Morphology of Bacteria PDF

Morphology of bacteria Dr Anusha G Professor Department Of Microbiology Pre- Test  Learning objectives Classification of bacteria depending on their morphology and Gram staining property Bacterial cell wall and...

Morphology of bacteria Dr Anusha G Professor Department Of Microbiology Pre- Test  Learning objectives Classification of bacteria depending on their morphology and Gram staining property Bacterial cell wall and cell wall appendages Bacterial Growth Curve  Shape of Bacteria Depending - on shape, bacteria - classified into: Cocci (meaning berry) - oval or spherical cells Bacilli or rods - rod shaped. Cocci - arranged in groups (clusters), pair or chains. Bacilli - arranged in chain, pair, and some bacilli are curved, comma shaped, or cuneiform shaped  Cocci and bacilli - further classified - based on Gram staining into: Gram-positive cocci Gram-negative cocci Gram-positive bacilli Gram-negative bacilli.  Bacterial cell anatomy The outer layer/envelope - consists - (1)rigid cell wall (2) plasma membrane Cytoplasm contains - cytoplasmic inclusions (mesosomes, ribosomes, etc) and a diffuse nucleoid containing single circular chromosome.  Some bacteria - possess additional cell wall appendages - capsule, flagella and fimbriae. Classification of bacteria depending on their morphology and Gram staining property Bacteria Example Gram-positive cocci arranged in Cluster Staphylococcus Chain Streptococcus Pairs, lanceolate shaped Pneumococcus Pair or in short chain, Enterococcus spectacle shaped Tetrads Micrococcus Octate Sarcina Gram-negative cocci arranged in Pairs, lens shaped Meningococcus Pairs, kidney shaped Gonococcus Bacteria Example Gram-positive bacilli arranged in Chain (bamboo stick Bacillus anthracis appearance) Chinese letter or cuneiform Corynebacterium diphtheriae pattern Palisade pattern Diphtheroids Branched and filamentous Actinomyces and Nocardia form Bacteria Example Gram-negative bacilli arranged in Pleomorphic (various shapes) Haemophilus, Proteus Thumb print appearance Bordetella pertussis Comma shaped (fish in stream Vibrio cholerae appearance) Curved Campylobacter (Gull-wing shaped) and Helicobacter Chain Streptobacillus Spirally coiled, flexible Spirochetes Rigid spiral forms Spirillum Bacteria that lack cell wall Mycoplasma BACTERIAL CELL WALL  Tough and rigid structure - surrounding the bacterium. 10–25 nm thickness Weighs about 20–25% of the dry weight of the cell.  Functions of cell wall Protection to cell against osmotic lysis Confers rigidity - presence of peptidoglycan layer in the cell wall Protect cell from toxic substances Site of action of several antibiotics  Contains - virulence factors (e.g. endotoxin) - contribute to pathogenicity Antibody against specific cell wall antigens (e.g. antibody to LPS) - provide immunity against bacterial infection.  Differences between gram-positive and gram negative cell wall Characters Gram-positive wall cell Gram-negative cell wall Peptidoglycan layer Thicker (16–80 nm) Thinner (2 nm) Lipid content Nil or scanty (2–5%) Present (15–20%) Lipopolysaccharide Absent Present (endotoxin) Teichoic acid Present Absent Variety of amino acids Few Several Aromatic amino acids Absent Present  Peptidoglycan Thicker (50–100 layers thick, 16–80 nm) than gram-negative cell wall Each layer - mucopeptide (murein) chain - alternate units of N-acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG) molecules - cross linked via tetrapeptide side chains and pentaglycine bridges 3 Tetrapeptide side chain - from NAM molecule - composed of L-alanine- D-glutamine - L-lysine - D-alanine Structure of Gram- Peptidoglycan layer of Gram- positive cell wall positive  Teichoic Acid Polymers of glycerol or ribitol - joined by phosphate groups. Maintain - structure of cell wall. Two types: (i) Cell wall teichoic acid and (ii) Lipoteichoic acid. Absent in gram-negative bacteria.  Peptidoglycan layer Very thin (1–2 layer, 2 nm thick) - composed of a mucopeptide chain - similar to gram-positive cell wall. Consists - alternate NAM and NAG molecules  Outer Membrane Phospholipid layer - lies outside the thin peptidoglycan layer Serves as - protective barrier to the cell Outer membrane proteins (OMP) or porin proteins - specialized proteins  Lipopolysaccharide (LPS) Consists of three parts: Lipid A or the endotoxin Core polysaccharide O side chain (or O antigen or somatic antigen) Periplasmic Space Space between the inner cell membrane and outer membrane. It encompasses the peptidoglycan layer.  CELL MEMBRANE Essential for - survival of the bacteria. 5–10 nm thick -bilayered phospholipid - several proteins are embedded - integral proteins and peripheral proteins  Lacks sterols -such as cholesterol (except in Mycoplasma). Contain pentacyclic sterol-like molecules - hopanoids  Functions of cell membrane Semi permeable membrane Transport system -nutrient uptake, and waste excretion Site for metabolic processes  CYTOPLASMIC MATRIX Bacterial cytoplasm, lacks membrane-bound organelles. Composed of water (70%) Packed with ribosomes, storage granules - inclusions and cell membrane invaginations - mesosomes. Plasma membrane and everything within it - called as protoplast.  Ribosomes Sites for protein synthesis. Composed of rRNA and ribosomal proteins. Integrated with the mRNA to form polysomes At this site - genetic codons of the mRNA - translated into peptide sequences. Each 70 S unit - consists of a 30 S and a 50 S subunits.  Intracytoplasmic Inclusions Storage sites of nutrients/energy Formed by bacteria under nutritional deficiency conditions and disappear when the deficient nutrients are supplied. Two types of inclusions: Organic inclusion bodies Inorganic inclusion bodies  Mesosomes Invaginations of plasma membrane - in the shape of vesicles, tubules, or lamellae. Prominent in gram-positive bacteria. Involved in: Bacterial respiration Cell wall formation Chromosome replication  Nucleoid Bacteria do not have a true nucleus - genetic material - located in an irregularly shaped region called - nucleoid. Bacteria possess a single haploid chromosome. Comprises of super coiled circular double stranded DNA Seen by electron microscopy or on staining with Feulgen stain Bacteria also possess extrachromosomal DNA - plasmids  CELL WALL APPENDAGES Composed of: Capsule and Slime Layer Flagella Fimbriae or Pili  Capsule and Slime Layer Layer of amorphous viscid material lying outside the cell wall called glycocalyx. Capsule -well organized and not easily washed off Slime layer - diffuse, unorganized loose material that can be removed easily Capsulated bacteria Composition Pneumococcus Polysaccharide Meningococcus Polysaccharide Haemophilus influenzae Polysaccharide Klebsiella pneumoniae Polysaccharide Pseudomonas aeruginosa Polysaccharide Bacteroides fragilis Polysaccharide Bacillus anthracis Polypeptide (glutamate) Streptococcus pyogenes (some Hyaluronic acid strains)  Functions of capsules Bacterial virulence Prevent cell from drying out (desiccation) Protects the bacterium from the action of lysozyme and bacteriophages. Toxic to the host cells and induces abscess formation (e.g. Bacteroides fragilis)  Capsules as vaccine Capsular vaccines are available for bacteria such as Pneumococcus, Meningococcus and Haemophilus influenzae serotype-b.  Biofilm Formation Biofilm - living ecosystem made of millions of adherent bacterial cells embedded within a self-produced matrix of extracellular polymeric substance Capable of adherence to damaged tissues and plastic surfaces. Adhesion is a first step in colonization and sometimes leads to disease  Demonstration of Capsule Capsule can be detected by various methods: Negative staining by India ink and nigrosin stain M’Faydean capsule stain Serological test Quellung reaction Capsular antigen  Flagella Thread-like appendages - protruding from the cell wall Confer motility to the bacteria 5–20 µm in length and 0.01 - 0.02 µm in thickness.  Arrangement of Flagella Monotrichous - e.g. Vibrio cholerae, Pseudomonas and Campylobacter Lophotrichous - e.g. Spirillum Peritrichous - e.g. Salmonella Typhi, Escherichia coli Amphitrichous - e.g. Alcaligenes faecalis.  Ultrastructure of Flagella On electron microscope - bacterial flagellum is - composed of three parts 1. Filament - longest portion of the flagellum that extends from the cell surface to the tip. 2. The basal body 3. Hook  Detection of Flagella Direct demonstration of flagella: Tannic acid staining (Leifson’s method) Electron microscopy. Indirect means by demonstrating the motility: Craigie tube method Hanging drop method Semisolid medium, e.g. mannitol motility medium Dark ground or phase contrast microscopy.  Types of motility shown by different bacteria Types of motility Bacteria Tumbling motility Listeria Gliding motility Mycoplasma Stately motility Clostridium Darting motility Vibrio cholerae, Campylobacter Swarming on agar plate Proteus, Clostridium tetani Corkscrew, motility lashing, flexion extension Spirochete  Fimbriae or Pili Short, fine, hair-like appendages - help in bacterial adhesion. Special type of pili (called sex pilus) - helps in conjugation. Pili - made up of protein called pilin Antigenic; but, the antibodies against pilin antigens are not protective. Not related to motility  Type of Pili According to functions, pili are of two types. 1. Common pili or fimbriae 2. Sex pili A B  Detection of Fimbriae Electron microscope Surface pellicle - thin layer formed at the surface of a liquid culture of strongly aerobic bacteria such as Pseudomonas.  Atypical Forms of Bacteria Involution forms: Swollen and aberrant forms of bacteria (e.g. gonococci and Yersinia pestis) formed in ageing cultures in high salt concentration Pleomorphic bacteria: Exhibit great variation in the shape and size of individual cells, e.g. Proteus and Haemophilus.  L Form (Cell Wall Deficient Forms) Discovered by E. Klieneberger, while studying Streptobacillus moniliformis. Named it as L form - after its place of discovery, i.e. Lister Institute, London (1935) L forms play a role in the persistence of pyelonephritis and other chronic infections.  Bacterial Spores Spores are highly resistant resting (or dormant) stage of the bacteria. Formed in unfavorable environmental conditions - as a result of the depletion of exogenous nutrients.  Bacterial spore comprises of several layers. From innermost towards the outermost, the layers are: core → cortex → coat → exosporium  Sporulation Refers to - process of formation of spores from vegetative stage of bacteria. Not a method of reproduction - because bacteria do not divide during sporulation. Complex process and takes about 10 hours. Mature spore formed is extremely resistant to heat and disinfectant  Germination Transformation of dormant spores into active vegetative cells when grown in a nutrient-rich medium.  Shape and Position of Spores For a given species, the precise position, shape and relative size of the spore are constant. Position: Central, subterminal or terminal Shape: Oval or spherical in shape Width: Diameter of spore may be same or less than the width of bacteria A. Non-bulging, oval and terminal; B. Non-bulging, round, and subterminal; C. Non-bulging, oval and central; D. Bulging, round and terminal; E. Bulging, oval and terminal; F. Bulging, oval, and central  Sporicidal Agents Spores are resistant to most of the routinely used disinfectants. Only limited agents called as sterilants are capable of killing the spores, e.g. autoclave, or ethylene oxide sterilizer, etc.  Demonstration of Spores Gram staining: Spores appear as unstained refractile bodies within the cells Modified Ziehl–Neelsen staining: Spores are weakly acid-fast and appear red color. Special techniques for endospore staining include the Schaeffer–Fulton stain and the Moeller stain  Applications of spores Indicators of proper sterilization. Spores of Geobacillus stearothermophilus - sterilization control for autoclave and plasma sterilizer Spores of Bacillus atrophaeus - sterilization control for hot air oven and ethylene oxide sterilizer. Used as agents of bioterrorism PHYSIOLOGY OF BACTERIA 64  Bacterial Growth Requirement ▪ Water constitutes - 80% of total bacterial cell. ▪ Minimum nutritional requirements - essential for growth and multiplication of bacteria include - sources of carbon, nitrogen, hydrogen, oxygen and some inorganic 65  Bacterial Cell Division ▰ Bacteria divide by a relatively simple form of cell division, i.e. by binary fission ⮚ Nuclear division ⮚ Cytoplasmic division 66  Rate of Multiplication in Bacteria Generation time is the time required for a bacterium to give rise to two daughter cells under optimum condition. ⮚ Escherichia coli and most of the other pathogenic bacteria -20 minutes; ⮚ Mycobacterium tuberculosis - 20 hours ⮚ Mycobacterium leprae - 20 days 67 Bacterial Count ▰ Total count: Indicates total number of bacteria (live or dead) in the specimen. This is done by counting the bacteria under microscope using counting chamber. ▰ Viable count: Measures the number of living (viable) cells in the given specimen. Viable count may be obtained by a technique called as pour plate method. 68 Bacterial Growth Curve ▰ When a bacterium is inoculated into a suitable liquid culture medium and incubated, its growth follows a definite course. ▰ When bacterial count of such culture is determined at different intervals and plotted in relation to time, a bacterial growth curve is obtained. 69 Bacterial growth curve comprises of four phases. ⮚ Lag phase ⮚ Log phase ⮚ Stationary phase ⮚ Phase of decline 70  Lag Log Stationary Decline Bacteria No Yes Yes No divide Bacterial No No Yes Yes death Total count Flat Raises Raises Flat Viable count Flat Raises Flat Falls Special Accumulation of Uniformly stained Gram variable. Produce: features enzymes and Metabolically active Produce: Involution forms metabolites Attains Small size Granules spores, maxi- mum size exotoxin, antibiotics, bacteriocin 71 Lag phase ▪ Period between inoculation and beginning of multiplication of bacteria. ▪ Bacteria increase in size due to accumulation of enzymes and metabolites. ▪ Bacteria reach their maximum size at the end of lag phase. 72 Log phase Bacteria divide exponentially so that the growth curve takes a shape of straight line. At this stage, the bacterium is: ▰ Smaller in size ▰ Biochemically active ▰ Uniformly stained 73 Stationary phase ▰ Number of viable cells remain stationary as there is a balance between the dying cells and the newly formed cells. But the total count keeps raising. 74 In this phase: ▪ Bacterium becomes gram-variable ▪ More storage granules are formed ▪ Sporulation occurs in this phase ▪ Bacteria produce exotoxins, antibiotics and bacteriocins 75 Decline phase ▰ Bacteria stop dividing completely; while the cell death continues due to exhaustion of nutrients, and accumulation of toxic products. ▰ Involution forms are seen. 76  Factors Affecting Growth of Bacteria There are several environmental factors that affect the growth of the bacteria. ▪ Oxygen ▪ Carbon dioxide ▪ Temperature ▪ pH ▪ Light ▪ Osmotic Effect ▪ Mechanical and Sonic Stresses ▪ Moisture and Desiccation 77 Oxygen Classification Explanation Examples Obligate aerobes Can grow only in the Pseudomonas, presence of oxygen Mycobacterium tuberculosis, Bacillus, Brucella and Nocardia Facultative anaerobes They are aerobes that can Most of the pathogenic also grow anaerobically bacteria, e.g. E.coli, S.aureus Facultative aerobes They are anaerobes that Lactobacillus can also grow aerobically 78 Classification Explanation Examples Microaerophilic bacteria Can grow in the presence Campylobacter, of low oxygen tension, i.e., Helicobacter, 5-10% of oxygen Mycobacterium bovis Obligate anaerobes These bacteria can grow Clostridium only in the absence of oxygen. Oxygen is lethal to these bacteria Aerotolerant anaerobe Tolerate oxygen for some Clostridium histolyticum time, but do not use it 79 Carbon dioxide ▪ Organisms that require higher amounts of carbon dioxide (5–10%) for growth are called capnophilic bacteria. ▪ Examples - Brucella abortus, Streptococcus pneumoniae, etc 80 Temperature ▪ Psychrophiles: Grow at temperatures below 20°C; example, most of the saprophytes, e.g. Pseudomonas ▪ Mesophiles: Grow within a temperature range 25°C - 40°C; example, most of the pathogenic bacteria ▪ Thermophiles: Grow at a high temperature range of 55°C–80°C, e.g. Geobacillus stearothermophilus. 81 pH ▪ Most pathogenic bacteria grow between pH 7.2 -7.6. ▪ Very few bacteria (e.g. lactobacilli) can grow at acidic pH below pH 4 ▪ Vibrio cholerae are capable of growing at alkaline pH (8.2–8.9). 82 Light ▪ Bacteria (except phototrophs) grow well in darkness. ▪ They are sensitive to ultraviolet rays and other radiations in light. ▪ Photochromogenic mycobacteria produce pigments only on exposure to light. 83 Osmotic Effect ▪ Bacteria are able to withstand a wide range of external osmotic variation because of the mechanical strength of the cell wall. ▪ Sudden exposure to hypertonic saline may cause cell shrinkage (plasmolysis) ▪ Exposure to distilled water may cause cell swelling and rupture (plasmoptysis). 84 Bacterial Metabolism ▪ Process by which a microbe obtains the energy and nutrients (e.g. carbon) for its survival and reproduction. 85 Bacterial Metabolism ▪ Autotrophs ▪ Heterotrophs ▪ Lithotrophs ▪ Organotrophs ▪ Chemotrophs ▪ Phototrophs 86 Post - Test Thank you