Microorganisms Lecture Notes PDF

Lecture 1 By  Microorganisms Microorganisms are tiny living organisms that can not be seen by the naked eye. Microorganisms: o Cellular (the whole organism is one cell) o Acellular (not formed of a cell). They include: 1) Bacteria 2) Viruses 3) Fungi ...

Lecture 1 By  Microorganisms Microorganisms are tiny living organisms that can not be seen by the naked eye. Microorganisms: o Cellular (the whole organism is one cell) o Acellular (not formed of a cell). They include: 1) Bacteria 2) Viruses 3) Fungi  BACTERIA Bacteria are unicellular prokaryotic organisms i.e. having a primitive nucleus. Properties of bacteria: - Approximately 1 μm in diameter. - Both DNA and RNA are present. - Growth on ordinary media. - Divide by binary fission. - Seen by the light microscope. - Sensitive to anti-bacterial agents.  VIRUSES Viruses are acellular microorganisms i.e. they have no cell structure. They are one of the smallest infectious agents. They are obligate intracellular parasites i.e. they use the biological machinery of a host cell for their survival and reproduction.  BACTERIAL MORPHOLOGY Size Shape Arrangement Staining  Bacterial Size Small but variable sizes (µm) µm = 10-3 millimetre  Bacterial Shapes Coccus Bacillus Spiral (spherical or ovoid) (rod-like) spirilla or spirochetes  Bacterial Arrangement. Same shape BUT different arrangement ↓ pairs, chains, clusters, or other groupings; Helps to differentiate bacteria into types ↓ Naming  Bacterial Arrangement Cocci Pairs Chains Irregular Clusters 10  Bacterial Arrangement Bacilli Pairs Chains No special arrangement 11  Staining of Bacteria Bacteria cells are almost colorless and transparent A staining technique is often applied to the cells to color them Their shape and size can be easily determined under the microscope. 12  Types of Stains 1. Simple stain: Single basic dye e.g. Methylene blue All bacteria take the color of the dye 2. Differential stain: Two dyes separated by a decolorizing agent e.g. Gram stain & Ziehl-Neelsen stain 3. Special stain: e.g. Fontana stain  Differential staining Principles of differential stain  Application of the first dye (primary stain) Decolorization Application of the second dye (counterstain) e.g. Gram stain & Ziehl-Neelsen (Z-N stain) 1. Gram stain  Differential Stain: Gram Stain Color of Color of Gram + ve cells Gram –ve cells Primary stain: Violet Violet Methyl violet & Gram’s Iodine Decolorizing agent: Violet Colorless Ethyl Alcohol 95% Counterstain: Violet Pink Diluted carbol fuchsin  Gram Stain Colorless Primary stain Methyl violet & Gram’s Iodine Decolorizing agent Ethyl Alcohol 95% Counterstain Diluted carbol fuchsin Gm +ve cocci Gm -ve bacilli 2. Ziehl-Neelsen Stain  Mycobacterium Not readily stainable with ordinary stains. (high lipid content of cell wall) A strong stain e.g., concentrated carbol fuchsin + HEAT Resist decolorization by strong mineral acids or acid- alcohol Acid-fast  Components Primary Stain: – Conc. Carbol fuchsin + HEAT (5 – 10 min) Decolorizing agent: – 20% H2SO4 or 3% HCL in alcohol Counter stain: – Methylene blue Acid Fast bacilli (Z-N stain)  Bacterial Ultrastructure and functions Bacterial Ultrastructure  Bacterial Ultrastructure Cytoplasm – Nucleoid – Plasmids – Ribosomes – Inclusion Granules Cytoplasmic membrane Cell wall – Some bacteria have an extra envelope – Some bacteria have appendages – Nucleoid (bacterial DNA): Genetic information of a bacterial cell is contained in a single chromosome formed of supercoiled double-stranded DNA. – Plasmids In many bacteria, additional genetic information is contained on plasmids – Small circular – ds DNA molecules – Replicate independently of the chromosome – Ribosomes The site of protein synthesis in the cell. – Inclusion Granules These are nutrient granules (e.g., carbohydrates and lipids)  Bacterial Ultrastructure Cytoplasm – Nucleoid – Plasmids – Ribosomes – Inclusion Granules Cytoplasmic membrane Cell wall – Some bacteria have an extra envelope – Some bacteria have appendages  Phospholipid protein bilayer similar to that of eukaryotic cells except that, in bacteria, it lacks sterols.  Functions of the Cytoplasmic membrane Selective transport. – Simple diffusion – Facilitated diffusion – Active transport – Mesosomes Complex invagination of the cytoplasmic membrane Involved in cell division and sporulation  Bacterial Ultrastructure Cytoplasm – Nucleoid – Plasmids – Ribosomes – Inclusion Granules Cytoplasmic membrane Cell wall – Some bacteria have an extra envelope – Some bacteria have appendages  Immediately surrounds the cytoplasmic membrane. It is strong and relatively rigid, though having some elasticity. Structure of the cell wall Its impressive strength due to peptidoglycan  Structure of the cell wall Peptidoglycan Gram-positive Gram-negative 40 sheets 1 – 2 sheets 50% of cell wall material 5-10% cell wall material Besides peptidoglycan, additional components in the cell wall divide bacteria into Gram-positive and Gram-negative. Gram-positive cell wall is composed of: 1. Peptidoglycan: comprising up to 50% of the cell wall material 2. Teichoic acids Gram-negative cell wall is composed of: 1. Peptidoglycan: 5-10% of the cell wall material. 2. Outer membrane: Phospholipid protein bilayer: present external to the peptidoglycan layer. The outer surface of the lipid bilayer is composed of lipopolysaccharide (LPS) 3. Periplasmic space: The space between the cytoplasmic and outer membranes. 1. Maintaining a characteristic shape for the bacterium. 2. Supporting the weak cytoplasmic membrane against the high internal osmotic pressure of the protoplasm. 3. Playing a major role in cell division. 4. Responsible for the staining affinity of the bacterium.  Mycoplasma: It is the only group of bacteria that exists naturally without cell wall. – Mycoplasmas do not assume a defined recognizable shape, because they lack a rigid cell wall. – These organisms are naturally resistant to cell wall inhibitors, such as penicillins and cephalosporins L. Forms: Develop from cells that normally possess cell wall, – when exposed to hydrolysis by lysozyme or by blocking peptidoglycan biosynthesis with antibiotics, such as penicillin, provided that they are present in an isotonic medium. L. forms resynthesize their walls once the inducing stimulus is removed. L. forms may survive antibiotic therapy. Their reversion to the walled state can produce relapses of the overt infection.  Bacterial Ultrastructure Cytoplasm – Nucleoid – Plasmids – Ribosomes – Inclusion Granules Cytoplasmic membrane Cell wall – Some bacteria have an extra envelope – Some bacteria have appendages  Capsule and Glycocalyx – Capsule Some bacteria synthesize (in-vivo) large amount of extracellular polysaccharides outside the cell wall to form an additional surface layer.  Capsule and Glycocalyx (cont.) - Capsule: adheres to the surface of the cell. - Glycocalyx: Loose meshwork of polysaccharide fibrils extending outwards from the cell  Capsule and Glycocalyx (cont.) Functions: 1. It protects the cell wall against various kinds of antibacterial agents. 2. It protects the bacterial cell from phagocytosis. Thus, it is an important virulence factor.  Capsule and Glycocalyx (cont.) Functions: 3. Some bacteria attach to the target surface by using their glycocalyx in order to establish infection.  Bacterial Ultrastructure Cytoplasm – Nucleoid – Plasmids – Ribosomes – Inclusion Granules Cytoplasmic membrane Cell wall – Some bacteria have an extra envelope – Some bacteria have appendages  Flagella Pili (fimbriae)  Flagella are long, whip-like appendages present in many genera of bacteria.  They can only be seen by the electron microscope.  Flagella (cont.) Function: – Flagella are responsible for bacterial motility; they move the bacteria towards attractants (e.g. nutrients) or away from repellants (e.g. disinfectants) by a process called chemotaxis.  Flagella Pili (fimbriae)  They are shorter and thinner than flagella, present in many genera. They can only be seen by the electron microscope. Functions: Adherence: Contributing to the establishment of infection (through attachment of bacteria to specific receptors on the host cell). Conjugation: Forming an attachment tube (sex pilus) between two bacterial cells Transfer of DNA  Bacterial Spores (Endospores) A bacterial spore is a highly resistant resting phase that does not grow or reproduce and is formed in-vitro by some bacteria (e.g., Bacillus and Clostridium). Sporulation is triggered by the onset of unfavourable environmental conditions e.g., - depletion of nutrients - accumulation of metabolites, or - changes in the growth requirements  Bacterial Spores (Endospores) Viability and resistance Spores are highly resistant to disinfectants, dryness, and heating. Moist heat at 121°C for 10-20 min. is needed to kill spores. Germination Endospores can respond quickly to changes in the environment returning to the vegetative state within 15 min. = Germination  Bacterial Spores (Endospores) Using Gram’s stain, the spore remains uncoloured. – stained using special stains.  BACTERIAL GROWTH AND PHYSIOLOGY  Bacterial Growth An increase in the size and number of bacterial cells. Bacterial Reproduction Binary Fission  Generation time (doubling time) Is the time between two successive divisions. – It may be as short as 13 min. and may reach 24 hrs.  Growth Requirements: These are essential factors needed for bacterial growth 1. Moisture: is an absolute requirement 2. Nutrients 3. Oxygen (O2). 4. Carbon dioxide (CO2). 5. Temperature.  Nutrients Bacteria can be classified according to the way by which they can obtain raw material into: Bacteria Autotrophs Heterotrophs Can use simple inorganic Require organic sources of materials as a source of carbon, as they can not Carbon and synthesize synthesize complex organic complex organic substances substances from simple from it. inorganic materials Most bacteria of medical importance are heterotrophic  Oxygen For bacteria to grow in the presence of oxygen (aerobic respiration), they require two enzymes: – Superoxide dismutase enzyme: detoxify superoxide (O2-) radicals – Catalase enzyme: detoxify hydrogen peroxide (H2O2)  Oxygen Strict aerobes Facultative anaerobes Strict anaerobes Require oxygen for Use oxygen when present Require complete absence growth of oxygen to grow Continue growth when because they lack oxygen is absent superoxide dismutase and catalase enzymes killed by oxygen  Carbon dioxide (CO2) The minute amount of CO2 present in air is sufficient for most bacteria. Certain species require higher concentrations (5-10%) of CO2 for growth.  Temperature and pH The optimal growing temperature is 37C The optimal pH is 7.2-7.6  Bacterial Growth Curve 1 2 3 4  No. of viable Causes of events of the phase organisms Constant at initial Adaptation to new environment 1. Lag phase number Formation of new enzymes and intermediates to permit growth 2. Exponential Markedly increase Growth rate is accelerated with time (logarithmic) Giving a characteristic linear plot on a Phase logarithmic scale Constant at a high Slow loss of cells through death which 3. Stationary level is balanced by the formation of new phase cells through growth and division. Due to exhaustion of nutrients and accumulation of toxic products Markedly reduced At the end of the stationary phase, the 4. Decline death rate increases and exceeds the phase multiplication rate Due to nutrient exhaustion and accumulation of toxic metabolic end products.  MCQs 1) Which structure is responsible for the staining affinity of bacteria? a) Flagella b) Cytoplasmic membrane c) Cell wall d) Mesosomes  MCQs 2) Which statement about L-forms is TRUE? a) Bacteria that exists naturally without a cell wall. b) It is naturally resistant to cell wall inhibitors. c) Develop from cells that normally possess a cell wall. d) They are unable to resume their cell wall.