Lecture 1 Microbiology 17-03-23 PDF

Lecture 1 Bacterial Morphology and Structures, Metabolism & Growth 1  Content Cell structure: basic structure and function of eukaryotic and prokaryotic cells. Techniques to study morphology of bacteria. Optical method...

Lecture 1 Bacterial Morphology and Structures, Metabolism & Growth 1  Content Cell structure: basic structure and function of eukaryotic and prokaryotic cells. Techniques to study morphology of bacteria. Optical methods (light, phase contrasting, dark-field, fluorescent, electron, confocal scanning laser microscope). Basics of bacterial cell wall structures and properties associated with bacterial cell walls. Staining: Gram positive/negative cell wall, acid-fast cell wall, cell walls of Archea. Protoplasts, spheroplasts, L-forms. Peptidoglycan, LPS, pathogenesis, and targets of antibiotics. Structures of the bacterial cell and the genetic makeup of bacteria (Chromosome, plasmid, ribosomes, target of antibiotics, roles in pathogenesis or drug resistance) Morphology of bacteria and the versatility of bacteria (in clinical diagnosis). Bacterial appendages, bacterial spores, capsules, Flagella, Pili etc.), and their relatedness to the clinical practice 2  History of Microbiology Micrographia Robert Hooke First report of cell structure 1665 ‘Little boxes’ in cork : CELL First illustrated book on microscopy  Anton Van Leeuwenhoek (1678) First person to see bacteria Single lens microscope 4  Spontaneous Generation Aristotle (384 a.C): any damp body gives rise to living things – Miasma – “bad air” – phoenix myths - In Greek mythology, a phoenix (Ancient Greek: φοῖνιξ phoînix) is a long-lived bird that cyclically regenerates or is otherwise born again. – “Golam” – a clay figure brought to life by magic. Herodotus (c. 484 – c. 425 BC): Living organisms could arise from non living matter – crocodiles from mud Van Helmont (17th century): small animals only – maggots from meat – mice from feed 5 Lazzaro Spallanzani (1729-1799) 1. OOpen 2.Closed 3. BBoiled, left open 4. BBoiled, closed 6 Swan Neck Flask Experiment Add broth to flask Bend the neck of the flask (air can enter but dust cannot) Heat broth No bacterial growth Break neck of flask dust enters growth occurs Conclusion: microbes in the dust not in air 7  Louis Pasteur 1822-1895 Germ theory Fermentation Pasteurization Rabies vaccine Streptococcus pneumoniae causes lobar pneumonia 8  Vaccination In 1885, while studying rabies, Pasteur tested his first human vaccine. Pasteur produced the vaccine by attenuating the virus in rabbits and subsequently harvesting it from their spinal cords in sterile air. Pasteur was first used to treat a human bite victim Joseph Meister 9 years old boy on 6 July 1885. 9  Louis Pasteur Added 20 years to the lifespan of every man woman and child improved the quality of life 10 Robert Koch (1843-1910) Confirmed germ theory Discovered cause of – anthrax – cholera – tuberculosis Developed – pure culture techniques – staining techniques – solid media 11  Koch’s postulates Rules to prove an organism causes a disease - four criteria designed to establish a causative relationship between a microbe and a disease Organism consistently isolated from diseased individuals Organism cultivated in pure form Signs and symptoms induced after inoculation Same organism isolated from experimentally infected individual 12 Koch’s postulates 13 Universal tree of life 14 Prokaryotic and Eukaryotic cells Cells are divided into two types according to the way their DNA is stored. 1. Prokaryotic cells, which lack a membrane covered nucleus, have their DNA located in a part of the cell called the nucleoid. 2. Eukaryotic cells have a membrane-covered nucleus which stores the cell's DNA. Prokaryotic cells live in a wide variety of environments and can be found in water, soil, and the air. Bacteria are a type of a prokaryotic cell. Some bacteria cause diseases like Strep throat and Staph infections, but many bacteria are beneficial. Beneficial bacteria: -decompose dead remains. -can be used to make chemicals for human use (e.g., industrial chemicals, medicines). -are import in the manufacturing process of some human foods (e.g., yogurt and cheeses). 15 EEUKARYOTIC CELLS -have a membrane bound nucleus that contains their DNA. -are larger than prokaryotic cells with a lower surface area to volume ratio. -have a number of membrane-bound inner compartments Organelles can be divided into four categories. 1. The nucleus and ribosomes. 2. Organelles of the endomembrane system. 3. The energy-related organelles. 16  Bacterial cell components The nucleus communicates with the ribosomes to control protein synthesis. It does this by making mRNA copies of the genes (recipes) for the proteins that the cell needs to build. The mRNA's are translated on the surface of the ribosome. Each organelle of the endomembrane system has its own enzymes and produces specific products. The products of the endomembrane system are moved around in the cells in transport vesicles. 17 Comparing of Prockaryotic and Eukaryotic cells 18  Bacterial cell morphology Cocci: sphere, 1μm Bacilli: rods, 0.5-1 μm in width -3 μm in length Spiral bacteria: 1~3. and 0.3- 0.6 μm in width. Unit for measurement Micron or micrometer, μm: 1μm=10 -3 mm Size: Varies with kinds of bacteria, and also related to their age and external environment. 19 Bacterial Shapes and Arrangements 20 Arrangement of Cocci Cocci may be oval, elongated, or flattened on one side. Cocci may remain attached after cell division. These group characteristics are often used to help identify certain cocci. Cocci that remain in chains after dividing are called streptococci Cocci that remain in pairs after dividing are called diplococci.. Cocci that divide in two planes and remain in groups of four are called tetrads. Cocci that divide in three planes and remain in groups cube like groups of eight are called sarcinae. Cocci that divide in multiple planes and form grape like clusters or sheets are called staphylococci. 21 Arrangement of Bacilli Since bacilli only divide across their short axis there are fewer groupings. Bacillus is a shape (rod shaped) but there is also a genus of bacteria with the name Bacillus. Most bacilli appear as single rods. Diplobacilli appear in pairs after division. Streptobacilli appear in chains after division. Some bacilli are so short and fat that they look like cocci and are referred to as coccobacilli 22 Arrangement of Spiral bacteria Spiral bacteria have one or more twists. Vibrios look like curved rods. Spirilla have a helical shape and fairly rigid bodies. Spirochetes have a helical shape and flexible bodies. Spirochetes move by means of axial filaments, which look like flagella contained beneath a flexible external sheath. 23 Other bacterial shapes Archea 24  Bacterial cell morphology Essential structures Cell wall Cell membrane Cytoplasm Nuclear material Particular structures Capsule Flagella Pili Spore 25  Bacterial structure Bacterial cytoplasm is surrounded by a cell membrane, and a cell wall. The cell membrane is similar to that of eukaryotic cells. The cell wall maintains the shape of the cell. The DNA of a bacterium is a single circular chromosome that resides in the nucleoid. The cytoplasm of a bacterium has thousands of tiny particles called ribosomes that make all of the proteins needed by the cell. Bacteria can have appendages with specific functions. Flagella can be used to help bacteria move in26 water.  Techniques to study morphology of bacteria - Microscopy Light microscopy Dark-field microscopy Phase-contrast microscopy Luminescent microscopy Electron microscopy Scanning Electron Microscopy 27  The light microscope The resolving power of the light microscope under ideal conditions is about half the wavelength of the light being used. Resolving power is the distance that must separate two point sources of light, if they are to be seen as two distinct images. In other words, RP is the smallest distance at which two objects can be successfully distinguished. With yellow light of a wavelength of 0.4 µm, the smallest separable diameters are thus about 0.2 µm, ie, one-third the width of a typical prokaryotic cell. The useful magnification of a microscope is the magnification that makes visible the smallest resolvable particles. Useful magnification is calculated as according to the following equation: m = mome, where mo is the magnification of the objective and me is the magnification of the eyepiece 28  The bright-field microscope This microscope is most commonly used in microbiology courses and consists of two series of lenses (objective and ocular lens), which function together to resolve the image. These microscopes generally employ a 100-power objective lens with a 10-power ocular lens, thus magnifying the specimen 1000 times. Particles 0.2 µm in diameter are therefore magnified to about 0.2 mm and so become clearly visible. With this microscope, specimens are rendered visible because of the differences in contrast between them and the surrounding medium. Many bacteria are difficult to see well because of their lack of contrast with the surrounding medium. Dyes (stains) can be used to stain cells or their organelles and increase their contrast so that they can be more easily seen in the bright-field microscope. 29  The phase contrast microscope This type of the microscope was developed to improve contrast differences between cells and the surrounding medium, making it possible to see living cells without staining them; With bright-field microscopes, killed and stained preparations must be used. The phase contrast microscope takes advantage of the fact that light waves passing through transparent objects, such as cells, emerge in different phases depending on the properties of the materials through which they pass. This effect is amplified by a special ring in the objective lens of a phase contrast microscope, leading to the formation of a dark image on a light background 30  The dark-field microscope The dark-field microscope is a light microscope in which the lighting system has been modified to reach the specimen from the sides only. This is accomplished through the use of a special condenser that both blocks direct light rays and deflects light off a mirror on the side of the condenser at an oblique angle. This creates a “dark field” that contrasts against the highlighted edge of the specimens and results when the oblique rays are reflected from the edge of the specimen upward into the objective of the microscope. Resolution by dark-field microscopy is quite high. Thus, this technique has been particularly useful for observing organisms such as Treponema pallidum, a spirochete which is less than 0.2 µm in diameter and therefore cannot be observed with a bright-field or phase contrast microscope 31  The fluorescence microscope The fluorescence microscope is used to visualize specimens that fluoresce, which is the ability to absorb short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible). Some organisms fluoresce naturally because of the presence within the cells of naturally fluorescent substances such as chlorophyll. Those that do not naturally fluoresce may be stained with a group of fluorescent dyes called fluorochromes. Fluorescense microscopy is widely used in clinical diagnostic microbiology. 32  Differential interference contrast microscopy Differential interference contrast microscopes employ a polarizer to produce polarized light. The polarized light beam passes through a prism that generates two distinct beams; these beams pass through the specimen and enter the objective lens where they are recombined into a single beam. Because of slight differences in refractive index of the substances each beam passed through, the combined beams are not totally in phase but instead create an interference effect, which intensifies subtle Yeast differences in cell structure. S. cerevisiae Structures such as spores, vacuoles, and granules appear three dimensional. DIC microscopy reveals internal cell structures that are less apparent by bright-field 33 techniques.  The electron microscope The superior resolution of the electron microscope is due to the fact that electrons have a much shorter wavelength than the photons of white light. There are two types of electron microscopes in general use: the transmission electron microscope (TEM), which has many features in common with the light microscope, and the scanning electron microscope (SEM). The TEM employs a beam of electrons projected from an electron gun and directed or focused by an electromagnetic condenser lens onto a thin specimen. As the electrons strike the specimen, they are differentially scattered by the number and mass of atoms in the specimen; some electrons pass through the specimen and are gathered and focused by an electromagnetic objective lens, which presents an image of the specimen to the projector lens system for further enlargement. 34  SEM microscopy The SEM generally has a lower resolving power than the TEM; It is particularly useful for providing three-dimensional images of the surface of microscopic objects. Electrons are focused by means of lenses into a very fine point. The interaction of electrons with the specimen results in the release of different forms of radiation (eg, secondary electrons) from the surface of the material, which can be captured by an appropriate detector, amplified, and then imaged on a television screen. An important technique in electron microscopy is the use of “shadowing.” When an electron beam is passed through the heavy metal coated preparation in the electron microscope and a positive print is made from the “negative” image, a three dimensional 35 effect is achieved Type of microscope Maximum useful Resolution Description magnification Bright field 1,500X 100-200 nm Extensively used for the visualization of micro organisms; usually necessary to stain specimens for viewing 1,500X 100-200 nm Used for viewing live microorganisms, particularly those with characteristic morphology; Dark field staining not required; specimen appears bright on a dark background Fluorescence 1,500X 100-200 nm Uses fluorescent staining; useful in many diagnostic procedures for identifying microorganisms Phase contrast 1,500X 100-200 nm Used to examine structures of living microorganisms; does not require staining TEM (trans- mission Used to view ultrastructure of electron – microscope) 500,000-1,000,000X 0.1 nm microorganisms, including viruses SEM (scanning electron 10,000-100,000X 1-10 nm Used for showing detailed surface microscope) structures of microorganisms, produces a three-dimensional image 36  Staining methods Simple staining - only one dye is used, differentiation between bacteria is impossible. Differential staining – more than one dye is used, differentiation between bacteria is possible (e.g. Gram stain, Fast-acid stain). Special staining – more than one dye is used, special structures are seen. 37  Differential Staining Methods - Acid-Fast Staining Acid-fast Stain: ACID-FAST Cell Color Cell color Mycobacterium and many STAIN Nocardia species are called acid-fast because during an Procedure Reagent Acid fast Non acidfast acid-fast staining procedure bacteria bacteria they retain the primary dye Primary dye Carbolfuchsin RED RED carbol fuchsin despite Decolorizer Acid-alcohol RED COLORLESS decolorization with the powerful solvent acid-alcohol. Couterstain Methylene blue RED BLUE Nearly all other genera of bacteria are nonacid-fast. The acid-fast genera have lipoidal mycolic acid in their cell walls. It is assumed that mycolic acid prevents acid-alcohol from decolorizing protoplasm. The acid-fast stain is a differential stain. 38  Special staining Capsule stain Spore stain 39 Flagella stain  Gram staining The Gram stain was developed by Christian Gram in 1884 and can differentiate between the two types of cell walls Gram positive and Gram-negative. Christian Gram 1884 40  Cell wall The cell wall surrounds the plasma membrane and protects the cell from changes in water pressure. The bacterial cell wall consists of peptidoglycan (or murein), a polymer consisting of NAG and NAM and short chains of amino acids. Penicillin interferes with peptidoglycan synthesis. 41  Structure of Peptidoglycan in a Celll Wall Alternating NAM and NAG molecules form a carbohydrate backbone (the glycan portion). Rows of NAG and NAM are linked by polypeptides (the peptido- portion). The structure of the polypeptide cross-bridges may vary but they always have a tetrapeptide side chain, which consists of 4 amino acids attached to NAMs. The amino acids occur in alternating D and L forms. While peptidoglycan is present in (most) all bacterial cell walls, there are two basic variations of structure seen in most bacterial cells, one described as Gram-positive and one described as Gram-negative 42  Gram-positive cell wall Gram-positive cell walls consist of many layers of peptidoglycan and also contain teichoic acids. Teichoic acids may: bind and regulate movement of cations into and out of the cell prevent extensive wall breakdown and possible cell lysis during cell growth provide much of the cell wall's antigenicity 43  The Gram-negative Cell Wall Gram-negative bacteria have a lipopolysaccharide-lipoprotein-phospholipid outer membrane surrounding a thin (sometimes a single) peptidoglycan layer. Gram-negative cell walls have no teichoic acids. 44  Gram+ Gram- Gram- Gram- Gram+ 45 Gram+  Cell Wall Functions The outer membrane protects the cell from phagocytosis and from penicillin, lysozyme, and other chemicals. Porins are proteins that permit small molecules to pass through the outer membrane; specific channel proteins allow other molecules to move through the outer membrane. The lipopolysaccharide component of the outer membrane consists of sugars (O polysaccharides) that function as antigens and lipid A, which is an endotoxin. Endotoxin causes fever and shock. 46  Gram stain mechanism The cells are first stained with the primary stain, crystal violet – 1 min. Wash with water Mordant, Gram's iodine – 1 min The iodine forms a complex with the crystal violet and the crystal violet-iodine complex becomes "trapped" inside the peptidoglycan. Wash with water Decolorize (using acetone-alcohol) – 5-10 sec The cells for a period of time long enough to dissolve the outer membrane of Gram-negative cells and pull the crystal violet-iodine complex through the thin layer of peptidoglycan. Counter-stain with safranin, to stain the Gram-negative cells pink. Gram-positive cells will also stain with safranin but it will not be seen on top of the purple crystal violet-iodine remaining in the cells. 47  Comparative Characters of Gram-positive and Gram-negative bacteria 48  Atypical cell wall Mycoplasma is a bacterial genus that naturally lacks cell walls; the presence of sterols in the plasma membrane protects from osmotic lysis. Mycobacterium is a genus that has mycolic acids in its cell Mycoplasma walls, giving it a "waxy" cell wall that is resistant to decolorization with acid-alcohol when stained with carbolfuschin (and so is designated "acid-fast"). Archea have pseudomurein; they lack peptidoglycan. 49  Damage of the cell wall In the presence of lysozyme, gram-positive cell walls are destroyed, and the remaining cellular contents are referred as a protoplast. In the presence of lysozyme (after disruption of the outer membrane), gram-negative cell walls are not completely destroyed, and the remaining cellular contents are referred to as spheroplasts. Protoplasts and spheroplast are subject to osmotic lysis. Proteus and some other genera can lose their cell walls spontaneously or in response to penicillin and swell into L forms (Lister Institute). L forms can live and divide and/or return to the normal walled state. Antibiotics such as penicillin interfere with cell wall (peptidoglycan) synthesis. 50  Structures external to the cell wall- Glycocalix The glycocalyx (capsule, slime layer, or extra cellular polysaccharide) is a gelatinous polysaccharide and/or polypeptide covering. The exact chemical composition varies depending on the species. Capsules are organized and firmly attached to the cell wall. Capsules may protect pathogens from phagocytosis. Capsules enable adherence to surfaces, prevent desiccation, and may provide nutrients. Slime layers are unorganized and loosely attached to the cell wall. Extracellular polymeric substance (EPS) is the description of a glycocalyx that is a component of a biofilm. 51  Structures external to the cell wall- Flagella Flagella are relatively long filamentous appendages consisting of a filament, hook, and basal body. Prokaryotic flagella rotate to push the cell. Motile bacteria exhibit taxis; positive taxis is movement toward an attractant, and negative taxis is movement away from a repellent. 52 Arrangement of Flagella 53  Movement of Bacteria There are many schemes for flagellation in bacteria, of which peritrichous flagella and a single polar (monotrichous) flagellum are two types. a | In the case of peritrichous flagella, such as those found in Escherichia coli, counter-clockwise (CCW) flagellar rotation results in the formation of a helical bundle that propels the cell forward in one direction in a smooth-swimming motion (a 'run'). By contrast, the presence of clockwise (CW) rotation causes unbundling of the helical bundle, allowing the bacterium to randomly reorient its direction (a 'tumble'). b | In the case of a single polar flagellum, CCW rotation propels the cell forward in a run, whereas CW rotation propels the cell backward with a concomitant random reorientation. 54  Structure of Flagella Flagella are anchored by pairs of rings associated with the plasma membrane and cell wall. Gram positive bacteria have only the inner pair of rings. The filament is composed of the globular protein flagellin, which is arranged in several intertwined chains that form a helix around a hollow core. Flagellin can vary in structure and is used to identify some pathogenic bacteria serologically. The flagellar antigens are referred to as H antigens. E. coli may express any of at least 50 different variants; serovars (serological variants) identified as O157:H7 are associated with food borne epidemics (O antigens are somatic antigens and are lipopolysaccharide complexes associated with the cell wall). 55  Axial filaments Spiral cells that move by means of an axial filament (endoflagellum) are called spirochetes. Axial filaments are similar to flagella, except that they wrap around the cell. 56  Fimbria and pili Fimbriae and pili are short, thin appendages. Cells may have many fimbriae, which help the cells adhere to surfaces. Cells that have pili have only one or two. Pili join cells either for the transfer of DNA from one cell to another (sex pili) or are used for special types of movement; twitching, seen in Pseudomonas aeurginosa, Neisseria gonorrhoeae and some strains of E. coli, or the gliding motility of myxobacteria 57  Structure of inner cell wall The plasma membrane encloses the cytoplasm and is a phospholipid bilayer with peripheral and integral proteins (the fluid mosaic model). The plasma membrane is selectively permeable. Plasma membranes carry enzymes for metabolic reactions, such as nutrient breakdown, energy production, DNA replication and photosynthesis in prokaryotes. These reactions take place in mitochondria, the nucleus, and chloroplasts in eukaryotic cells. 58  Chromatophores Chromatophores are the infoldings of the plasma membrane that contain pigments involved in photosynthesis Mesosomes, irregular infoldings of the plasma membrane, are artifacts, not true cell structures. Plasma membranes can be destroyed by alcohols and polymyxins. 59  Comparison of Prokaryotes and Eukaryotes Characteristic Prokaryotes Eukaryotes Size of cell Typically 0.2-2.0 m m in diameter Typically 10-100 m m in diameter Nucleus No nuclear membrane or nucleoli (nucleoid) True nucleus, consisting of nuclear membrane & nucleoli Membrane-enclosed organelles Absent Present; examples include lysosomes, Golgi complex, endoplasmic reticulum, mitochondria & chloroplasts Flagella Consist of two protein building blocks Complex; consist of multiple microtubules Glycocalyx Present as a capsule or slime layer Present in some cells that lack a cell wall Cell wall Usually present; chemically complex (typical When present, chemically simple bacterial cell wall includes peptidoglycan) Plasma membrane No carbohydrates and generally lacks sterols Sterols and carbohydrates that serve as receptors present Cytoplasm No cytosketeton or cytoplasmic streaming Cytoskeleton; cytoplasmic streaming Ribosomes Smaller size (70S) Larger size (80S); smaller size (70S) in organelles Chromosome (DNA) arrangement Single circular chromosome; lacks histones Multiple linear chromosomes with histones Cell division Binary fission Mitosis Sexual reproduction No meiosis; transfer of DNA fragments only Involves meiosis (conjugation) Size of cell Typically 0.2-2.0 m m in diameter Typically 10-100 m m in diameter 60  Cytoplasm and nucleus Cytoplasm Cytoplasm is the fluid component inside the plasma membrane. The cytoplasm is mostly water, which inorganic and organic molecules, DNA, ribosomes, and inclusions. Nucleus The Nuclear Area The nuclear area contains the DNA of the bacterial chromosome. Bacteria can also contain plasmids, which are circular, extra-chromosomal DNA DNA molecules. 61  Ribosomes The cytoplasm of a prokaryote contains numerous 70s ribosomes; ribosomes consist of rRNA and protein. Protein synthesis occurs at ribosomes; it can be inhibited by certain antibiotics. The difference between prokaryotic (70s= 30 S+ 50S) and eukaryotic (80s = 60 S + 40 S) ribosomes allows antibiotics to selectively target the prokaryotic ribosomes while sparing eukaryotic ribosomes. S = Sedimentation coefficient 62  Difference between prokaryotic and eukaryotic ribosomes S = Sedimentation coefficient Ratio of rRNA vs proteins in prokaryotic cells is 60% & 40 % (RNA rich) Ratio of rRNA vs proteins in eukaryotic cells is 40% & 60% (Protein rich) 63  Inclusions Inclusions are reserve deposits found in prokaryotic and eukaryotic cells. Among the inclusions found in bacteria are metachromatic granules (inorganic phosphate), polysaccharide granules (usually glycogen or starch), lipid inclusions, sulfur granules, carboxysomes (ribulose 1,5-diphosphate carboxylase), magnetosomes (Fe3O4), and gas vacuoles. Magnetosomes are : present in several gram-negative bacteria (Aquaspirillum magnetotacticum for example) act like magnets may be used to move downward until they reach a suitable attachment site have been demonstrated to decompose hydrogen peroxide in vitro so it has been suggested that magnetosomes may protect cells from hydrogen peroxide accumulation 64  Endospores Endospores are resting structures formed by some bacteria for survival during adverse environmental conditions. The process of endospore formation is called sporulation; The return of an endospore to its vegetative state is called germination. Two genera that commonly form endospores are Bacillus and Clostridium. 65 Targets of antibiotics 66 Targets of antibiotics and roles in pathogenesis or drug resistance 67 Molecular approach in clinical diagnostic 68 Your manual The Science of Microbiology 1 CHAPTER Cell Structure CHAPTER 2 69