Introduction to Microbiology for PC-I Students PDF

Introduction to microbiology for PC-I students By:- Yisiak Oumer (BSc, MSc) 1  Introduction Microbiology is a subject which deals with living organisms that are individually too small t...

Introduction to microbiology for PC-I students By:- Yisiak Oumer (BSc, MSc) 1  Introduction Microbiology is a subject which deals with living organisms that are individually too small to be seen with the naked eye It considers the microscopic forms of life and deals about their reproduction, physiology, participation in the process of nature, helpful and harmful relationship with other living things, and significance in science and industry Sub divisions of microbiology  Bacteriology – which deals with bacteria  Mycology- which deals with fungi  Virology- studies about viruses  Protozology – which deals with Protozoa.  Phycology - Which deals with Algae. 2 Introduction… Medical Bacteriology: - it involves the study of bacterial pathogens, the disease caused by them, and the body’s defenses against these diseases It is also concerned with epidemiology, transmission of pathogens, disease prevention measures, and aseptic techniques, treatment of bacterial infectious diseases, immunology, and the production of vaccines to protect against infectious disease. 3  Distribution of microorganisms in nature Microorganisms can be found in every ecosystem and in close association with every type of multi-cellular organism They exist in soil, water, air, in our food, in our clothing, in and on our body etc.  Can also survive in most unlikely environment like in cold air, in hot springs at temperatures of 900C. Microorganisms Inhabit the surface of living human and animal bodies and grow abundantly in the mouth and intestinal tract. 4  Distribution of microorganisms in nature… Actually only a small percentage microbes are pathogenic, few are able to cause disease. The others cause disease only if they accidentally invade the wrong place at the right time such as when the host immunity is low.  These microbes are considered opportunistic. Most of this microorganisms that live on the human body with out causing disease and apparent physiological response comprise the normal flora. 5 The Germ Theory of diseases It was hard for people to believe that diseases were caused by tiny invisible “by animalcules”. Diseases, they thought, were caused by:  Demons  Witchcraft  Bad luck  The wrath of God  Evil spirits 6  Koch’s Postulate: - proof of germ theory of disease In 1876 germ theory of disease is coined by German scientist, Robert Koch. Koch’s postulates to identify the causative agent of a particular disease are; 1. The microorganism (pathogen) must be present in all cases of the disease. 2. The pathogen can be isolated from the diseased host and grown in pure culture. 3. The pathogen from the pure culture must cause the same disease when inoculated into a healthy, susceptible laboratory animal. 4. The pathogen must be reisolated from the new host and shown to be the same as the originally inoculated pathogen. 7  Fig. Koch’s postulate 8 Exceptions to Koch’s postulate 1. Many healthy people carry pathogens but do not exhibit symptoms of the disease. 2. Some microbes are very difficult or impossible to grow in vitro (in the laboratory) in artificial media. Eg. Treponema pallidum. 3. Many pathogens are species specific. Eg. Brucella abortus cause abortion in animals but not in humans. 4. Certain diseases develop only when an opportunistic pathogen invades imuno-compromised host. 9  Taxonomic classification of organisms 1. Taxonomy: Taxonomy is the science of classification, identification, and nomenclature.  For classification purposes, organisms are usually organized into subspecies, species, genera, families & higher orders. 2. Classification  Classification is the orderly arrangement of bacteria into groups.  There is nothing inherently scientific about classification and different groups of scientists may classify the same organisms differently. 10 Taxonomic classification of organisms… 3. Identification  Identification is the practical use of classification criteria to distinguish certain organisms from others. 4. Nomenclature  Nomenclature (naming) is the means by which the characteristics of a species are defined and communicated among microbiologists.  A species name should mean the same thing to all microbiologists yet some definitions vary in different countries or microbiologic specialty groups.  For example, the organism known as Clostridium perfringens in the United States is called Clostridium welchii in England. 11 Taxonomic classification of organisms…  The biologists have adopted a standard method of expressing these names that identify a specific organism.  They are using binomial nomenclature involving genus and species name.  To express genus capitalize the fist letter of the word and underline or italicize it for example Esherchia/ Esherchia.  To express genus & species together, capitalize the first letter of the genus name (and then underline or in italicize the entire name- for example Eschelchia coli /Esherchia coli  The genes can be abbreviated as E. coli 12 Kingdoms of Living Organisms There 5 kingdoms living organisms. 1. Animalia – Helminth 2. Plantae – Algae 3. Fungi – Fungus 4. Protista - e.g., protozoa 5. Monera - Bacteria and Cyanobacteria 13 Eukaryotes & Prokaryotes Cells have evolved into two fundamentally different types:  Eukaryotic  Prokaryotic They can be distinguished on the basis of their structure and the complexity of their organization. Fungi and protozoa are eukaryotic, whereas bacteria are prokaryotic. 14 Table 1: Characteristics of Prokaryotic and Eukaryotic Cells. Characteristic Prokaryotic Cells Eukaryotic Cells DNA within a nuclear membrane No Yes Mitotic division No Yes Histones No Yes Chromosome number single Multiple Membrane-bound organelles, such No Yes as mitochondria Size of ribosome 70S 80S Peptidoglycan Yes No Sexual reproduction No Yes Endoplasmic reticulum No Yes 15  General properties of Bacterial Cell:  Typical prokaryotic cell  Contain both DNA and RNA  Most grow in artificial media  Replicate by binary fission  Almost all contain rigid cell wall  Sensitive to antimicrobial agent 17 Bacterial Classification A) Morphologically bacteria can be classified as:  Spherical or coccoid/cocci- (singular –coccus)  Rods or bacilli (singular - bacillus)  Spirals or spirilla (Singular - Spirillum) Some bacteria are variable in shape and are said to be pleomorphic (many-shaped). The shape of a bacterium is determined by its rigid cell wall. 18  A B C Fig. Different bacterial morphologies A. Cocci, B. Bacilli, C. Spiral shape 19  The cells of cocci may be found in various arrangements depending on the species and the way they divide  e.g – Micrococcus:-Cocci occurring singly. Diplococci- Pairs of cocci Strepto cocci – Cocci in chain Staphylococci- Cocci in cluster Tetrads – Four cocci as in box Octads(sarcina) – Eight cocci as in box Bacilli (rods) may be short or long, thick or thin, pointed or with blunt ends,  Some rods resemble cocci and are often called coccobalilli because they are very short small bacilli.  Some bacilli stack up next to each other eg. Diphteroids, Some are coma shaped e.g. V.cholara Spiralls usually occur singly.  The different species of spirilla varies in size, length, rigidity, number and amplitude of their coils. 20 Bacterial Classification… B) Based on staining characteristic classified as;  Gram positive  Gram negative Other characteristic  Acid fast bacilli C) Based on presence of capsule covering  Capsulated /non capsulated D) Based on spore formation  Spore former /non spore former E) Motility  Motile and non motile 21  Fig. different bacterial arrangements 22 23  Bacterial Cell structure Bacterial structure is considered at three levels. 1. Cell envelope proper: Capsule, cell wall and cell membrane 2. Cellular element enclosed with in the cell envelope: Mesosomes, ribosome, nuclear material, polyamines and cytoplasmic granules. 3. Cellular element external to the cell envelope (appendages) : Flagellum, Pilus 24  1. Cell Envelop proper A. Capsule and slim layer  Capsules are often regarded as portion of the cell envelope  Capsular constituents vary among the different species of prokaryotes.  Many bacteria have slimy layers, consisting of polysaccharid only; others have proteins within the polysaccharide capsule  Both serve to enable the bacteria to attach to tissues and to resist phagocytic digestion.  The basic difference between the capsule and the slim is their property of firm attachment to the cell.  The gels formed by the capsule adhere to the cell where as 25 the slim can easily be washed off.  Capsule can be detected by Indian ink staining in which the capsule stands out as a halo.  Some of the important characteristics of capsule include:  Usually it is weekly antigenic  Not necessary for viability  Endows virulence  Protects from phagocytosis  Capsulated strains are invariably non motile  The organism in which capsules have been demonstrated include; Pneumococci, klebsella, Escerchia coli, hemophilus influenza, etc 26  Cell Envelop proper… B. Cell wall (back bone of bacteria) – a non-living secretion of the cell membrane, – Multi layered structure and constitutes about 20% of the bacterial dry weight – Average thickness is 0.15-0.5 m Chemical Composition of cell wall  The major component of cell wall is peptidoglycan (PG) layer  The rigidity of the cell wall is due to the presence of this a unique substance called peptidoglycan layer  It is composed of N-acetyl Muramic acid and N-acetyl Glucosamine backbones, cross-linked with peptide 27 chain and pentaglycine bridge  Chemical Composition of cell wall..  This PG is found only in bacteria and not found in other Micro organisms  Some times called back bone of bacteria.  PG is made up of two alternate repeating amino sugar and peptides. 1. The amino sugars are A. N – acetyl glucose amine (NAG) B. N – acetyl muramic acid (NAM) 2. Peptides – which consist of 4 amino acids – L -alanin – D -isoglutamic acid – L -lysine (or di aminopimelic acid) 28 – D - alanin  Function of cell wall  Provides shape to the bacterium  Gives rigidity to the organism  Protects from destructive environmental factors  Provides staining characteristics to the bacterium  Contains receptor sites for phages  Site of action of antibody  Contains components toxic to host (endotoxins). 29  Function of cell wall  It contains unique and varied surface antigens (Ags) the serologic identification of these Ags is a major diagnostic tool’ and cell wall biosynthesis is the site of action of many antibiotics.  The gram stain divide bacteria in to two classes, which differ in their ability to retain a basic dye after fixation. – The difference is based on major variation in the structure of the cell wall. 30  Types of cell wall I. Gram positive cell wall of bacteria – has layers (Peptidoglycan (PG) cross linked with teichoic acid) – The PG layers is much thicker than Gram negative bacteria and is 15 – 50 nm thick – The PG layer comprises 50 – 90% of the cell wall and 20 to 40% of the cell wall weight 31 Fig. Gram-Positive the cell wall of bacteria 33  Gram positive cell wall …Cont’d  The large amount of peptideglycan make gram- positive bacteria susceptible to the enzyme lysozyme & penicillin.  Lysozyme hydrolyzes peptidoglycan by specific cleavage between N-acetyl muramic acid and the N-acetyl glucosamine of the glycon strand.  Penicillin specifically inhibits peptidoglycan synthesis  Teichoic acids and cell wall-associated protein are the major surface antigens of the gram-positive cell well. 34  Gram positive cell wall …Cont’d Teichoic acid – These polymers of glycerol phosphate or ribitol phosphate are located in the outer layer of the GP cell wall. Function of Teichoic acid – Used to bind (keep) Mg+2 concentration in the cell wall – Used to activate autolytic enzyme (enzymes which are secreted by bacteria usually when it dies) – To bind bacteriophage in the cell wall. 35 II. Gram negative Cell wall of bacteria  Is some what complex than Gram positive bacterial cell wall  Has thin peptidoglycan layer (3 – 8nm)  Has high lipid content (lipopolysaccharied) in the outer membrane  Has periplasmic space. 36 Fig. Gram-negative cell wall of 38 bacteria  Cell Wall-less Forms of bacteria Bacteria with defective cell walls include:  Protoplasts - Cells of bacteria, from which the cell wall has been completely removed by chemical or enzymatic treatment.  Spheroplast- Gram-negative bacterial cell in which the cell wall has been partially removed by an enzymatic or chemical treatment.  L-forms –Bacterium- Referring to a defective, spherical or irregular shaped Bacterial cell whose cell wall is partially or totally absent because of adverse growth conditions as observed in Proteus,Streptococcus, &Vibrio N.B Mycoplasma- A genus of cell wall-less, sterol requiring, 39 catalase- Negative bacteria (family of Mycoplasmataceae).  C. Cytoplasmic membrane (Plasma membrane)  It is the actual barrier between the interior and exterior of the bacteria cell.  The cytoplasmic membrane exhibits a well-defined selective permeability, excretion of enzyme, and biosynthesis of cell well and other proteins  The bacterial transport system and the principal energy system (oxidative phosphorylation) are located in the cytoplasmic membrane.  It accounts for 30% of the dry weight of bacterial cell  Chemically, the plasma membrane consists of proteins and phospholipids. 41  Cytoplasmic membrane …  It is 60% protein, 20 – 30% lipid and 10-20% carbohydrate  Although procaryotes lack any intracellular organelles for respiration or photosynthesis, many species possess the physiologic ability to conduct these processes, usually as a function of their plasma membrane. Function of Cell Membrane  Regulates the transport of nutrients and waste products into and out of the cell (It is selectively permeable)  Assists DNA replication  Energy generation by oxidative phosphorylation,  Secretion of enzymes and toxins 42  D. Cytoplasm  The semi liquid cytoplasm that surrounds the nucleoid and is contained within the plasma membrane.  The cytoplasm consists of water, enzymes, oxygen, waste products, essential nutrients, proteins, carbohydrates, lipids and a complex mixture of all the materials required by the cell for its metabolic functions. 43  2. Cellular Element Enclosed with in the Cell Envelope A. Mesosomes  Are complex invaginations of cytoplasmic membrane in to the cytoplasm seen in many bacteria, but not in all. – Increase in the total surface area of the membrane. – Mesosomes are attached to chromosomes and are involved in DNA segregation during cell division – It is involved in respiratory enzyme -activity (Site of oxidative phosphorulation) 44 B. Ribosomes  Cytoplamic particles, which are the sites of protein synthesis  It is composed of ribosomal RNA (rRNA) (70%) and proteins (30%)  The ribosomes of prokaryotes are smaller than cytoplasmic ribosomes of eukaryotes.  Procaryotic ribosomes are 70s in size, being composed of 30S and 50S subunits. – S or Svedberg unit designates the sedimentation coefficient of the rRNA 45 C. Cytoplasmic inclusions  Are distinct granules that may occupy a substantial part of the cytoplasm.  The granules in bacteria represent the accumulated food reserve.  For example, – Glycogen granules (polysaccharides) – Poly-beta hydroxy butyrate granules (lipid) – Polyphosphate inclusions (reserves of PO4) – Volutin granules (poly metha sulphate) 46 D. Nuclear material  The prokaryotic nucleoid is considered primitive nucleus, – it is not surrounded by a nuclear membrane – It does not have a definite shape, and has little or no protein material.  Is concentrated in the cytoplasm as a nucleoid  The nucleoid consists of one long double-stranded circular DNA molecule (chromosome).  The chromosome serves as the control center of the bacterial cell, carries the genetic information needed for producing several thousand enzymes and other proteins.  The size of the chromosome varies according to species.  Apart from nucleus, the bacteria may have some extra chromosomal genetic material in the form of DNA, which47 is known as Plasmid. 3. Cellular Element External to the Cell Envelope A. Flagellum  It is the organ of locomotion in bacterial cell and consists of filament  is free on the surface of bacterial cell  Size: 3-20m in length and 0.01-0.013m in diameter.  It is composed of protein named as flagellin  The flagellar antigen in motile bacterium is named as H (Hauch) antigen. 48  Flagellum … Cont’d  The presence of fllagella in bacterial cell is detected by – Hanging drop preparation – Swarming phenomenon on surface of plate agar – Motility in semi solid media Flagellar arrangements 1. Atrichous: Bacteria with no flagellum: Eg. All cocci 2. Monotrichous: Bacteria with single polar flagellum: Eg. V.cholara 3. Lophotrichous: Bacteria with bunch of flagella at one pole: Eg. Pseudomonas flouresense 4. Amphitrichous: Bacteria with flagella at both poles: Eg. Alcaligenes faecales 5. Peritrichous: Bacteria with flagella all over their surface: 49 Eg. S.typhi FLAGELLUM … Fig. Different flagellar arrangements 50 Importance of flagella Used for motility. Flagella may play a role in bacterial pathogenesis. Some species of bacteria are identified by their flagellar antigen. e.g., Salmonella species, are identified in the clinical laboratory by the use of specific antibodies against flagellar proteins. 51 B. Pilli (Fimbriae)  short, hair-like structures (finer filaments) on the surfaces of procaryotic cells.  Are extruding from the cytoplasmic membrane  Are shorter and stiffer than flagella, and slightly smaller in diameter.  Like flagella, they are composed of protein called pilin arranged in helical strand 52  Two functional types of pili may occur independently or together on some cell. I. Common pili – almost always called fimbriae – Help for attachment of bacteria to epithelial cell – They considered as virulence factor in some species of bacteria, because they allow pathogens to attach to (colonize) tissues and to resist attack by phagocytic white blood cells. II. Sex pili or F pili – occur less commonly – appear to be specifically involved in bacterial conjugation, i.e transfer of genetic material (DNA) from one bacterium to another. 53 C. Bacterial spores These highly resistant structures formed in response to adverse conditions. Under conditions of limited supply of nutrition, vegetative forms of certain bacteria especially gram-positive bacilli and actinomycets form highly resistant and dehydrated forms, which are called endospores. These endospors are capable of survival under adverse conditions such as heat, drying, freezing, radiation, and actions of toxic chemicals. Spores are usually smooth walled and ovoid, in some species it is spherical. It does not take up ordinary stains 54  It looks like areas of high refractivity under light microscope  Spores may have different arrangements in the bacteria some bacteria may have spores at the end (terminal) some near the end (sub terminal),some at the center (central ) Fig. Different arrangements of spores in the bacteria 55  Growth, nutrition & multiplication of Bacteria Bacterial growth refers to an increase in bacterial cell numbers (multiplication) which results from a programmed increase in the biomass of the bacteria. Bacteria multiply by binary fission. Bacteria have well defined requirements of proper nutrients, oxygen, pH, and temperature. 56 Growth, nutrition & multiplication of Bacteria…  The rate of bacterial growth are greatly influenced by the following environmental parameters 1- Nutrition 2- Temperature 3- Oxygen 4- PH 5-salentery 6- Pressure 7- Light radiation 57 NUTRITION OF BACTERIA  For their optimal growth bacteria have well defined requirements of proper nutrients, oxygen, PH and temperature. All bacteria need some form of the element Carbon, H, O2, S, P, and N for growth. Important ions such as K, Ca, Fe, Mn, Mg, Co, Cu, Z, Ur are needed by certain bacteria. Some have specific vitamin, and growth factor requirements and others need organic substances secreted by other microorganisms during their growth. 58 Nutrition … Cont’d 1. Nutrient requirement Depending on their nutritional requirement bacteria can be classified  Autotrophs: - are free-living, non-pathogenic bacteria, most of which can use carbon dioxide as their carbon source.  Heterotrophs are generally parasitic bacteria which require more complex, organic compounds than carbon dioxide as their source of carbon and energy e.g. sugar 59 2. Temperature requirement Optimum temperature is the temperature at which growth occur best. Based on temperature requirement, microorganisms can be broadly classified into Psycrophylic- are those bacteria, which grow in the range of -5 to 200C  These bacteria include those which cause spoilages of food at refrigeration temperature (2-8oc). Mesophilic- are those bacteria, which grow at 20-450C and show optimum growth at 37oC.  all medically important bacteria (pathogenic bacteria) belong to this group. 60 2. Temperature requirement …Cont’d Thermophilic – are those organisms which prefer high temperature (50-800C)  May cause spoilage of under processed canned food Hyperthermophilic  Those which grow at a temperature of above 800C  Some of them grow even at 2500C  arefound in hot springs, geysers and industrial heated wastes 61 3. Oxygen requirement The need of oxygen for particular bacterium reflects its mechanism to meet the requirement of energy. On the basis of this requirement, bacteria have been divided in to:  Obligate Anaerobes-these grow only in the environment devoid of oxygen. e.g. clostridium  Facultative aerobes- these can grow under both aerobic and anaerobic conditions, e.g. enterobacteriaceae  Obligate aerobes- these cannot grow unless oxygen is present in the medium e.g. pseudomonas  Microaerophilic- these organisms can grow under conditions with low oxygen tension e.g. Hemophilus, 62 4. pH requirement Most pathogenic bacteria require a pH of 7.2-7.6 for their optimal growth. Based on pH requirement bacteria can be classified as  Neutrophilic:- bacteria grow best at neutral pH (pH=7) Most pathogenic micro-organism best grow at neutral pH (pH=7)  Acidophilic Bacterial grow best at acidic pH (pH7) E.g. Vibrio cholerae grow at a pH of 8.6 63 5. Salinity (salt concentration) In most case bacteria need small amount of salt concentration to grow. Halophytes are bacteria which need high concentration of salt for their growth. E.g. Staphylococcus epidermidis 64 Bacterial growth Bacteria reproduce by binary fission, a process by which one parent cell divides to form two progeny cells. Because one cell gives rise to two progeny cells, bacteria are said to undergo exponential growth Growing bacterial population doubles at regular interval. Exponential growth;1, 2, 4, 8, (20, 21, 22, 23,….2n)(where n =the number of generations) Fig. Bacterial binary fission 65 Generation time or population doubling time. The interval of time between two cell division, or the time required for a bacterium to give rise to two daughter cells under optimum conditions The generation time of bacteria ranges from as little as 20 minutes for E.coli to more than 20 hrs for Mycobacterium tuberculosis. The generation time varies not only with the species but also with the amount of nutrients, the temperature, the pH, and other environmental factors. 66 Bacterial growth curve The growth cycle of bacteria has four major phases. 1. The Lag phase 2. The log phase (exponential phase) 3. The stationary phase 4. The decline phase If a small number of bacteria are inoculated into a nutrient medium and the bacteria are counted at frequent interval, the typical phase of a standard growth curve can be demonstrated. 67  The Lag Phase this phase is of short duration in which bacteria adapt themselves to new environment in such away that the bacterial machinery brings itself in conformity with the nutrition available. This is a period of active macro molecular synthesis like DNA, RNA, various enzymes & other structural components It is the preparation time for division. No increase in cell number occurs, however, vigorous metabolic activity occurs. This can last for a few minutes up to many hours. The duration of lag phases varies with the species, nature of culture medium, temperature of incubation etc. 68 The log, logarithmic, or exponential phase During this phase, the population can double approximately every 30 minutes with fast growing bacteria It has limited duration because of:-  Exhaustion of nutrients  Accumulation of toxic metabolic end products  Rise in cell density  Change in pH and  Decrease in oxygen tension (in case of aerobic organisms) 69 Stationary Phase Occur when nutrients depletion or toxic products cause growth to slow until the number of new cells produced balances the number of cells that die resulting in a steady state The number of viable cell remain constant There is almost a balance between the bacterial reproduction and bacterial death The death/decline phase Due to severe depletion of nutrients and accumulation of toxic end products the number of bacteria dying is much more than those dividing and hence there is gradual decline in the total number of organism. There is drastic decline in viable cells. 71 Fig. Bacterial growth curve 72