Methodological handbook was worked out in accordance with standard study program on «Microbiology and virology» on speci
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MINISTRY OF EDUCATION AND SCIENCE OF THE REPUBLIC OF KAZAKHSTAN AFTER AL-FARABY KAZAKH NATIONAL UNIVERSITY
I.S. Savitskaya, A.S. Kistaubayeva, L.V. Ignatova, I.V. Blavachinskaiya
MICROBIOLOGY AND VIROLOGY Educational manual
Almaty «Kazakh University Press» 2014 1
UDK 579:578 BBK 28.4+28.3 M 76 Recommended by the Academic Council of the Faculty biological and Editorial and Publishing Council of KazNU after al-Faraby
Reviewers: Doctor biological scince, Professor J.A. Siniyavskyi Doctor biological scince, Professor A.A. Zhubanova Doctor biological scince, Professor K.H. Zhumatov
M 76 Microbiology And Virology: educational manual / I.S. Savitskaya, A.S. Kistaubayeva, L.V. Ignatova, I.V. Blavachinskaiya. − Almaty: Kazakh University Press, 2014. ‒ 158 c. ISBN 978-601-04-0322-21 Methodological handbook was worked out in accordance with standard study program on «Microbiology and virology» on specialty «5В070100 – Biotechnology». Methodological handbook «Microbiology and virology. Lectures and Labs» was written in English and is to be used not only by university students, master degree students, teachers and science staff, who study and conduct the course «Microbiology and virology» in English language, but by everybody interested in the problems of biology and virology.
UDK 579:578 BBK 28.4+28.3 © Savitskaya I.S. at all, 2014 © KazNU after al-Faraby, 2014
CONTENTS Preface .............................................................................................5 Short Lectures Of The Course "Microbiology And Virology" ...........................................................6 Chapter I INTRODUCTION TO MICROBIOLOGY .................................6 Chapter II BACTERIAL CELL STRUCTURES & FUNCTIONS PROKARYOTIC AND EUKARYOTIC CELLS, AND THEIR DIFFERENCES .............................................................................15 Eukaryotic Cells ...............................................................................15 Prokaryotic Cells ..............................................................................17 Structure Of The Bacterial Cell Biochemical Composition ................................................................19 Cellular Architecture Of Procaryotic Cell ........................................21 Surface Structures Of The Bacterial Cell. The Cell Envelope ............................................................................23 Internal Structures Of The Bacterial Cell. Internal Structures .........34 Chapter III GENERATION AND DIFFERENTIATION OF THE PROKARYOTES .................................................................. 40 Asexual reproduction .......................................................................40 Generation Time (Doubling Time) ...................................................42 Sexual reproduction or genetic recombination ....................................... 43 Reproduction Forms .........................................................................51 Dormant (resting) form of bacteria ..................................................52 Cells with specialized metabolic functions – nitrogen Fixation ............................................................................................57 Levels Of Cellular Organization Of Eubacteria. Microbial Communities. Biofilm ......................................................................57 3
Chapter IV SYSTEMICS AND TAXONOMY OF MICROORGANISMS....63 Taxonomy of Microorganisms .........................................................65 Chapter V VIROLOGY. GENERAL CHARACTERISTIC OF VIRUSES ..................................................................................73 Viral Structure ..................................................................................75 Shapes Of Viruses ............................................................................76 Unconventional agents .....................................................................77 Multiplication Of Viruses .................................................................79 Integrative Infection .........................................................................81 Bacterial Viruses – Bacteriophages ..................................................82 Methodical Instructions To Laboratory Works On Discipline "Microbiology And Virology" .......................................................... 86 Student Independent Work ...............................................................137 Test yourself .....................................................................................144 Case-Study 1. To Develop The Laboratory Regulations Of Obtaining Enrichment Cultures Of Clostridium Groups Of Bacteria...................................................147 Exam Questions ...............................................................................152 Print and online resources ................................................................155
PREFACE Microbiology is one of the most applied of all the biological sciences. Since this fact the discipline «Microbiology and virology» is included in the cycle “Basic disciplines” of study curriculum on specialty «5В070100 – Biotechnology”. Methodological handbook was worked out in accordance with standard study program on «Microbiology and virology» on specialty «5В070100 – Biotechnology». Methodological handbook «Microbiology and virology. Lectures and Labs» was written in English and is to be used not only by university students, master degree students, teachers and science staff, who study and conduct the course «Microbiology and virology» in English language, but by everybody interested in the problems of biology and virology. In the lectures given in this study handbook is included the main information about features and properties of microorganisms and viruses; their role in the nature and human life; principles of their classification; structures of procaryotic cells; features of growth and reproduction of microorganisms and viruses. Logically proposed information, enough large number of diagrams and tables makes it easy to percept and comprehend theoretical section of the work. The instructions of performing of all labs on microbiology and virology are given in the study handbook in details. They include such basic techniques as мicroscopic examination technique; staining methods; detection of inclusions, reserve nutrients, capsule, endospores, motility. They also contain information for studying morphology of bacteria, actinomycetes, and eukaryotic microorganisms. It is also important to state that the authors of the handbook conduct lectures on microbiology,virusology and biotechnology and Blavachinskaiya I.V. is a teacher of English of Biology faculty in Al-Farabi University. Thus all the materials for students are regularly renovated and adapted.
SHORT LECTURES OF THE COURSE "MICROBIOLOGY AND VIROLOGY"
INTRODUCTION TO MICROBIOLOGY
Microorganisms and Microbiology. Features of microorganisms. Microbiology is one of the most applied of all the biological sciences which did not exist as a true science before the later part of 19th century. Microbiology is the study of microorganisms. Microorganisms: − a large and diverse group of microscopic organisms that exist as single cells or cell clusters also include non-cellular life forms such as viruses and prions. Microbiology is the study of microorganisms that is the organism which of microscopic dimensions. These organisms are too small to be clearly perceived by the unaided human eye. Microorganisms are living organisms that are usually too small to be seen clearly with the naked eye. Organism with a diameter of 1 mm or less is microorganisms and fall into the broad domain of microbiology. Because most of the microorganisms are only a few thousands of mm in size, they can only be seen with the aid of microscope. Due to the invisibility of microbes the naked eye and the need for special techniques to study them, microbiology was the last of the three 6
major divisions in biology (the other two are botany and zoology) to develop.
Figure 1 - Extension of microorganisms
At present there is general agreement to include five major groups as microorganisms. The subdivisions are: 1. Bacteriology (Bacteria) 2. Mycology (Fungi) 3. Phycology (Algae) 4. Protozoology (Protozoa) 5. Virology (Viruses) Microorganisms are present everywhere on Earth which includes humans, animals, plants and other living creatures, soil, water and atmosphere. Microbes can multiply in all three habitats except in the atmosphere. Together their numbers far exceed all other living cells on 7
this planet. Microorganisms are relevant to all of us in a multitude of ways. Factors ubiquitous microorganisms: − Small cell size − easy to transport; − High the rate of reproduction; − Easy adaptation and greater resistance to environmental factors; − A wide variety of power sources and methods of obtaining energy. The influence of microorganism in human life is both beneficial as well as detrimental also.
Figure 2 − Importance of microorganisms
Microorganisms are involved in the global cycle of elements. Microorganisms are indispensable components of our ecosystem. Microorganism play an important role in the recycling of organic and inorganic material through their roles in the C, N and S cycles, thus playing an important part in the maintenance of the stability of the biosphere. They are also the source of nutrients at the base of all 8
ectotropical food chains and webs. In many ways all other forms of life depend on the microorganisms. Nitrogen Fixation − outside the body, good bacteria play an important role in helping plant roots absorb nitrogen from the air. Plants with these nitrogen-fixing bacteria are able to make their own fertilizer and populate soils that are deficient in nutrients. In the absence of microorganisms, higher life forms would never have arisen and could not now be sustained (oxygen). Humans, plants, animals are closely tied to microbial activities for the recycling of key nutrients and for degrading organic matters. No other life forms approach the importance of microorganisms in supporting and maintaining all life on Earth. Microorganisms are used to clean the environment from various natural and anthropogenic contaminants. The use of microbes to reduce or degrade pollutants, industrial waste and household garbage, a new area referred to as bioremediations being given substantial importance these days. A common edible mushroom contain a protein lectin that can stop cancer cell multiplication. This discovery of 21st century could lead to new targets for therapy. Similarly an endophytic Fungus Taxomyces andreanae is being used to produce taxol, an antitumor diterpenoid used in the treatment of some cancers. Taxon was originally obtained from the bark of Taxus brevifolia. Due to the activity of microorganisms we can take a number of important manufactures necessary for people (baking, brewing, wine making, receiving milk products, the production of different individual chemicals, antibiotics, hormones, enzymes, etc.). Particular attention is paid to the microorganisms used in biotechnology industries for the production of valuable products (antibiotics for human and veterinary medicine, enzymes, alcohols, organic and amino acids, vitamins, hormones, etc.). The objects of biotechnology are viruses, bacteria, fungi (micromycetes and macromycetes), protozoan organisms, cells, tissues, plants, animals and humans, some nutrients and functional similarities with substances (enzymes, lectins, nucleic acids, etc.). At present time, the microorganisms are really the main objects of biotechnology. 9
Cells of microorganisms − a kind of biofactory which in the course of its activity produces a variety of valuable products (proteins, fats, carbohydrates, vitamins, amino acids, antibiotics, hormones, antibodies, antigens, enzymes, alcohols, etc.). These products are urgently necessary in human life, they are not yet available for non biotechnological production because of the complexity of the technology or economical inexpedience, especially in large-scale production. Microorganism cells are reproduced very quickly, which allows for a relatively short time to artificially increase the relatively low-cost raw materials on an industrial scale large amounts of microbial biomass. The highest rate of metabolism and plasticity, the unique ability to perceive and express foreign genes responsible for production of practically useful compounds isolated microorganisms among other objects of biotechnology. So, the influence of microorganism in human life is both beneficial as well as detrimental also. For example microorganisms are required for the production of bread, cheese, yogurt, alcohol, wine, beer, antibiotics (e.g. penicillin, streptomycin), vaccines, vitamins, enzymes and many more important products. Microorganisms can serve as tools and model systems for other disciplines, such as genetic engineering. But, many microorganisms are pathogens of humans, animals, plants, and also cause spoilage of food products and various industrial materials. Microorganisms also have harmed humans and disrupted societies over the millennia. Microbial diseases undoubtedly played a major role in historical events such as decline of the Roman empire and conquest of the new world. It was in the year 1347 when plague or 'black death' struck Europe and within 4 yers killed 25 million people that is 1/3 of the population. This dreaded disease is believed to have changed european culture and prepared the way for renaissance. Pathogenic bacteria are responsibile for cholera, syphilis, anthrax, leprosy, and the bubonic plague. Respirtory infections inlcuding tubercolis are caused by bacteria. Examples of diseases caused by viruses include the common cold, influenza, cold sores, and chicken pox. Ebola, Aids, and Avian Influenza are caused by viruses. 10
Microbiology Complex of biological sciences studying:
Figure 3 − Tasks and subject matter of microbiology
But, Indigenous Flora − microbes that inhabit our bodies. The microorganisms that live in our digestive tracks help stimulate the digestive system, and prevent the bad bacteria from colonizing our gut. In addition to health threat from some microorganisms many microbes spoil food and deteriorate materials like iron pipes, glass lenses, computer chips, jet fuel, paints, concrete, metal, plastic, paper and wood pilings. So, Microbes may be classified as Friends or Enemies. Microbiology is one of the largest and most complex of the biological sciences as it deals with many diverse biological disciplines. In addition to studying the natural history of microbes, it deals with every aspects of microbe-human and environmental interaction. 11
These interactions include: ecology, genetics, metabolism, infection, disease, chemotherapy, immunology, genetic engineering, industry and agriculture. The branches that come under the large and expanding umbrella of microbiology are categorized into pure and applied sciences. The branch microbiology has two major aspects: the theoretical and the applied.
Figure 4 − Microbiology research areas
Some of the most accessible research tools. Excellent model systems for understanding basic life processes. Industrial Microbiology − Concerned with industrial uses of microbes in production of alcoholic beverages, vitamins, NH2-acids, enzymes, antibiotics and other drugs. Agricultural Microbiology − Study of relationships of microbes and crops and on control of plant diseases and improvement of yields. Food Microbiology − Deals with interaction of microorganisms and food in relation to food processing, food spoilage, food borne disease and their prevention 12
Dairy Microbiology − Deals with pro-duction and maintenance in quality control of dairy products. Aquatic Microbiology − Study of microorganisms found in fresh estuarine and marine waters. Air Microbiology − Deals with the role of aerospora in contamination and spoilage of food and dissemination of plant and animal diseases through air. Exomicrobiology − Deals with exploration for microbial life in outer space. Medical Microbiology − Causative agents of disease, diagnostic procedure for identification of causative agents, preventive measures. Public Health Microbiology − Concerns with monitoring, control and spread of diseases in communities.
Theoretical aspects of microbiology Field
Study of detailed structure of microorganism
Concerned with classification, naming and identification of microorganism
Study of metabolism of microbes at cellular and molecular levels
Study of interrelationships between microbes and environment
Microbial genetics and Molecular Biology
Study of genetic material, structure and function and biochemical reactions of microbial cells involved in metabolism and growth
Figure 5 − Major Fields Theoretical Microbiology
Biotechnology − Scientific manipulation of living organisms especially at molecular and genetic level to produce useful products. Agricultural Microbiology − Study of relationships of microbes and crops and on control of plant diseases and improvement of yields. Microorganisms are relevant to all of us in a multitude of ways. Microbiology today is a dynamic science with ramifications in virtually all aspects of human life.
BACTERIAL CELL STRUCTURES & FUNCTIONS PROKARYOTIC AND EUKARYOTIC CELLS, AND THEIR DIFFERENCES The living organisms are divided into two groups on the basis of their cellular stucture. The two groups are known as prokaryotic and eukaryotic. There are two types of cellular microorganisms (prokaryotes and eukaryotes). All prokaryotes are microorganisms, but only some eukaryotes are microorganisms. We have microorganisms of non-cellular, pre-cellular (such as viruses − the world of Vira, viroids, prions) and cellular shapes (bacteria, archaea, microscopic fungi, algae and protozoa). Currently, it is being developed a classification of all living beings, in which it is identified three domains (worlds): Bacteria, Archae and Eukarya on the basis of the nucleotide sequence of 16S rRNA. The domain Eukarya includes all eukaryotic organisms as unicellular, so multicellular ones. Domain Bacteria includes prokaryotic organisms that are typical signs of bacteria, in particular cellular membranes containing peptidoglycane. Archaebacteria that are different from bacteria and eukaryae in a number of significant features belong to a relatively new domain Archae. EUKARYOTIC CELLS A eukaryotic cell is larger and more complex than a prokaryotic cell and found in animals, plants, algae, fungi, and protozoa. When you look at a eukaryotic cell with a microscope you'll notice a highly organized structure of organelles that are bound by a membrane. Each organelle performs a specialized function for the cell's metabolism. Eukaryotic cells also contain a membrane-bound nucleus where the cell's DNA is organized into chromosomes. Depending on the organism, a eukaryotic cell may contain external projections called flagella and cilia. These projections are used for moving substances along the cell's surface or for moving the entire cell. Flagella move the cell in a wavelike motion within its environment. Cilia move substances along the cell's surface and also 15
aid in movement of the cell. Flagella and cilia are comprised of axoneme microtubules. An axoneme microtubule is a long, hollow tube made of protein called a tubulin. Many eukaryotic cells have a cell wall. The composition of the cell wall differs with each organism. For example, the cell walls of many fungi are composed of chitin cellulose. Chitin is a polysaccharide, which is a polymer of N-acetylglucosamine (NAG) units. The cell wall of other fungi is made of cellulose, which is also a polysaccharide. Cellulose is also found in the cell wall of plants and many algae. Yeast has a cell wall composed of glucan and mannan, which are two polysaccharides. In contrast, protozoa have no cell wall and instead have a pellicle. A pellicle is a flexible, proteinaceous covering. Eukaryotic cells of other organisms (such as animals) that lack a cell wall have an outer plasma membrane that serves as an outside cover for the cell. The outer plasma membrane has a sticky carbo hydrate called glycocalyx on its surface. Glycocalyx is made up of covalently bonded lipids and proteins in order to form glycolipid and glycoprotein in the plasma membrane. Glycolipid and glycoprotein anchor the glycocalyx to the cell, giving the cell strength and helping the cell to adhere to other cells. Glycocalyx is also a molecular signature used to identify the cell to other cells. White blood cells use this to identify a foreign cell before destroying it. A eukaryotic cell lacks peptidoglycan, which is critical in fighting bacteria with antibiotics. A bacterium is a prokaryotic cell. Peptidoglycan is the framework of a prokaryotic cell's cell wall. Antibiotics such as penicillin attack peptidoglycan resulting in the destruction of the cell wall of a bacterium. Eukaryotic cells invaded by the bacterium remain unaffected because eukaryotic cells lack peptidoglycan The cytoplasm of a eukaryotic cell contains cytosol, organelles, and inclusions, which is similar to the cytoplasm of the prokaryotic cell. Eukaryotic cytoplasm also contains a cytoskeleton that gives structure and shape to the cell and assists in transporting substances throughout the cell. The nucleus of a eukaryotic cell contains DNA (hereditary information) and is contained within a nuclear envelope. DNA is also 16
found in the mitochondria and chloroplasts. Depending on the organism, there can be one or more nucleoli within the nuclear envelope. A nucleolus (little nucleus) is the site of ribosomal RNA synthesis, which is necessary for ribosomes to function properly. In the nucleus, the cell's DNA is combined to form several proteins called histones. The combination of about 165 pairs of DNA and nine molecular of histones make up the nucleosome. When a eukaryotic cell is not in the reproduction phase, the DNA and its proteins look like a threaded mass called chromatin. When the cell goes through nuclear division, the strands of chromatin condense and coil together, producing rod-shaped bodies called chromosomes. A eukaryotic cell uses a method of cell division during reproduction called mitosis. This is the formation of two daughter cells from a parent cell. The mitochondrion is an organelle that is comprised of a series of folds called cristae that is responsible for the cell's energy production and cellular respiration. Chemical reactions occur within the center of the mitrochondrion, called the matrix; it is filled with semifluid in which adenosine triphosphate (ATP) is produced. ATP is the energy molecule in the cell. The mitochondrion is the powerhouse of the cell. Eukaryotic cells of green plants and algae contain plastids, one of which is chloroplast. Chloroplasts are organelles that contain pigments of chlorophyll and carotenoids used for gathering light and enzymes necessary for photosynthesis. Photosynthesis is the process that converts light energy into chemical energy. The pigment is stored in membranous sacs called thylakoids that are arranged in stacks called grana. PROKARYOTIC CELLS A prokaryotic cell is a cell that does not have a true nucleus. The nuclear structure is called a nucleoid. The nucleoid contains most of the cell's genetic material and is usually a single circular molecule of DNA. Karyo- is Greek for "kernel." A prokaryotic organism, such as a bacterium, is a cell that lacks a membrane-bound nucleus or 17
membrane-bound organelles. The exterior of the cell usually has glycocalyx, flagellum, fimbriae, and pili. The main difference between these two cell types is that Prokaryotic cells do not have a nuclear membrane. Instead, their genetic material (DNA) is found within a dense region of the cytoplasm called the nucleoid. Membrane-bound organelles are absent. Prokaryotic cells lack membranous structures such as an endoplasmic reticulum, a Golgi apparatus, lysosomes, peroxisomes and mitochondria. Table 1 Differences between Prokaryotic and Eukaryotic Cells Characteristics Cell wall Plasma membrane Glycocalyx Flagella Cytoplasm Membrane-bound organelles Ribosomes Nucleus Chromosomes Cell division Sexual reproductions
Prokaryotic Cells Include peptidoglycan Chemically complex No carbohydrates No sterols Contain a capsule or a slime layer Protein building blocks No cytoplasmic streaming
Eukaryotic Cells Chemically simple
Contain carbohydrates Contain sterols Contained in cells that lack a cell wall Multiple microtubules Contain cytoskeleton Contain cytoplasmic streaming None Endoplasmic reticulum Golgi complex Lysomes Mitochondria Chloroplasts 70S 80S Ribosomes located in Organelles are 70S No nuclear membrane No Have a nucleus Have a nuclear nucleoli membrane Have a nucleoli 10-100 0.2-2.0 mm in diameter mm in diameter Single circular chromosome Multiple linear chromosomes Have No histones histones Binary fission Mitosis No meiosis Meiosis DNA transferred in fragments
So, Prokaryotic cells lack internal membranes. 18
The main similarities between prokaryotic and eukaryotic cells is survival of the organism and carrying out same process of life. There are many other similarities between prokaryotic and eukaryotic cells. − prokaryotic and eukaryotic cells have in common the genetic material, that is, presence of DNA − prokaryotic and eukaryotic cells have in common the presence of RNA − prokaryotic and eukaryotic cells, both have a cell membrane covering them − prokaryotic and eukaryotic cells similarities are seen in their basic chemical structures. Both are made up of carbohydrates, proteins, nucleic acid, minerals, fats and vitamins − prokaryotic and eukaryotic cells have in common ribosomes, that are the structures that make up proteins − prokaryotic and eukaryotic cells regulate the flow of nutrients and waste matter that enters and leaves the cells − prokaryotic and eukaryotic cells both carry out the basic life process, that is, photosynthesis and reproduction. − prokaryotic and eukaryotic cells need energy supply to survive − prokaryotic and eukaryotic cells both have ''chemical noses'' that keeps them updated and aware of all the reactions that occur within them and in the surrounding environment. Some prokaryotic and eukaryotic cells have in common glycocaly. This is a sugar based structure that is sticky and helps the cells in anchoring to each other, thus, giving them some protection. Prokaryotic and eukaryotic cells, both have lipid bilayer known as the plasma layer that forms the boundary between the inside and outside of the cell. In short, prokaryotic and eukaryotic cells are the smallest units of life without which their would be no life. STRUCTURE OF THE BACTERIAL CELL BIOCHEMICAL COMPOSITION A bacterial cell has five essential structural components: chromosome (DNA), ribosomes, cell membrane, cell wall, and some 19
sort of surface layer which may be an inherent part of the cell wall. The biochemical composition of these structures are macromolecules such as DNA, RNA, protein, polysaccharide, phospholipid, or some combination there of. The macromolecules are made up of primary subunits such as nucleotides or amino acids (Table 2). It is the arrangement or sequence in which the subunits are put together, called the primary structure of the molecule, that often determines the exact properties that the macromolecule will have. Thus, at a molecular level, the primary structure of a macromolecule determines its function or role in the cell, and the functional aspects of bacteria are related directly to the structure and organization of the macromolecules in their cell make-up. Diversity within the primary structure of these molecules accounts for the diversity that exists among procaryotes. Table 2 Macromolecules that make up cell material Macromolecule
Where found in cell
Flagella, pili, cell walls, cytoplasmic membranes, ribosomes, cytoplasm
Capsules, inclusions (storage), cell walls
Nucleic Acids (DNA/RNA)
DNA: nucleoid (chromosome), plasmids rRNA: ribosomes; mRNA, tRNA: cytoplasm
The overall chemical composition of a bacterial cell may be inferred by chemical analysis of a bacterium such as E. coli, which is displayed in Table 3. The macromolecules comprise about 96 percent of the dry weight of the cell ("dry weight" is the remaining material after all water is removed). Small molecules and inorganic 20
ions, which are constituents of the cytoplasm, comprise the remaining 4 percent. Table 3 Molecular composition of E. coli under conditions of balanced growth Molecule
Percentage of dry weight
Small molecules: precursors, metabolites, vitamins, etc Inorganic ions
Total dry weight
CELLULAR ARCHITECTURE OF PROCARYOTIC CELL At one time it was thought that bacteria were essentially "bags of enzymes" with no inherent cellular architecture. The development of the electron microscope in the 1950-s revealed the distinct anatomical features of bacteria and confirmed the suspicion that they lacked a nuclear membrane. Structurally, a bacterial cell has three architectural regions: appendages (attachments to the cell surface) in the form of flagella and pili or fimbriae; a cell envelope consisting of a capsule, cell wall and plasma membrane; and a cytoplasmic region that contains the cell chromosome (DNA) and ribosomes and various sorts of inclusions. 21
Table 5 Characteristics of typical bacterial cell structures Structure Flagella
Function(s) Swimming movement
Predominant chemical composition Protein
Pili Sex pilus Common fimbriae
Capsules (includes "slime layers" and glycocalyx)
Mediates DNA transfer during conjugation Attachment to surfaces; protection against phagotrophic engulfment Attachment to surfaces; protection against phagocytic engulfment, occasionally killing or digestion; reserve of nutrients or protection against desiccation
Protein Protein Usually polysaccharide; occasionally polypeptide
Cell wall Gram-positive bacteria
Prevents osmotic lysis of cell protoplast and confers rigidity and shape on cells
Peptidoglycan prevents osmotic lysis and confers rigidity and shape; outer membrane is permeability barrier; associated LPS and proteins have various functions
Plasma membrane Ribosomes
Permeability barrier; transport of solutes; energy generation; location of numerous enzyme systems Sites of translation (protein synthesis)
Peptidoglycan (murein) complexed with teichoic acids Peptidoglycan (murein) surrounded by phospholipid proteinlipopolysacchari de "outer membrane" Phospholipid and protein RNA and protein Highly variable; carbohydrate, lipid, protein or inorganic
Often reserves of nutrients; additional specialized functions
Genetic material of cell
Extrachromosomal genetic material
SURFACE STRUCTURES OF THE BACTERIAL CELL. THE CELL ENVELOPE The cell envelope is a descriptive term for the several layers of material that envelope or enclose the protoplasm of the cell. The cell protoplasm (cytoplasm) is surrounded by the plasma membrane, a cell wall and a capsule. The cell wall itself is a layered structure in Gram-negative bacteria. All cells have a plasma membrane, which is the essential and definitive characteristic of a "cell". Almost all procaryotes have a cell wall to prevent damage to the underlying protoplast. Outside the cell wall, foremost as a surface structure, may be a polysaccharide capsule or glycocalyx. Cell Wall. The prokaryotic cell's cell wall is located outside the plasma membrane and gives the cell its shape and provides rigid structural support for the cell. The cell wall also protects the cell from its environment. Pressure within the cell builds as fluid containing nutrients enters the cell. It is the job of the cell wall to resist this pressure the same way that the walls of a balloon resist the build-up of pressure when it is inflated. If pressure inside the cell becomes too great, the cell wall bursts, which is referred to as lysis. The cell wall of bacteria deserve special attention for several reasons: 1. They are an essential structure for viability, as described above. 2. They are composed of unique components found nowhere else in nature. 3. They are one of the most important sites for attack by antibiotics. 4. They provide ligands for adherence and receptor sites for drugs or viruses. 5. They cause symptoms of disease in animals. 6. They provide for immunological distinction and immunological variation among strains of bacteria. Most procaryotes have a rigid cell wall. The cell wall is an essential structure that protects the cell protoplast (the region bound by and including the membrane) from mechanical damage and from osmotic rupture or lysis. Bacteria usually live in relatively dilute environments such that the accumulation of solutes inside the cell 23
cytoplasm greatly exceeds the total solute concentration in the outside environment. Thus, the osmotic pressure against the inside of the plasma membrane may be the equivalent of 10-25 atmospheres. Since the membrane is a delicate, plastic structure, it must be restrained by an outside wall made of porous, rigid material that has high tensile strength. Such a material is murein, the ubiquitous component of bacterial cell walls. The cell wall of many bacteria is composed of peptidoglycan, which covers the entire surface of the cell. Bacterial murein is a unique type of peptidoglycan. Peptidoglycan is a polymer of sugars (a glycan) cross-linked by short chains of amino acids (peptide). All bacterial peptidoglycans contain N-acetylmuramic acid, which is the definitive component of murein. Peptidoglycan is made up of a combination of pepide bonds and carbohydrates, either Nacetylmuramic acid, commonly referred to as NAM, or Nacetylglucosamine, which is known as NAG. The cell walls of archaea may be composed of protein, polysaccharides, or peptidoglycan-like molecules, but never do they contain murein. This feature distinguishes the bacteria from the archaea. The wall of a bacterium is classified in two ways: • Gram-positive. A gram-positive cell wall has many layers of peptidoglygan that retain the crystal of violet dye when the cell is stained. This gives the cell a purple color when seen under a microscope. In Gram-positive Bacteria (those that retain the purple crystal violet dye when subjected to the Gram-staining procedure) the cell wall is thick (15-80 nanometers), consisting of several layers of peptidoglycan. Running perpendicular to the peptidoglycan sheets are a group of molecules called teichoic acids (TA) which are unique; to the Gram-positive cell wall. Function of TA: antigenic determinant participate in the supply of Mg to the cell by binding Mg++ ; regulate normal cell division. So, the main futures of Gram positive bacterial cell wall : 1. Thick peptidoglycan layer 2. Pentaglycin cross linkage (interpeptide bridge) 3. Teichoic acid: ribitol TA & glycerol TA 4. All have lipoteichoic acid. 24
• Gram-negative. The cell wall does not retain the crystal of violet dye when the cell is stained. The cell appears pink when viewed with a microscope. In E.coli and other Gram-negative bacteria, the glycan backbone of peptidoglycan is made up of alternating molecules of N-acetylglucosamine (G) and Nacetylmuramic acid (M). The N-acetylmuramic acid (M) attaches a peptide side chain that contains the amino acids L-alanine, (L-ala), D-glutamate (D-glu), Diaminopimelic acid (DAP), and D-alanine (D-ala). The glycan backbone of the peptidoglycan molecule can be cleaved by an enzyme called lysozyme that is present in animal serum, tissues and secretions, and in phagocyte granules. The function of lysozyme is to lyse (rupture) bacterial cells as a defense against bacterial pathogens. Some Gram-positive bacteria are very sensitive to lysozyme and the enzyme is quite active at low concentrations. Lachrymal secretions (tears) can be diluted 1:40 000 and retain the ability to lyse certain bacterial cells. Gram-negative bacteria are less vulnerable to attack by lysozyme because their peptidoglycan is shielded by the outer membrane. The exact site of lysozymal cleavage is the beta 1,4 bond between N-acetylmuramic acid (M) and N-acetylglucosamine (G). In the Gram-negative Bacteria (which do not retain the crystal violet in the Gram-stain procedure) the cell wall is relatively thin (10 nanometers) and is composed of a single layer of peptideglycan surrounded by a membranous structure called the outer membrane. Table 5 Correlation of the Grams stain with cell wall properties of Bacteria Property
Thickness of wall
thick (20-80 nm)
thin (10 nm)
Number of layers
Peptidoglycan (murein) content
Teichoic acids in wall
Lipid and lipoprotein content
Sensitivity to Penicillin G
Sensitivity to lysozyme
The formation of the peptide bond between nearby chains of peptidoglycan is blocked by a group of antibiotics of the beta lactam class, which includes penicillin and cephalosporin and their relatives. Hence, the beta lactam antibiotics are effective against many bacteria because they prevent the assembly of the bacterial cell wall. In the presence of these antibiotics, the bacterium grows and synthesizes cell wall material but is unable to assemble the peptidoglycan sheet. Hence, the wall becomes progressively weaker and weaker until the cell lyses or ruptures. The cell wall is one of the most effective targets in bacterial cells for antibiotics because the wall material is unique to bacteria, and the animal taking the antibiotic totally lacks the target against which the antibiotic is directed. The most significant difference in the Gram-positive wall is the occurrence of an interpeptide bridge of amino acids that connects nearby side chains to one another. Assembly of the interpeptide bridge in Gram-positive murein is inhibited by the beta lactam antibiotics in the same manner as the interpeptide bond in Gram-negative murein. Gram-positive bacteria are more sensitive to penicillin than Gram-negative bacteria because the peptidoglycan is not protected by an outer membrane and it is a more abundant molecule. The Outer Membrane of Gram-negative Bacteria. The outer membrane is composed of phospholipids and lipopolysaccharides. The outer membrane of Gram-negative bacteria invariably contains a unique component, lipopolysaccharide (LPS or endotoxin), which is 26
toxic to animals. In Gram-negative bacteria the outer membrane is usually considered as part of the cell wall.Special interest as a component of the Gram-negative cell wall is the outer membrane, a discrete bilayered structure on the outside of the peptidoglycan sheet. For the bacterium, the outer membrane is first and foremost a permeability barrier, but primarily due to its lipopolysaccharide content, it possesses many interesting and important characteristics of Gram-negative bacteria. The outer membrane superficially resembles the plasma membrane except the outer face contains a unique type of Lipopolysaccharide (LPS) referred to by medical microbiologists as endotoxin because of its toxic effects in animals. Bacterial lipopolysaccharides are toxic to animals. When injected in small amounts LPS or endotoxin activates several host responses that lead to fever, inflammation and shock. Endotoxins may play a role in infection by any Gram-negative bacterium. The toxic component of endotoxin (LPS) is Lipid A. The O-specific polysaccharide may provide for adherence or resistance to phagocytosis, in the same manner as fimbriae and capsules. The O polysaccharide (also referred to as the O antigen) also accounts for multiple antigenic types (serotypes) among Gram-negative bacterial pathogens. Thus, E. coli O157 of the different antigenic types of E. coli and may be identified on this basis. So, the main futures of Gram-negative bacterial cell wall : 1. Thin peptidoglycan 2. Tetrapeptide cross linkage 3. A second membrane structure : protein+lipopolysaccharide = Lipoprotein Finally, the cell walls of most bacteria are made up of the polysaccharide murein (a polymer of amino sugars), which is found only in prokaryotes. Prokaryotic cells are found in bacteria and cynobacteria (blue-green algae). Cell wall-less forms. A few bacteria are able to live or exist without a cell wall. The Mycoplasma are a group of bacteria that lack a cell wall. Mycoplasma have sterol-like molecules incorporated into their membranes and they are usually inhabitants of osmoticallyprotected environments (contain a high concentration of external solute). 27
The study of Mycoplasma has become important in the understanding of chronic diseases. As both an extracellular and intracellular pathogen, a better understanding of the virulence mechanisms of mycoplasma will provide fresh understanding of how to diagnose and combat this pathogen. Mycoplasma pneumoniae is the cause of primary atypical bacterial pneumonia, known in the vernacular as "walking pneumonia". For obvious reasons, penicillin is ineffective in treatment of this type of pneumonia. Mycoplasma species are often found in research laboratories as contaminants in cell culture. Mycoplasmal cell culture contamination occurs due to contamination from individuals or contaminated cell culture medium ingredients. Mycoplasma are flask-shaped and are most likely descended from Gram-positive bacteria. Due to their seriously degraded genome they cannot perform many metabolic functions, such as cell wall production or synthesis of purines. As such stripped down organisms they are considered the perfect model of the minimalist cell. Meaning they are believed to contain the absolute minimum machinery necessary for survival and are considered the model organisms for the essential functions of all living cells. The Mycoplasma cell is built of a minimum set of organelles including a plasma membrane, ribosomes, and a highly coiled circular chromosome. Sometimes, under the pressure of antibiotic therapy, pathogenic streptococci can revert to cell wall-less forms (called spheroplasts) and persist or survive in osmotically-protected tissues. When the antibiotic is withdrawn from therapy the organisms may regrow their cell walls and reinfect unprotected tissues. As a part of their natural life cycle, bacteria can transform into a variety of forms. One of those phases is the L-form. L-form bacteria, also known as cell wall deficient bacteria, are a phase of bacteria that are very small and lack cell walls. Though the subject of a great deal of research over the last 100 years and implicated in a variety of diseases, L-forms remain largely misunderstood - or at the very least, underappreciated - by the medical research community. According to the Marshall Pathogenesis, L-forms are part of a metagenomic microbiota responsible for chronic disease. 28
Thus far, researchers have identified over 50 different species of bacteria capable of transforming into the L-form and it is likely that more species will be found in the coming years. L-form bacteria are pleomorphic, that is, they can change size and shape. During much of their lifetimes they are tiny, about 0.01 microns in diameter. Since they are smaller than viruses or fungal particles, they cannot be seen with a normal optical microscope. The small, individual forms of L-form bacteria are often referred to as coccoid bodies. Coccoid bodies sometimes group together, assuming the appearance of a string of pearls. Occasionally L-form bacteria break out of the cells. In the lab they can grow into long, thin biofilm filaments that can reach 60-70 microns in length. The biofilm filaments are composed of L-form bacteria and a protective protein sheath. For reasons still unknown, L-forms can also grow into large “giant” bodies. L-form bacteria, also known as cell wall deficient bacteria. Occasionally wall-less bacteria that can replicate are generated by the treatments (L forms). Two types of L-forms are distinguished: unstable L-forms, that are capable of dividing, but can revert to the original morphology; stable L-forms, that are unable to revert to the original bacteria. Protoplast - is all of the cell from the plasma membrane inward (i.e., the plasma membrane plus the cytoplasm). Spheroplast - The gram-negative equivalent of protoplasts. In spheroplasts cell wall is partially preserved. Flagella. Flagella are filamentous protein structures attached to the cell surface that provide the swimming movement for most motile prokaryotes. Prokaryotic flagella are much thinner than eukaryotic flagella; the diameter of a prokaryotic flagellum is about 20 nanometers, well-below the resolving power of the light microscope. The flagellar filament is rotated by a motor apparatus in the plasma membrane allowing the cell to swim in fluid environments. The flagellar apparatus consists of several distinct proteins: a system of rings imbedded in the cell envelope (the basal body), a hook-like structure near the cell surface, and the flagellar filament. The innermost rings, the M and S rings, located in the plasma 29
membrane, comprise the motor apparatus. As the M ring turns, powered by an influx of protons, the rotary motion is transferred to the filament which rotates thereby propelling the bacterium. Flagella attach to the cell by hook and basal body which consists of set(s) of rings and rods. Gram - : 2 sets of ring and rods, L, P, S, M rings and rods. Gram + : S, M rings and rods. Spirochete, has flagella on both ends and found around the cell’s circumference (endoflagella), which forms the axial filament that wraps around the cell membrane and an outer membrane (run between the spirochaetes outer membrane and their peptidoglycan layer). This particular structure is what allows the bacteria to move as the flagellum create a screw-like effect to propel the organism. Procaryotes are known to exhibit a variety of types of tactic behavior, i.e., the ability to move (swim) in response to environmental stimuli. For example, during chemotaxis a bacterium can sense the quality and quantity of certain chemicals in its environment and swim towards them (if they are useful nutrients) or away from them (if they are harmful substances). Other types of tactic response in procaryotes include phototaxis, aerotaxis and magnetotaxis. The occurrence of tactic behavior provides evidence for the ecological (survival) advantage of flagella in bacteria and other procaryotes. Flagella are made of protein and appear "whip-like." They are used by the prokaryotic cell for mobility. Flagella propel the microorganism away from harm and towards food in a movement known as taxis. Movement also occurs in response to a light or chemical stimulus. Movement caused by a light stimulus is referred to as phototaxis and a chemical stimulus causes a chemotaxis movement to occur. Flagella can exist in the following forms: − Monotrichous: One flagellum. − Lophotrichus: A clump of flagella, called a tuft, at one end of the cell. − Amphitrichous: Flagella at two ends of the cell. − Peritrichous: Flagella covering the entire cell. 30
− Endoflagellum: A type of amphitrichous flagellum that is tightly wrapped around spirochetes. A spirochete is a spiral-shaped bacterium that moves in a corkscrew motion. Borrelia burgdorferi, which is the bacterium that causes lyme disease, exhibits an endoflagellum. Differences in flagella among bacterial flagella varies by strains, serovars. Also contains globular proteins called flagellin. Flagellin can vary in structure and is used to identify some pathogenic bacteria serologically. The flagellar antigens are referred to as H antigens. Fimbriae and pili. Fimbriae are proteinaceous, sticky, bristle-like projections used by cells to attach to each other and to objects around them. Neisseria gonorrhoeae, the bacterium that causes gonorrhea, uses fimbriae to adhere to the body and to cluster cells of the bacteria. Pili are tubules that are used to transfer DNA from one cell to another cell similar to tubes used to fuel aircraft in flight. Some are also used to attach one cell to another cell. The tubules are made of protein and are shorter in length than flagella and longer than fimbriae. Fimbriae and pili are interchangeable terms used to designate short, hair-like structures on the surfaces of procaryotic cells. Like flagella, they are composed of protein. Fimbriae are shorter and stiffer than flagella, and slightly smaller in diameter. Generally, fimbriae have nothing to do with bacterial movement (there are exceptions, e.g. twitching movement on Pseudomonas). Fimbriae are very common in Gram-negative bacteria, but occur in some Archaea and Gram-positive bacteria as well. Fimbriae are most often involved in adherence of bacteria to surfaces, substrates and other cells or tissues in nature. In E. coli, a specialized type of pilus, the F or sex pilus, apparently stabilizes mating bacteria during the process of conjugation, but the function of the smaller, more numerous common pili is quite different. Common pili (often called fimbriae) are usually involved in specific adherence (attachment) of procaryotes to surfaces in nature. In medical situations, they are major determinants of bacterial virulence because they allow pathogens to attach to (colonize) tissues 31
and/or to resist attack by phagocytic white blood cells. For example, pathogenic Neisseria gonorrhoeae adheres specifically to the human cervical or urethral epithelium by means of its fimbriae; enterotoxigenic strains of E. coli adhere to the mucosal epithelium of the intestine by means of specific fimbriae; the M-protein and associated fimbriae of Streptococcus pyogenes are involved in adherence and to resistance to engulfment by phagocytes. Glycocalyx. Glycocalyx is a sticky, sugary envelope composed of polysaccharides and/or polypeptides that surround the cell. Glycocalyx is found in one of two states. It can be firmly attached to the cell's surface, called capsule, or loosely attached, called slime layer. A slime layer is water-soluble and is used by the prokaryotic cell to adhere to surfaces external to the cell. Glycocalyx is used by a prokaryotic cell to protect it against attack from the body's immune system. This is the case with Streptococcus mutans, which is a bacterium that colonizes teeth and excretes acid that causes tooth decay. Normally the body's immune system would surround the bacterium and eventually kill it, but that doesn't happen with Streptococcus mutans. It has a glycocalyx capsule state, which prevents the Streptococcus mutans from being recognized as a foreign microorganism by the body's immune system. Capsules. Most bacteria contain some sort of a polysaccharide layer outside of the cell wall polymer. In a general sense, this layer is called a capsule. A true capsule is a discrete detectable layer of polysaccharides deposited outside the cell wall. A less discrete structure or matrix which embeds the cells is a called a slime layer or a biofilm. A type of capsule found in bacteria called a glycocalyx or microcapsule is a very thin layer of tangled polysaccharide fibers on the cell surface. Capsules have several functions and often have multiple functions in a particular organism. Like fimbriae, capsules, slime layers, and glycocalyx often mediate adherence of cells to surfaces. Capsules also protect bacterial cells from engulfment by predatory protozoa or white blood cells (phagocytes), or from attack by antimicrobial agents of plant or animal origin. Capsules in certain soil bacteria protect cells from perennial effects of drying or 32
desiccation. Capsular materials (e.g. dextrans) may be overproduced when bacteria are fed sugars to become reserves of carbohydrate for subsequent metabolism. Capsules are considered protective structures. Various functions have been attributed to capsules including: 1) Adherence to surface, tissue or substrate in nature. 2) Capsules also often play a role in pathogenicity acting as virulence factors to protect cells from phagocytosis and/or complement-mediated killing. 3) Protect cells from perennial effects of drying or desiccation. 4) Capsular materials (e.g. dextrans) may be overproduced when bacteria are fed sugars to become reserves of carbohydrate for subsequent metabolism. 5) Important plant pathogens such as strains of Pseudomonas, Rhizobium, and Erwinia require capsules for pathogenicity. Some bacteria produce slime materials to adhere and float themselves as colonial masses in their environments. Other bacteria produce slime materials to attach themselves to a surface or substrate. Bacteria may attach to surface, produce slime, divide and produce microcolonies within the slime layer, and construct a biofilm, which becomes an enriched and protected environment for themselves and other bacteria. A classic example of biofilm construction in nature is the formation of dental plaque mediated by the oral bacterium, Streptococcus mutans. The bacteria adhere specifically to the pellicle of the tooth by means of a protein on the cell surface. The bacteria grow and synthesize a dextran capsule which binds them to the enamel and forms a biofilm some 300-500 cells in thickness. The bacteria are able to cleave sucrose (provided by the animal diet) into glucose plus fructose. The fructose is fermented as an energy source for bacterial growth. The glucose is polymerized into an extracellular dextran polymer that cements the bacteria to tooth enamel and becomes the matrix of dental plaque. The dextran slime can be depolymerized to glucose for use as a carbon source, resulting in production of lactic acid within the biofilm (plaque) that decalcifies the enamel and leads to dental caries or bacterial infection of the tooth. 33
INTERNAL STRUCTURES OF THE BACTERIAL CELL. INTERNAL STRUCTURES The cytoplasmic constituents of bacterial cells invariably include the procaryotic chromosome (nucleoid), ribosomes, and several hundred proteins and enzymes. The chromosome is typically one large circular molecule of DNA, more or less free in the cytoplasm. Procaryotes sometimes possess smaller extrachromosomal pieces of DNA called plasmids. The total DNA content of a procaryote is referred to as the cell genome. The cell chromosome is the genetic control center of the cell which determines all the properties and functions of the bacterium. During cell growth and division, the procaryotic chromosome is replicated in to make an exact copy of the molecule for distribution to progeny cells. Cytosol and Cytoplasm. The cytosol is the intracellular fluid of a prokaryotic cell that contains proteins, lipids, enzymes, ions, waste, and small molecules dissolved in water, commonly referred to as semifluid. Substances dissolved in cytosol are involved in cell metabolism. The cytosol also contains a region called the nucleoid, which is where the DNA of the cell is located. Unlike human cells, a prokaryotic microorganism has a single chromosome that isn't contained within a nuclear membrane or envelope. Cytosol is located in the cytoplasm of the cell. Cytoplasm also contains the cytoskeleton, ribosomes, and inclusions. Ribosomes. A ribosome is an organelle within the cell that synthesizes polypeptide. There are thousands of ribosomes in the cell. You'll notice them as the grainy appearance of the cell when viewing the cell with an electron microscope. A ribosome is comprised of subunits consisting of protein and ribosomal RNA, which is referred to as rRNA. Ribosomes and their subunits are identified by their sedimentation rate. Sedimentation rate is the rate at which ribosomes are drawn to the bottom of a test tube when spun in a centrifuge. Sedimentation rate is expressed in Svedberg (S) units. A sedimentation rate ref34
lects the mass, size, and shape of a ribosome and its subunits. It is for this reason why the sedimentation rates of subunits of a ribosome do not add up to the ribosome's sedimentation rate. Ribosomes in prokaryotic cells are uniquely identified by the number of proteins and rRNA molecules contained in the ribosome and by sedimentation rate. Prokaryotic ribosomes are relatively small and less dense than ribosomes of other microorganisms. For example, bacterial ribosomes have a sedimentation rate of 70S compared to the 80S sedimentation rate of a eukaryotic ribosome. Ribosomes and their subunits are targets for antibiotics that kill a bacterium by inhibiting the bacterium's protein synthesis. These antibiotics only kill cells that have a specific ribosome sedimentation rate. Cells with a different ribosome sedimentation rate are unaffected by the antibiotic. This enables the antibiotic to kill bacterium and not the body that is infected by the bacterium. The distinct granular appearance of procaryotic cytoplasm is due to the presence and distribution of ribosomes. The ribosomes of procaryotes are smaller than cytoplasmic ribosomes of eucaryotes. Procaryotic ribosomes are 70S in size, being composed of 30S and 50S subunits. The 80S ribosomes of eucaryotes are made up of 40S and 60S subunits. Ribosomes are involved in the process of translation (protein synthesis), but some details of their activities differ in eucaryotes, bacteria and archaea. Protein synthesis using 70S ribosomes occurs in eucaryotic mitochondria and chloroplasts, and this is taken as a major line of evidence that these organelles are descended from procaryotes. For example, erythromycin and chloramphenicol, popular antibiotics, kill bacteria whose subunits have a sedimentation rate of 50S. Streptomycin and gentamycin affect bacteria whose subunits have a 30S sedimentation rate. Nucleoid: Is the region of the cytoplasm that contains DNA. It is not surrounded by a nuclear membrane. DNA is always a closed 35
loop (i.e. a circular), and not associated with any proteins to form chromatin. • The chromosome of a typical bacterial cell is free in the cytoplasm and located in an area called the nuclear region or nucleoid. Prokaryotic cells contain DNA called the bacterial chromosome that is haploid (composed of a single DNA copy) and circular. • Most cells have a single chromosome, but some species may have two. Vibrio cholerae and Agrobacterium tumefaciens are examples of bacterial cells having two chromosomes. Bacterial chromosomes a single large circular double stranded DNA no histone proteins. The molecule is further twisted by the enzyme gyrase (topoisomerase). The only proteins associated with the bacterial chromosomes are the ones for DNA replication, transcription etc. Since the amount of DNA necessary to provide the genetic information necessary for the life of a bacterial cell far exceeds the actual volume of the cytoplasm, the chromosome is folded into loops about 50,000 to 100,000 bp in length. Bacterial Genome is consisted from 2 subsystems: 1. Bacterial chromosomes (part of the nucleoid) 2. Plasmids subsystem Plasmids are extrachromosomal rings of DNA found in some bacterial cells. These are considerably smaller than the bacterial chromosome, ranging in size from a few thousand to several million bp. Cell Properties Carried by Plasmids: 1. Drug resistance − Resistance (R) factors carry genes that allow a cell to be resistant to antibiotics or other antimicrobial compounds. 2. Fertility (F) factors are plasmids that carry genes necessary for a cell to transfer DNA to another compatible cell through a process called conjugation. 3. Production of antimicrobial agents − Bacteriocidin factors carry genes for the production of toxins that kill other bacteria that might compete with the cell for nutrients and space. 36
4. Metabolic activities - Examples of these genes are those that allow bacteria to utilize unique or unusual materials for carbon or energy sources. Many of the genes for these metabolic pathways are on transmissible plasmids. 5. Virulence Toxins − Enterotoxins (Escherichia coli, Vibrio cholerae), exfoliative toxin (Staphylococcus aureus), dermotoxin of Bacillus anthracis, the neurotoxin of Clostridium tetani, and the pesticide toxin of Bacillus thuringiensis. Adhesins − such as produced by the plasmids of Yersinia enterocolitica, Shigella flexneri, Escherichia coli strains that produce dysentery, and Yersinia pestis. Growth factors - Other plasmid borne virulence factors act to directly aid the bacteria in competing with mammalian host cells for growth. Col V of Escherichia coli contains genes for iron sequestering compounds. The acquisition of iron is essential for the survival of Escherichia coli in mammalian infections. Inclusions. An inclusion is a storage area that serves as a reserve for lipids, nitrogen, phosphate, starch, and sulfur within the cytoplasm. Scientists use inclusions to identify types of bacteria. Inclusions are usually classified as granules. Often contained in the cytoplasm of procaryotic cells is one or another of some type of inclusion granule. Inclusions are distinct granules that may occupy a substantial part of the cytoplasm. Inclusion granules are usually reserve materials of some sort. For example, carbon and energy reserves may be stored as glycogen (a polymer of glucose) or as polybetahydroxybutyric acid (a type of fat) granules. Polyphosphate inclusions are reserves of PO4 and possibly energy; elemental sulfur (sulfur globules) are stored by some phototrophic and some lithotrophic procaryotes as reserves of energy or electrons. Some inclusion bodies are actually membranous vesicles or intrusions into the cytoplasm which contain photosynthetic pigments or enzymes.
Table 6 Some inclusions in bacterial cells Cytoplasmic inclusions
many bacteria e.g. E. polyglucose coli
Polybetahydroxy- many bacteria butyric acid (PHB) e.g. Pseudomonas
Function reserve carbon and energy source
polymerized hydroxy butyrate
reserve carbon and energy source reserve phosphate; polyphosphate many bacteria linear or cyclical possibly a reserve of (volutin granules) e.g. Corynebacterium polymers of PO4 high energy phosphate phototrophic purple reserve of electrons and green sulfur (reducing source) in sulfur globules bacteria and elemental sulfur phototrophs; reserve lithotrophic colorless energy source in sulfur bacteria lithotrophs aquatic bacteria buoyancy (flotation) protein hulls or shells gas vesicles especially in the vertical water inflated with gases cyanobacteria column endospore-forming unknown but toxic to parasporal crystals bacilli protein certain insects (genus Bacillus) orienting and certain aquatic magnetite (iron magnetosomes migrating along geobacteria oxide) Fe 3 O4 magnetic field lines enzymes for many autotrophic carboxysomes autotrophic CO2 site of CO2 fixation bacteria fixation light-harvesting phycobilisomes cyanobacteria phycobiliproteins pigments light-harvesting lipid and protein and chlorosomes Green bacteria pigments and bacteriochlorophyll antennae
• Granule inclusion. Membrane-free and densely packed, this type of inclusion has many granules each containing specific substances. For example, polyphosphate granules, also known by the names metachromatic granules and volutin, have granules of polyphosphate that are used to synthesize ATP and are involved in 38
other metabolic processes. A polyphosphate granule appears red under a microscope when stained with methylene blue. • Vesicle inclusion. This is a protein membrane inclusion commonly found in aquatic photosynthetic bacteria and cyanobacteria such as phytoplankton, which suspends freely in water. These bacteria use vesicle inclusions to store gas that give the cell buoyancy to float at a depth where light, carbon dioxide (CO2), and nutrients all required for photosynthesis are available.
GENERATION AND DIFFERENTIATION OF THE PROKARYOTES Asexual reproduction. Bacteria generally reproduce very commonly by vegetative and asexual mode of reproduction. No sexual reproduction was reported by many microbiologist but electron microscopic study reports the unidirectional genetic recombination among certain bacteria. Reproduction in bacteria includes the following methods: Vegetative reproduction It includes the following types: − Binary fission; − Budding; − Cyst formation; − Gonidia or segmentation. Binary fission. The most common and most important mode of cell division which occur in bacteria when the environmental factor such as light, moisture, temperature are favourable is transverse binary fission. In this a single cell divides after developing a transverse septum (cross wall). Bacilli and spiral bacteria divide along the longitudinal axis of the cell while in coccus this division can be on any axis. Mesosomes play an important role in binary fission. Binary fission occurs in following steps: 1) Division of nuclear or genetic material : When bacterial cell attains its maximum size, it generally increase longitudinally. After that its circular DNA undergo replication and results into two DNA components. This replication is of semi conservative type. Now these two DNA moves to two opposite poles with the help of mesosomes. Because no spindle fibres are formed during this entire process, this division is known as amitosis. 2) Division of cytoplasm and septum formation : By the end of the division of nuclear material, the cytoplasmic membrane start invagination in the middle of the cell. This invagination is of centripetal direction and the inner most layer of cell wall also invaginate along with the L2 layer of plasma 40
membrane and it forms the septum initial. This invagination appears as constriction on the cell surface. This constriction continuously deepens and results into two daughter cells. Under favourable conditions a single binary fission is completed within 18-20 minutes. The generation time is the amount of time needed for a cell to divide. This varies among organisms and depends upon the environment they are in and the temperature of their environment. Some bacteria have a generation time of 24 hours, although the generation time of most bacteria is between 1 to 3 hours. Bacterial cells grow at an enormous rate. For example, with binary fission, bacteria can double every 20 minutes. In 30 generations of bacteria (10 hours), the number could reach one billion. It is difficult to graph population changes of this magnitude using arithmetic numbers, so logarithmic scales are used to graph bacterial growth. So, Bacteria normally reproduce by a process called binary fission: 1. The cell elongates and chromosomal DNA is replicated. 2. The cell wall and cell membrane pinch inward and begin to divide. 3. The pinched parts of the cell wall meet, forming a cross wall completely around the divided DNA. 4. The cells separate into two individual cells. Budding. Some bacteria reproduce by budding. A small outgrowth or bud emerges from the bacterium and enlarges until it reaches the size of the daughter cell. It then separates, forming two identical cells. Some bacteria, called filamentous bacteria (or actinomycetes), reproduce by producing chains of spores located at the tips of the filaments. The filaments fragment and these fragments initiate the growth of new cells. In this type of process the bacterial cell wall gets thinned at the end of cell, and it develops a cytoplasmic growth or protuberance which is covered by thin membrane. This structure is known as bud. It contain the part of genetic material of the parent cell. This out growth increases in size and develops a constriction at its base and ultimately it gets separated from parent cell. Now this 41
bud cell increases in size and attain the size of parent cell eg. Hypomicrobium. Cyst. Cyst formation is very rare in bacteria eg. Azotobacter. Cyst is a spherical cell which is formed under unfavourable conditions. Here the entire protoplast of the bacterial cells rounds up, shortened, constricts and separated from the cell wall. After that a thick cell wall is formed around this entire structure. This structure is known as cyst and on germination it gives rise to a single vegetative cell. Gonidia or Segmentation. Bacteria which produces extensive filamentous growth form gonidia. Here such bacterial filament produces small bacillary or coccoid cells each of which give rise to new growth. By Conidia. Many bacterial species viz Streptomyces produces small minute disc like rounded bodies in chains at the tip of their filamentous structure. They are formed in chains. The filamentous bearing conidia is known as conidiophores. Conidia are formed in basipetal succession. Each conidium germinate and produce new filamentous bacterium. By Oidiospores. The entire filamentous structure of certain species of Actinomyces become septate at its end. Thus numerous micro size reproductive units are formed which are known as oidiospore. Each oidium on germination give rise to new filamenttous bacteria. By Sporangiospores. Many branched filamentous bacteria become swollen at its terminal end and form sporangia. The cytoplasm of these sporangia divided to form small sized sporangiospore which on germination give rise to new filamenttous bacteria under favourable condition. GENERATION TIME (DOUBLING TIME) • The time required for a cell to divide or a population to double is known as the generation time. • Doubling time is the time it takes a bacterium to do one binary fission starting from having just divided. And ending at the point of having just completed the next division. 42
• Most bacteria have a doubling time of 1-3 hours, although some may be greater than 24 hours. • E. coli may have a doubling time of 20 minutes; get 20 generations in 7 hours, going from one cell to one million cells. • Bacterial division occurs according to a logarithmic progression (two cells, four cells, eight cells, etc.). But such a rapid rate of cell division cannot continue for a long time because the rapid increase in growth rate of bacterial population is inhibited due to following reasons: 1. Lack of space, food, water, oxygen other salts and accumulation of their own harmful waste products in the medium. 2. Environmental factors like light, temperature, moisture becomes unfavourable. 3. Death due to senescence and sometimes they are eaten by microscopic animals and viruses. Therefore survival rate of bacteria in nature is only 1%. SEXUAL REPRODUCTION OR GENETIC RECOMBINATION Unlike other prokaryote no true sexual reproduction is found in bacteria because: they lack sexual structures and no gametic fusion takes place. Karyogamy and meiosis is also absent in bacteria. Bacteria are haploid organisms. Gene transfer in bacterial cell do not produce zygotes but partial diploid called mero-zygotes. The original genome of recipient is named as endogenote. While the portion of DNA introduced from donor cell into recipient cell is called exogenome. So, Features of the bacterial recombination: − Gene transfer in bacterial cell do not produce zygotes but partial diploid called mero-zygotes. − The original genome of recipient is named as endogenote. − While the portion of DNA introduced from donor cell into recipient cell is called exogenote. However three different mechanism were later discovered for transferring gene or genetic material from one bacterial cell to another. These mechanisms in order of their discovery are: 1. Transformation 43
2. Conjugation 3. Transduction Transformation. Here there is transfer and expression of naked DNA from donor to recipient cell of bacteria takes place. Transformation was discovered by Fredrick Griffith (1928) an English microbiologist while working on two strains of Diplococcus pneumoniae (new name Streptococcus pneumoniae). He reported that DNA is a genetic material. Griffith used two strains of D. pneumoniae 1) bacteria with smooth and capsulated cell wall called as SIII (virulent or pathogenic). 2) Bacteria with rough and non capsulated cell wall called as RII avirulent or non pathogenic). SIII is found responsible for causing death in mice while RII does not cause pneumonia in mice. He carried out the following experiments on mice with these two strains. These are as follows : 1) No death of mice occur while injecting the RII bacterial strain in mice. 2) Death of mice occur while injecting the SIII strain of bacteria. 3) No death occur in mice while injecting it with the heat killed (heated at 85°C) SIII strain. 4) Death of mice occurred while injecting it with a combination of heat killed SIII strain + live RII strain of Diplococcus pnhenumoniae. The virulent SIII and non virulent RII strains were isolated from killed mice. Thus it was concluded that by receiving the genetic material of heat killed virulent SIII strain, the living RII strains produces the progeny of virulent and pathogenic SIII strain. Although Griffith was unable to identify the transforming principle. Based on the Griffith experiment O.T. Avery, Macleod and Maccarty performed further experiment in vitro system. They identified the transforming substance in 1944 as the polysaccharide present in the capsule of virulent strain SIII of pneumococcus which was absent in non virulent RII strain. Thus it was concluded that the DNA fragments isolated from dead mice get transformed into pathogenic and virulent SIII strain. This experiment explained that DNA is a genetic material. 44
For transformation the donor DNA must be single stranded and of specific size which transforms very easily in the RII strain of bacteria. During transformation the DNA fragment of SIII strain, bind and finally integrate in the bacterial chromosome of RII strain. The replaced portion of DNA of RII strain get destroyed. Three step of transformation: 1. High molecular weight DNA must bind to the cell surface. 2. The bound DNA is taken up through the cell membrane. 3. The donor DNA fragment is then integrated into the host chromosome or replicates autonomously as a plasmid. Transformation is entirely a laboratory procedure and never occur in nature and it can lead to increased virulence. It is found in various bacterial spp. viz Bacillus, Haemophilus, Salmonella, Rhizobium etc. In addition transformation is widely used in recombinant DNA technology. Conjugation. Conjugation is the commonest process of sexual reproduction in bacteria. In conjugation two parental cells physically contact between two genetically different cells of the same or closely related species and transfer their genetic material through a small tube like projection called conjugation tube. The genetic material from one cell (donor or male) is transferred to other (recipient or female). Lederberg and Tautum (1946) used two mutant of the E.coli strain K12. Both mutant require certain growth factors while culturing on minimal media. These bacterial strain are known as Auxotrophs. Strains A of E. coli require methionine and biotin for there growth. There genotype is Met- Bio- while strain B require Thrionine, Leucine and Thiamine for their growth. There genotype is Thr-, Leu-, Thi-. The culture of both, strain A and strain B of E. coli were mixed and centrifuged and washed to remove the previous culture media. After that they were cultured in minimal medium. It was found that both the strains were able to grow in minimal medium or it was concluded that both strains (previously auxotrophs) get converted into prototrophs or it was confirmed from the above experiment that the conjugation recombination of both auxotrophic strain viz. Strain A (met- bio-) and strain B (Thi-, Leu-, Thr-) 45
resulted in the formation of prototrophic strain (Met+ Bio+ Thr+ Leu+ Thi+). This experiment explain the genetic recombination by conjugation. Fertility factor or F factor in conjugation was first of all discovered by William Hayes (1950) in E. coli. He reported that in E.coli a plasmid is present in the form of fertility factor. E.coli is classified in two strains on the basis of presence and absence of F factor. They are as follows : 1) F+ strain bearing the fertility factor, also known as donor cell. It always bears sex pili or F pili on its surface. 2) F- strain lacking the fertility factor, also known as recipient cell. It lacks sex pili or F pili on its surface. 3) According to Hayes in E. coli this sexual recombination is unidirectional where the genetic material is transferred to the recipient cell from the donor cell. This process occurs in following steps : (a) In a group of bacterial cell the cells of two opposite strains viz. F+ and F- comes towards each other get attached by the sex pili. After that a tubular structure called as conjugation tube is formed. (b) The DNA of bacterial plasmid is double stranded which become single stranded by the activity of enzyme endonuclease which create a nick as a result a 5' to 3' end of single stranded DNA becomes free. (c) This single stranded DNA of the donor cell moves towards the recipient cell through the conjugation tube. The donor DNA moves by its 5' end into the recipient cell. (d) The conjugation is completed after the transfer of single stranded DNA into the recipient cell. As a result both F+ and F- cells are separated. (e) The single stranded plasmid DNA of donor cell combine with the recipient DNA strand with the help of enzyme ligase. (f) The single stranded DNA of donor and recipient cell synthesize its complementary strand and becomes double stranded. Thus the recipient F- strain change into F+ donor strain. Higt frequency recombination or HFR tranver. Jacob & Wollman (1951) reported, when F+ plasmid of donor reaches to the 46
F- plasmid of recipient cell in the newly formed F+ cell (previously F- recipient cell) this plasmid occur in two stages either it lie independently in the cytoplasm of F+ cell or it get combined with the bacterial chromosome. This later stage where the F+ factor combines with the bacterial chromosome is known as episome. The term episome was used by Lederberg et al. (1952). This type of bacterial cell convert into high, reproductive ability donor or male cell. This process is known as high frequency recombination or Hfr male. The reproductive ability of Hfr strain is 1000 times more than of F+ strain. The integration of plasmid DNA with this genophore (F bacterial DNA) can occur in 20 parts. Conjugation in HFr male : During conjugation a conjugation tube is formed between the Hfr male and F recipient cell. After that the donor DNA opens near the F+ factor and become single stranded. Now this single stranded DNA moves slowly from the donor cell to the recipient cell. This transfer process is continued until, both the Hfr and F- cells are separated naturally. After this separation some part of DNA of Hfr strain remain inside the Frecipient cell and get combined with the DNA of recipient cell as a result new genes are integrated into the recipient F" cell. The combination of both the DNA results into the formation of a genetic hybrid which is partially diploid. This type of reproduction is found in Salmonella, Pseudomonas, Vibrio and E. coli. Sexduction. Jacob Adelberg (1959) reported this process in bacteria. In general in Hfr strains the F+ factor is integrated with the bacterial chromosome but some times this F+ factor separate from the bacterial chromosomes and becomes fully autonomous and replicate independently. Sometimes during separation this F+ factor contains some genes of the bacterial chromosome and now it is called as F+ prime. When this F+ prime cells comes in contact with the F" recipient cell it transfer some of the genes are taken from the DNA of the previous bacterial cell. This process is known as sexduction and as a result the recipient cell become partly diploid and the structure is called as merozygote. Significance of Conjugation. Among the Gram - bacteria this is the major way that bacterial genes are transferred. Transfer can 47
occur between different species of bacteria. Transfer of multiple antibiotic resistance by conjugation has become a major problem in the treatment of certain bacterial diseases. Since the recipient cell becomes a donor after transfer of a plasmid it is easy to see why an antibiotic resistance gene carried on a plasmid can quickly convert a sensitive population of cells to a resistant one. Gram + bacteria also have plasmids that carry multiple antibiotic resistance genes, in some cases these plasmids are transferred by conjugation while in others they are transferred by transduction. The mechanism of conjugation in Gram + bacteria is different than that for Gram -. In Gram + bacteria the donor makes an adhesive material which causes aggregation with the recipient and the DNA is transferred. Transduction. Transport of bacterial DNA of donor cell to the recipient cell with the help of bacteriophage or transduction is the bacteriophage mediated transfer of genetic material of donor bacterial cell to the recipient bacterial cell. This mode of gene exchange or reproduction was reported by Zinder and Lederberg (1952) in those forms of bacteria which are responsible for mouse typhoid (Salmonella typhimurium). Then transduction has been reported in E.coli, Proteus, Schizella and Staphylococcus. Zinder and Lederberg (1952) initially began their experiments with the objective of discovering whether the E. coli type of genetic exchange also existed in S. typhimurium. They cultivated two a uxotrophic strains of S. typhimurium. The strain A was unable to synthesize the amino acids, phenylalanine and tryptophan (Phe-, Try-) but could synthesize methionine and Histidine (Phe- TryMet+ His+). The other strain was unable to synthesize methionine and histidine but able to synthesize phenylalanine and tryptophan (Phe+ Try+ Met- His-). Crossing or combine culturing of strain A and strain B resulted in a wild type prototroph which could synthesize all four amino acids. (Phe+ Try+ Met+ His+). Each auxotrophic strain, A and B was placed in both the arms of Davis Utube. The two arms of tube were separated by a sintered glass filter which was impervious to bacterial cells but allowed the free passage of nutrient media and other molecules particles smaller 48
than O.lu. The culture medium was made to pass through the filter from one arm to the other by alternating suction and pressure. Thus the two auxotrophic strain although physically separate were grown in the same medium. A large number of prototroph appeared in the experiment. Thus it was concluded that these protrotrophs are obtained by the method other than conjugation. Because this process was resistant to DNA enzyme activity, transformation process can't be involved in the synthesis of these prototroph. Thus the production of prototrophic Salmonella strain is due to the activity of certain filterable agent which was later called as Bacteriophage P22. The transducing frequency is low and our only one in 105 to 107 cells undergo transduction. Hershey and Chase (1952) at the same time discovered bacteriophage and explained that during the infection of bacteria by bacteriophage there is a transfer of nucleic acid of bacteriophage into the bacterial cell. Transduction is generally of two types: 1. Generalized transduction 2. Specialized transduction. Generalized Transduction: It is completed in following steps: 1) This type of transduction starts with the infection of bacteria with the bacteriophage. This process is controlled by the DNA segments called as prophage particle present in the cytoplasm of bacterial cell. 2) During the infection of the lysogenic bacterial cell by bacteriophage, the DNA of the bacteria breaks down into small fragments and the nucleic acid of the bacteriophage utilizes the bacterial enzymes and synthesize new phage components. 3) At the same time when these progeny phage particles are form the DNA fragments of the bacteria incorporate into these DNA particles of the phage. 4) The genetic material or DNA fragments of the previous bacterial cell is transferred to the new bacterial cell infected by these progeny phage particles.
Thus generalized transduction is the process where bacteriophage plays an active role in the transfer of DNA fragments of the bacterial cell. Specialized Transduction: Andre Lwoff (1953) reported that certain bacterial strains are able to survive for a long time even after infected by the bacteriophage and these is no lysis of bacterial cell. Here in these bacteria there is a joining of bacterial DNA with the phage DNA and both DNA i.e. bacterial DNA and phage DNA replicate commonly. This bacteria is known as lysogenic bacteria and the phage is called as prophage. This bacterial cells can survive in lysogenic stage for many generations which is due to the synthesis of a special repressor protein. This protein inhibits the synthesis of phage particle inside the bacterial cell. As the synthesis of this protein is stopped the bacterial cell start the synthesis of phage components. The DNA of both i.e. of phage DNA and bacterial DNA breaks down before the synthesis of the phage particles starts. At the same time some bacterial genes are carried out by phage DNA and replicate with the phage DNA. These resultant progeny phage particles are entirely different from the parent one when these progeny phage particles infect a new bacterial cell, some of the gene (of the previous bacterial cell) are also transmitted to the newly infected bacterial cell. In this type of transduction only those special genes are transmitted which are attached very closely to the phage DNA. Significance. Lysogenization that results in a change in the phenotype of the host cell is called lysogenic conversion. Lysogenic (phage) conversion occurs in nature and is the source of virulent strains of bacteria. So, Bacteria are known to exchange genes in nature by three fundamental processes: conjugation, transformation and transduction. Conjugation requires cell-to-cell contact for DNA to be transferred from a donor to a recipient. In transformation, DNA is acquired directly from the environment, having been released from another cell. During transduction, a virus transfers the genes between mating bacteria. 50
Types of differentiation Bacteria can form specialized, morphologically differentiated structure: − forms that serve to reproduce; − resting forms; − cells with specialized metabolic functions. REPRODUCTION FORMS Baeocytes – formed by repeated multiple divisions of the mother cell (Cyanobacteria). Baeocyte production in the cyanobacterium Stanieria. Stanieria never undergoes binary fission. It starts out as a small, spherical cell approximately 1 to 2 µm in diameter. This cell is referred to as a baeocyte (which literally means small cell). The baeocyte begins to grow, eventually forming a vegetative cell up to 30 µm in diameter. As it grows, the cellular DNA is replicated over and over, and the cell produces a thick extracellular matrix. The vegetative cell eventually transitions into a reproductive phase where it undergoes a rapid succession of cytoplasmic fissions to produce dozens or even hundreds of baeocytes. The extracellular matrix eventually tears open, releasing the baeocytes. Hormogonium – adaptation in filamentous forms of Cyanobacteria for reproduction by fragmentation of filaments. Hormogonium called trichome fragments that are subsequently degraded. Hormogonium not just a mechanical separation of a group of two, three or more cells. Each Hormogonium can give rise to a new individual. If a group of cells, similar to Hormogonium, wearing thick coat, it is called gormospores (gormocysts), which also serves the function and reproduction, and the shifting of adverse conditions. Gonidium – broad cell of Algae; each of which give rise to new growth. Gonidium are produced within the mother cell, or are formed from the top of the mother cell. Gonidium is an obsolete form of conidium. Gonidium also called Cocci or Planococci. Gonidium 51
retain mucosa. Cocci lack clearly defined coats. Planococci too naked, but are capable of active movement. Exospores - (Actinomycetes). Types of Actinomycetes exospores: − Single Exospores (Micromonospora); − Chains of spores on the end of hypha of aerial mycelium (Streptomyces); − Formed inside the sporangium (Streptosporangium). DORMANT (RESTING) FORM OF BACTERIA − Cysts; − Microcysts (Mixocysts); − Akinetes; − Endospores. Cysts − Bacterial cells that have lost mobility turned into resting forms. Cysts have two coats: Intine and Exine. Contain poly-beta-hydroxybutyric acid (Azotobacter). Cysts form some methanotrophic and oligotrophic bacteria, Spirochetes, Rickettsia. Mixocysts − Resting forms in Myxobacteria (Myxococcus, Chondromyces, Stigmatella). Have a thick coat. Aggregate, forming fruiting bodies. Fruiting bodies are a mass of mucus, which shipped cysts. Raised above the surface of the substrate by simple or branched stems. Fruiting body contains 10000-1000000 mixospores. Akinetes − a form of resting spore. Large, thick-walled, resistant cells. Akinetes containing a food reserve: granules of cyanophycin, polyphosphates, glycogen in Cyanobacteria. Its thick cell wall allows it to withstand adverse conditions. They can germinate immediately after the formation, with no rest period, when placed in favorable conditions. In the absence of such conditions spores can survive for a long time.During germination of spores produced one germ, to make a break through the coat. Endospore - endospore formation occurs in bacteria to tide over unfavourable environmental conditions. They are produced under conditions of limited supply of carbon, nitrogen and phosphorous. This process was first of all reported by Cohn (1817) 52
and late by Koch (1877). Endospores are generally formed in bacteria pathogenic to plants, animals and human beings. Endospores are heat, chemical, drying, freezing and radiation resistant bodies. They can survive under dormancy even upto 50 years and on getting favourable environmental conditions they may germinate to start a new bacterial life. Endospores are found in bacteria like Bacillus, Clostridium, Sporolactobacillus, Sporosarcina and Desidfotomacidum. Generally a single cell transform into a single endospore but in certain cases two endospores are also reported from a single cell. They may be oval, ellipsoidal or spherical in shape and usually in central, terminal or sub-terminal in position. Thus endospore is a highly resistant structure. The resistant nature is due to following reasons: 1. Lowest metabolic activities. 2. Very few amount of water. 3. Impermeable and protective nature of spore coat. 4. Lack of active enzymes. 5. High percentage of Ca+2 ion in spore composition. 6. Presence of stabilizer compound i.e. Picolenic acid. The structure of endospore is variable in different species of bacteria. The cell wall is multi layered and acquire more than half of the volume of spore. The bacterial protoplast core is mainly made up of DNA and is surrounded by dense cytoplasm. Protein percentage is about 90% of the total volume of the protoplasm. An diocholinic acid act as stabilizer is found in cytoplasm. The amount of enzyme is very low in the spore. The protoplast of spore is surrounded by a very thin membrane known as core membrane. Membrane in turn is surrounded by spore wall made up of disulphide rich protein called Keratin. The spore wall is about 30-60% of the dry weight of the spore. This spore wall provide protection to the spore from unfavourable conditions and harmful chemicals. 53
An electron dense cortex is present between the spore wall and inner membrane. This cortex is made up of modified peptides. The endospore of Bacillus sphaericus lack the cortex. While in some bacteria an extra membrane, exosporium is found. Spore wall is generally divided into outer and inner coat. Under unfavourable condition when there is no cellular division and there is scarcity of ATP or energy in the cell, a special gene become active and is found responsible for the formation of endospore. In the presence of RNA polymerase enzyme this gene synthesize a special protein which initiate the formation of endospore in the cell. Fitz-James & Young (1969) studied the process of formation of endospore in Clostridium Electron microscopically. The developmental stage of endospore formation in Bacillus subtilis and Clostridium have been studied in detail and consist of stage O (Vegetative stage) to VII stage. This can be divided into six main steps which are as follows : 0 Stage : In this stage the cell which is going to form endospore, enlarges in size and its chromatin material condenses attain the shape of an axial filament reaching from one end to the another end of the cell. I Stage : Axial filament develop completely. There is a change in the metabolic activity of the cell. II Stage: The plasma membrane start invaginating towards the one end of the cell. This invagination grows centripetally and both the end unite to form a spore septum. Septum formation ends by the formation of a small spore known as fore spore. The genetic material also get transported to this structure. III Stage: Spore septum of both i.e. mother cell and fore spore grows around the protoplasm of fore spore by a process called engulfment. Thus fore spore lies freely in the cytoplasm of mother cell. IV Stage: Cell wall of peptidoglycan is synthesized outside the plasma membrane of fore spore. This structure is called as spore wall. 54
V Stage: There is a deposition of peptidoglycan cortex between the spore wall and cell membrane. The protoplasm condenses and synthesize dipiconilic acid whose deposition occurs on cell membrane. There is an increase in the concentration of Ca+2, arginine and glutamic acid in the spore cytoplasm. Spore wall become multilayered. VI Stage: The spore mature into spore mother cell, the mother cell is called as sporangium. VII Stage: There is autolysis of sporangium and the spore is released free in the environment and it is transmitted by air. The Endospore formation in Clostridium requires 2 hours while Bacillus subtilis requires 7 hours. After transmission these endospore lie dormant for many years. Germination of endospore. The process of germination start under favourable environmental conditions. Spore coat becomes soft by the imbibition of water. The cytoplasm of spore swells up by absorbing salts, nutrients and water. Thus as a result the upper spore coat breaks up and the developing cell comes out. Generally a single spore is formed from a single bacterial cell and on germination of a single spore a single bacterial cell is formed. Thus endospore formation is considered to be the perennation method of bacterium. A bacterial structure sometimes observed as an inclusion is actually a type of dormant cell called an endospore. Endospores are formed by a few groups of Bacteria as intracellular structures, but ultimately they are released as free endospores. Biologically, endospores are a fascinating type of cell. Endospores exhibit no signs of life, being described as cryptobiotic. They are highly resistant to environmental stresses such as high temperature (some endospores can be boiled for hours and retain their viability), irradiation, strong acids, disinfectants, etc. They are probably the most durable cell produced in nature. Although cryptobiotic, they retain viability indefinitely such that under appropriate environmental conditions, they germinate back into vegetative cells. 55
Table 7 Differences between endospores and vegetative cells Property
Typical Gram-positive murein cell wall polymer
Thick spore coat, cortex, and peptidoglycan core wall
Calcium dipicolinic acid
Present in core
Cytoplasmic water activity
Resistance to chemicals and acids
Sensitivity to lysozyme
Sensitivity to dyes and staining
Endospores are formed by vegetative cells in response to environmental signals that indicate a limiting factor for vegetative growth, such as exhaustion of an essential nutrient. They germinate and become vegetative cells when the environmental stress is relieved. Hence, endospore-formation is a mechanism of survival rather than a mechanism of reproduction. Functions of resting forms: 1) Preservation of DNA. 2) Protect the population from the harmful effects of the environment. 3) Are a way of transmitting infective host to host (pathogenic). 56
CELLS WITH SPECIALIZED METABOLIC FUNCTIONS – NITROGEN FIXATION. Heterocysts – thick coat, polar thylakoids, no nucleoid, lack of photosystem II. Heterocysts are special cells for N2 fixation: thick cell wall, low oxygen concentration, photosystem II (light reaction) inactive but photosystem I active to provide ATP; connected to vegetative cells by cell wall pores. The enlarged cells are heterocysts, specialised cells within which N2-fixation takes place but photosynthesis doesn't. Bacteroides – are inside the knob, polymorphic, have leggemoglobin (binds oxygen). So, the manifestations of morphological differentiation of Eubacteria aimed at improving their survival, it is shown in: − Formation of special cells with increased resistance (endospores, cysts). − Formation of structures for efficient reproduction of the form (baeocytes, hormogonium, gonidium, exospores). − Formation of structures with additional unique metabolic abilities (nitrogen fixation by heterocysts and bacteroides). LEVELS OF CELLULAR ORGANIZATION OF EUBACTERIA. MICROBIAL COMMUNITIES. BIOFILM. Most prokaryotic cells do not reside on the Earth surface but Lie underground in the oceanic and terrestrial subsurfaces! Because these habitats are relatively unexplored, there is much left for microbiologists to discover and understand about the life forms dominating Earth. Cells live in nature in populations. In an environment where microbial population lives − habitat. Population of cells interact with other population of cells in assemblages − microbial communities. The communities may: − consist of free-swimming cells (in aquatic environment); 57
− form on living and non-living surfaces – biofilms. The effects of organisms on each other and on their habitat. Populations in microbial communities interact in various way, either harmfully or beneficially. Organisms together with the physical and chemical constituents of their environment ecosystem. Major microbial ecosystems include: − Terrestrial (soil, rock) − Higher organisms (both plants and animals) − Aquatic (oceans, ponds, lakes, streams, hot springs). The properties of an ecosystem are often controlled by microbial activities the extent of microbial life. Prokaryotic cells constitute the major portion of biomass on Earth, as key reservoirs of essential nutrients for life. Careful estimates of total microbial cell No 5 x 1030 cells total amount of carbon equals that of all plants on Earth the collective contents of nitrogen and phosphorus in prokaryotic cells is over 10 times that of all plant biomass. There is much left for you to discover. Most prokaryotic cells do not reside on the Earth surface but Lie underground in the oceanic and terrestrial subsurfaces. Because these habitats are relatively unexplored, there is much left for microbiologists to discover and understand about the life forms dominating Earth. Levels of cellular organization of Eubacteria 1) Single- celled organisms; 2) Homologous associations; 3) Heterologous associations; 4) Biofilms; 5) Multicellular organisms. Unicellular organism. Unicellulate – the ability to perform all the functions inherent in the body, regardless of the neighboring cells. Colony − a random union of cells. Homologous associations. Formed as a result of incomplete division or cell separation by: 1) Coagglutination of external coat containing polysaccharides: − Zooglea (slimy matrix) Zooglea ramigera 58
− Sheath (vagina)- Sphaerotilus natans 2) Egestion of cellulose – mikoderma (cell skin) Acetobacter xylinum 3) Binding by cellular appendages protein nature − Fimbriae and pili Heterologous associations. Formed with: 1) other prokaryotes; 2) unicellular eukaryotes; 3) tissues of eukaryotes. A biofilm is a living layer of bacteria that is attached to a surface. Biofilm – community of bacteria permanently attached to the substrate and to each other and protected these cells produced by the extracellular polymerous matrix. Biofilm development occurs in five stages: 1. Reversible attachment: Cells transiently affix to substratum, and surface induced gene expression results in a protein profile significantly different from planktonic bacteria. 2. Irreversible attachment (fixation): Cells reorient themselves, clusters develop, motility is lost, and the quorum sensing regulon becomes activated. At this stage, the microbes produce extracellular polymers that provide strong adhesion. 3. Maturation I: Cell clusters become thicker than 10 υm and the quorum sensing system becomes active. Cells that attach to the surface, facilitate subsequent attachment of cells, extracellular matrix holds together the entire colony. Accumulate nutrients, the cells begin to divide. 4. Maturation II (growth): Cell clusters reach maximum thickness (100 υm) with a protein profile most different from planktonic cells. Formed a mature biofilm, and now it changes its size and shape. Extracellular matrix is a protection of cells against external threats. 5. Dispersion (release of bacteria): Cluster structures change, and pores and channels form. Motile and non-motile bacteria are present as the protein profile begins to resemble planktonic cells once again. By dividing the periodic break away from the biofilm, 59
individual cells that can over time attach to the surface and form a new colony. Within the biofilm bacteria combined intercellular contacts of two types: 1) Cytoplasmic bridges − membrane tubes that connect the cytoplasm of various cells 2) Close clumping of cells, which in certain parts of the bacteria have a common cell wall. This allows for the generation of common responses to external stimuli and exchange by signaling molecules. The view of bacteria as unicellular organisms has strong roots in the tradition of culturing bacteria in liquid media. However, in nature microbial activity is mainly associated with surfaces where bacteria form highly structured and cooperative consortia which are commonly referred to as biofilms. The ability of bacteria to organize structurally and to distribute metabolic activities between the different members of the consortium demands a high degree of coordinated cell-cell interaction. Recent work has established that many bacteria employ sophisticated intercellular communication systems that rely on small signal molecules to control the expression of multiple target genes. In Gram-negative bacteria, the most intensively investigated signal molecules are N-acyl-Lhomoserine lactones (AHLs), which are utilized by the bacteria to monitor their own population densities in a process known as 'quorum sensing'. These density-dependent regulatory systems rely on two proteins, an AHL synthase, usually a member of the LuxI family of proteins, and an AHL receptor protein belonging to the LuxR family of transcriptional regulators. At low population densities cells produce a basal level of AHL via the activity of an AHL synthase. As the cell density increases, AHL accumulates in the growth medium. On reaching a critical threshold concentration, the AHL molecule binds to its cognate receptor which in turn leads to the induction/repression of AHL-regulated phenotypes (e.g. bioluminescence in Vibrio fischeri). Quorum sensing is a method of bacterial communication and allows the coordination of gene expression in response to 60
fluctuations in cell density. This enables populations of cells to control a diverse array of biological processes in synchrony, from biofilm formation to bioluminescence. In order to quorum sense individual cells produce and respond to small signalling molecules, termed autoinducers, that accumulate in the cell’s external environment. The concentration of autoinducer increases, both intra and extra-cellularly, with increasing cell density and once a minimal threshold concentration is reached, gene expression is altered. Cell-to-cell communication allows bacteria to coordinate their activity and thus enjoy benefits otherwise reserved for multicellular organisms. Within biofilms, cell-cell communication occurs through a phenomenon known as quorum sensing, in which signaling molecules called autoinducers, specific for that species, are produced and exchanged among bacterial neighbors. The autoinducer, produced by many bacterial species and believed to be a universal signaling molecule. They release molecules that act as signals, basically creating a group discussion that helps the bacteria decide when to form a biofilm, how to react to a threat, or when to move on to a new spot if the nutrient level begins to drop. In order to quorum sense individual cells produce and respond to small signalling molecules, termed autoinducers, that accumulate in the cell’s external environment. The types of signalling molecules – autoinducers: 1) Acyl homoserine lactones (AHLs) – (Gram-negative bacteria); 2) Peptides (Gram-positive bacteria); 3) Furanozildiested boron (Gram-negative and Gram-positive bacteria) The Basic properties of biofilms: − Interaction of different types of common bacteria; − Microorganisms collected in microcolonies; − Microcolonies surrounded by a protective matrix; − Inside microcolonies - different media; − Microorganisms are primitive system of communication. 61
Biofilms are everywhere. These colonies of bacteria and other microorganisms can be found in mundane places, such as shower curtains, sink drains, and toothbrushes, as well as in more exotic locales, from chilly Arctic waters to scalding deep-sea vents. For example, biofilms are commonly found in showers, toilets, catheters, medical equipment, and even in your mouth! Dental plaque is an example of a biofilm commonly found in humans. Modern biotechnology can successfully use the best community of microorganisms to perform specific functions. This is important in the production of food, medicines and food supplements, all kinds of waste disposal, to neutralize water and soil pollution by oil products. Practice has shown manifold increase in the efficiency of microorganisms in such an organization. Formation of biofilms has important biological significan, because the microbial cells are better protected from: − antimicrobial immunity factors; − antibiotics; − adverse environmental factors. Thus, the tendency to mechanically join eubacterial cells - the necessary preconditions for the emergence of simple variants of true multicellularity. Consequence of this cellular organization − promoting the viability of multicellular complex than the single cell of the same species in the same conditions.
SYSTEMICS AND TAXONOMY OF MICROORGANISMS Systematics is the study of organisms in order to place organisms having similar characteristics into the same group. Systemics and Taxonomy is the science of organisms based on a presumed natural relationship. Scientists observe each organism, noting its characteristics. Organisms that have similar characteristics are presumed to have a natural relationship and therefore are placed in the same group. Classification tries to show this natural relationship. Taxonomy is a subset of systemics. Systemics is the study of organisms in order to place organisms having similar characteristics into the same group. Using techniques from other sciences such as biochemistry, ecology, epidemiology, molecular biology, morphology, and physiology, biologists are able to identify characteristics of a organism. Taxonomy organizes large amounts of information about organisms whose members of a particular group share many characteristics. Taxonomy lets scientists make predictions and design a hypothesis for future research on the knowledge of similar organisms. A hypothesis is a possible explanation for an observation that needs experimentation and testing. If a relative of an organism has the same properties, the organism may also have the same characteristics. Taxonomy puts microorganisms into groups with precise names, enabling microbiologists to communicate with each other in an efficient manner. Taxonomy is indispensable for the accurate identification of microorganisms. For example, once a microbiologist or epidemiologist identifies a pathogen that infects a patient, physicians know the proper treatment that will cure the patient. By the 1900s, scientists had discovered microorganisms that had characteristics that were dramatically different than those of plants and animals. Therefore, Linnaeus' taxonomy needed to be enhanced to encompass microorganisms. 63
In 1969 Robert H. Whitteker, working at Cornell University, proposed a new taxonomy that consisted of five kingdoms. These were Monera, Protista, Plantae (plants), Fungi, and Animalia (animals). Monera are organisms that lack a nucleus and membrane-bounded organelles, such as bacteria. Protista are organisms that have either a single cell or no distinct tissues and organs, such as protozoa. This group includes unicellular eukaryotes and algae. Fungi are organisms that use absorption to acquire food. These include multicellular fungi and single-cell yeast. Animalia and plantae include only multicellular organisms. Before Woese's six-kingdom taxonomy, scientists grouped organisms into eukaryotes animals, plants, fungi, and one-cell microorganisms (paramecia) and prokaryotes (microscopic organisms that are not eukaryotes). Woese's six-kingdom taxonomy consists of: − Eubacteria (has rigid cell wall) − Archaebacteria (anaerobes that live in swamps, marshes, and in the intestines of mammals) − Protista (unicellular eukaryotes and algae) − Fungi (multicellular forms and single-cell yeasts) − Plantae − Animalia Woese determined that archaebacteria and eubacteria are two groups by studying the rRNA sequences in prokaryotic cells. Woese used three major criteria to define his six kingdoms. These are: − Cell type. Eukaryotic cells (cells having a distinct nucleus) and prokaryotic cell (cells not having a distinct nucleus). − Level of organization. Organisms that live in a colony or alone and one-cell organisms and multicell organisms. − Nutrition. Ingestion (animal), absorption (fungi), or photosynthesis (plants). In the 1990s Woese studied rRNA sequences in prokaryotic cells (archaebacteria and eubacteria) proving that these organisms should be divided into two distinct groups. Today organisms are 64
grouped into three categories called domains that are represented as bacteria, archaea, and eukaryotes. The domains are placed above the phylum and kingdom levels. The term archaebacteria (meaning from the Greek word archaio "ancient") refers to the ancient origin of this group of bacteria that appears to have diverged from Eubacteria. The archaea and bacteria came from the development of eukaryotic organisms. The evolutionary relationship among the three domains is: − Domain Bacteria (Eubacteria); − Domain Archaea (Archaebacteria); − Domain Eucarya (Eukaryotes). Different classifications of organisms are: − Bacteria (Eubacteria); − Archaea (Archaebacteria) − Eukarya (Protista, Fungi, Plantae, Animalia). Archaea lack muramic acid in the cell walls. Bacteria have a cell wall composed of peptidoglycan and muramic acid. Bacteria also have membrane lipids with ester-linked, straight-chained fatty acids that resemble eukaryotic membrane lipids. Most prokaryotes are bacteria. Bacteria also have plasmids, which are small, double-stranded DNA molecules that are extrachromosomal. Eukarya are of the domain eukarya and have a defined nucleus and membrane bound organelles. TAXONOMY OF MICROORGANISMS Taxonomy has three components: − Classification. The arrangement of organisms into groups based on similar characteristics, evolutionary similarity or common ancestry. These groups are also called taxa. − Nomenclature. The name given to each organism. Each name must be unique and should depict the dominant characteristic of the organism. − Identification. The process of observing and classifying organisms into a standard group that is recognized throughout the biological community. 65
A taxonomy and classification has an overlapping hierarchy that forms levels of rank or category similar to an organization chart. Each rank contains microorganisms that have similar characteristics. A rank can also have other ranks that contain microorganisms. In the taxonomy of prokaryotes, the most commonly used rank (in order from most general to most specific) is: Domain - Kingdom – Phylum – Class – Order - Family – Genus – Species.
Figure 6 – Classification categories of microorganisms
The basic taxonomic group in microbial taxonomy is the species. Taxonomists working with higher organisms define their species differently than microbiologists. Prokaryotic species are characterized by differences in their phenotype and genotype. Phenotype is the collection of visible characteristics and the behavior of a microorganism. Genotype is the genetic make up of a microorganism. 66
Species − a set of organisms that share a common evolutionary origin, a close genotype (a high degree of genetic homology, 60%) and as close phenotypic characteristics. Species − collection of individuals of the same genotype, have a pronounced phenotypic similarity. This is the lowest taxonomic rank that has official standing in nomenclature and should also be italicized when used. The prokaryotic species are collections of strains that share many properties and differ dramatically from other groups or strains. A strain is a group of microorganisms that share characteristics that are different from microorganisms in other strains. Strain – any particular sample (isolate) of the species.
Figure 7 − Schematic diagram of basic classified conceptions
Each microorganism within a strain is considered to have descended from the same microorganism. For example, Biovars is a species that contains strains characterized by differences in its biochemistry and physiology. Morphovars is also a species whose strains differ morphologically and structurally. Serovars is another species that has strains that are characterized by distinct antigenic properties (substances that stimulate the production of antibodies). Culture – the entire set of microorganisms grown on solid or liquid media. Pure culture- set (population) of microorganisms, consisting of the same species. 67
Clone – culture, were isolated from 1 cell, i.e. clone − progeny of 1 cell. A taxonomy is based on scientists' ability to characterize organisms into a classification system.
Figure 8 − Taxonomic systems of classification
The most widely used classification system is called the natural classification. The natural classification requires that an organism be grouped with organisms that have the same characteristics. In the mid-eighteenth century, Linnaeus developed the first natural classification using anatomical characteristics of organisms. Other natural classifications use classical characteristics to group organisms. These characteristics are: − Morphological. Morphological characteristics classify organisms by their structure, which normally remain the same in a changing environment and are good indications of phylogentic relatedness. − Ecological. Ecological characteristics classify organisms by the environment in which they live. For example, some microorganisms live in various parts of the human intestines and others live in marine environments. Ecological characteristics include the ability to cause disease, temperature, pH, and oxygen requirements of an organisms, as well as an organism's life cycle. − Genetic. Genetic characteristics classify organisms by the way in which they reproduce and exchange chromosomes. For example, eukaryotic organisms reproduce sexually by conjugation where two cells come together and exchange genetic material. Prokaryotic organisms do not reproduce sexually and instead use transformation to reproduce. Transformation occurs between strains 68
of prokaryotes if their genomes are dissimilar but rarely between genera.
Figure 9 − Characteristics of genetic taxonomy
In the early 1990-s, T. Cavalier-Smith developed the two-empire and eight-kingdom taxonomy based on phentic and phylogenetic characteristics. Phentic measures the physical characteristics of an organism using a process called numerical taxonomy. Numerical taxonomy is a phentic classification based on physical measurements of an organism.
Figure 10 − Numerous characteristics, used for taxonomic classification of microorganisms
Phylogenetic measures the evolutionary relationship among organisms. Polyphase taxonomy: − Find groups of similar strains; − Determination of the phylogenetic position of these groups; − Measuring the differences between the groups and its closest neighbors; − Collection of data differentiating groups, preferably at levels of cellular organization. Nomenclature − The name given to each organism. Nomenclature is the assignment of names to taxa according to international rules. In reference to any organism, their official name consists of two parts: the genus and the species (or specific epithet) and should always be italicized. Microorganisms are given a two-part name. The first part is the Latin name for the genus. The second part is the epithet. Together these parts uniquely identify the microorganism. The first part of the name is always capitalized and the second part of the name is always lowercase. Both parts are italicized.
Figure 11 − Diagram of binomial name of microorganisms development
For example, Escherichia coli is a bacterium that is a member of the Escherichia genus and has the epithet coli. Sometimes the name is abbreviated such as E. coli. 70
However, the abbreviation maintains the same style as the full name (uppercase, lowercase, italic). Identification. The process of observing and classifying organisms into a standard group that is recognized throughout the biological community. Step 1 − Streak Plate Isolation: − The most commonly used isolation technique in microbiological laboratories is the streak plate method. − The goal of streak plate isolation is to separate each of individual in a population for further study. − Pure cultures (axenic cultures) composed of cells arising from a single progenitor called a colony-forming unit (CFU). Step 2 − Microscopy and Staining: Microscopy and Staining: one of the major characteristics that divide bacteria into different groups is their cell wall (Gram stain), shape, and arrangement. Step 3 − Biochemical Tests: Microbiologists also use biochemical tests, noting a particular microbe's ability to utilize or produce certain chemicals. Step 4 − Molecular Techniques: DNA based properties (G+C content, ribosomal gene se-quences, protein sequences and total gene sequences). The sciences of genomics and bioinformatics have led to a radical reclassification of procaryotes based on comparative analysis of organismal DNA. Genomics involves the study of all of the nucleotide sequences, including structural genes, regulatory sequences, and noncoding DNA segments, in the chro-mosomes of an organism. − Metagenomics. Sequencing of 16S rRNA genes obtained from environmental samples produces a broad profile of microbial diversity and reveals that the vast majority of microbes present have been missed by reliance on cultivation-based methods. This observation has given rise to the field of metagenomics. Metagenomics (also called environmental genomics) is the application of modern genomics techniques to the study of communities of microorganisms directly in their natural envi71
ronments, bypassing the need for isolation and lab cultivation of individual species. Metagenomics provides a means to identify and quantify microbes from environmental samples based on the presence of distinctive genes. This enables studies of organisms that are not easily cultured in the laboratory, as well as studies of organisms in their natural environment.
VIROLOGY. GENERAL CHARACTERISTIC OF VIRUSES A virus (Latin for "poison") is an obligate intracellular parasite that can only replicate inside a living host cell. Once inside the living host cell, a virus becomes integrated in the metabolism of its host, making a virus difficult to control by chemical means. You cannot kill a virus with antibiotics. Drugs that destroy the host's ability to be used by a virus for replication tend to also be highly toxic and have a negatively and sometimes deadly effect on the host cell.
Figure 12 − Common feature of viruses and some bacteria
Before a virus enters a cell, it is a free virus particle called a virion. A virion cannot grow or carry out any biosynthetic or biochemical function because it is metabolically inert. Viruses are not cells. They vary in size from 20 nanometers (polio virus) to 300 nanometers (smallpox virus) and cannot be seen under a light microscope. 73
In 1933, microbiologist Wendell Stanley of the Rockefeller Institute for Medical Research showed that viruses could be regarded as chemical matter rather than as living organisms.
Figure 13 – Fundamental difference between viruses and bacteria
The main properties of viruses: 1. Ultramicroscopic size (measured in nanometers). 2. Viruses is contain only one type of nucleic acid − DNA or RNA. 3. Special fragmented method of reproduction. 4. Viruses do not have of their own protein-synthesizing systems, they are obligate intracellular parasites. 5. Habitat viruses are living cells − bacteria, plants, animals and humans. 74
VIRAL STRUCTURE The major components of a virus are: Nucleic acid core. The nucleic acid core can either be DNA (1.5 150 000 000 D) or RNA that makes up the genetic information (genome) of the virus. RNA genomes only occur in viruses. Proteins core. The first group − the internal histones − ribonucleoproteins or dezoxynucleoproteins. The second group − viral enzymes: − Replication and transcription enzymes (DNA polymerase, RNA polymerase, reverse transcriptase in retroviruses); − Enzymes involved in the penetration of the virus into the cell and the output of virions (neurominidase, lysozim, ATP-ase).
Figure 14 − Enzyme classification of virus
Capsid. Capsid − case. A capsid is the protein coat that encapsulates a virus and protects the nucleic acid from the environment. It also plays a role in how some viruses attach to a host 75
cell. A capsid consists of one or more proteins that are unique to the virus and determine the shape of the virus. Capsid + nucleic acid = nucleocapsid. Envelope. An envelope is a membrane bilayer that some viruses have outside their capsid. If a virus does not have an envelope, the virus is called a naked virus. Examples of diseases caused by naked viruses are chickenpox, shingles, mononucleosis, and herpes simplex. A naked virus is more resistant to changes and is less likely to be affected by conditions that can damage the envelope. Environmental factors that can damage the envelope are: − Increased temperature; − Freezing temperature; − pH below 6 or above 8; − Lipid solvents; − Some chemical disinfectants (chlorine, hydrogen peroxide, and phenol). Naked viruses are more resistant to changes in temperature and pH. Examples of diseases caused by naked viruses include poliomyelitis, warts, and the common cold. Additional lipoprotein envelope – supercapsid. He acquired the virus at the time of exit from the cell. The outer envelop consists of a complex virus proteins, they are part of glycoproteins and glycolipids. They appear in the form of spikes or appendages on the surface of supercapsid. Capsid and superkapsid protect virions from environmental influences are involved in the adsorption of virus to the cell, determine the antigenic and immunogenic properties. So, the Enveloped viruses = nucleocapsid + surepcapsid SHAPES OF VIRUSES A virus can have one of two structures. These are: − Helical virus. A helical virus is rod or thread-shaped. The virus that causes rabies is a helical virus. − Icosahedral virus. An icosahedral virus is spherically shaped. Viruses that cause poliomyelitis and herpes simplex are icosahedral viruses. 76
There are two ways of packing of capsomere in the capsid − spiral (helical viruses) and cub (spherical viruses). When the helical symmetry of the type of protein subunits arranged in a spiral. Between them, too, laid in a spiral nucleic acid (filamentous viruses). For cubic symmetry type of virions they can be in the form of polyhedron. Just have arranged virus nucleocapsid only − naked viruses. Viral classification criteria − Type of nucleic acid; − The size and morphology of the virions; − Type of symmetry; − Having superkapsid; − Type of Host-cells; − The antigenic properties; − Type of infection transmission. Nomenclature of viruses Kingdom – Vira Family – Viridae Subfamily – Virinae Genus − Virus Species − a special name (influenza virus H5N1, rubella virus, herpes virus, etc. UNCONVENTIONAL AGENTS There are also the 'unconventional agents' sometimes known as 'unconventional viruses' or 'atypical viruses' − Up to now, the main kinds that have been studied are viroids and prions. Viroids– small supercoiled circular RNA molecules. In 1971, plant pathologist O.T. Diener discovered an infectious RNA particle smaller than a virus that causes diseases in plants. He called it a viroid. Viroids infect potatoes, causing potato, spindle tuber disease. They infect chrysanthemums, stunting their growth. Viroids cause cucumber pale fruit disease. Millions of dollars are lost each year in crop failures caused by viroids. 77
A viroid is similar to a virus in that it can reproduce only inside a host cell as particles of RNA. However, it differs from a virus in that each RNA particle contains a single specific RNA. In addition, a viroid does not have a capsid or an envelope. Some viruses do not have an envelope. Do not have a capsid. Viroids contain RNA only. They are small (less than 400 nucleotides), single stranded, circular RNAs. The RNAs are not packaged, do not appear to code for any proteins, and so far have only been shown to be associated with plant disease. However, there are some suggestions that somewhat similar agents may possibly be involved in some human diseases - Hepatitis delta virus. Main futures of Viroids: − Naked RNA (no capsid); − 300 – 400 nucleotides long; − Closed, folded, 3-dimensional shape (protect against endonucleases ?); − Plant pathogens; − Base sequence similar to introns. Prions proteinlike infectious particle. Devoid of DNA or RNA. Prions contain protein only (although this is somewhat controversial). They are small, proteinaceous particles and there is controversy as to whether they contain any nucleic acid, but if there is any, there is very little, and almost certainly not enough to code for protein. A prion is a small infectious particle that contains a protein. Some researchers believe that a prion consists of proteins without nucleic acids because a prion is too small to contain a nucleic acid and because a prion is not destroyed by agents that digest nucleic acids. Prions Diseases: − Scrapie (sheep); − Creutzfeldt-Jacob disease (CJD); − Kuru (Tribes in New Guinea); − Bovine Spongiform Encephalopathy (BSE). Prion diseases referred to as transmissible spongiform encephalopathies (TSEs) are progressive neurological diseases that are fatal to humans and animals. Researchers believe that prions cause Creutzfeldt-Jakob disease. Creutzfeldt-Jakob disease is a neuro78
logical disease that causes progressive dementia first observed by Hans Gerhard Creutzfeldt and Alfon Maria Jakob in the 1920s. In 1976, Carlton Gajdusek won the Nobel prize for his work with the TSE Kuru. Kuru is characterized by progressive ataxia incapacitation and death. In 1982, neurobiologist Stanley Prusiner proposed that proteins cause the neurological disease Scrapie, which is a degenerative neural conditon in sheep. Prusiner named this infectious protein prion. Prions also cause other neurological diseases such as Kuru and Gerstmann-Strausler-Sheinker syndrome. However, scientists are still studying prions to learn their origins and how prions replicate and cause disease. MULTIPLICATION OF VIRUSES Animal viruses infect and replicate animal cells. They differ from bacteriophages in the way they enter a host cell. For example, DNA viruses enter an animal host in this way: 1. Attachment. Animal viruses attach to the host cell's plasma membrane proteins and glycoproteins (host cell receptors). Types of receptors: Glycoproteins − contain neuraminic acid Glycolipids (gangliosides) − contain sialic acid. Viruces attachment proteins: Filaments (adenoviruce) or Spikes (orthoviruces, paramyxoviruces). 2. Penetration. Animal viruses do not inject nucleic acid into the host eukaryotic cell. Instead, penetration occurs by endocytosis, where the virion attaches to the microvillus of the plasma membrane of the host cell. The host cell then enfolds and pulls the virion into the plasma membrane, forming a vesicle within the cell's cytoplasm. Fusion with membrane of host. Nucleocapsid penetrates into the cell. Superkapsid integrated into cytoplasmic membrane. 3. Transport of the virus into the cell. It occurs with intracellular membranous vesicles, in which the virus is transferred to the ribosomes, endoplasmic reticulum, or in the cell nucleus.
4. "Undressing“ (Uncoating) of the virus - Deproteinization and removal of the capsid and superkapsid - separation of the Viral Genome from the capsid. 5. Biosynthesis of viral components. It starts with a reproduction of the virus. It has the separate character - virion components are synthesized in different parts of the cell: − The proteins on ribosomes; − Nucleic acids in the nucleus (DNA) or (RNA) on cytoplasm. When the virus has taken over the cell, it immediately directs the host to begin manufacturing the proteins necessary for virus reproduction. The host produces three kinds of proteins: early proteins-enzymes used in nucleic acid replication, late proteins-proteins used to construct the virus coat, and lytic proteins-used to break open the cell for viral exit. The capsoid material is made from the host's resources. The final viral product is assembled spontaneously, that is, the viruses do not grow their parts as most organisms do, their protein parts are made separately by the host and come together by chance. 6. Assembly of viral particles. The principles of the assembly of viral particles. − Formation of virus is a multistage process with the formation of intermediate forms. − The assembly of simple viruses is the formation of nucleocapsid. Sophisticated viruses initially formed nucleocapsid proteins that interact with capsid shells. − Formation of virus occurs in the nuclear or cytoplasmic membranes of cells. − Sophisticated viruses include in its membership the components of the host cell. 7. Release viruces from cell. By lysis or exocytosis. Lysis - The virus particles burst out of the host cell into the extracellular space resulting in the death of the host cell. Once the virus has escaped from the host cell it is ready to enter a new cell and multiply. Exocytosis (Budding) - As the newly formed viral particle pushes against the host cell’s plasma membrane a portion adheres to it. The plasma membrane envelops the virus and becomes the viral 80
envelope. The virus is released from the cell. This process slowly uses up the host’s cell membrane and usually leads to cell death. Multiplication of RNA Viruses: 1. Attachment to host cell receptor. 2. Fusion with membrane of host. 3. Nucleocapsid enters the cytoplasm. 4. Transcription in cytoplasm by viral RNA polymerase. 5. Translation by host cell ribosomes. 6. Assembly of viral particles. 7. Release from cell. INTEGRATIVE INFECTION Virogeny – (integration of incorporation) viral nucleic acid in the cell genome; Provirus − a virus that is integrated in the cellular genome. Viruses leave the host DNA intact and incorporate their own into the host's chromosomes. When viral DNA is incorporated into the host's chromosome as a latent form, this is called a provirus. The herpesvirus can remain latent as a provirus in a host indefinitely only to be excised from the host's genome in times of stress. This is why someone can have recurrent infections long after he has seemingly been cured. Defective viruses. Defective (imperfect, faulty) viruses, viruses that have lost in the process of replication of its genome: 1) Defective interfering particles − virions containing only part of the genetic information of the source of the virus. Reproduced only in the presence of a related helper viruses. 2) Viruses satellites − for reproduction requires the involvement of any helper virus. 3) Integrated genomes − proviruses. 4) Pseudovirion − virion with normal capsid containing a part of their nucleic acid and nucleic acid fragments of the host or another virus. The clinical consequences of persistent activation of human viruses: recurrences of acute infection. The chronic course of infection, intrauterine infection, tumorigenicity. 81
BACTERIAL VIRUSES − BACTERIOPHAGES Viruses that use bacterial cells as hosts are called bacteriophages. There is hardly a single species of bacteria where sufficient investigation has not found a phage. The presence of bacteriophage is recognized by the appearance of 'plaques' or lytic holes in a continuous bacterial lawn. Phage nucleic acid occurs either as double or single stranded nucleic DNA or as a double or single stranded RNA. Phages are distinguished: 1. On the morphology; 2. Structural organization; 3. Type of nucleic acid; 4. The nature of interaction with the microbial cell.
Figure 15 − Practical importance of Bacteriophage
Distinctive properties complicated bacteriophage: − binary symmetry; − contractile sheath on the rod; − The introduction of nucleic acid of bacteriophage into the bacterial cell by injection (an injection mechanism). According to specificity of action are distinguished: 1) Polyvalent phage − able to interact with related bacterial species. 2) Monovalent phage − interact with certain types of bacteria. 3) Typical phages − interact with individual variations (types) of this bacterial species. Multiplication in Phage Bacteriophages replicate by either a lytic cycle or a lysogenic cycle. The difference in these two cycles is that the cell dies at the end of the lytic cycle and remains alive in the lysogenic cycle. The first two scientists to observe bacteriophages were Frederic Twort of England and Felix d'Herelle of France in the early 1900s. The name bacterio-phage is credited to d'Herelle and means "eaters of bacteria." Lytic Cycle. The most studied bacteriophage is the T-even bacteriophage. The virions of T-even bacteriophages are big, complex, and do not contain envelopes. The T-even bacteriophages are composed of a head-and-tail structure and contain genomes of double-stranded DNA. The lytic cycle of replication begins with the collision of the bacteriophage and bacteria, called attachment. The tail of the bacteriophage attaches to a receptor site on the bacterial cell wall. After attachment, the bacteriophage uses its tail like a hypodermic needle to inject its DNA (nucleic acid) into the bacterium. This is called penetration. The bacteriophage uses an enzyme called lysozyme in its tail to break down the bacterial cell wall, enabling it to inject its DNA into the cell. The head or capsid of the bacteriophage remains on the outside of the cell wall. After the DNA is injected into the host's bacterial cell's cytoplasm, biosynthesis occurs. Here the T-even bacteriophage uses the host bacterium's 83
nucleotides and some enzymes to make copies of the bacteriophage's DNA. This DNA is transcribed to mRNA, which directs the synthesis of viral enzymes and capsid proteins. Several of these viral enzymes catalyse reactions that make copies of bacteriophage DNA. The bacteriophage DNA will then direct the synthesis of viral components by the host cell. Next maturation occurs. Here the T-even bacteriophage DNA and capsids are put together in order to make virions. The last stage is the release of virions from the host bacterium cell. The bacteriophage enzyme lysozyme breaks apart the bacterial cell wall (lysis) and the new virus escapes. The escape of this new bacteriophage virus will then infect neighboring cells and the cycle will continue in these cells. The Lysogenic Cycle. Some viruses do not cause lysis and ultimate destruction of their host cells which they infect. These viruses are called lysogenic phages or temporate phages. These bacteriophages establish a stable, long-term relationship with their host called lysogeny. The bacterial cells infected by these phages are called lysogenic cells. The most studied bacteriophage, which multiplies using the lysogenic cycle, is the bacteriophage Lambda. This bacteriophage infects the bacterium E. coli. When the bacteriophage Lambda penetrates an E. coli bacterium, the bacteriophage DNA forms a circle. The circle recombines with the circular DNA of the bacteria. This bacteriophage DNA is called a prophage. Every time the bacteria host cell replicates normally, so does the prophase DNA. On occasion, however, the bacteriophage DNA can break out of the prophage and initiate the lytic cycle. Lysogenicity- symbiosis of microbial cells with prophage. Prophage − temperate phage: lysis not all cells in a population and phage DNA integrated into the bacterial chromosome. Lysogenic culture − the culture of bacteria containing a prophage. Prophage Induction − produce phage by lysogenic bacteria. The phage 84
(lysogenic) Convergence − changing the properties of microorganisms under the influence of prophage (Corynebacterium diphtheriae; Streptococcus pyogenes) or Scarlet Fever (Clostridium botulinum). Application of of bacteriophages: 1) Identification of bacteria − phage typing; 2) Phagotherapy − treatment of infections and dysbacterioses; 3) Epidemiological analysis − identification of the source of infection; 4) Genetic engineering - the creation of vectors to production of recombinant DNA
Figure 16 − Morphology types of phages
Growing Viruses; 1. Bacteriophages − Lawn of Bacteria on a Spread Plate; 85
− Add Bacteriophages; − Infection will result in “Plaques”. 2. Animal Viruses − Living Animals (mice, rabbits, guinea pigs); − Chicken Embryos (Eggs). Used to be most common method to grow viruses. Still used to produce many vaccines (Flu Vaccine). − Cell Cultures − most common method to grow viruses today.
METHODICAL INSTRUCTIONS TO LABORATORY WORKS ON DISCIPLINE "MICROBIOLOGY AND VIROLOGY" Lab work №1. Subject: Rules of Applied Microbiology Laboratory. Session Purpose: Know the rules of the laboratory work and safety. Objectives: 1. To review the safety precautions in the microbiology laboratory. 2. Sign the page after you have reviewed the safety procedures. Rules of Applied Microbiology Laboratory. The signature page of this lab manual, following these lab rules, must be complete prior to your participation in any laboratory for this class. During Laboratory Procedures: 1. Do not bring any outerwear into lab. This includes cell phones! Absolutely NO cell phone use during lab. 2. Wear a lab coat, close-toed shoes, gloves and goggles at all times during the experiment. 3. Disinfect your work space before and after each lab. 4. Using a sterilized and cooled inoculation loop. The bacteriological loops and needles are disinfected by baking in a burner flame before and after the screening of microorganisms. 5. Tie back long hair. 6. Do not consume food and/or beverages, apply cosmetics, touch your face or put anything into your mouth in the lab. 7. Clean and store microscopes properly after each use. Scope will be checked and points deducted for improper microscope care. 8. Turn off your Bunsen burner when it is not in use. 9. Do not pick up broken glass. Dispose of broken glass using the dust pan and broom, in the box designated for ‘glass’. 10. If you spill a culture, cover the spill with a paper towel, soak the towel in disinfectant and allow it to sit for 20 minutes. Dispose of the toweling in a biohazard bag. 87
11. Know the location of the fire extinguisher, fire blanket and eye wash station before you begin your work. 12. Report any accident or injury to the instructor. 13. If you are feeling ill, do not work with live microbes. 14. If you are pregnant or taking immunosuppressant drugs you may need to postpone taking this class. Please have this discussion with your physician. 15. Adhere to the directions for disposal of all items used in the lab. 16. The research results are recorded in exercise book. Lab work №2. Subject: The Laboratory Microscope. Session Purpose: How to Use a Compound Light Microscope. Objectives: 1. Learn proper use and care of a compound light microscope. 2. Use the terminology (listed) correctly. 3. Perform exercises 1-3. Parts of the Microscope. Basically, the microscope consists of a support system, a light system, a lens system, and a focusing system. Each of these systems works together to produce a magnified image of the specimen. Support System. The support system consists of the base, arm, and stage. The base and arm are structural elements which hold the other parts of the microscope in place while the stage holds the slide. Depending on the microscope, the slide can be positioned under two spring clips and moved by the fingers, or it can be held in place by a mechanical stage and moved by means of two control knobs. Light System. The light system passes light through the specimen using the light source, the condenser, and the iris diaphragm. In a bright-field microscope, an incandescent bulb is usually used as the source of illumination. Light from the light source then passes through the condenser which focuses the light on the specimen. An iris diaphragm is used to control the intensity, or brightness, of light which passes through the specimen, thus allowing the operator to adjust the intensity and achieve an optimum viewing contrast. Lens System. The lens system forms the actual image which you will see when you look through a microscope. A typical compound 88
microscope has two lenses - an objective lens near the specimen and an ocular lens at the top - each of which magnifies the image of the specimen by a certain amount. The ocular lens on most microscopes magnifies 10x (meaning that the image produced by the ocular lens is ten times as large as the specimen). In contrast, the typical microscope has at least three objective lenses mounted on a revolving nosepiece to allow for different magnifications. The low-power objective is the shortest and generally magnifies 10x; the middle-sized lens is the high-dry objective which usually magnifies between 40x and 45x; and the longest lens is the oil-immersion lens which usually magnifies between 97x and 100x. To determine the total magnification (TM) of the image, simply multiply the magnification power of the ocular lens by the magnification power of the objective lens which is being used. For example, if you are using a high-dry objective with a magnification power of 40x, then the total magnification will be 10 × 40 = 400x. Alternatively, using the 10x low-power objective, the total magnification would be simply 10 × 10 = 100x. As you can see, you would use the higher power objective lenses to magnify smaller objects and the low-power objective lens to magnify large objects. There is a limit to the amount of useful magnification one can achieve with a light microscope. The maximum resolving power normally possible with a light microscope is about 0.2 micrometers (um), or 1/100,000 inch. Smaller objects can be viewed using an electron microscope. Focusing System. The final system at work in the microscope is the focusing system. So far, we have learned how all of the components of the microscope are held together by the support system, how the light system sends light through the specimen, and how the lens system uses that light to magnify the specimen's image and transmit it to our eyes. The focusing system adjusts the distance between the slide and the objective lens so that the image comes into focus. The focusing system consists of two knobs − the coarse adjustment knob and the fine adjustment knob. When focusing, the operator first turns the coarse adjustment knob (which is the larger focus knob) in order to move the stage a large distance and bring the 89
image into the focal plane of the objective lens. At this stage, the image will be visible but fuzzy. Then the operator turns the smaller knob, known as the fine focus knob, to fine tune the focus and to make the image sharply focussed.
Figure 17 − Parts of the Microscope
Care of the Microscope. Microscopes are delicate pieces of equipment, so you should follow a few basic rules to prevent damage to the microscope. These rules are meant to prevent you from: − dropping the microscope − damaging the lenses; − storing the microscope improperly. Dropping a microscope can break the lenses or can alter the alignment of the lenses. To prevent this damage, you should always carry the microscope with two hands - one hand under the base and the other hand on the arm of the microscope. When using the microscope, keep the instrument at least six inches from the edge of the lab table and keep any excess electrical cord on the table top to keep the microscope from being pushed or pulled off the table. The 90
microscope's lenses are very delicate and can easily be scratched or damaged by oils. Lenses should be cleaned before and after each use with special lens paper. (Cleaning with paper towels or cloth can damage the lenses.) In addition, you should refrain from touching the glass lens with your finger to avoid depositing oils or scratching the glass. When using the microscope to view a specimen, you should follow common sense rules of behavior. Do not tamper with any part of the microscope unless you understand its purpose. A common mistake is to focus quickly while looking through the eyepiece of the microscope so that the objective lens bumps into the slide. To prevent damage to the lens, you should always make large focus changes slowly while observing the movement of the objective lens from the side of the microscope. Finally, the microscope should be stored carefully. Unplug the electrical cord by pulling on the plug instead of the cord. Remove oil from the oil-immersion objective using lens paper, then turn the nosepiece so that the low-power objective is in place. Carefully lower the objective to its lowest position by turning the coarse adjustment knob. Then store the microscope under a dust cloth. Lab Exercise 1. Parts of the Microscope. Examine your microscope carefully. Find the location of each of the parts listed below: Ocular Lens Nosepiece Objectives Low-dry High-dry Oil-immersion Mechanical Stage Coarse Adjustment Knob Fine Adjustment Knob Condenser Condenser Adjustment Knob Microscope Base 91
Arm Iris Diaphragm Lever Light Source
Lab Exercise 2. Observe the Letter “e”. To familiarize yourself with the workings of the microscope, perform the following exercises: 1. Cut a small case letter "e" from the newspaper. Prepare a "wet mount" using the following technique: a. Get a clean microscope slide b. Place a drop of water on the slide c. Place the "e" in the drop of water d. Apply a cover slip 2. Observe the "e" under scanning, low and high power. Draw or take a photomicrograph (depending on which version of the lab report you are completing) of what you see at 100xTM and 400xTM and label with the magnification power. (Note: Always label micrographs with the total magnifying power). 3. What happened to your field of view as you increased your magnification? (Think about how much of the '"e" you see as you go from scanning to high power.) 4. Compare the '"e" that you observe with your unaided eye to the view through the microscope. What is different about it? (i.e. Look at the "e" the way it is mounted on your slide and then view it through the microscope. Does its orientation change?) 5. Create a micrograph of your “e” at 100xTM and 400xTM. A micrograph is a photo of what you see through the microscope. Lab Exercise 3. Equipment: Microscope Slide Coverslip Dropper Dropper bottle of water Newspaper print 92
Complete the Table. PART Base Light Source Iris Diaphragm Condenser Stage Arm Coarse Adjustment Knob Fine Adjustment Knob Low Power Objective High Power Objective Ocular Lens Revolving Nosepiece
Lab work № 3. Subject: Viewing Specimens. Session Purpose: Learn of how to make a wet mount slide of a specimens. To view microorganisms learning to operate a microscope. Objectives: 1. Learn how to make a wet mount slide «The crushed drop» from liquid microbic culture. 2. Learn how to make a wet mount slide «The crushed drop» from agar microbial culture. 3. Perform exercises 1,2. «The crushed drop» from liquid and agar microbial culture. The preparation «the crushed drop» quickly prepares and allows to establish the form of cells, their sizes, character of congestions, mobility, but is short-lived. Lab Exercise 1. «The crushed drop» from liquid microbial culture. Methodical instructions: Prepare a specimen of liquid microbial culture. Observe the cells of algae. Observe the following and describe what you see: a) Chlorella vulgaris; b) Spirulina platensis. 93
Procedure: 1. Obtain a clean microscope slide. 2. Using the dropper, place a drop of liquid microbial culture onto the center of a clean, dry slide.
3. Hold the side edges of the coverslip and place the bottom edge on the slide near the drop of specimen.
4. Slowly lower the coverslip into place. The water should spread out beneath the coverslip without leaving any air bubbles. If air bubbles are present, you can press gently on the coverslip to move the air bubbles to the sides.
5. Set up the microscope. 6. Remove the dust cover from the microscope. Plug in the microscope. Turn on the microscope's light source. Place the condenser at its highest position and close the iris diaphragm somewhat to prevent overbrightness. If the microscope has two ocular lenses, adjust the width between the lenses to your eyes. You will know the width is correct when you see one bright circle of light rather than two. 94
7. View the specimen with the low-power objective. Turn the nosepiece until the low-power objective locks into place. Place the slide on the stage and center it over the condenser. Looking from the side, turn the coarse adjustment knob until the lowpower objective is in its lowest possible position. Looking through the ocular lens, slowly turn the coarse adjustment knob in the other direction. This raises the low-power objective away from the slide. Continue until a clear image appears. Slowly turn the fine adjustment knob until the object comes into sharp focus. Adjust the light for maximum contrast using the iris diaphragm. Move the slide around on the stage using your fingers or the control knobs until you find a microorganism. 8. Sketch a picture of the microorganism. 9. Sign the picture and specify Total Magnification (TM). 10. View the microorganism with the high-power objective. Center the microorganism exactly in the center of the field of view. Turn the nosepiece until the high-power objective locks into place. At this point, the microorganism should still be roughly in focus. Turn the fine adjustment knob slowly to focus the image. (Never use the coarse adjustment when the high power objective is in place!). Open the iris diaphragm until optimum contrast is achieved. 11. Sketch a picture of the microorganism. Sign the picture and specify Total Magnification (TM). Lab Exercise 2. «The crushed drop» from agar microbial culture. Methodical instructions: Prepare a specimen of agar microbial culture. Observe the cells of Yests and describe what you see. Procedure: 1. Obtain a clean microscope slide. 2. Place one drop of water on the slide.
3. Sterilize the transfer loop in the in the burner flame (NOTE: Never pick up the loop to use it or put it down without sterilizing it first!) 4. Allow loop to cool. 5. Using your sterilized inoculation loop, obtain a small sample of microbial culture from the source tube. Note: 1) If obtaining microbe sample from slant tubes: − Never pick up test tube by the cap; − DO NOT set cap down on lab bench; − Flam neck of the test tube before and after obtaining sample. 2) Be gentile! The most common error is transferring too much inoculum (bacteria) when making smears from solid media. The microbial colonies are found growing on the surface of the agar medium. DO NOT remove agar with your sample! 3. After mixing the inoculum in the water, flame the loop to sterilize it before setting it down. 4. Hold the side edges of the coverslip and place the bottom edge on the slide near the drop of specimen.
5. Slowly lower the coverslip into place.
6. View the microorganism with the low-power objective (x10) and high-power (x40) objective. 7. Sketch a picture of the microorganism. 8. Sign the picture and specify Total Magnification (TM). Proper Storage of Your Microscope To practice proper care of your microscope, make certain to clean it and put it away properly at the end of this exercise. 1. Make certain the slide is removed from the stage. 2. Clean all lens with Lens Paper. Obtain a clean sheet of lens paper. Rub oculars to clean. 3. Pull the body tube away from the stage (i.e. lower the stage as far as possible) 4. Turn off the microscope. Turn the nosepiece of the microscope until the low-power objective locks into place. Carefully lower the objective to its lowest position by turning the coarse adjustment knob. 5. Turn off the light source. 6. Remove your slide. Clean the slide and coverslip with water. 7. Unplug the microscope and store it under a dustcloth. 8. Wrap the cord. 9. Return the proper storage location in the cabinet. Equipment: Microscope Slide Coverslip Dropper Dropper bottle of water Disinfectant tray Liquid culture of Chlorella vulgaris Liquid culture of Spirulina platensis Culture of Yeasts in slant tubes Inoculation loop Burner flame 97
Lab work № 4. Subject: Staining Methods. Simple Staining with Methylene Blue. Session Purpose: Learn of how to make a Simple Staining with Methylene Blue. How to Prepare & Heat Fix a Microbial Smear for Staining. Objectives: 1. Learn how to make a stained vital "crushed drop". 2. Learn how to make a smear using the heat fixation process. 3. Learn how to make a stained fixed smear. 4. Perform exercises 1, 2. Not all specimens can be clearly seen under a microscope. Sometimes the specimen blends with other objects in the background because they absorb and reflect approximately the same light waves. You can enhance the appearance of a specimen by using a stain. A stain is used to contrast the specimen from the background. A stain is a chemical that adheres to structures of the microorganism and in effect dyes the microorganism so the microorganism can be easily seen under a microscope. Stains used in microbiology are either basic or acidic. Basic stains are cationic and have positive charge. Common basic stains are methylene blue, crystal violet, safranin, and malachite green. These are ideal for staining chromosomes and the cell membranes of many bacteria. Acid stains are anionic and have a negative charge. Common acidic stains are eosin and picric acid. Acidic stains are used to stain cytoplasmic material and organelles or inclusions. There are two types of Stains: simple and differential. A simply stain has a single basic dye that is used to show shapes of cells and the structures within a cell. Methylene blue, safranin, carbolfuchsin and crystal violet are common simple stains that are found in most microbiology laboratories. There are two ways to prepare a specimen to be observed under a light compound microscope. These are a wet mount and a smear. A wet mount is a preparation process where a live specimen in culture fluid is placed on a concave glass side or a plain glass slide. 98
The microorganism is free to move about within the fluid, although the viscosity of the substance slows its movement. This makes it easier for you to observe the microorganism. The specimen and the substance are protected from spillage and outside contaminates by a glass cover that is placed over the concave portion of the slide. Obviously if trouble has been taken to isolate pure cultures of bacteria, one wants to ensure that only these bacteria are viewed under the microscope. Glass slides are not sterile and are often coated with numerous bacteria as well as dust and spray cellulose fibres. Slides should therefore be wiped clean with tissue paper and passed once or twice through a bunsen flame. Cover slips when used should be wiped with tissue paper. Always hold slides either in a slide holder or on the edge surfaces not on the planar surface. Cover slips should always be held be the edge surface. A smear is a preparation process where a specimen that is spread on a slide. You prepare a smear using the heat fixation process. For most staining procedures one need only air dry the film by holding the slide high above a bunsen flame, when dry pass the slide film side up, three times through the flame to kill and fix the cells. Too much heat will distort the shape of the organism and could alter its uptake of stain - the slide should feel warm but not hot. Bear in mind that staining procedures kill the cells and that some of the cells characteristics may inevitably be altered. All stainings are to be carried out over the staining rack or in the sinks. The purpose of simple staining technique is to determine cell shape, size and arrangement of bacterial cells. It is a simplest staining technique to get knowledge about bacterial shape, is either coccus or bacillus or spiral in shape. Lab Exercise 1. Vital "crushed drop"specimen - wet mount. Methodical instructions: Prepare a stained vital "crushed drop"specimen of agar microbial culture. Observe the cells of Yests and describe what you see. 99
Procedure: 1. Obtain a clean microscope slide. 2. Place one drop of water on the slide.
3. Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony. 4. Gently mix the bacteria into the water drop. 5. Place one drop of a weak solution (1:1000) dye (methylene dark blue or fuchsin). 6. Hold the side edges of the coverslip and place the bottom edge on the slide near the drop of specimen.
7. Slowly lower the coverslip into place.
8.View the microorganism with the high-power (x40) objective. 9. Sketch a picture of the microorganism. 10. Sign the picture and specify Total Magnification (TM). Lab Exercise 2. Heat Fixing the Microbial Sample. Stained fixed smear. Before staining, the sample must be heat fixed. This process accomplishes three things. It functions to: kill the bacteria 100
firmly affix the smear to the microscope slide allow the sample to more readily take up the stain.
In order to heat fix a bacterial smear, it is necessary to first let the bacterial sample air dry. Then either place the slide in the slide holder of a microincinerator, or pass the dried slide through the flame of a Bunsen burner 3 or 4 times, smear side facing up. Once the slide is heat fixed, it can then be stained. Methodical instructions: Prepare a stained fixed smear of agar microbial culture. To go for Simple staining the first thing you should have to do, is to prepare a thin bacterial film i.e. Bacterial smear on a clean and dry glass slide and fix it. Make it by mixing a small amount of Microbial culture in small water droplet and spread it. Just fix it by heating carefully. You can follow the part two of this series to make Microbial smears perfectly and quickly fixed. Application of staining reagent (dye). When your smear is ready go with any basic dye (cationic dyes e.g. crystal violet, carbol fuchsin, methylene blue etc.) and flood it on heat fixed Microbial smear for few seconds. Different dyes have different exposures time to stain cell, for example-crystal violet, 2-60 seconds; carbol fuchsin,15-30 seconds and methylene blue,15-20 seconds. The increased time will result in increase in darkness. Removal of excess dye and observation. You applied the stain, some amount of which get bound to the cells while some amount is freely moving in solution. To eliminate or say minimize the background noise of stains it is necessary to Remove remaining amount. It is simply done by washing out the slide with water and then dry it either in air or by blotting the surface now apply a drop of oil-immersion on it and examine under 100X of microscope to see colourful cells under transparent background. Procedure: a) Heat Fixing the Microbial Sample. 1. Use a clean glass slide. 2. Take a loop of the culture. 3. Place the live microorganism on the glass slide. 101
4. The slide is air dried then passed over a Bunsen burner about three times. The heat causes the microorganism to adhere to the glass slide. This is known as fixing the microorganism to the glass slide.
b) Stain the microorganism with an appropriate stain: 1. Place a slide on the centre supports of the staining rack, and flood the smear with a few drops of the methylene blue stain, and allow to act for 1-3 min. 2. Wash the smear with water (either from a wash bottle or a slow running tap) to remove dye. 3. Dry the slide using absorbent paper pressed lightly over the surface. 4. Examine the stained preparation under the x40 objective. 5 .Sketch a picture of the microorganism. 6. Sign the picture and specify Total Magnification (TM). 102
Notice: No coverslip is required with stained preparations, but take due care when using high magnification that the objective lens does not touch the smear. Proper Storage of Your Microscope. Equipment: Microscope Slide Coverslip Dropper Dropper bottle of water Disinfectant tray Culture of Yeasts in slant tubes Inoculation loop Burner flame Staining material - Methylene blue stain
Lab work № 5. Subject: Morphology of the bacteria. Session Purpose: Acquaintance with morphology of bacteria and methods of its studying under a microscope fixed stained smear. Objectives: 1. How to Prepare & Heat Fix a Bacterial Smear for Staining 2. Practice simple staining of bacteria. 3. How to use oil immersion lens. 4. View bacteria under oil immersion. 5. Perform exercises 1, 2. 103
Lab Exercise 1. Stained fixed smear of rod-shape bacteria. Methodical instructions: To observe prokaryotic cells, and practice oil immersion techniques, obtain a sample from the source plates or tubes on your lab bench (Escherichia coli and Bacillus subtilis) according to instructions below. Each lab partner should prepare a slide. Execute aseptic technique, as outlined below, to transfer bacteria to a slide. Observe the cells of rod-shape bacteria (Escherichia coli and Bacillus subtilis), describe what you see. Procedure: 1. Use a clean glass slide. Draw two circles with pencil. 2. Put a drop of water in each circle. 3. Inoculate circle of water which each of following: Escherichia coli; Bacillus subtilis. 4. After heat fixing, stain with Methylene blue and allow to act for 60 sec., rince. Notice: No coverslip is required with stained preparations, but take due care when using high magnification that the objective lens does not touch the smear. 5.Then observe under oil immersion lens. 6. Sketch a picture of each type of bacteria under oil immersion Observing bacteria under oil immersion lens: 1. Make sure that your bacterial smear is clearly in focus at 100 x TM. 2. Put a drop of immersion oil directly on each of the two bacterial smears on your slide, then switch to the oil immersion lens. 3. Only use fine focus adjustment 4. When done, use lens paper to clean up your lens and the stage.
Lab Exercise 2. Stained fixed smear of sphere-shape bacteria. Methodical instructions: Prepare a stained fixed smear of agar bacterial culture. Observe the cells of sphere-shape bacteria (Staphylococcus aureus and Sarcina lutea), describe what you see. Procedure: 1. Prepare fixed smears of the two bacterial strains as outlined (Staphylococcus aureus; Sarcina lutea). 2. Place a slide on the centre supports of the staining rack, and flood the smear with a few drops of the Fuchsine stain, and allow to act for 60 sec. Wash the smear with water (either from a wash bottle or a slow running tap) to remove dye. 3. Dry the slide using absorbent paper pressed lightly over the surface. 4. Examine the stained preparation under oil-immersion lens. Notice: Remember to wipe the oil-immersion lens with tissue whenever it is removed from the immersion oil. Proper Storage of Your Microscope To practice proper care of your microscope, make certain to clean it and put it away properly at the end of this exercise. 1. Make certain the slide is removed from the stage. 2. Clean all lens with Lens Paper if there is excess oil. a. Obtain a clean sheet of lens paper. b. Rub oculars to clean as demonstrated. c. After use, carefully clean all lens surfaces with lens tissue. If by chance oil gets onto the x10 or x40 objectives wipe off immediately. Do not leave immersion oil on the immersion objective when you put the microscope away. 3. Put the scanning power objective in place. 4. Pull the body tube away from the stage (i.e. lower the stage as far as possible) 5. Wrap the cord. 6. Return the proper storage location in the cabinet. Equipment: Microscope Slide Dropper bottle of water 105
Disinfectant tray Culture of Bacteria in slant tubes Inoculation loop Burner flame Staining material - Methylene blue and Fuchsine stains Immersion oil
Lab work № 6. Subject: Differential Staining. Gram Staining. Session Purpose: To study the organization of the bacterial cell wall, determining the relationship of microorganisms to Gram staining. Objectives: 1. Determine the ratio of Gram staining an unknown culture, to take control cultures of Staphylococcus aureus and Escherichia coli. 2. Master the technique of making the short test with 3% KOH. 3. Perform exercises 1-3. Differential Vs Simple Staining: Simple staining use single dyes while differential staining procedures take use of more than a single dye for visualization. As there are more than one dyes are used for differential staining purposes the cell appears multicoloured and cellular organization can be visualized easily and more confidently. Simple staining technique can only give informations about cell shape, cell size clustering or arrangement of the cells while differential procedures can give additional informations in terms of presence or absence of any specific cellular part, or substance in cells, making characterization and identification of microorganisms easy. Differential staining techniques are for making cellular differences visible, giving opportunity to distinctly compare or characterize microorganisms. There are two major powerful applications of differential staining, viz. characterization and identification. Characterization of microorganisms: every microbial cell is not alike and the classification and characterization of these 106
microbial cells requires revelation of these differing characters. If these differences are based on structural levels, it can be easily traced using differential staining techniques. Gram’s staining technique is one of the most important and versatile technique of differential staining which is necessarily applied for classification of bacteria at initial levels of study. This differential staining is the most significant technique in whole microbial staining approaches. Acid-fast staining, Endospore staining etc are some other differential staining procedures used for identification and characterization of microorganisms. Locating structural variations: Every microorganism is cellular and contains similar features but they also have some distinct variations. These variations may be in form of chemical composition of any specific cellular part or complete presence or absence of any cellular component. Differential staining can also locate these structural variations such as endospores, flagella, capsules, pilli etc. In 1884 Hans Christian Gram, a Danish physician, developed the Gram stain. Gram-stain is a method for the differential staining of bacteria. Gram-positive microorganisms stain purple. Gram-negative microorganisms stain pink. Staphylococcus aureus, a common bacterium that causes food poisoning, is Gram-positive. Escherichia coli is Gram-negative. Lab Exercise 1. Gram Stain Procedure (variant A). Methodical instructions: In one glass of skim make strokes of different microorganisms in the center − a smear of cells studied culture, left and right - the control of microorganisms. The cells of one of the test organisms (eg, Staphylococcus aureus) Gram stain, and the cells of another (for example, Escherichia coli) − not colored. Smears must be thin so that the cells are uniformly distributed over the surface of the glass and did not form clusters, since the thickness of the stroke depends on the results of staining. Smears air-dried and fixed over the burner flame. Procedure: 1. Prepare the specimen using the heat fixation process. 2. Place a drop of crystal violet stain on the specimen for 1-2 min. Poured into the dye, not washing the smear with water. 107
3. Apply iodine on the specimen using an eyedropper for 1-2 minutes. The iodine helps the crystal violet stain adhere to the specimen. Iodine is a mordant, which is a chemical that fixes the stain to the specimen. 4. Wash the specimen with an ethanol during 0.5-1 min. 5. Wash the specimen with water to remove the dye. 6. Apply the fuchsin stain to the specimen using an eyedropper. 7. Wash the specimen. 8. Use a paper towel and blot the specimen until the specimen is dry. The specimen is ready to be viewed under the microscope. Grampositive bacteria appear blue-violet and gram-negative bacteria appear pink. When stained by Gram, the following errors: a) All cells are blue due to lack of bleaching; b) All cells are pale pink, gentian violet staining due to insufficient or excessive treatment with alcohol. For comparison, you can use an accelerated test for determining membership gram-positive bacteria or gram-negative species. Lab Exercise 2. Gram Stain Procedure (variant B). Methodical instructions: In one glass of skim make strokes of different microorganisms in the center − a smear of cells studied culture, left and right − the control of microorganisms. The cells of one of the test organisms (eg, Staphylococcus aureus) Gram stain, and the cells of another (for example, Escherichia coli) − not colored. Smears must be thin so that the cells are uniformly distributed over the surface of the glass and did not form clusters, since the thickness of the stroke depends on the results of staining. Smears air-dried and fixed over the burner flame. Procedure: 1. Prepare the specimen using the heat fixation process. 2.Place a drop of crystal violet stain on the specimen for 1-2 min, and then the dye is washed off. 3. Apply iodine on the specimen using an eyedropper for 1-2 minutes and washed off. The iodine helps the crystal violet stain adhere to the specimen. Iodine is a mordant, which is a chemical that fixes the stain to the specimen. 108
4. Wash the specimen with an alcohol-acetone decolorizing solution during 0.5-1 min. 5. Wash the specimen with water to remove the dye (now the gram-positive cells are purple and the gram-negative cells are colorless). 6. Apply the safranin stain to the specimen using an eyedropper. 7. Wash the specimen. 8. Use a paper towel and blot the specimen until the specimen is dry. 9. And examined microscopically. Gram-positive bacteria retain the purple dye, even through alcohol wash. Gram-negative bacteria appear pink because they pick up the safranin counterstain. Lab Exercise 3. Rapid method for determining membership gram-positive bacteria or gram-negative species. Procedure: 1. The culture of the bacteria studied by means of a loop is transferred to the dense medium on a glass slide in a drop of 3% KOH solution and mix thoroughly. 2. After 10 sec loop drops sharply raised above. 3. Under these conditions, Gram-negative bacteria is characterized by mucus, which stretches for a loop at 0.5-1.0 cm mucus occurs as a result of the destruction of the cell walls of gramnegative bacteria and leaving the nucleic acids. If the mucus is not produced, the bacterium belongs to the gram-positive. Equipment: Microscope Slide Dropper bottle of water Disinfectant tray Culture of Bacteria in slant tubes Inoculation loop Burner flame Staining material - Methylene blue and Fuchsine stains 3% KOH solution Immersion oil 109
Lab work № 7. Subject: Staining Microbial Structures: Capsule. Session Purpose: Negative staining. Capsule staining. Objectives: 1. Practice negative staining of bacteria. 2. Determine the capsule of Azotobacter. 2. Perform exercises 1,2. Capsules are mucilaginous substance, majorly composed of polysaccharides which are secreted by bacteria during their active growth and form a viscous coat around the cell. When this mucilaginous structure is irregularly arranged and loosely bound to the cell, referred by slime layer. Capsule is partially synthesized in cytoplasm and composed of mainly polysaccharide but may contain other material like poly-Dglutamic acid. The ability of capsule formation is genetically determined. The capsule is well develop in some bacteria like Azotobacter, Streptococcus pneumoniae, Clostridium perfringes and Klebsiella pneumoniae. To dertermine the presence or absence of capsule for classification and identification of bacteria is done by staining. There are two widely used methods for staining of bacterial capsule, which we will see below in detail: Capsule staining by negative staining method and Anthony staining method. Negative staining. Simply flooding the smears with an appropriate basic dye is not always the best option to stain the microbial specimens. The fixation of smears results in shriveled or distorted cells which are not appropriate for measuring the size of the cells. Additionally presence of refractile bodies such as Endospores, storage granules and capsule forming microorganisms are not easy to stain by dyes hence it is devised not to stain the cells but the background. As in this modification the background is stained not the cells hence they appear transparent against the dark background and the process is referred as indirect staining. This is simplest and often quickest staining technique used for microorganisms. 110
The microbial cells contain various components in its outer layer (cell membrane, cell wall etc) which results in development of overall negative charge at surface of microbial cells. When a basic dye is applied, positive chromophore group get attached to the cell surface and provides colour to the cells. Indirect staining uses the just opposite mechanism which is the use of acidic dyes. Acidic dyes contain chromophores with negative charge hence repelled by the cell surface and causes darkening of the background against colorless cells. As this indirect method of staining uses acidic or negatively charged dyes, the technique is most commonly referred as negative staining. Nigrosin or India ink) is the most commonly used acidic dye for this purpose. How negative staining is done? Application of stain: The first step is to take a drop of nigrosin (a negative stain) and place at one end of a clean glass slide. Inoculum transfer: After placement of stain, transfer a loop full of inoculum from a broth culture by inoculating loop on the applied stain. Then mix gently with the loop. If culture is taken from an agar medium then mix a drop of water in the nigrosin and try to emulsify without spreading the mix too much.
Spreading the smear: Take another clean slide and put it at 3045 degree angle, on the mixed amount.When it starts to spread on the junction of slides swipe the slide to another corner forming a thin smear. Drying the thin smear and observation: Allow the smear to air dry. After that apply a drop of oil-immersion on it and examine under 100X of microscope. The technique of negative staining can be easily applied for: 111
− Getting quick informations about the cell shape, arrangement without any heat distortion. − This technique is very useful to detect the presence of cell breakage and refractile inclusions in cells such as sulphur and polyβ-hydroxybutyrate granules and endospores without using any specific procedure. − The most common and routine application of negative staining is in determining the size of the microbial cells using microbial technique. Lab Exercise 1. Capsule staining by negative staining method. Methodical instructions: In this technique bacterial background is stained byusing an acidic dye (e.g. nigrosin dye or India ink), which carry a negative charge as a result is repel by bacterial cell that have also bears a negative charge as a result of which bacterial capsule that surrounded the cell is appears as clear zone between cell wall and dark background. Procedure: 1. Apply the nigrosin dye or India ink on glass slide: Take a clean glass slide and put a drop of nigrosin dye close to one end of glass slide. 2. Apply the bacterial culture: Add a loopfuls of a broth culture into the drop of the stain and mix with the loop. 3. Prepare a thin smear: Place a edge of second slide at 30° angle on first slide and pushed away to other end of the slide to prepare thin smear of suspended organism. 4. Air dry the smear. 5. Observation the slide: Place a drop of oil-immersion on the slide and examine under 100x magnification. Lab Exercise 2. Capsule staining by Anthony staining method. Methodical instructions: This method is devised by E.E. Anthony in 1931 for capsule staining in bacteria. In this method two stains viz. crystal violet (1% aqueous) work as primary stain and copper sulphate act as a decolorizing agent as well as counter 112
stain. Crystal violet color the both, cell wall as well as capsule and appears dark blue in color but capsule being non ionic will not absorb primary stain. On applying copper sulphate, excess stain removes out and capsular material become decolorized. The capsule finally appears light blue in contrast to the deep blue color of the cell. Procedure of this technique is:
Procedure: 1. Prepare the smear: Take a loop full of microbial culture broth at one end of slide and prepare a heavy smear of the bacterium. Then allow the smear to dry. 2. Apply crystal violet: Flood the smear with crystal violet for 2minutes. 3. Wash off the dye: Wash off the dye with 20% copper sulphate. 4. Drain copper sulphate: Drain out the copper sulphate and gently blot dry the smear. 5. Observe it: Place a drop of oil-immersion on the slide and examine under 100 x magnification. Equipment: Microscope Slide Dropper bottle of water Disinfectant tray Culture of Bacteria in slant tubes Inoculation loop Burner flame 113
Nigrosin or India ink Crystal violet (1% aqueous) Copper sulphate 20% Immersion oil
Lab work № 8. Subject: Staining Microbial Structures: Inclusions. Session Purpose: Methods for detection and visualization of intracellular polymers stored. Objectives: 1. To determine volutin in the cells of Saccharomyces. 2. To determine the granules of polyglucose in the cells of Bacteria and Yeast. 3. Detection of fat inclusions in Bacillus. 4. Stained fat inclusions. 5. Perform exercises 1-4. Lab Exercise 1. Detection and visualization of Polyphosphate (volutin granules) in Yeast. Methodical instructions:To identify volutin in yeast usually used the following method. Procedure: 1. Fixed smear stained with methylene blue (Loeffler's methylene blue staining) for 3 min. 2. The dye is poured, the drug is washed with water and without drying, applied to smear a small drop of 1% solution of sulfuric acid. 3. A smear covered with a coverslip. Volutin appears the form of drops of blue-purple color on the little-blue background of the cytoplasm. Lab Exercise 2. Detection and visualization of Polyphosphate (volutin granules) in Bacteria. Methodical instructions:Volutin detected by the method of coloring Omelyansky. Coloring is based on the metachromatic granules of low solubility in acid solutions. 114
Procedure: 1. To skim the slide is prepared thin smear of bacteria, it is dried in air and fixed over the burner flame. 2. On the fixed smear Ziehl's solution is poured and stained the cells for 0.5 min without heating. 3. The dye is poured, the drug was washed with water and additionally stained with methylene blue (1:40) for 20-30 sec. 4. The drug is again washed with water and dried. When properly stained grains volutin are red and clearly visible agains the background of blue cytoplasm. Lab Exercise 3. Granules of polyglucose. (Glycogen, starch, granulosis). Methodical instructions: Glycogen inclusions in cells of the well to investigate in Saccharomyces cerevisiae and Bacillus mycoides one-two-day age. To detect an object in granulosis cane use enrichment culture of Clostridium. These substances are detected microchemical processing cells Lugol's iodine solution. 3 a. Glycogen, starch (in Saccharomyces cerevisiae). Procedure: 1. A drop of cell suspension test organisms on a slide add a drop of Lugol's solution. 2. The drug is covered with a coverslip. Granules of starch substances stained blue, and the pellets glycogen − a russet. 3 b. Granulosis (in Clostridium butyricum, Cl. butylicum, Cl. Pasteurianum). Procedure: 1. Apply a drop of microbial culture on a glass slide. 2. Add a drop of enrichment cultures Lugol's solution . 3. Covered with a coverslip, on which is placed a drop of immersion oil. In places the cells, which contain granulosis, there is a blue color. 115
Lab Exercise 4. Stained fat inclusions. Procedure: 1. Apply a drop of microbial suspension on a slide. 2. Add a drop of solution of Sudan III. Sudan III was dissolved in fat inclusions of the bacterial cell, turning them into an orange-red, the cytoplasm of the cell remains colorless. Equipment: Microscope Slide Several cover glasses Dropper bottle of water Disinfectant tray Culture of Yeats and Bacteria in slant tubes Inoculation loop Burner flame Immersion oil Staining material: − Loeffler's methylene blue − Ziehl's solution − Lugol's solution − Sudan III. Lab work № 9. Subject: Staining Microbial Structures: Endospore (Spore). Session Purpose: Methods for detection and visualization of intracellular polymers stored. Objectives: 1. Acquaintance with the methods of Endospore staining. 2. Endospore staining by Ozheshko method. 3. Perform exercises 1, 2. An endospore is a special resistant, dormant structure formed within a cell that protects the microorganism from adverse environmental conditions. Although endospores are relatively 116
uncommon in bacterial cells, they can be formed by seven genera of bacteria. Because endospore are highly refractive, they can be detected under the light microscope but cannot be differentiated from inclusions of stored material. Endospores cannot be stained by ordinary methods, such as simple staining and Gram staining, because the dyes do not penetrate the wall of the endospore. The most commonly used endospore stain is the Schaeffer-Fulton endospore stain and Ozheshko method. Lab Exercise 1. Schaeffer-Fulton endospore staining. Methodical instructions: Malachite green, the primary stain, is applied to a heat-fixed smear and heated to steaming for about five minutes. The heat helps the stain to penetrate the endospore wall. SO, when malachite green is applied to a heat-fixed smear of bacterial cells, the stain penetrates the endospores and stains them green. Then the penetration is washed for about 30 seconds with water to remove the malachite green stain from all of the sell`s parts except the endospore. Next, safranin, a counterstain, is applied to the smear to stain portions of the cell other than endospore. When safranin (red) is then applied, it stains the remainder of the cells red or pink. In a properly prepared smear, the endospore appear green within red or pink cells. Procedure: 1. Prepare the smear-Initially make a thin smear of bacteria on a clean, non-greasy dirt free slide, dry it and heat fix the smear on slide. 2. Apply the dye-Flood the smear with malachite green and heat the slide by steaming, which will cause enhanced penetration of the impermeable spore coat of endospore by malachite green. Steam for 5 minutes, without letting the smear to dry by adding more stain to the smear from time to time. 117
3. Time to Counter-stain-Wash the slides under slowly running tap to remove excess dye and then apply counter stain safranin for 30 seconds. After then wash the slide and make it complete dry using absorbent/ blotting paper. 4. Colourfull cells are waiting-Place a drop of oil-immersion on slide at smear area and enjoy viewing green colored endospore in pink coloured cells. Do not forget to use 100x objective else you will not be able to see the beauty of these tiny creatures. The endospore appear green within red or pink cells. Lab Exercise 2. Ozheshko endospore staining. Methodical instructions: The spores are stained by using special process that help dyes to penetrate the spore wall known as Ozheshko staining procedures and can be performed following these sequential steps. Procedure: 1 step. Stained cells with spores. 1. On fat-free glass prepare thin bacterial smear. 2. The slide is air dried, without heat fixation. 3. 0.5% solution of hydrochloric acid (HCl) is applied to a smear and heated for 2 min, holding high above the burner flame until vapors. 4. Then the smear is washed for about 30 seconds with water to remove the acid. 5. Smear cover filter paper. The fuchsin Ziehl's solution, the primary stain, is applied to a smear and heated about 7 minutes. Until the vapor (not to boiling). As evaporation of dye added to it periodically, not giving the drug to dry. It is important that the dye evaporates, but the paper does not dry out. 6. Remove the paper. Wash the specimen with water to remove the dye. Carefully blotted with filter paper. As a result of this treatment, cells were stained with the spores evenly. Next, desaturate the cytoplasm of cells, but not spores. 2 step. Discoloration of the cytoplasm of cells. 1. 1 % solution of hydrochloric acid (HCl) or sulfuric acid (H2SO4) is applied to a smear for decolorizing of cytoplasm of cells during 15-30 seconds. 118
2. Wash the decolorized smear by water to remove the acid. 3 step. Counterstain. 1. Stained the decolorized smear with methylene blue for 2 minutes. In a properly prepared smear, the endospore appear red within blue cells. Equipment: Microscope Slide Several cover glasses Dropper bottle of water Disinfectant tray Culture of sporulation Bacteria in slant tubes Inoculation loop Burner flame Immersion oil Gloves Filter paper. Staining material: − Malachite green − Safranin − Loeffler's methylene blue − Methylene blue − Ziehl's solution (fuxin) Mordant ant decolorizing agents: − 0.5% HCl −1% HCl −1 % H2SO4 Lab work № 10. Subject: Investigating bacterial motility by flagella. Session Purpose: Motility procedures & testing. Objectives: 1. To observe bacteria in a «hanging drop», study their morphology, and determine their motility. 2. To determine bacterial cultures grown in motility test medium. 3. Perform exercises 1, 2. 119
Methodical instructions: The motility of bacteria can be determined by several methods: 1) It can be determined microscopically by observing cells in a wet mount. This method is called the hanging drop technique. In this procedure a drop of cells is placed on a cover slip which is then placed on a special slide with a concave depression in its center. The coverslip is held in place with petroleum jelly. This creates an enclosed glass chamber that prevents drying. It is important to distinguish between cells that are moving due to the vibrations of the table and microscope and cells that are actually motile.
2) An alternative method, one that is safer when working with potential pathogens, is the motility stab. In order to determine if an organism is motile in semi-soft medium (SSM), examine the stab line where the tube was inoculated. If an organism is non-motile, it will be concentrated along the stab line. If the organism is motile, uniform turbidity is observed throughout the media. This distribution of cells within the agar is an indication the organism possessed flagella which allowed movement into the media.
Figure 18 − Reaction of motile and non-motile microbes in SSM
Lab Exercise 1. «Нanging drop» or a simple «wet mount». Procedure: 1.Using a toothpick applying a circular "ring" of Vaseline around the edge of depression on the slide. 2. Take a cover glass and clean it thoroughly. It may be dipped in alcohol and polished dry with tissue. 3. Using good aseptic technique, sterilize the wire loop, remove the cap of the tube, and take up a loopful of culture. Be certain the loop has cooled to room temperature. Close and return the tube to the rack. 4. Place the loopful of agar culture in drop of water in the cover glass as in figure 3, step 2 (do not spread it around). (Place 5-10 loopfuls of the mixed overnight incubated broth onto a coverslip. Sterilize the loop and put it down. 5. Hold the hollow-ground slide inverted with the well down over the cover glass, then press it down gently so that the petroleum jelly adheres to the cover glass. Now turn the slide over. You should have a sealed wet mount, with the drop of culture hanging in the well. 121
6. Place the slide on the microscope stage, cover glass up. Make your examination with the high-dry and oil-immersion objectives (be very careful not to break the cover glass with the latter). Reduce your Iris to very LOW LIGHT! Focus under oil immersion and look for "tumbling" behavior.
Figure 19 − Hanging drop setup for observing motility under the microscope
Note: 1) To make visualization easier you can be added to the bacterial suspension drop of a weak solution of methylene blue. 2) Vibrating Microbes are NOT motile. You MUST focus within 3 minutes of preparation of the slide as by 5 minutes all the microbes are NON-MOTILE as they are dead - killed from the heat produced by the light source. 122
Result: Make drawings in the following circles to show the shape and grouping of each organism. Indicate below the circle whether it is motile or nonmotile.
Lab Exercise 2. The motility gel deep/STAB test. Procedure: Period 1. 1. Inoculate each bacterial culture into a separate tube of the semisolid Motility Medium. 2. Use the needle and carefully stab-inoculate the medium about half-way down through the center. 3. The wire is then pulled out of the media as close as possible to the location where it entered. 4. The tube is incubated for approximately 1-2 days at 30 °C and observed for evidence of motility. Period 2. 1. Observe the tubes of Motility Medium for growth away from the line of inoculation and the subsequent cloudiness throughout the medium as discussed in the introduction. In a well-lit room, hold all of the tubes together against a darker part of the ceiling (such as the space between the fluorescent light units) so that degrees of growth can be discerned easily. Ignore all surface growth and any growth 123
that might be creeping down from the surface along the inner wall of the tube. 2. Tabulate your results. Complete the table. Confirm your Positive Hanging-drop Motility with a Stab Motility Test. Bacterial cultures grown in motility test medium Bacterial culture
Motility in Hangingdrop
Motility in Stab Test
Equipment: Microscope Slide Several cover glasses Several hollow-ground slides Dropper bottle of water Disinfectant tray Inoculation loop Burner flame Immersion oil Young broth cultures (12-15 hours) of Enterobacter aerogenes (or other motile organism) and Staphylococcus epidermidis. These will serve as positive and negative controls (respectively) for the unknown. Young broth culture of an unknown organism Several tubes of semisolid medium such as O.75% agar Lab work № 11. Subject: Morphology of eukaryotic. Session Purpose: To familiarize with the basic characteristics of microscopic fungi, yeasts to study the morphology of fungi and yeasts, to determine the genus of fungi. 124
Objectives: 1. In vivo preparations view of culture of fungi Mucor, Penicillium and Aspergillus. Sketch the mycelium and spore bodies. 2. Determine the generic identity of unknown fungi, using the provided key. 3. View the colonies of fungi grown on solid nutrient medium in Petri dishes at low magnification microscope. 4. Prepare stained with methylene blue preparation "crushed drop" the yeast Saccharomyces genus and Rhodotorula, determine the shape of cells, to identify budding, living and dead cells. Sketch the yeast cells. 5. Perform exercises 1, 2. Lab Exercise 1. The study of the morphology of Mold. Methodical instructions: Colonial morphology, the structure of the mycelium and sporophores location can be studied by looking at the colonies of fungi grown on solid nutrient medium in Petri dishes at low magnification microscope. Morphological structure of hyphomycetes studied in squashed droplet, which is separated by dissecting needles small portion of mycelium from fruit-bearing hyphae and putting it in a drop of water, straighten, cover with a coverslip and mikroskopiruyut. Procedure: 1. First, browsing the drug 8x lens, but after the discovery of fruiting hyphae establish a 40x lens. Morphology blastomycetes studied in smears under immersion lens. 2. Fixing and painting of the aerial mycelium of fungi. 3. On the aerial mycelium of the fungus that developed on solid nutrient medium in a petri dish, put 2 drops of 96o alcohol, then immediately 2 drops of concentrated acetic acid. 4. Stained with alcohol-water solution of gentian violet in 1:40 2-3 minutes. 5. Then poured into the dye, washed with water and covered with colored mycelium coverslip area, consider it under low magnification microscope. To familiarize yourself with the form of spores and mycelia of fungi make the drug "mark" (see above). This drug is also useful for 125
studying natural arrangement of cells in colonies of microorganisms. Genus of fungus determined by a key Nikitinsky − Aleeva, the results described in the form: Genus
Type of mycelium
Type of spores
The location of spores
The key to the genera of fungi belonging: 1. Fungi reproduce sporangiosporami inside the sporangia − 2. Fungi reproduce conidia formed on the outside of the special conidiophores, sometimes directly on the mycelium − 5. 2. Sporangiophores bearing sporangia, usually simple, rarely simple branching. Sporangia are all the same − 3. Sporangiophores branching. Sporangia of two kinds: large − on the main axis, and small (sporangioli) − on the side branches − 4. 3. Sporangiophores single, simple, sometimes branched. Sporangia small or large, is always homogeneous, colorless or colored. Spores roundish or elliptical, smooth, colorless or greyish − Mucor. Sporangiophores are shrubs growing on stolons from one center, with large black heads. Spores, rounded ovate, wrinkled − Rhizopus. 4. Bush branches are whorled on the main axis Sporangiophores in one or more tiers. Designated branches nerazduty. Spores cylindrical, and ellipsoidal, colorless − Thamnidium. Bush branches diverging from the blistering on the main axis Sporangiophores. The branches of the second order is also moving away from swollen areas. Sporangioli sit on small swellings of the terminal branches. Spores ellipsoidal or spherical, colorless − Chaetostilum. 5. Conidia are formed on conidiophores much different from the ordinary vegetative hyphae − 6. Conidiophores and differ little from ordinary hyphae, or none at all, and conidia are formed directly on the mycelium − 14. 6. Conidiophores branched profusely in various ways − 7. Conidiophores not branched or sometimes branched, but only slightly. Branching is simple, forked or kustoobraznoe − 9.
7. Branching tree − 8. Branching racemose or repeatedly forked, conidia are in chains, smooth, colorless or colored, round − Penicillium. 8. Conidiophores treelike branching, large. The branches are arranged randomly, the conidia at the ends of the branches of shrubs developed, colorless, ovoid, smooth − Botrytis. Conidiophores treelike branched. The branches are whorled. Conidia formed singly or in batches, elongated-ovate or elliptical, colorless or slightly colored − Verticillium. 9. Conidiophores unbranched, long, with large pear-shaped clumps of conidia at the end. Conidia two-celled, hyaline or pinkish. Colonies of the fungus a yellowish-pink − Trichotecium. Conidiophores and conidia of some form − 10. 10. Conidiophores branched. Branching rarely simple forked −11. Simple (just as an exception branching) are at the end of conidiophores clavate or vesicular swelling and sometimes swelling is absent − 12. 11. Conidia large, irregularly shaped, dotted with warts, arranged in chains, painted in brown. Colonies at first and then the mucous fluffy, with a strong odor of hydrogen arsenic − Acaulium. Conidia hyaline, long, serpoobraznye, multicellular (with transverse septa), sometimes there are a chain of one another or appear directly on the mycelium. Colonies of the fungus are colored in pink (especially the underside) − Fusarium. 12. The ends of the conidiophores clavate or swollen and saccately range covered by sterigmata bearing conidial chains − 13. Extension on the end is often missing, sterigmata are only at the top of konidkonostsev but do not grow on the sides. Conidia small rounded, smooth, hyaline, sterigmata are in chains − Cytromyces. 13. Sterigmata covering club-shaped swellings, simple, unbranched, bear conidial chains. Conidia round, smooth or prickly, stained or discolored − Aspergillus. Sterigmata branching form to saccately inflated conidiophore two tiers. The top is a chain of conidia. Conidia smooth, rounded, painted − Sterigmatostis. 14. Conidia are formed directly on the mycelium − 16. 127
Conidia are formed on conidiophores, slightly different from the ordinary vegetative hyphae − 15. 15. Conidiophores are visible only when cultured in the (hanging drop). Conidia easily fall apart − 17. Place of formation of conidia are visible in ordinary microscopic preparations − 18. 16.Odnokletochnye conidia hyaline, veretenovkdnye or roundoblong. Conidia slimy, black − Dematium. Conidia unicellular, ovoid, drozhzheobraznye. Young colonies are similar to yeast, then become shaggy − Monilia. 17. Conidia are obtained simply by dividing the mycelium and is easy to fall off, colorless, rectangular, sometimes united in short chains − Oidium. Conidiophores long, multicellular. Conidia irregular(Long, rounded or limonoobraznye), painted in a light olive-green color − Cladosporium. 18. On the slow-growing colonies no real conidiophores. Small, shiny, yellow-brown conidia formed very long chains at the ends of ordinary hyphae − Catenularia. Large multicellular, round-pear-shaped or acuminate-oblong conidia are formed singly or in short chains on short lateral branches of vegetative hyphae, conidiophores play a role − Alternaria. Lab Exercise 2. Morphology of yeast. Methodical instructions: The shape of vegetative cells of yeast varied: round, oval, ovoid, cylindrical, lemon, etc. Many yeast reproduce by budding, in which the cell surface formed small bump − kidney, which was gradually increased to the size of the mother cell. At the same time or successively formed buds on different sides of the mother cell is plural or multilateral budding. Procedure: 1. Prepare the yeast preparations "crushed drop". 2. Stained with methylene blue. When the microscope with 40x objective to determine the shape of yeast cells, the presence of 128
the living and the dead, and the number of budding cells. Stained yeast cells are dead. 3.The results recorded in Table. Table The morphology of yeast cells The shape of cells
The number of budding cells,%
The number of dead cells,%
Equipment: Microscope Slide Several cover glasses Dropper bottle of water Disinfectant tray Inoculation loop Burner flame Culture of Yeast in slant tubes Petri dishes with cultures of Mold Gloves Filter paper Staining material: − Gentian violet 1:40 − Methylene blue Lab work № 12. Subject: Genetic recombination. Conjugation in Escherichia coli K-12. Session Purpose: To implement Conjugation in Escherichia coli K-12. Objectives: 1. Learn how to make a Suspension hybridization. 2. Learn how to make a Spot hybridization. 3. Perform exercises 1, 2. 129
Conjugation requires cell-to-cell contact for DNA to be transferred from a donor to a recipient. Bacterial conjugation is plasmid-mediated gene transfer. A plasmid that can mediate gene transfer is termed the F (fertility) plasmid. A bacterial cell containing the F plasmid is called an F+cell. A bacterial cell not containing a F plasmid is called the F-cell. A bacterial cell containing a F plasmid integrated into the bacterial DNA is termed a Hfr (high frequency of recombination) cell. Lab Exercise 1. Suspension hybridization. Methodical instructions: Use the Met His Val Ser Strains of Escherichia coli K-12. Strain 1 2 3
Genotype Met – His- Val+ Ser+ Met + His+ Val- SerMet + His+ Val- Ser-
Make crossing between strains: F+ x F- and Hfr x F-. Conjugation is carried out in liquid medium. The conjugation mix for this purpose is prepared. Procedure: 1. To culture of a strain 1 add culture of a strain 2. 2. To culture of a strain 1 add culture of a strain 3. 3. Place test tubes in the thermostat, 370, 1hour. 4. Divide Petri's cup with the minimum medium into 2 sectors. 5. Sign each. 6. From the first test tube place 100 mcl of a conjunction mix in sector 1. 7. Distribute a mix with a spatial on surface of nutrient medium. 8. From the second test tube place 100 mcl of a conjunction mix in sector 1. 9. Distribute a mix with a spatula on surface of nutrient medium. 10. Place cups in the thermostat, 370, 48 hours. Lab Exercise 2. Spot hybridization. Methodical instructions: Make crossing between strains: F+ x F- and Hfr x F-. Transfer of a factor occurs not only by crossing in a liquid broth, but also on a cup surface with agar. 130
Procedure: 1. Divide a Petri dish with the minimum nutrient medium into 5 sectors. 2. Sign each. 3. Drop suspension on sector 1 of a strain 1. 4. Drop suspension on sector 2 of a strain 2. 5. Drop suspension on sector 3 of a strain 3. 6. On sector 4, firstly a drop of a strain 1 and when it dry a drop of suspension a strain 2. 7. On sector 4, firstly a drop of a strain 1 and when it dry a drop of suspension a strain 3. 8. Put Petri dish in the thermostat on 370, 48 hours. Write the results of experiment in the table. Make a conclusion. Results of conjugation in E. Coli Method of hybridization Spot
Type of hybridization F- x F-
Presence of recombinants
F- x Hfr Suspension
F- x F+ F- x Hfr
Equipment: Broth Culture of F-, F+, Hfr strains Escherichia coli K-12 Petri dishes with minimum nutrient medium Disinfectant tray Inoculation loop Burner flame Spatula Lab work № 13. Subject: Isolation of bacteriophage from pathologic material and objects of the environment. Session Purpose: Introduction to qualitative and quantitative methods of detection of bacteriophages. 131
Objectives: 1. Isolate bacteriophage from the samples of water and soil with varied methods. 2. Make qualitative analyzing of bacteriophage in liquid and solid nutrient mediums. 3. Make phage-typing of Staphylococcus aureus. 3. Perform exercises 1-4. It is known that bacteriophages have parasitic properties that contribute the existence and reproduction only in homologous cultures of microorganisms. On this basis, the presence of the bacteriophage can be regarded as an indirect measure of infection of the test material by relevant microbe. In practice of Microbiology bacteriophage can be detected when the isolation of microbe culture doesn’t give a positive result, for example due to an excessively small amount of microbe in the material or contamination by foreign microflora. Currently were developed various methods for the qualitative detection of phage in the material, which are based on seeding the test material on solid and liquid nutrient media with bacterial strain which is homologous to the required phage. Microbial cultures, sensitive to bacteriophage, are called the test cultures. An important condition to the effectiveness of treatment with the phage medication is to identify the sensibility to phage by pathogen. Lab Exercise 1. Isolation of bacteriophage from the test material. Methodical instructions: Tested liquid is filtered from germs. The solid material (soil samples, food, feces) is grained in a mortar and emulsified, then filtered through paper, and through a bacterial filter. The presence of phage in obtained filtrate is identified on solid and liquid mediums with the use of test-cultures. 1a. Isolation of bacteriophage with enrichment method "without seeding." This method is based on the principle of creating the most favorable conditions for reproduction. 132
Procedure: 1. The test material is inoculated into a liquid medium, which is optimal for the growth of microorganisms. 2. Cultures are incubated in thermostat with the optimum temperature for 18-20 hours. 3. After this period cultures are filtered through paper, and then with bacterial filter. 4. The filtrates are tested for the presence of bacteriophage on the solid and liquid nutrient media. 1b. Isolation of bacteriophage with enrichment method with "seeding". The essence of this method is the enrichment of the material tested cells of the microorganism to which we search the bacteriophage. Procedure: 1. Liquid material is investigated directly, and solid material is grained in mortar and seeded in meat-peptone broth or into other liquid medium. 2. Simultaneously with the test material 3 0.5-1 ml 6 hour broth culture or flush with the agar culture bacteria which are homologous to the isolating phage. 3. For the control,bacteria culture is inoculated into a sterile nutrient medium. 4. Seeds are put into a thermostat at 370 C for 18 - 24 hours. 5. After incubation of the test and control tubes,we should take several milliliters of the liquid and filtered through a bacterial filter. 6. The filtrates are tested for the presence of bacteriophage which are homologous to the bacteria, applied for enrichment. Lab Exercise 2. Detection of bacteriophage on solid nutrient medium with the Ott’s method. Methodical instructions: Beef-extract 1.5% agar is poured into Petri dishes (higher concentration of agar inhibits the growth of negative colonies of bacteriophage). We can put several samples on 133
the one dish. We should divide the bottom of the dish in sectors, and insert in each sector drops of filtrate. Interpretation of results: evidence of a bacteriophage is the lack of growth in culture filtrate falling drops (active bacteriophage) or the appearance of this small plot of bald spots - colonies of bacteriophage (phage weak activity). Account or the results: presence of bacteriophage is shown by the absence of culture growth where the drops of filtrate (active bacteriophage) were inserted or appearing in this places little sterile spots-colonies of bacteriophage (bacteriophage of weak activity). Procedure: 1. Dishes with agar are inoculated with 3 -6 –hour broth culture or washout with daily agar culture of bacteria , which are homologous to the phage. 2. For deriving continuous growth 2-3 drops of culture is pounded with a pallet over its entire area and accurately distributed the broth culture (approximately 2ml) on the plate surface, the excess is removed by pipette. 3. Then dry it for 30-40 minutes at 370C, and cover it with sterile filter paper. 4. On the dried surface of the seeding we must put drops of the test-filtrate. 5. When the liquid is absorbed in the medium, the cup turned upside down and placed in a thermostat at 370C for 18-24 hours. Lab Exercise 3. Titration of bacteriophage on solid nutrient media with the method of agar layers by Gracia. Methodical instructions: After detection of bacteriophage in test-material ,it is necessary to identify it’s quantitative content or find phage’s titre. To express the titer of bacteriophage we can use two indicators: the number of active phage particles contained in 1 ml of the test material, or the value of the highest dilution at which exerts its bacteriophage lytic action. The resulting value is expressed with the negative 10 logarithm, where the rate indicates the dilution of phage. There are several methods of bacteriophage titration, but the most popular is the titration method of phagein liquid nutrient media, 134
offered by Appelman and method of agar layers by Gracia. It is based on inserting various dilutions of titrated bacteriophage in corresponded bacteria culture and seeding of mixture on solid nutrient media with the purpose of deriving negative colonies of bacteiophage. During the titration with the method of agar layers, seeding should be done no fewer than two dishes of phage with same dilution. Phage titration is detected by the counting of quantity of negative colonies on parallel dishes and multiplying arifmetic verage on dilution index. For example, seeding of 1ml of phage in 105 dilution we can observe 128 negative colonies on the one dish, and 146 on the second titration phage is equal to 1,37*107. Procedure: 1. Nutrient Media are prepared the previous day. 25-30 ml of 1.5% of beef-extract agar is poured into Petri dishes. Before the pouring we should add 0,004 % alcohol solution of gentian violet: 0,1 of dye in each 100 ml of beef-extract agar. 2. Dished with medium is covered with sterile blotting paper and dried in thermostat for 30 min, close it with lid and stay for night. 3. Prepared medium must be dried, because even little moisturization can change quantitative index of particles of phage in testes liquid. 4. Beef-extracted 0,7 % agar, poured into tubes for 2,5 ml can be prepared a several days before the experiment. 5. On the day of making an experiment, we should prepare consequent dilutions in tubes. For the dilution of phage it is necessary to use sterile broth or isotonic solution of sodium chloride. 6. After that add 1 ml of dilutions of titrated phage into tubes with 0,7 % of beef-extracted agar, which is melted in water-bath and cooled till 44-460C. 8. Simultaneously with phage put on each tube 0,1 ml of similar and sensitive to phage culture. 9. Content of tubes is mixed by rolling tubes between the palm, and pour out with 2nd layer into the 1,5% agar dishes. 10. After the cooling of medium, dishes are incubated at 370C for 18-20hrs. 135
Lab Exercise 4. Staphylococcus phage-typing. Methodical instructions: Bacteriophages are used for the diagnostics of infective diseases, determination of specific appliance of agent, identification of bacteria from environment. Phage-type allows to realize intraspecific differentiation of bacteria. Since phage-typoing characterization of bacterial strains is stable enough, it’s extremely important for epidemiological analysis. Using phage labeling,it’s impossible to establish links between individual cases and identify the sources of infection, ways of its expansion. Widespread in the study received a diagnosis of staphylococcal infections of staphylococcal bacteriophages for coagulase positive staphylococcus isolated from human (International dialing − England) and from cattle (Davidson). These sets contain 23 and 7 types of bacteriophages. Typing is carried by all phage sets simultaneously. Account and registration of results. Extent of Staphylococcus culture lysis is registred by the following scheme: ++++ - confluent(full) lysis; +++ - semi-confluent (insignificant growth of culture in the zone of lysis); ++ - presence of more than 50 colonies of phage on the place of inserting the phage drops); + - from 20 till 50 colonies of phage; +- - less than 20 colonies of phage; - - full absence of lysis. First 3 extent of lysis is called strong reaction. Extent of lysis (+,+-)-weak reaction. Strain of Staphylococcus can be called typed, if one of the phage could give a strong reaction. If during the typing 1 TP were derived only weak reactions or lysis is absent, then this strain should be tested again with the same phages in 100 TP dilution. Staphylococcus can have lysis with several phages, which helps to identify phage-puzzle to each strain. If the strain identifies one phage-puzzle or has difference to one or two strong reactions, they will be identical. 136
Procedure: 1. Lyophilized contents of each ampuls with a typical phage were dissolved in 1 ml of Hottinger broth, Martin or beef- broth with 0.4 % glucose and 0.02% calcium chloride - the main dilution 10-1. 2. Next, prepare dilutions corresponding to 1 dilution test (TP) and TP 100. 3. Daily agar culture of the test strain is inoculated into a test tube with a beef- broth (you can use Hottinger or March broth ), pH 7.27.4 , and grown at 37 ° C till the appearing of visible turbidity (3- 5h). 4. Pour into Petri dishes 1.2% of Hottinger agar on fresh meat water with 0.4 % glucose and 0.02% calcium chloride (the latter is added directly to the molten medium ready before filling the dishes) . 5. Dishes with cooled agar is dried for 40-60 minutes at 370C. With the help of pasteur pipette irrigate the surface of agar. 6. Excess of culture is deleted and dried for 30-40 minutes at 370C. 7. Bottom of the seeded dish is divided in the correspondence of the quantity of used phage. Drop of corresponded phage should be inserted into the seeded medium. 8. After the drying of the drops of the phage, dishes should be turned over and incubated 18-20 hrs at 30 C or 5-6 hrs at 37 C and stayed in room temperature for 18-20 hours. Equipment: Cultures of microorganism on solid and liquid mediums Beef-extracted broth Beef-extracted agar Hottinger’s broth Petri dishes Disinfectant tray Inoculation loop Burner flame Spatula Bacterial pipette
STUDENT INDEPENDENT WORK Student Independent Work (SIW) 1. Subject: Bacterial Shapes. Bacteria can have any of the following three shapes: rod-, sphereand curved – haped bacteria. The most basic method used for identifying bacteria is based on the bacterium's shape and cell arrangement. This section will explain the three morphological categories which all bacteria fall into - cocci, bacilli, and spirilla. You should keep in mind that these categories are merely a way of describing the bacteria and do not necessarily refer to a taxonomic relationship. The task for 1 group. Rod-haped bacteria. The task for 2 group. Sphere -haped bacteria. The task for 3 group. Curved-haped bacteria. Method of SIW − A group task: Analytical review with presentation . Methodical instructions: In the presentation should to include the following: − a variety of forms; − their arrangement; − examples; − a brief description. Student Independent Work (SIW) 2. Subject: Structures Internal to the Cell Wall. Plazma (cytoplasmic) membrane. 1. A group task: Analytical review with presentation . The task for 1 group. Plazma (cytoplasmic) membrane. Structure. Functions. Destruction of the plasma membrane by antimicrobial agents. The task for 2 group. 138
Movement of materials across membranes. Simple diffusion.Facilitated diffusion.Osmosis. The task for 3 group. Movement of materials across membranes. Active transport. Group translocation. 2. Make up the questions for study outline. Plazma membrane. 1. The plasma membrane encloses the cytoplasm and is a phospholipid bilayer with protein (fluid mosaic). 2. The plasma membrane is selectively permeable. 3. Plasma membranes carry enzymes for metabolic reactions, such as nutrient breakdown, energy production, and photosynthesis. 4. Mesosomes – irregular infoldings of the plasma membrane – are now considered artifacts. 5. Plasma membranes can be destroyed by alcohols and polymyxins. Movement of materials across membranes 1. Movement across the membrane may be by passive processes, in which materials move from higher to lower concentration and no energy is expended by the cell. 2. In simple diffusion, molecules and ions move until equilibrium is reached. 3. In facilitated diffusion, substances are transported by permeases across membranes from high to low concentration. 4. Osmosis is the movement of water from high to low concentration across a selectively semipermeable membrane until equilibrium is reached. 5. In active transport, materials move from low to high concentration by permeases. And the cell must expend energy. 6. In group translocation, energy is expended to modify chemicals and transport them across the membrane. 2. Prepare the answers to these tasks. 1. Explain what would happen in the following experiments. (a) A suspension of bacteria is placed in distilled water. (b) A suspension of bacteria is placed in distilled water with lysozyme. 139
(c) A suspension of bacteria is placed in an aqueous solution of lysozyme and 10% sucrose. (d) A suspension of gram-negative bacteria is placed in distilled water with penicillin. 1. Compare and contrast the following: (a) Simple diffusion and facilitated diffusion. (b) Active transport and facilitated diffusion. (c) Active transport and group translocation. 3. Starch is readily metabolized by many cells, but a starch molecule is too large to cross the plasma membrane. How does a cell get the glucose molecules from a starch polymer? How does the cell get these glucose molecules across the plasma membrane? Student Independent Work (SIW) 3. Subject: History of microbiology. The discovery of microbiology as a discipline could be traced along the following historical eras : 1. Discovery Era. Morphological period. 2. Golden Age. 3. In 20 th Century − Era of Molecular Biology Homework: Prepare the answers to these tasks: 1Variant. Discovery era. Transition period. Methodical instructions: Your answer should be noted: The emergence of microbiology. Discovery and description of microorganisms. Studies of Antony Van Leeuvenhoek. experimentations mainly of Girolamo Fracastoro, Robert Hooke, Francesco Redi, Lazzaro Spallanzani, Theodor Schwan. 2 Variant. Physiological period. Golden age. Methodical instructions: The Golden age of microbiology began with the work of Louis Pasteur and Robert Koch who had their own research institute. During this period, we see the real beginning of microbiology as a discipline of biology.
Your answer should be noted: The discoveries of Pasteur and Koch. Their significance for microbiology, biotechnology and medicine. 3 Variant. Ecological and physiological approach to microbiology. Methodical instructions: The development of microbial ecology associated with the names of Sergei Winogradsky and Victor Omelyansky. Your answer should be noted: The work of these scientists. Microecological principle in the study of microorganisms. Chemolythotrophic microorganisms. Nitrogen-fixing bacteria. Research and discovery of Martin Beijerinck. 4 Variant. Immunological period. Methodical instructions: Notable contribution to the antiseptic treatment for the prevention and cure of wound infections. Phagocytic and humoral theory of immunity. Era of chemotherapy and antibiotics. Your answer should be noted: The works of scientists: Lord Joseph Lister, Edward Jenner, Emile Roux , Elie Metchnikoff, Paul Ehrlich, Gerhard Domagk, Alexander Fleming. Their significance for microbiology, biotechnology and medicine. 5 Variant. History of Microbiology in 20-th century. Era of molecular biology. Methodical instructions: In the later years the microorganism were picked up as ideal tools to study various life processes and thus an independent discipline of microbiology, molecular biology was born. The relative simplicity of the microorganism, their short life span and the genetic homogeneity provided an authentic simulated model to understand the physiological, biochemical and genetical intricacies of the living organisms. Your answer should be noted: Here your will highlight briefly only few of the important microbiological accomplishments. The works of pioneers in the area of microbial genetics: George W. Beadle and Edward L. Tatum, Max Delbruck and Salvadore Luria, Joshua Lederberg, Francois Jacob, Jacques Monod. Remarkable discovery in genetics by discovering the molecular structure of DNA providing framework for understanding molecular basis' of 141
inheritance and expression of genetic information: Colin. M. Maclead and Maclyn McCarty, Watson and Crick, Ochoa and Kornberg, Hargovind Khorana, Arber, Smith and Nathans. Student Independent Work (SIW) 4. Subject: Diversity of prokaryotes. Homework: Prepare the answers to these tasks: 1Variant. Gracilicutes. Characterization of Spirochetes. Your answer should be noted: 1. The features of their morphology and structure of cells. 2. The habitat. 3. Representatives. 2Variant. Gracilicutes. Characterization of anaerobic spiral Gram- bacterium. Your answer should be noted: 1. The features of their morphology and structure of cells. 2. The habitat. 3. Representatives. 3Variant. Gracilicutes. Characterization of sliding bacteria. Your answer should be noted: 1. The features of their morphology and structure of cells. 2. The habitat. 3. Representatives. 4 Variant. Gracilicutes. Characterization of budding bacteria. Your answer should be noted: 1. The features of their morphology and structure of cells. 2. The habitat. 3. Representatives. 5 Variant. Firmicutes. Characterization of Corynebacterium. Your answer should be noted: 1. The features of their morphology and structure of cells. 2. The habitat. 3. Representatives. 6 Variant. Firmicutes. Characterization of Mycobacterium and Nokardia forms. Your answer should be noted: 1. The features of their morphology and structure of cells. 142
2. The habitat. 3. Representatives. 7 Variant. Firmicutes. Characterization of Actinomyces. Your answer should be noted: 1. The features of their morphology and structure of cells. 2. The habitat. 3. Representatives. Student Independent Work (SIW) 5. Subject: Viruses and Prions. Morphologies and biological characteristics of viruses, viroids and prions. Control questions on the topic of the lecture 1. Nature of viruses 2. Structure and main properties of viruses. Signs of living and inanimate matter. 3. Chemical composition of viruses. Receptor and reproductive apparatus. Virus reproduction. 4. Virus classification. Examples of causes. 5.Viroids and prions. Prion proliferation. Prion diseases. 6. Methods of diagnostics and virology. Independent work for students 1. Demonstration of types of symmetry in viruses (according to electronic microscopy observations). 2. Demonstration of immunoluminescent microscopy for virus detection (by slides and tables) 3. Demonstration of cultivating methods of viruses (virology method.) on egg embryos. Record. 4. Demonstration of cultivating methods of viruses (virology method) tissue culture. Draw it down. 5. Demonstration: cytopathic effect of viruses on tissue culture. Record (tables and slides.). 6. Demonstration of hemagluttinacin and hemabsorbtion’s reactions for detecting viruses. 7. Layout of the research activity journal.
Methodic recommendation Making good use of demonstrational tables you should research morphology, structure of viruses, spiral and cubic types of symmetries. According to international classification all viruses can be separated in two by the type of nucleic acid they are carrying – DNA & RNA. Following duplication of viruses happens on the basis of the size of the virus, its symmetry when forming capsids, presence or absence of outer shells and quantity of capsomeres in it. Virusoscopic method of research In a research material it allows to detect characteristic for certain virus’s pathological changes in cells, sometimes even outer and inner clusters of viruses could be detected. It’s worth to mention, that success of this technic is tied closely to the level of development of microscopic technology (immunofluorescent, electronic microscopy). Virilogy method of research Virology method of research is the basic and most credible; it allows releasing virus from research material and its following identification. Chicken embryos and tissue cultures (artificially cultivated cells of certain kind of tissues) are used for the sake of gathering virus containing material. Tissue cultures can be either supported on natural, or synthetic nutrient mediums, which are prepared on the basis of Hank’s and Earl’s solutions. Tissue cultures divide into surviving and growing; being cultivated in normal test-tubes, Carrel cups, Barsky test-tubes. Growing cultures are divided to primarily trypsinized non-digestible one layer cultures – like tissues of monkeys’ kidney, human embryo’s kidney, chicken embryo, and transplantable cultures – human amnion cells (A8, F1), malignant tumor (Hela, Hep1, Hep2). Viruses can only reproduce in living organism cell. Therefore, viruses are being cultivated by infection of chicken embryos, tissue cultures, and breast fed animals. For indication of virus in material different ways of evaluation of cytopathic effect of viruses are used, color test in tissues culture, hemagglutination and hemadsorption. 144
TEST YOURSELF Choose only one true answer: 1) One of the difference between viruses and bacteria? a. have 70S and 80S ribosomes b. have a lysosome c. have a Golgy apparatus d. have a system of microtubes e. have only one type of nucleic acid 2) One of the difference between viruses and bacteria? a. doesn't have a cellular structure b. have a CPM c. propagated by division d. sporulate e. have energy systems 3) What is intracellular form of the existence of viruses? a. virion b. plasmid c. transposon d. virus e. prion 4) What is nucleocapsid? a. Complex viral capsid and genome b. DNA complexed with an internal protein c. RNA complexed with an internal protein d. complex of proteins and lipids e. fragmented DNA 5) What function does "plus" RNA do on viruses? a. ribosomal RNA b. transport RNA c. information RNA 6) What is а provirus? a. infectious protein b. extracellular virus c. intracellular virus d. the viral genome that integrated into the genome of the cell e. "naked" nucleic acid of the virus in the cell
7) What stage do phase of ion attraction and physical attachment form? a. undressing of virus b. penetration of the virus into the cell c. reproduction d. viral yield of cells e. adsorption 8) Which way does the release of simple virus happen? a. by endocytosis b. by viropeksis c. by explosion of cell d. by budding e. by membrane fusion 9) Which way does the release of complex virus happen? a. by budding b. by explosion of cell c. by endocytosis d. by viropeksis e. by phagocytosis Choose all true answers: 10) Which structures do viruses have? a. capsid b. cytoplasm c. supercapsid d. mesosome e. lysosome 11) What ways does reproduction of viruses come to light? a. by cytopathic effect b. in complement fixation reaction c. in reaction of hem adsorption d. in agglutination tests e. by the presence of spots beneath sterile agar f. in the color sample 12) What forms of infections are viruses characterize? a. Integrative b. Abortive c. Conservative d. Productive e. all the answers are correct
13) Match its: Types of infectious agents: Structure: 1. Viruses a. cell 2. Prions b. protein infections particle 3. Viroids c. acellular nucleoprotein structure 4. Bacterias d. small RNA molecules 14) Match its: Forms of infection Mechanisms 1. Productive form of infection a. inclusion of viral DNA into cell DNA 2. Integrative form of infection b. reproduction of the virus and the virus out of the cells 3. Abortive form of infection c. violation of the reproduction of the virus in cell
CASE-STUDY 1. TO DEVELOP THE LABORATORY REGULATIONS OF OBTAINING ENRICHMENT CULTURES OF CLOSTRIDIUM GROUPS OF BACTERIA Purpose: the efficiency of comprehention of lecture material and application of practical skills while allocation of microorganisms from environment. Smart Goal: allocate the enrichment culture of Clostridium group of bacteria within 2 weeks. Requirements: 1. To characterize the enrichment cultures and principles of electivity while allocating of microorganisms from various environmental objects. 2. To characterize Clostridium groups of bacteria. 3. To list the elective conditions, arrange them in order of importance. 4. To specify the steps of obtaining cumulative culture and provide the necessary materials and equipment. 5. To obtain the enrichment culture. 6. To carry out the required analysis, that confirms the receipt of this very culture, and to know how to distinguish it from others. 7. To fill in the table. The name of culture
Note (outgassing, the presence of a film, turbidity, specific odor, presence of mucus)
8. To fill in the laboratory journal, to give a presentation. The evaluation criteria Evaluation parameters
Number of points 3 points
Student demonstrate s a deep understandi ng of the theoretical material, he logically expounds it, using in practice.
The submissio n contains some inconsiste ncies and minor mistakes.
There is no logical sequence, there are gaps in the knowledge of the material, there is no proper reasoning and the ability of using knowledge in practice.
Ignorance of theoretical material of the program.
Completeness of the content
3 or more elective conditions are revealed, a complete algorithm of obtaining the enrichment culture is developed, enrichment culture is obtained, entries in the lab notebook are made, presentation is prepared
2 elective conditions are revealed, a complete algorithm of obtainning the enrichmen t culture is developed, enrichmen t culture is obtained, entries in the lab notebook are made, presentatio n is prepared
1elective condition is revealed, algorithm of obtaining enrichment culture is not complete, the obtained enrichment culture does not match the requirements, entries in the laboratory journal are not complete , the presentation is not complete
Elective conditions are not identified, there is no algorithm of obtaining enrichment culture, the enrichment culture is not obtained
Visualization of proposed information
Not bewer than 6 illustrations (models, schemes, tables, figures etc.)
3-5 illustration s (models, schemes, tables, figures etc.)
1-3 illustrations (models, schemes, tables, figures etc.)
Analysis of obtained results
The obtained enrichment culture is confirmed by three qualitative reactions
The obtained enrichmen t culture is confirmed by two qualitative reactions
The obtained enrichment culture is confirmed by one qualitative reactions
No qualitative reactions
Accuracy and literacy in filling the table
The table is full, the analyzes are indicated
There are small inaccuraci es and minor mistakes in the table
Some points of table are not filled
The table is not filled
CASE-STUDY 1. THE INFECTIOUS PROCESS. Work in small groups (3-4 people) and organized between the two groups of students: 1) The group − "a patient" Roles distributed in the group − an emergency doctor, a patient (1 or 2, according to the number of participants), an accompaning person. 2) The group − "a doctor" Roles distributed in the group − a doctor of a hospital reception (1 or 2, according to the number of participants), a nurse, a doctor assistant. After this task the roles are changed. Assignments for the "patient" group: 1. The patient shows symptoms and clinical signs of some disease, according to the issued version. 2. An emergency doctor and an attendant answer the questions asked by physician doctor, describing the symptoms of the disease. 3. Emergency doctor makes a preliminary diagnosis and puts the preliminary (not necessarily correct) diagnosis. The task for the "doctor" group: 1. Сarry out an anamnesis. 2. Has the emergency room physician correctly diagnosed the patient? 150
3. If not, then what kind of diagnosis is spoken about? 4. Which microorganisms can cause the disease? 5. What material should be sent to the laboratory for bacteriological research, to what purpose? 6. Select the method of laboratory diagnosis and carry out the research. 7. How could the infection of a patient happen? 6. Assign a treatment regimen to patient. The rest of the students belong to the group of "observers". The task for observers: 1. Has the doctor correctly diagnosed the patient? 2. Make the evaluation of the actions of a physician with the prior conclusion of bacteriological laboratory. 3. What methods of laboratory diagnosis should be used to confirm the correct diagnosis? 4. Was the method of laboratory research chosen correctly or not. Possible variants of infectious diseases: 1. gram-positive cocci 2. facultative anaerobic spore-forming gram-positive rods 3. facultative anaerobic not spore-forming gram-positive rods 4. anaerobic gram-positive rods 5. aerobic gram-negative cocci and coccobacteria 6. non-fermentative gram-negative anaerobic rods 7. facultative anaerobic fermentative gram-negative rods 8. whimsical aerobic gram-negative rods and coccobacteria 9. mycobacteria 10. mycoplasma The work is done in small groups (3 - 4 students) according to variants. The work is evaluated according to the following criteria. The evaluation criteria Points Evaluation parameters
There is no logical
Ignorance of the
0 -5 points
tes a deep understan ding of the theoretical material, he logically expounds it, using in practice.
contains some inconsistencies and minor mistakes.
sequence, there are gaps in the knowledge of the material, there is no proper reasoning and the ability of using knowledge in practice.
theoretica l material of the program.
Completeness of the content
A detailed descriptio n of the clinical picture of the disease is given, the diagnosis is correct argument ated, performed laboratory tests are true, pathogen is specified, answers are given to all the questions of a task
A detailed description of the clinical picture of the disease is given, the diagnosis is correct argumenttated, performed laboratory tests are true, pathogen is specified, answers are given not to all the questions of a task
The description is given not to all items, the clinical picture is blurred, the diagnosis is not definite, laboratory research is not full.
The description is not complete, according to the clinical picture the diagnosis can not be identified, the diagnosis is not correct, the laboratory study is not correct
There are drawings,
There are drawings
Illustratio ns and
diagrams in the "patient" group There is a diagram of making diagnosis and laboratory research on the blackboar d in the "doctor" group.
and diagrams, but they do not fully describe the diagnosis
schemes are insufficient for making a diagnosis
Novelty of approach
The work is original, illustrating schemes and drawings are colorful and clear
The work is original, schemes and drawings illustrate the diagnosis, but not colorfully enough
The work is originnal, but schemes and pictures are not colorful, small, illegible
The work is not original
EXAM QUESTIONS 1. The basic properties of microorganisms. Factors ubiquitous of microorganisms. 2. The role of microorganisms in natural processes and human activities. 3. Major fields of theoretical and applied Microbiology 4. Major Characteristics of Eukaryotes and Prokaryotes. 5. Rod-haped bacteria. The variety of forms, their arrangement, examples, a brief description. 6. Sphere -haped bacteria. The variety of forms, their arrangement, examples, a brief description. 7. Curved-haped bacteria. The variety of forms, their arrangement, examples, a brief description. 8. Plazma (cytoplasmic) membrane. Structure. Functions. Destruction of the plasma membrane by antimicrobial agents. 9. Movement of materials across membranes. Simple diffusion. Facilitated diffusion.Osmosis. 10. Movement of materials across membranes. Active transport. Group translocation. 11. The emergence of microbiology. Discovery and description of microorganisms. Studies of Antony Van Leeuvenhoek. experimentations mainly of Girolamo Fracastoro, Robert Hooke, Francesco Redi, Lazzaro Spallanzani, Theodor Schwan. 12. The Golden age of microbiology. The discoveries of Pasteur and Koch. Their significance for microbiology, biotechnology and medicine. 13. Ecological and physiological approach to microbiology. Microecological principle in the study of microorganisms. Chemolythotrophic microorganisms. Nitrogen-fixing bacteria. Research and discovery of Sergei Winogradsky, Victor Omelyansky and Martin Beijerinck. 14. Immunological period of microbiology . Notable contribution to the antiseptic treatment for the prevention and cure of wound infections. Phagocytic and humoral theory of immunity. Era of chemotherapy and antibiotics. 15. Bacterial cell envelop. The composition and functions of Bacterial Envelope. 16. Cell Wall of Gram positive bacteria. 17. Cell Wall of Gram negative bacteria. The Outer Membrane of Gramnegative Bacteria. 18. Cell Wall-less Forms. Protoplasts. Spheroplasts. L-forms of the bacterium. Mycoplasma. 154
19. Appendages structures of bacterial cell. Pili and fimbriae. Properties and functions of pili and fimbriae. 20. The structure and function of the bacterial flagella and axial filaments. 21. Different arrangements of bacterial flagella. Flagella movement. Correlation of swimming behavior and flagellar rotation. Taxis. 22. Glycocalyx structure. Capsules, slime Layers. Their functions. Vegetative reproduction. Binary fission of Gram positive and Gram negative bacteria. The stage of binary fission. Generation time. 23. Vegetative reproduction. Budding. Multiply fission. The types of grown cycle. Asexual Reproduction of Actinomycetes. 24. Resting cell shape in prokaryotes. Cysts. Endospore. The structure and function. 25. The stage of endospore formation. Germination of endospore. 26. Quorum sensing-social lives of bacteria. Biofilms. Cell-to-cell communication. Signalling molecules. 27. Genetic Exchange in Bacteria. Transformation. 28. Genetic Exchange in Bacteria. Conjugation. 29. Genetic Exchange in Bacteria. Transduction. Types of transduction. 30. Systemics and Taxonomy of microorganisms. Classification. Types of taxonomy: numerical, phylogenetic, polyphase. Nomenclature. 31. The characteristic features of Archaebacteria. Сlassification of Archaea. 32. Viruses. The composition and structure of the virion. 33. Unconventional viruses. Defective viruses. 34. Diversity of viruses. Classification criteria. Nomenclature of viruses. 35. The interaction of the virus with the cell. Reproduction of viruses. 36. Bacteriophages. Types of morphology. The chemical composition. 37. The types of interaction of phage with the bacterial cell. Lysogenicity.
PRINT AND ONLINE RESOURCES 1. Abramson, J.; Smirnova, I.; Kasho, V.; Verner, G.; Kaback, H. R.; and Iwata, S. 2003. Structure and mechanism of the lactose permease of Escherichia coli. Science 301: P. 610–615. 2. Becton, Dickinson and Co. 2005. Difco and BBL manual: Manual of microbiological culture media, 1st ed., Franklin Lakes, NJ: BD. 3. Davidson, A. L., and Chen, J. 2004. ATP-binding cassette transporters in bacteria. 4. Bartlett, D. H., and Roberts, M. F. 2000. Osmotic stress. In Encyclopedia of microbiology, 2d ed., vol. 3, J. Lederberg, editor-in-chief, P. 502515. San Diego: Academic Press. 5. Cavicchioli, R., and Thomas, T. 2000. Extremophiles. In Encyclopedia of microbiology, 2d ed., vol. 2, J. Lederburg, editor-in-chief. − P. 317 –337. San Diego: Academic Press. 6. Cotter, P. D., and Hill, C. 2003. Surviving the acid test: Responses of gram-positive bacteria to low pH. Microbiol. Mol. Biol. Rev. 67(3): P.429453. 7. Gitai, Z.; Thanbichler, M.; and Shapiro, L. 2005. The choreographed dynamics of bacterial chromosomes. Trends Microbiol. 13(5): P. 221-228. 8. Hall-Stoodley, L.; Costerton, J.W.; and Stoodley, P. 2004. Bacterial biofilms: From the natural environment to infectious diseases. Nature Rev. Microbiol. 2:95 − P. 108. 9. Hoskisson, P. A., and Hobbs, G. 2005. Continuous culture-making a comeback? Microbiology 151: P. 3153-3159. 10. Gilbert, P., and McBain, A. J. 2003. Potential impact of increased use of biocides in consumer products on prevalence of antibiotic resistance. Clin. Microbiol. Rev.16(2): − P. 189-208. Internet Resources 1. American Society for Microbiology. Available online. URL: www.asm.org. Accessed October 3, 2009. The main international association for microbiologists. 2. Australian Society for Microbiology. Available online.URL: www.theasm.org.au. Accessed October 3, 2009. A good supplementary resource for international topics. 3. Centers for Disease Control and Prevention. Available online. URL: www.cdc.gov. Accessed October 3, 2009. The main source of updated information on diseases. 4. Doctor Fungus. Available online. URL: www.doctorfungus. org. Accessed October 3, 2009. An excellent resource on mycology. 156
5. International Society of Protistologists. Available online. URL: www.uga.edu/protozoa. Accessed October 3, 2011. A resource on protozoa. 6. J. Craig Venter Institute. Available online. URL: www.jcvi. org. Accessed November 15, 2012. An institute focused on molecular aspects of microorganisms. 7. Microbe World. Available online. URL: www.microbeworld.org. Accessed October 3, 2012. Contains useful educational materials in basic microbiology. 8. Microbiology Network. Available online. URL: www.microbiol.org. Accessed October 25, 2012. A clearinghouse for other online resources in microbiology plus microbiology news. 9. Microscopy Society of America. Available online. URL:www.microscopy.org. Accessed October 24, 2012. A good resource for the latest technologies. 10. National Institute of Allergy and Infectious Diseases. Available online. URL: www3.niaid.nih.gov. Accessed October 3, 2013. An essential resource for infectious diseases with some international content. 11. Pasteur Institute. Available online. URL: www.pasteur.fr/ip/easysite /go/03b-00002j-000/en. Accessed October 23, 2013. An interesting site that describes current research. 12. Society for Anaerobic Microbiology. Available online. URL: www.clostridia.net/SAM. Accessed October 26, 2013.This Web site covers a unique specialty in microbiology. 13. Society for General Microbiology. Available online. URL: www. sgm.ac.uk. Accessed October 3, 2013. Contains excellent references on a variety of subjects regarding microorganisms.
Savitskaya Irina Stanislavovna Kistaubayeva Aida Serikovna Ignatova Ludmila Viktorovna Blavachinskaiya Irina Valeryevna
MICROBIOLOGY AND VIROLOGY Educational manual Выпускающий редактор Г. Бекбердиева Компьютерная верстка А. Калиева Дизайн обложки: Р. Скаков ИБ № 7086 Подписано в печать 27.02.14. Формат 60х84 1/16. Бумага офсетная. Печать цифровая. Объем 9, 87 п.л. Тираж 200 экз. Заказ № 197. Издательство «Қазақ университетi» Казахского национального университета им. аль-Фараби. 050040, г. Алматы, пр. аль-Фараби, 71. КазНУ. Отпечатано в типографии издательства «Қазақ университетi».