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Bacteriology Bacteriology Definition: • A branch of microbiology dealing with the identification, study, and cultivation of bacteria and with their applications in medicine, agriculture, industry, and biotechnology OR • The branch of science concerned with the study of bacteria • Bacteria: Microscopic living organisms normally are unicellular with no distinct nuclei , reproduce by fission or forming spore found everywhere and can exist independently(Free living) or dependently(parasitic) can be dangerous as cause disease or beneficial as process of fermentation(Wine production) Morphology and Classification • Bacteria are Prokaryotes as they lack nuclei and other organelles which characterize the true cell(Eukaryotes ie : Algae ,Fungi , Animal and plant cells). • Size: Bacteria cells range from 0.2µm-3 µm in diameter • Compared to RBC which is 8µm in diameter • Major classes: Are based on staining properties some are GRAM stains other are ACID - FAST stains • Those staining Gram positive show Purple colour • Gram negative show Red Colour • Gram negative have thin cell wall • However Gram resistant bacteria stain poorly or no stain at all. ie: Mycobacterium Morphology and Classification • Ultrastructure: • Generally Gram +ve and –ve have similar internal structures but dissimilar cell envelopes A. Internal Structure: Nucleoid: Central region not surrounded by nuclear membrane but contains DNA Ribosome: Differ in size and shape with eukaryotic ribosomemajor target for antibiotic action Plasmids: Small and circular fragments of extrachromosomal DNA may carry antibiotic resistance genes Morphology and Classification • B. Cell Envelope(Cytoplasmic membrane+Cell wall) Cytoplasmic(cell,plasma)membrane Structurally similar to eukaryotic membrane but lacks sterols. Associated with enzymes and other proteins that carry out energy production , transportation of nutrients and synthesis of structural components Cell wall of Gram +ve bacteria: Has thick peptidoglycan layer forming meshlike exoskeleton -This is porous for Antibiotics Morphology and Classification cont: Teichoic and lipoteichoic acids : Associated with peptidoglycan are antigenic may promote adhesion to host tissue. Cell wall of Gram +ve bacteria has: Peptidoglycan layer: adjacent to cytoplasmic membrane Outer membrane :maintains bacterial structure acts as permeability barrier protects against adverse environment conditions Periplasmic space: Is btn outer membrane and cytoplasmic membrane has degradative enzymes and nutrient binding proteins Morphology and Classification cont: Lipoproteins: Covalently linked to peptidoglycan layer are inserted into the outer membrane connecting the two structures. Cell wall of Mycobacterium: Structure of peptidoglycan differs slightly from other bacteria Waxlike lipid coat containing mycolic acid surrounds the peptidoglycan –like layer(This is responsible with virulence and antiphagocytic activity of mycobacteria). Mycobacteria and other mycolic acid producing bacteria can be identified acid-fast stains. Morphology and Classification cont: • Other external structures: Bacterial capsule: Loose layers of polysacharide or protein surrounds cell wall of some bacteria Pili(fimbriae) :Short , hairlike appendages contains protein subunits anchored in plasma membrane of some bacteria – promotes adhesion of bacteria to host cells Fagella long, ropelike appendages –useful in motility Difference btn Prokaryotic and Eukaryotic cells Charecteristics Size: Cell wall: Prokaryotic cells 0.5 - 3µm Plasma Membrane: Has no sterols except Mycoplasma Has protein,peptidoglycan Only Plant and fungal & lipids differ Nuclear membrane: Absent Single,Circular DNA Genome: molecule in nucleoid Organelles: Cell division: Eukaryotic cells >5µm Absent Binary fission Has sterols Present Multiple,linear DNA molecules in nucleus Present Mitosis and Meiosis Comparison of Gram +ve and Gram –ve Bacteria Characteristics Gram +ve Gram-ve Absent Thick Absent Present P/nt in some sp Present Thin Present Absent P/nt in some spp Very Sensitive Very permiable Some species Some spp. Largely resistant Structure: •Outer membrane •Peptidogycan layer •Lipopolysaccharides •Teichoic Acids •Capsule,pili,flagella Functional : •Lysozyme sensitivity •Antibiotic permiable •Sporulation •Exotoxin production Impermeable to many None Some spp. Bacterial Growth • Bacterial growth curve: When placed in a new media show a pattern of growth and multiplication in four phases(Lag,Long (Exponential),Stationary and Death) • Lang phase: Bacteria adapt themselves to growth condition,they mature but unable to divide,synthesis of RNA,enzymes and other molecules takes place. • Long phase(Exponential): There is cell doubling ,if growth not limited doubling continues at constant rate growth condition determine growth. Bacterial Growth Cont.. • Stationary phase: Growth rate and death rate are equal, The number of new cells created is limited by the growth factor • Due to a growth-limiting factor such as the depletion of an essential nutrient, and/or the formation of an inhibitory product such as an organic acid. • Death(Decline) phase: Bacteria die this can be to lack of nutrients , environmental factors ie: high or low temperature below the tolerance band for the species, or other injurious conditions. Growth Requirements • Different bacteria show variation in the condition conducive to their growth. Temperature: Low temperatures tend to reduce growth rates which has led to refrigeration hence useful in food preservation(pathogenic bacteria grow at 35 ˚ᶜ-37˚ᶜ(near normal body temperature) Acidity: Optimal acidity for bacteria tends to be around pH 6.5 to 7.0 with the exception of acidophiles. Oxygen requirement: Obligate aerobes require O2 for growth ie: Mycobacteria tuberculosis,Neisseria etc. Anaerobes do not require O2 for growth Growth Requirements • Obligate anaerobes are killed by O2 ie:Treponema,Actinomyces • Aerotolerant anaerobes: Can survive in the presence of O2 but grow best in its absence (ie: Clostridium spp) • Facultative anaerobes: Can grow under anaerobic or aerobic conditions( E.coli,Staphylococcus aureus) Growth Requirements Nutrient Requirements • Undemanding eaters: Can be cultured on simple media(e.g.E.coli,Salmonella) • Demanding eaters: Require complex media with numerous growth factors(e.g Haemophilus,Neisseria) Cell Division • Most bacteria divide by binary fission Chromosome duplication is initiated at specific sequence (replication origin) in the DNA Synthesis of new membrane and cell wall in the centre of the cell forms a septum that eventually divides the cytoplasm into two daughter cells with complete chromosome. Bacterial Spores(Endospores) • Spores are formed by some Gram – positive bacteria representing a dormant state that is resistant to heat , dry and chemicals. • Spore formation : a variant type of cell division is induced by depletion of essential nutrients needed for normal growth-Antibiotics and Toxins are produced during sporogenesis prior to release of the spore from the bacterial cell. • Germination of spores: Cell germinated into vegetative form starts by demage on the spore coat by trauma , water or aging and requires special nutrients once spore demaged the cell becomes susceptible to agents. Normal Flora Microorganisms that normally reside at a given site and under normal circumstances do not cause disease. , even beneficial, in their usual sites, and they inhibit the growth of pathogens, but can cause infection if introduced into unusual sites. Non sterile sites in the body(e.g: lumen of GIT, UGT, Mouth , Saliva , Pharynx and Skin) harbor certain organisms Skin: Staphylococcus epidermis,Proprionibacterium acnes Upper respiratory tract:Staphylococcus aureus, Staphylococcus pneumoniae,Haemophilus influenzae Colon:Escherichia coli (E.Coli),Bacteroides fragilis Normal Flora cont… Urogenital Flora: Candida spp Conjunctival Flora: Haemophilus and Staphylococcus Oral and Upper Respiratory Tract Flora:Neisseria, Bordetella, Corynebacterium, and Streptococcus spp. Sterilization • The removal of all microorganisms and other pathogens from an object or surface by treating it with chemicals or subjecting it to high heat or radiation. Uses of Sterilization: Sterilization for Surgical Procedures: Gloves, aprons, surgical instruments, syringes etc. Sterilization in Microbiological works like preparation of culture media, reagents and equipments where a sterile condition is to be maintained. Sterilization • Classification of Sterilization methods: Physical and Chemical Agents Physical Agents: 1. Heat 2. Radiation 3. Filtration Chemical Agents: Physical Agents: • STERILIZATION BY HEAT: This is cheaper and rapid method of sterilization achieved by heating above 100˚ᶜ Types of Heat : A. Sterilization by moist heat B. Sterilization by dry heat Sterilization by Moist Heat: Acts by denaturation and coagulation of protein, breakage of DNA strands, and loss of functional integrity of cell membrane. Sterilization by Moist Heat: • Sterilization at 100°C: Boiling: Boiling at 100°C for 30 minutes in a water bath( Syringes, rubber goods & surgical instruments) Steaming: Steam (100°C) more effective than dry heat at the same temperature(Steam has more penetrating power, more heat is given up during condensation). Sterilization Cont.. • Sterilization above 100°C Autoclaving: Sterilization is by steam under pressure. temperature higher than 100°C, higher temperature is obtained by employing a higher pressure . Autoclave is closed and made air-tight, and water starts boiling. Inside pressures increases and now the water boils above 100°C. At 15 ib per sq. inch pressure, 121°C temperatures is obtained kept for 15 minutes to kill spores. -like a pressure cooker. Sterilization Cont.. Sterilization below 100°C Pasteurization:Heating of milk to temperature & for a period of time to kill pathogens presenting in milk without changing colour, flavour & nutrities (Mycobacterium bovis, Salmonella species, E.coli and Brucella species be present in milk present in milk)-Spore forming are not destroyed. Methods of Pasteurization: (i) Flash Method(Heating is done at 72°C for 15 seconds-short time method). (ii) Holding Method(Heating is done between 63°C and 66°C for 30 minutes). Sterilization Cont.. Inspissation: Stiffening of protein without coagulation as the temperature is below coagulation temperature. Sterilization by dry heat: How it works? (1) Protein denaturation, (2) Oxidative damage, (3) Toxic effect of elevated electrolyte (in absence of water). Dry heat at 160°C (holding temperature for one hour is required to kill the most resistant spores) unsuitable for clothing –can be spoiled. Red Heat(Wire loops used in microbiology laboratory sterilized by heating to red on bunsen burner or spirit lamp flame ). Sterilization Cont.. Flaming: Involves passing instrument without allowing to turn red-incomplete sterilization-scarpel Sterilization by Hot Air: Hot Air Oven (Sterilizer) Most common method used for sterilization(Glass wares, swab sticks, all-glass syringes, powder and oily substances) holding temperature of 160°C for one hour is recommended to kill spores. Sterilization holding time: Is the time for which the entire load in the autoclave requires to be exposed. Disinfection Disinfection: A procedure of treatment that eliminates many or all pathogenic microorganisms with the exception of bacterial spores. Disinfectants: A germicide that inactivates virtually all recognized pathogenic microorganisms but not necessarily all microbial forms. They may not be effective against bacterial spores. Antiseptics: Mild forms of disinfectants that are used externally on living tissues to kill microorganisms, e.g. on the surface of skin and mucous membranes. Disinfection cont.. • Factors for successful disinfectants: • • • • • • Type and level of microbial contamination Concentration of active ingredients pH of disinfectant Temperature Humidity Duration of contact btn disinfectant and item to be disinfected • Pressure of organic matter or soil load Disinfection cont… • Chemical Disinfectant Groups: a. Aldehydes(Formaldehyde,Paraformaldehyde & Glutaraldehyde): Formaldehyde & Paraformaldehyde: Have broad spectrum biocidal activity effective for both surface and space contamination-implicated to cancer. Paraformaldehyde: Generates formaldehyde when heated –useful in space contaminations. Glutaraldehyde: Colourless liquid with sharp pungent smell useful when exposed for many hours-effective to virus,Spore and vegetative bacteria Disinfection cont… b. Chlorine and Idophores: Chlorine: Good on clean surface but inactivated by organic matter they are broad spectrum, inexpensive and fast acting ie: Calcium hypochlorite Idophores: An Iodine and solubilizing agent –For both antiseptic and disinfection c. Quaternary Ammonium Compounds: They are odorless,colourless they have detergent action Disinfection cont… d. Heavy Metals: Soluble salts of mercury,Silver lactate and Mercuric compouds,they are good bactericidal but they are hazardous should be used with care. e. Alcohols: They disrupt cellular membrane, solubilization of lipids and denaturation of proteins. Effective against lipid cotaining virus and broad spectrum bacterial spp.,innactivate spore forming bacteria but they evaporate rapidly. References: 1. Microbiology and Immunology By : Rosenthal K.S 2. Introduction to Diagnostic Microbiology By: Delost M.D 3. Review of Medical Microbiology(15th edition) By:E.Jawetz,J.L.Melnick,E.A.Adelberg 4. District Laboratory Practice in Tropical Countries(Part 1&2) By : Monica Cheesbrough 5. Biology(4thEdition) 6. By: Sylvia S.Mader 7 . Wikipedia 8. Medical Microbiology(Updated 3rd Edition) By:Mims,Dockrell,Goering,Roitt,Wakelin,Zuckerman) . THE END TANK U