. Scanned probe microscope b. Fluorescent microscope d. Confocal microscope 18 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences 6. You are given a new slide to study in the laboratory. Which part of the microscope will you use to put the specimen into general focus? a. Iris diaphragm c. Objective lenses b. Coarse adjustment d. Fine adjustment 7. Which among the following classes of culture media is used to isolate fungi? a. Thayer Martin agar c. Saboraud dextrose agar b. Löwenstein Jensen agar d. Chocolate agar 8. Which among the following reagents used in Gram staining will enhance the uptake of the primary stain? a. Crystal violet c. 95% alcohol b. Gram’s iodine d. Safranin 9. You culture an organism using blood agar and after 24 hours of incubation you noted complete hemolysis of red blood cells surrounding the colonies. This is classified as what type of hemolytic reaction? a. Alpha hemolysis c. Delta hemolysis b. Beta hemolysis d. Gamma hemolysis 10. Which among the following reagents is used as the counterstain in the Ziehl Neelsen method of acid fast staining? a. Safranin c. Malachite green b. Carbol fuchsin d. Methylene blu  CHAPTER Prokaryotic and 2 Eukaryotic Cells LEARNING OBJECTIVES At the end of this chapter, the student should be able to: 1. differentiate prokaryotes from eukaryotes; and 2. characterize the different medically important microorganisms. Living Cells can be classified into two general categories—prokaryotesand eukaryotes. Prokaryotes are organisms that do not possess a true nucleus and membrane bound organelles (e.g., bacteria). Eukaryotic organisms are those that possess a true nucleus and membrane bound organelles. They are usually multicellular organisms and include plants, animals, fungi, parasites, and algae. Viruses are acellular organisms that possess only DNA or RNA. They are dependent on host cells for their replication and are considered as obligate intracellular parasites 20 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Comparison between Prokaryotes and Eukaryotes 1–10 μm 10–100 μm chloroplast mitochondrion circle of DNA nucleus linear DNA Typical Prokaryotic Cell Typical Eukaryotic Cell Figure 2.1 Diagrammatic representation showing the difference between a prokaryotic cell and a eukaryotic cell Table 2.1 Comparison between prokaryotic and eukaryotic cells Feature Prokaryotic Eukaryotic Genetic material Not enclosed within a membrane; Enclosed within a membrane; not associated with histones; usually associated with histones; usually circular linear Size Smaller (1–2 μm by 1–4 μm or less) Greater than 5 μm in diameter Cell type Mostly unicellular Mostly multicellular Nucleus No true nucleus and nuclear With true nucleus enclosed by membrane; called nucleoid nuclear membrane Cell wall Simple Complex Cell division Budding or binary fission Mitosis Sexual No meiosis; transfer of DNA only Meiosis reproduction Cytoskeleton Absent Present Mesosome Functions as mitochondria and Golgi Absen complex  Prokaryotic and Eukaryotic Cells 21 Feature Prokaryotic Eukaryotic Ribosomes 70S; located in cytoplasm 80S; located in membranes such as in the endoplasmic reticulum 70S; found in organelles such as mitochondria or chloroplast Membrane bound Absent Present organelles Extrachromosomal Present Absent plasmid Duration of cell Short (20–60 minutes) Long (12–24 hours) cycle Adapted from http://www.microbiologynotes.com/differencesbetween prokaryotic and eukaryotic cells Medically Important Microorganisms Organisms that are considered medically important are those that have the potential or the ability to produce significant clinical disease in humans. They may be part of the normal flora of the body or are true pathogenic organisms. These may be categorized into bacteria, viruses, fungi, algae, and parasites (protozoa and helminths). Viruses are acellular organisms. Their outer surface is called capsid, which is composed of repeating sub units called capsomeres. Viruses possess only a single nucleic acid, either DNA or RNA, but never both. In addition, viruses lack the necessary cellular parts that can allow them to replicate independent of the host cell. They also lack the genes and enzymes that are necessary for energy production. They rely on the cellular machinery of the host cell for protein and energy production. Hence, viruses are considered obligate intracellular parasites. Viruses are classified based on the following: (1) type of nucleic acid they possess; (2) shape of the capsid (icosahedral, helical, polyhedral, or complex); (3) number of capsomeres; (4) size of the capsid; (5) presence or absence of an envelope; (6) type of host they infect (humans, plants, or animals); (7) type of disease they produce; (8) target cell or tropism (e.g., T helper cells for HIV); and (9) immunologic or antigenic properties 22 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Herpesvirus Orf virus Vaccinia virus Paramyxovirus (mumps) Adenovirus Rhabdovirus T even coliphage Influenza virus Flexuous tailed phage Polyomavirus Picornavirus ΦX174 phage Tubulovirus 1 μm Figure 2.2 Diagrammatic representation of various forms and sizes of viruses Bacteriophages are a special type of viruses that primarily infect bacteria. They are similar to other viruses in that: (1) they are obligate intracellular parasites; (2) they are similarly shaped like other viruses; and (3) they may also be classified based on the type of nucleic acid they possess. They play a role in the acquisition of virulence factors of certain bacteria (e.g., diphtheria toxin of Corynebacterium diphtheriae), as well as in the transfer of genetic material from one bacterium to another (as in transduction). Bacteria are prokaryotic cells with majority having an outer covering called the cell wall that is composed mainly of peptidoglycan. Unlike viruses, they possess both DNA and RNA. Unlike eukaryotic organisms, bacteria possess a nucleoid instead of a true nucleus, smaller ribosomes, and lack mitochondria. Based on their physical characteristics, bacteria may be broadly categorized into (1) gram negative bacteria with cell wall (e.g., Escherichia coli); (2) gram positive bacteria with cell wall (e.g., Staphylococcus aureus); (3) acid fast bacteria with lipid rich cell wall (e.g., Mycobacterium tuberculosis); and, (4) bacteria without cell wall (e.g., Mycoplasma)  Prokaryotic and Eukaryotic Cells 2 Fungi are eukaryotic cells with an outer surface composed mainly of chitin. Their cell membrane is made up mostly of ergosterol. Like bacteria, fungi possess both DNA and RNA. Unlike bacteria, they possess a true nucleus that is enclosed by a nuclear membrane and mitochondria that function for ATP production. Fungal ribosomes are also larger than bacterial ribosomes (80 Svedberg units). Table 2.2 summarizes the major differences between fungi and bacteria. Protozoa are the representatives for parasites. Like bacteria and fungi, these are also eukaryotic cells that have an outer surface called a pellicle. These are unicellular organisms that usually divide through binary fission, similar to bacteria. Majority exist in two morphologic forms—cysts and trophozoites. The infective stage is the cyst while the pathogenic stage is the trophozoite. Protozoa possess both DNA and RNA as well as other cellular features seen in typical eukaryotic cells. Table 2.2 Comparison between fungi and bacteria Features Bacteria Fungi Cell type Prokaryotic; unicellular Eukaryotic; unicellular or multicellular Role in ecosystem Can be both producers and Mainly decomposers decomposers Optimal pH Neutral pH (6.5–7.0) Slightly acidic (4.0–6.0) Cell structures No true nucleus and membrane Possess true nucleus and bound organelles membrane bound organelles Main component Peptidoglycan, except in Chitin of cell wall archaebacteria Sterols in cell Absent except in Mycoplasma Present membrane Mode of nutrition Heterotrophic, chemoautotrophic, Heterotrophic; majority aerobic photoautotrophic, aerobic, and facultative anaerobic anaerobic, facultative anaerobic Reproduction Binary fission Sexual and asexual spores Adapted from https://www.majordifferences.com/2017/07/10differences between bacteria and.html Algae are eukaryotic organisms whose outer surface consists primarily of cellulose. They are described as plant like organisms because most of them have chlorophyll and are thus capable of photosynthesis. Unlike plants, they do not possess true roots, stems, and leaves. Table 2.3 summarizes the major differences between algae and plants. Algae vary in size from the single celled phytoplanktons to the large seaweeds found in the ocean floor. Algae do not produce significant disease in humans. Most algae are beneficial in that they are important sources of food, iodine, and other minerals. They may also be used as fertilizers, emulsifiers for puddings, and stabilizers for ice cream and salad dressings. 24 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Table 2.3 Comparison between algae and plants Features Algae Plants Taxonomic classification Kingdom Protista Kingdom Plantae Cellular structure Unicellular, multicellular or Multicellular colony forming Photosynthetic Yes Yes Energy source Carbon dioxide Carbon dioxide Storage form of energy Starch Starch Vascular system* Absent Present Habitat Mostly water Mostly rooted to the ground Composed of roots, stems, No Yes and leaves Method of reproduction Both asexual and sexual Sexual (complex) *Allow for dispersion of nutrients throughout the entire plant Diatoms are unicellular algae that inhabit both fresh and saltwater. Their cell wall contains silicone dioxide that may be utilized in filtration systems, insulation, and as abrasives. Dinoflagellates are also unicellular algae that are important members of the phytoplankton group. They contribute greatly to the oxygen in the atmosphere and serve as important links in the food chain. On the other hand, they are also responsible for what is known as “red tide.” These small organisms produce a powerful neurotoxin which, when ingested in significant amounts, is responsible for the potentially fatal disease called paralytic shellfish poisoning. Cyanobacteria Diatom Dinoflagellate Green algae Coccolithophore Figure 2.3 Various structures of phytoplanktons that are usually found floating on wate  Prokaryotic and Eukaryotic Cells 25 CHAPTER SUMMARY Living cells can be classified as either prokaryotic or eukaryotic. Prokaryotic cells, as exemplified by bacteria, are usually unicellular, do not possess a true nucleus and membrane bound organelles, and multiply by means of binary fission. Eukaryotic cells vary from unicellular (e.g., protozoa) to multicellular (e.g., fungi). They possess a true nucleus surrounded by a nuclear membrane as well as membrane bound organelles. Viruses are not classified as cells since they only possess an outer covering called capsid and a nucleic acid (either DNA or RNA). As such, they are dependent on the host cell machinery for their replication and are thus considered as obligate intracellular parasites. Medically important organisms are those which produce significant disease in humans. These may take the form of viruses, bacteria, fungi, protozoa, and algae. › Viruses are acellular, obligate intracellular parasites possessing only DNA or RNA and may be classified based on: (1) type of nucleic acid they possess; (2) shape of the capsid (icosahedral, helical, polyhedral, or complex); (3) number of capsomeres; (4) size of the capsid; (5) presence or absence of an envelope; (6) type of host they infect (humans, plants, or animals); (7) type of disease they produce; (8) target cell or tropism (e.g., T helper cells for HIV); and (9) immunologic or antigenic properties. › Bacteria are prokaryotic organisms that possess both DNA and RNA. Most possess a of cell wall composed predominantly peptidoglycan. › Fungi are eukaryotic organisms with a cell wall composed mainly of chitin and cell membrane that contains ergosterol. › Protozoa are mostly unicellular parasites that are eukaryotic. Most divide by binary fission similar to bacteria. › Algae are eukaryotic, aquatic, plant like organisms. Similar to plants, they are photosynthetic but unlike plants, they do not have true roots, stems, or leaves This page is intentionally left blank  Prokaryotic and Eukaryotic Cells 27 SELF ASSESSMENT QUESTIONS Name: Score: Section: Date Matching Type. A. Cell Type Column A Column B 1. Reproduce by meiosis and mitosis a. Prokaryotic cell 2. With 70S ribosomes b. Eukaryotic cell 3. Do not have membrane bound organelles 4. DNA associated with histones 5. Has true nucleus surrounded by nuclear membrane B. Organism Group Column A Column B 6. Obligate intracellular parasite a. Bacteria 7. Outer covering made up mostly of chitin b. Viruses 8. Possess only one type of nucleic acid c. Fungi 9. Capable of photosynthesis d. Protozoa 10. Unicellular parasite e. Algae 11. Include bacteriophages 12. Reproduction is through sexual and asexual spores 13. Outer surface consists primarily of cellulose 14. Possess chlorophyll 15. Use carbon dioxide as energy source This page is intentionally left blank  CHAPTER Bacterial 3 Morphology LEARNING OBJECTIVES At the end of this chapter, the student should be able to: 1. distinguish among the various general shapes of bacteria, citing examples for each; and 2. compare the external and internal structures of gram positive, gram negative and acid fast bacteria. Bacteria, which are prokaryotic, have simpler structures compared to eukaryotic organisms. In terms of morphology, bacteria may be classified into three basic shapes: coccus (pl. cocci), bacillus (pl. bacilli), and spiral shaped or curved. Cocci can be described as spherical or round shaped organisms (e.g., Staphylococcus, Streptococcus). They may be arranged singly, in pairs (diplococci), in chains (streptococci), in clusters (staphylococci), in groups of four (tetrad), or in groups of eight (octad). Rod shaped organisms are called bacilli (e.g., Escherichia coli, Salmonella). Some may be very short, resembling elongated cocci called coccobacilli (e.g., Haemophilus influenzae). Curved and spiral shaped organisms may show variations in their morphology. Vibrio cholerae, the organism causing cholera, is described as comma shaped. The causative agent of syphilis, Treponema pallidum, is spiral in shape while the causative agent of diphtheria, Corynebacterium diphtheriae, is club shaped 30 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Fundamental Shapes of Bacteria Flagella Pseudomonas Salmonella typhi Pneumococci Streptococci Treponema Spores Mycobacterium Clostridium tuberculosis tetani Staphylococci Leptospira Spheres (Cocci) Rods (Bacilli) Spirals (Spirochetes) Figure 3.1 Fundamental shapes of bacteria Envelope Structures Prokaryotic cells are surrounded by a complex envelope that may vary in composition. The envelope serves to protect the bacteria from harsh environmental conditions. Glycocalyx This is the outermost covering of some bacteria. It is a gelatinous substance that is located external to the cell wall, composed of polysaccharide or polypeptide, or both. It is called capsule if it is strongly attached to the cell wall and slime layer if it is loosely attached. The presence of the capsule is indicative of the virulence of an organism, aiding the organism in the evasion of phagocytosis. It can stimulate an antibody response from the immune system. The capsule serves to protect the organism from dehydration. Cell Wall The bacterial cell wall is sometimes called the murein sacculus. Its principal component is peptidoglycan, which is also called murein or mucopeptide. It is multi layered in gram positive bacteria and single layered in gram negative bacteria. The cell wall provides rigid support and gives shape to the bacteria. It protects the bacteria from osmotic damage and plays an important role in cell division  Bacterial Morphology 31 Special components of gram positive cell walls 1. Teichoic acids – comprise major surface antigens of gram positive organisms and can elicit antibody response. In some gram positive organisms such as Staphylococcus aureus, teichoic acids function for the attachment of the organism to the host cell. These also provide tensile strength to gram positive bacterial cell walls. 2. Polysaccharides – polysaccharide molecules include neutral sugars such as mannose, arabinose, rhamnose, and glucosamine. It also includes some acidic sugars such as glucuronic acid and mannuronic acid. Teichoic acid Wall associated protein Lipoteichoic acid Peptidoglycan Cytoplasmic membrane Figure 3.2 Diagrammatic representation of a typical gram positive bacterial cell wall Special components of gram negative cell walls 1. Outer membrane – a bi layered structure where the inner leaflet is composed of a lipopolysaccharide (LPS). It has special protein channels that allow the passage of small or low molecular weight hydrophilic substances such as sugars and amino acids. LPS has a complex glycolipid called lipid A, responsible for its endotoxin activity. It is located in the outer leaflet of the outer membrane. The inner core is a polysaccharide made up of repeat units. This repeat unit is also called the O antigen, which is unique for every species of bacteria. 2. Lipoprotein – functions to anchor the outer membrane to the peptidoglycan layer and stabilizes the outer membrane. 3. Periplasmic space – a fluid filled space between the outer membrane and the inner plasma membrane. It contains enzymes for the breakdown of large non transportable molecules into transportable ones and enzymes that serve to detoxify and inactivate antibiotics  32 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Lipoteichoic acid Teichoic acid Porin O specific side chains Lipopolysaccharide Outer membrane Peptidoglycan Broun's lipoprotein Periplasmic space Peptidoglycan Periplasmic space Plasma membrane Plasma membrane and integral proteins and integral proteins Gram (+) cell wall Gram (–) cell wall Figure 3.3 A comparison between gram positive and gram negative cell walls showing the differences in their constituents Acid fast cell wall Unlike gram positive and gram negative bacteria, acid fast organisms such as Mycobacterium tuberculosis possess an outer layer that is lipid rich. The cell wall of acid fast organisms is composed of large amounts of waxes that are known as mycolic acids. The inner layer of the cell wall is also made up of peptidoglycan but because the outermost layer is lipid rich, cell walls of acid fast organisms are hydrophobic. This is the reason why they cannot be stained using the reagents used in gram staining. The hydrophobic nature of their cell wall protects them from harsh chemicals such as strong acids and detergents. LAM Lipoteichoic acid Glycolipid LPS Mycolic acid Lipoprotein Porin PeptidoglycanPeptidoglycan Peptidoglycan Galactan Mannophosphoinositide Gram positive Bacteria Gram negative Bacteria Mycobacteria Figure 3.4 Schematic representation comparing gram positive, gram negative, and acid fast cell wal  Bacterial Morphology 33 Projecting Structures Flagella These are thread like structures made up entirely of molecules of the protein sub unit flagellin. They project from the capsule and are organs for motility. Flagella are classified into four types, namely: (a) monotrichous (single polar flagellum); (b) lophotrichous (a tuft of flagella at one end of the bacterium); (c) amphitrichous (flagella at both ends of the bacterium); and (d) peritrichous (flagella all around the bacterium). Bacteria without flagella are called atrichous. a b c d Figure 3.5 Typical arrangement of bacterial flagella. a Peritrichous, b monotrichous and polar, c lophotrichous and polar, and d amphitrichous and polar. Pili or Fimbriae These are rigid surface appendages found on many gram negative bacteria. They are fine and short in comparison with flagella. Their structural protein sub units are called pilins. Pili may also function for motility. They function for adherence to cell surface (common pili) or attachment to another bacterium during a form of bacterial gene exchange called conjugation (sex pili). Axial Filaments Axial filaments are also called endoflagella and are found in spirochetes (e.g., Treponema pallidum causing syphilis). These are composed of bundles of fibrils, the structures of which are similar to flagella. They arise from the ends of the bacterial cell and spiral around the cell. The filaments rotate producing movement of the outer sheath of the spirochetes propelling them forward 34 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Cytoplasmic Membrane Also called cell membrane or plasma membrane, the cytoplasmic membrane is located beneath the cell wall. It is sometimes called the cell sac because it encloses the cytoplasm of the cell. The cytoplasmic membrane is a selectively permeable membrane that allows for transport of selected solutes. In aerobic organisms, it is the site of the electron transport chain and serves as the site of ATP production. It therefore serves the function of the mitochondria, which are not found in prokaryotic cells. The cytoplasmic membrane also contains the enzymes needed for the biosynthesis of DNA, cell wall components, and membrane lipids. Internal Structures Nucleoid Bacteria have no true nucleus that is surrounded by a nuclear membrane. Its genetic material is packaged in a structure called the nucleoid. Bacteria possess a single, circular, double stranded DNA. Mesosomes The mesosome functions for cell division. It is also involved in the secretion of substances produced by bacteria. Ribosomes The ribosomes function for protein synthesis. Unlike eukaryotic ribosomes, bacterial ribosome is smaller (70S). Granules or Inclusion Bodies These are found in certain bacteria and serve for storage of food and energy (e.g., metachromatic granules of Corynebacterium diphtheriae or Much granules of Mycobacterium tuberculosis)  Bacterial Morphology 35 Endospores Endospores are structures produced by many bacteria when they are placed in a hostile environment. It is composed of dipicolinic acid which confers resistance to heat, drying, chemical agents, and radiation; making it very difficult to destroy. The process of spore production is called sporulation, and this occurs when the environmental conditions are detrimental to the bacteria. When environmental conditions become favorable, the endospores revert to their vegetative state through a process called germination. Some gram positive, but never gram negative, bacteria form spores. Pilus Capsule Cytoplasm Inclusion Ribosomes Cell wall Capsule Plasma membrane Nucleoid Cell wall containing DNA Plasmid Plasma membrane Fimbriae Flagella Figure 3.6 Parts of a typical prokaryotic cell a b c Figure 3.7 Spores showing a terminal and b central location, c as well as metachromatic granules of Corynebacterium diphtheria 36 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences CHAPTER SUMMARY There are three basic shapes of bacteria: (a) spherical or cocci; (b) rod shaped or bacilli; and (c) curved or spiral. Awithtypical prokaryotic cell is composed of three major components—an outer envelope its projecting structures, the cell membrane, and the internal structures. The envelope is composed of the following: » The outermost covering is the glycocalyx, also known as the capsule if it is adherent to the cell wall and slime layer when it is loosely attached to the cell wall. » The cell wall or the murein sacculus provides rigid support and shape to the bacteria. Its main component is peptidoglycan, which is multilayered in gram positive bacteria and monolayered in gram negative bacteria. › Gram positive cell wall contains teichoic acids which may function for the attachment of the bacterium to the host cell, as well as polysaccharide molecules. › Gram negative cell walls contain lipopolysaccharide made of a lipid A molecule A is and polysaccharides. The lipid component responsible for the endotoxic activity of gram negative bacteria. The lipopolysaccharide is an integral part of the outer membrane of gram negative bacteria. Gram negative bacteria also have a periplasmic space where important enzymes are found. › Acid fast organisms possess a cell wall that is also made up of an inner layer of peptidoglycan and an outer layer rich in waxes composed of mycolic acid and other lipids. This is responsible for the hydrophobic nature of its cell wall and the main reason why acid fast organisms cannot be stained using the reagents for Gram staining. Structures projecting from the bacterial capsule include pili or fimbriae of gram negative organisms, flagella, and axial filaments of spirochetes. » There are two types of pili: common pili which functions for attachment and sex pili which participates in gene exchange among bacteria in a process called conjugation. » Flagella may be of four patterns: (1) lophotrichous (a tuft of flagella on one end of the bacterium), (2) amphitrichous (a single flagellum on each end of the bacterium), (3) peritrichous (flagella surrounding the bacterium), and (4) monotrichous (only one flagellum at one end of the bacterium). » Axial filaments are similar in structure to flagella and help propel the spirochetes forward  Bacterial Morphology 3 Bacterial cytoplasmic membrane is the functional analogue of the mitochondria. It is selectively permeable and is the site of ATP production of aerobic bacteria. Bacteria do not have a true nucleus. Its genetic material is packaged in a structure called nucleoid. Bacterial ribosome is smaller than a typical eukaryotic ribosome. Bacteria possess structures that enable them to withstand adverse environmental conditions. These structures are the endospores which are mainly composed of dipicolinic acid. Other structures found in bacterial cells are the mesosomes, which play a role in cell division, and inclusion bodies or granules in some bacteria which serve as storage for food. This page is intentionally left blank  Bacterial Morphology 39 SELF ASSESSMENT QUESTIONS Name: Score: Section: Date: Multiple Choice. 1. What bacteria are partly round and partly rod shaped? a. Cocci c. Spirochetes b. Bacilli d. Coccobacilli 2. Which among the following bacterial structures acts as a functional analogue of the mitochondria? a. Capsule c. Cell membrane b. Nucleoid d. Outer membrane 3. Which among the following cell wall components is found only in gram positive bacteria? a. Lipopolysaccharide c. Mycolic acid b. Teichoic acid d. Muramic acid 4. Which among the following statements is correct regarding the cell wall of acid fast bacteria? a. It can be stained with crystal violet. b. Its outer layer is hydrophobic due to the presence of lipids. c. Its outer layer is hydrophilic due to the presence of a multi layered peptidoglycan. d. The major component is lipoteichoic acid. 5. A bacterium that has a tuft of flagella on one end is called: a. Lophotrichous c. Peritrichous b. Monotrichous d. Amphitrichou 40 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences 6. Which of the following structures is utilized by bacteria in exchanging genetic material from one bacterium to another? a. Pilus c. Mesosome b. Flagella d. Axial filament 7. Which among the following provides rigid support to bacteria and gives shape to the bacteria? a. Cell membrane c. Outer membrane b. Cell wall d. Capsule 8. The bacterial endospore is resistant to heat and drying due to the presence of this component: a. Teichoic acid c. Mycolic acid b. Muramic acid d. Dipicolinic acid 9. Which among the following is a structure utilized by some bacteria for food storage? a. Much granules d. A, B, and C b. Metachromatic granules e. A and B only c. Endospores 10. Which of the following contain enzymes used by bacteria to break down large molecules into smaller, easy to transport molecules? a. Cell wall c. Outer membrane b. Cell membrane d. Periplasmic spac  CHAPTER Bacterial Growth 4 Requirements LEARNING OBJECTIVES At the end of this chapter, the student should be able to: 1. define microbial growth; 2. discuss the various nutritional and physical requirements of bacteria for growth; and 3. illustrate the bacterial growth curve with explanation of the events occurring in each phase of the bacterial growth curve. Growth as defined in medical dictionaries involves an orderly and organized increase in the sum of all components of the organism. The process entails the replication of all cellular structures, organelles, and components. Microbial growth is concerned with the increase in the number of cells and not an increase in the size of the organism. A bacterial colony is composed of thousands of cells; hence, colonies in culture are actually composed of billions of cells. As in any living organism, bacteria require certain nutrients and physical conditions that will promote their growth. This chapter discusses the various nutritional and physical requirements of bacteria for growth. Nutritional Requirements Carbon Carbon makes up the structural backbone or skeleton of all organic molecules. Based on their carbon source, microorganisms may be classified into autotrophs (lithotrophs) and heterotrophs (organotrophs) 42 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Autotrophs are microorganisms that utilize inorganic compounds (e.g., carbon dioxide) and inorganic salts as their sole carbon source. Organotrophs are organisms that make use of organic substances like sugars or glucose as their carbon source. For both autotrophs and heterotrophs, their energy may be derived from either light (photolithotrophs and photoorganotrophs) or the oxidation of inorganic substances (chemolithotrophs and chemoorganotrophs).Most medically important bacteria are chemoorganotrophs. Nitrogen, Sulfur, Phosphorus These are necessary for the synthesis of cellular materials like proteins and nucleic acids. Nitrogen and sulfur are required for the synthesis of proteins. Nitrogen and phosphorus are essential for the synthesis of nucleic acids and ATP. Approximately 14% of the dry weight of a bacterial cell is nitrogen and about 4% is sulfur and phosphorus. Inorganic Ions These include magnesium, potassium, calcium, iron, and trace elements (e.g., manganese, zinc, copper, cobalt). Magnesium stabilizes ribosomes, cell membranes, and nucleic acids. It also serves as a co factor in the activity of many enzymes. Potassium is required for the normal functioning and integrity of ribosomes and participates in certain enzymatic activities of the cell. Calcium is an important component of gram positive bacterial cell wall and contributes to the resistance of bacterial endospores against adverse environmental conditions. Iron is a component of cytochrome, a component of the electron transport chain, and functions as a co factor for enzymatic activities. Trace elements are components of enzymes and function as co factors. Some are necessary for the maintenance of protein structure. Growth Factors Growth factors are essential to promote the growth and development of the bacterial cell. These include vitamin B complex and amino acids  Bacterial Growth Requirements 4 Physical Requirements Moisture/Water The bacterial cell is composed mainly of water. It serves as the medium from which bacteria acquire their nutrients. Oxygen Oxygen is used by aerobic bacteria for cellular respiration and serve as the final electron acceptor. Microorganisms are classified as either aerobes or anaerobes based on their oxygen requirements. Microorganisms that utilize molecular oxygen for energy production are referred to as aerobes. Strict aerobes are organisms that strictly require oxygen for growth. Microbes that cannot survive in the presence of oxygen are called obligate anaerobes. These organisms do not have the enzymes that break down free radicals produced in the body (i.e., catalase and superoxide dismutase). There are organisms that can grow and survive under both aerobic and anaerobic conditions. These are called facultative organisms. Most medically important bacteria are facultative. Some organisms are able to grow at low oxygen tension but their rate of growth is diminished. These are called microaerophiles. There are some organisms though that may require the addition of carbon dioxide to enhance their growth. These are called capnophiles. Temperature Enhanced enzyme activity requires certain temperatures. Microbes are classified into three groups based on their temperature requirements, namely: (1) thermophiles, which grow best at temperatures higher than 40 °C; (2) mesophiles, which require an optimal temperature of 20 °C–40 °C; and, (3) psychrophiles, which require an optimum temperature of 10 °C–20 °C. Most medically important bacteria are mesophiles. 44 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences 90 °C 80 °C Thermophile 70 °C 60 °C Pasteurization (62.8 °C) 50 °C 40 °C Mesophiles 30 °C Human body (37 °C) Psychrophiles 20 °C 10 °C 0 °C Refrigerator (4 °C) Figure 4.1 Classification of bacteria into three groups based on their optimum temperature requirements pH Another requirement of bacteria is the extent of acidity or alkalinity of their environment, which is referred to as the pH. Microorganisms that grow best in pH 8.4–9.0 are called alkalophiles. Those that grow best in pH 6.5–7.5 are called neutrophiles. Most medically important bacteria are neutrophiles. The pH of most human tissues are 7.0–7.2. Certain bacteria require a pH less than 6.0. These bacteria are called acidophiles. Osmotic Conditions Most organisms grow best under ideal conditions of osmotic pressure, which is determined by the salt concentration. The normal microbial cytoplasmic salt concentration is approximately 1%. The optimum condition is if the external environment also has the same salt concentration. If the extracellular salt concentration is increased (e.g., when food is salted), water will flow out of the microbial cell and the organism will shrink and die. On the other hand, if the external environment does not contain salt, water will flow into the bacterial cell causing the organism to swell and rupture. Organisms that require high salt concentrations for growth are called halophiles (e.g., diatoms and dinoflagellates) and those that require high osmotic pressure for optimal growth are called osmophiles.  Bacterial Growth Requirements 45 Bacterial Growth Curve The bacterial growth curve illustrates the phases in the growth of the population of bacteria when they are grown in a culture of fixed volume. It reflects the different stages in the growth of the organism and is divided into four phases: lag phase, log phase, stationary phase, and death or decline phase. microorganism Stationary Log, or phase exponential growth, Death, or phase decline, phase of Numbers Survival Lag phase phase Time Figure 4.2 Bacterial growth curve Lag Phase This is the period of adjustment for the bacteria in the new environment. During this phase, there is no appreciable increase in the number of microorganisms. The organisms will show increased metabolic activity in order to synthesize DNA as well as secrete enzymes which might not be present in their new environment but which are needed by the organism. Bacteria attain their maximum size toward the end of the lag phase. This phase may last for 1 to 4 hours. Log/Logarithmic/ExponentialPhase This period is characterized by rapid cell division, resulting in an increase in the number of bacteria. The organism exhibits high metabolic activity. This is the period when the generation time or doubling time of the organism (i.e., the time required for the bacterial cells to double in number) is determined. A generation time of 10 minutes means that the bacteria will double in number every 10 minutes showing exponential growth. The average duration of this phase is about 8 hours. 46 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Stationary Phase This is considered as the period of equilibrium. During this period, the rate of growth slows down, nutrients start to deplete, and toxic wastes begin to accumulate. As a consequence, some bacterial cells may die. However, since there are still bacterial cells undergoing cell division, the number of living cells equals the number of dead cells. Gram positive organisms may become gram negative organisms in this phase. Sporulation occurs towards the end of this phase, or in the case of spore forming organisms, during the beginning of this phase. Death or Decline Phase This is the period of rapid cell death where the number of dead cells is greater than the number of living cells. This is due to the continuous depletion of nutrients and accumulation of waste materials. Sporulation continues to occur during this stage. The duration of this phase varies from a few hours to a few days  Bacterial Growth Requirements 47 CHAPTER SUMMARY Bacteria require optimum nutrient and physical conditions for their growth. Nutritional requirements of bacteria include adequate supply of carbon, nitrogen, sulfur, phosphorus, inorganic ions, and growth factors. Bacteria are classified into two groups based on their carbon source: autotrophs/ lithotrophs and heterotrophs/organotrophs. » Autotrophs utilize inorganic compounds for their carbon source while organic compounds such as glucose serve as the carbon source of heterotrophs. Bacteria derive energy by two means: from sunlight or from oxidation of inorganic substances. Physical requirements of bacteria include moisture, oxygen, temperature, pH, and osmotic conditions. » Bacterial cell is made up mostly of water, which serves as the medium from which bacteria derive their nutrients. » Organisms that require oxygen for optimal growth are called aerobes while those that cannot survive in the presence of oxygen are called anaerobes. » Facultative organisms are those which can grow in the presence or absence of oxygen. » Bacteria may be grouped into three based on their temperature requirements: (1) those that require high temperature (thermophiles); (2) those that require temperature of 20 °C–40 °C (mesophiles); and (3) those that require temperature of 10 °C–20 °C (psychrophiles). » Acidophiles are organisms that grow best in pH < 6.0. Neutrophiles grow best at pH of 7.0–7.2 while alkalophiles are those that grow best at pH of 8.4–9.0. » Organisms that require salt for growth are called halophiles. Osmophiles are those that need high osmotic pressure for maximal growth. Based on their nutritional and physical requirements, most medically important bacteria are chemoorganotrophs,facultative, mesophiles, and neutrophiles. The bacterial growth curve illustrates the phases of growth of a bacterial population grown in culture of fixed volume. It is divided into a lag phase, log phase, stationary phase, and death or decline phase This page is intentionally left blank  Bacterial Growth Requirements 49 SELF ASSESSMENT QUESTIONS Name: Score: Section: Date Multiple Choice. 1. Microorganisms that utilize organic compounds as sole carbon source are called: a. Phototrophs c. Chemotrophs b. Heterotrophs d. Autotrophs 2. Which among the following is essential for the synthesis of nucleic acids and proteins? a. Iron c. Calcium b. Nitrogen d. Potassium 3. Organisms that strictly require oxygen for growth are called: a. Facultative c. Obligate anaerobes b. Obligate aerobes d. Microaerophiles 4. Most medically important bacteria are: a. Photoorganotrophs c. Mesophiles b. Alkalophiles d. Halophiles 5. Bacteria that require an optimum temperature of more than 40 °C are called: a. Thermophiles c. Psychrophiles b. Mesophiles d. Neutrophiles 6. Microorganisms that require carbon dioxide for growth are called: a. Halophiles c. Capnophiles b. Mesophiles d. Psychrophiles 50 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Matching Type. Column A Column B 7. Nutrients are depleted and toxic wastes accumulate a. Lag phase 8. Period of adjustment for the bacteria b. Log phase 9. Period of rapid cell division c. Stationary phase d. Death or 10. Period when spores begin to form decline phas  5 Normal Flora CHAPTER of the Human Body LEARNING OBJECTIVES At the end of this chapter, the student should be able to: 1. define “normal flora;” 2. differentiate between resident flora and transient flora; 3. explain the role of normal flora in the body; and 4. give examples of organisms that normally inhabit different sites in the body. Microbial Ecology is the study of the relationships between microorganisms and their environment. Among these relationships is the relationship of microbes with humans, and such include the normal flora (or indigenous flora) of the human body. Normal flora consists of the group of organisms that inhabit the body of a normal healthy individual in the community. These indigenous flora may be non pathogenic or pathogenic and may at times behave as opportunistic pathogens. There are two types of flora, namely: (1) resident flora and (2) transient flora. Resident flora are organisms that are relatively of fixed types and are regularly found in a given area of the body at a given age. Transient flora are those that inhabit the skin and mucous membrane temporarily for hours, days, or weeks and are derived from the environment. Normal flora are beneficial to the human body because they can inhibit the growth of pathogenic organisms by priming the immune system of newborns. At the same time, normal flora protects the body’s organs and systems that are in direct contact with the external environment and are therefore subject to the attack of invasive organisms. Normal flora do this by either competing with invasive organisms for nutrients essential for their growth or by producing substances that can kill them. Normal flora synthesize important vitamins that are essential to humans 52 Microbiology and Parasitology: A Textbook and Laboratory Manual for the Health Sciences Normal intestinal flora secrete vitamin K that is needed for the activity of some clotting factors. Other beneficial effects of normal flora include the following: 1. Normal flora can prevent pathogenic organisms from attaching to and penetrating the skin and other tissues by producing mucin which make it difficult for the pathogenic organisms to attach to the tissues to produce disease. 2. Normal flora in the intestines aid in the digestion of food by producing enzymes such as cellulase, galactosidase, and glucosidase. 3. Intestinal flora also help in the metabolism of steroids. The healthy fetus is normally sterile until birth, following the rupture of the bag of water. Once born, the newborn normal flora is derived from the mother’s genital tract during delivery, from the skin and respiratory tract of individuals who handled the newborn, and from the environment. There are certain body tissues and fluids that are normally sterile. Body fluids that are sterile include the cerebrospinal fluid (CSF), synovial fluid, and blood. In the blood, there may be low transient bacteremia brought about by physiologic trauma. The sterile tissues include the urinary bladder, uterus, fallopian tubes, middle ear, and paranasal sinuses. Presence of bacteria in these tissues and body fluids may lead to serious infections in these areas. For example, bacteria in the CSF can gain entry into the central nervous system, leading to a potentially fatal encephalitis. Normal Flora on Different Sites of the Body Skin The skin is the part of the human body that is in constant contact with the environment, making it the most exposed to microorganisms. There are certain factors that eliminate non resident flora from the skin, namely: (1) lysozyme in the skin; (2) acidic pH of the skin due to sweat; (3) free fatty acids in sebaceous secretions; and (4) the constant sloughing off of the skin. The normal flora of the skin consists mainly of bacteria and fungi. The microorganisms vary depending on the region of the skin. The skin may be divided into three regions: (1) axilla, perineum, and toe webs; (2) hand, face, and trunk; and (3) upper arms and legs. Skin of the axilla, perineum, and toe webs is characterized by having higher moisture levels, higher body temperature, and higher levels of surface lipids. These regions have more microorganisms compared to the others and are predominantly inhabited by gram negative bacilli. Dry sites (e.g., hands, forearms, feet, legs) have diverse flora because of their exposure to the environment. Predominant flora in these areas include Staphylococcus epidermidis and Staphylococcus hominis  Normal Flora of the Human Body 53 Most microorganisms in the skin are found in its superficial layers (stratum corneum) and hair follicles. Anaerobes inhabit the deeper structures and layers of the skin, such as hair follicles, sebaceous glands, and sweat glands. Table 5.1 summarizes the various microorganisms that inhabit the skin. Table 5.1 Normal flora found on the skin Organism Remarks Staphylococcus epidermidis Major skin inhabitant, comprising approximately 90% of resident aerobic flora Staphylococcus aureus Most commonly found in nose and perineum; in the nose, number varies with age (greater in newborns than in adults) Micrococci (Micrococcus luteus) Accounts for 20%–80% of micrococci in the skin Diphtheroids (Coryneforms) Classified into: lipophilic (common in axilla)