Bacterial Growth And Metabolism

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  • Enterobacteriacea Metabolic capabilities
  • Synthesized Break down
  • Oxidation Reduction
  • Photosynthesis Cellular Respiration
  • Bacterial Growth And Metabolism

    1. 1. Bacterial Growth and Metabolism Dr Kamran Afzal FCPS Microbiology
    2. 2. Introduction <ul><li>Bacterial growth </li></ul><ul><ul><li>Increase in size and number of an organism </li></ul></ul><ul><li>Net effect </li></ul><ul><ul><li>Increase in the total mass (biomass) of the culture </li></ul></ul><ul><li>Bacterial physiology </li></ul><ul><ul><li>Production of enzymes, biochemical and biophysical processes in a cell represent a sound genetic and biochemical control mechanism </li></ul></ul>
    3. 3. <ul><li>Bacteria: Binary fission to produce identical offspring, which cannot be distinguished as a parent or offspring </li></ul><ul><li>Yeasts: Budding </li></ul><ul><li>Moulds: Hyphal septation </li></ul><ul><li>Growth of bacteria in lab is used to serve many purposes </li></ul><ul><ul><li>Clinical perspective </li></ul></ul><ul><ul><ul><li>Detection, Identification, Antibiotic susceptibility </li></ul></ul></ul><ul><ul><li>Industrial objectives </li></ul></ul><ul><ul><ul><li>Biochemical analysis of growth in biomass </li></ul></ul></ul><ul><ul><ul><li>To produce desirable products in brewing and biotechnology industries </li></ul></ul></ul>
    4. 4. Types of growth <ul><li>In a lab bacterial growth is seen in three forms </li></ul><ul><li>Colonies (Sessile growth) </li></ul><ul><ul><li>Microscopic product of 20-30 cell divisions of a single cell </li></ul></ul><ul><li>Turbid suspension (Planktonic growth) </li></ul><ul><ul><li>Transformation of a clear broth medium to a turbid suspension of 10 7 -10 9 cells per ml </li></ul></ul><ul><li>Biofilm (Sessile growth) </li></ul><ul><ul><li>Growth is thinly spread over an inert surface, nutrition is obtained from surrounding fluid </li></ul></ul>
    5. 5. Doubling time <ul><li>Time reqd for any bacterial strain to double in number under a given set of growth conditions </li></ul><ul><ul><li>Vibrio cholerae : 13 min </li></ul></ul><ul><ul><li>MTB: 24 hrs </li></ul></ul><ul><ul><li>V. Cholerae can kill a man within 12 hrs, whereas </li></ul></ul><ul><ul><li>MTB takes months to develop </li></ul></ul>
    6. 6. Growth phases in broth culture <ul><li>Idealized growth curve of any given pure culture of a single organism can be obtained in broth culture </li></ul><ul><li>Four main growth phases </li></ul><ul><ul><li>Lag phase </li></ul></ul><ul><ul><li>Exponential (Log) phase </li></ul></ul><ul><ul><li>Stationary phase (Post-exponential) </li></ul></ul><ul><ul><li>Decline phase </li></ul></ul>
    7. 7. Lag phase <ul><li>When growth is initiated by inoculation into broth conditions, the number of cells present appears to remain constant in Lag phase </li></ul><ul><li>The cells are believed to be preparing for the growth </li></ul>
    8. 8. Exponential (Log) phase <ul><li>The increase in cell no. then becomes detectable and its rate accelerates rapidly </li></ul><ul><li>The no. of cells increase exponentially with time. When plotted on a ‘Log scale’ there is linear increase in log cell no. with time </li></ul>
    9. 9. Stationary phase <ul><li>Eventually growth slows down, the total bacterial cell no. reaches to a maximum and stabilizes </li></ul>
    10. 10. Decline phase <ul><li>A divergence begins to emerge towards the end of Stationary phase, the total cell no. remains constant while colony count declines </li></ul><ul><li>Death of cells is due to nutrient exhaustion and accumulation of detrimental end-products </li></ul>
    11. 11. Media for bacterial growth <ul><li>For identification of bacteria, a culture is obtained by growing the organisms on artificial media </li></ul><ul><li>Central features of media in medical microbiology </li></ul><ul><ul><li>Protein or protein hydrolysate, often derived from casein, or an infusion of brain, heart or liver </li></ul></ul><ul><ul><li>pH control, after sterilization </li></ul></ul><ul><ul><li>Salt content </li></ul></ul><ul><ul><li>Solidifying agents </li></ul></ul><ul><ul><ul><li>“ Agar” an indigestible polysaccharide extract of seaweed </li></ul></ul></ul><ul><ul><ul><li>gelatine </li></ul></ul></ul><ul><ul><ul><li>albumin </li></ul></ul></ul>
    12. 12. Types of culture media <ul><li>Simple media </li></ul><ul><li>Enriched media </li></ul><ul><li>Selective media </li></ul><ul><li>Differential media </li></ul><ul><li>Enrichment media </li></ul>
    13. 13. a. Simple media <ul><li>Contain basic nutrients for bacterial growth like broth with peptone </li></ul><ul><ul><li>Nutrient broth </li></ul></ul><ul><ul><li>Peptone water </li></ul></ul>
    14. 14. b. Enriched media <ul><li>Enriched by some substances like: </li></ul><ul><ul><li>Blood 5-10% </li></ul></ul><ul><ul><li>Serum 10% </li></ul></ul><ul><ul><li>Ascitic fluid 10% </li></ul></ul><ul><ul><li>Glucose 1-2% </li></ul></ul><ul><ul><li>Plasma 5-10% </li></ul></ul><ul><li>Examples </li></ul><ul><ul><li>Blood agar </li></ul></ul><ul><ul><li>Chocolate agar </li></ul></ul>
    15. 15. c. Selective media <ul><li>Contain substances such as bile salts or antibiotics that inhibit the growth of some organisms but have little or no effect on the required organism </li></ul><ul><li>Used for clinical specimens such as faeces </li></ul><ul><ul><li>APW and TCBS for Vibrio cholerae </li></ul></ul><ul><ul><li>Salmonella Shigella agar </li></ul></ul><ul><ul><li>Deoxycholate Citrate agar </li></ul></ul>
    16. 16. d. Differential media <ul><li>Combination of selective + differential properties which effectively colour code the colonies according to their biochemical properties </li></ul><ul><li>Colonies can be differentiated from each other </li></ul><ul><ul><li>CLED agar </li></ul></ul>
    17. 17. e. Enrichment media <ul><li>Broth media with selective properties, enriching the desired range of organisms and inhibiting others </li></ul><ul><li>Examples </li></ul><ul><ul><li>TT Broth </li></ul></ul><ul><ul><li>Selenite F Broth </li></ul></ul><ul><li>Cold enrichment </li></ul><ul><ul><li>Listeria monocytogenes </li></ul></ul><ul><li>Heat enrichment </li></ul><ul><ul><li>Legionella species </li></ul></ul>
    18. 18. Identification of microbes <ul><li>API-20E </li></ul><ul><li>The API-20E test is used to identify Gram-negative bacilli </li></ul><ul><li>System of 20 individual, miniaturized tests used to determine the biochemical activity of the organism </li></ul><ul><li>We can identify up to the genus and species level </li></ul><ul><li>API-10S/NE/Staph/Strep/Candida </li></ul>
    19. 19. Bacterial Physiology Nutritional types Physical conditions required for growth Bacterial metabolism Adaptive response Bacterial viability
    20. 20. 1. Nutritional types <ul><li>All living organisms in two main groups on the basis of carbon source </li></ul><ul><ul><li>Heterotrophs </li></ul></ul><ul><ul><li>Autotrophs </li></ul></ul><ul><li>Bacterial metabolism is so diverse that it cannot be encompassed by these two terms </li></ul>
    21. 21. <ul><li>Phototrophic: </li></ul><ul><ul><li>Energy for Adenosine triphosphate (ATP) may be obtained from light </li></ul></ul><ul><li>Chemotrophic </li></ul><ul><ul><li>Chemical oxidation </li></ul></ul><ul><li>Organotrophic </li></ul><ul><ul><li>Require organic sources of hydrogen </li></ul></ul><ul><li>Lithotrophic </li></ul><ul><ul><li>They can use inorganic source of hydrogen </li></ul></ul><ul><ul><li>(Ammonia, H 2 S) </li></ul></ul>
    22. 22. <ul><li>Chemo-organotrophs </li></ul><ul><ul><li>Vast majority of currently recognized medically important organisms </li></ul></ul><ul><li>Chemo-lithotrophs </li></ul><ul><ul><li>Pseudomonas spp </li></ul></ul><ul><li>Photo-lithotrophs </li></ul><ul><ul><li>Some medically important organisms </li></ul></ul>
    23. 23. Nutritional types
    24. 24. 2. Physical conditions <ul><li>Growth atmosphere </li></ul><ul><li>Growth temperature </li></ul><ul><li>pH </li></ul>
    25. 25. a. Growth atmosphere Descriptive term Property Example Growth atmosphere Strict (Obligate) aerobe Requires O 2 Pseudomonas aeruginosa Strict (Obligate) anaerobe Will not tolerate O 2 Bacteroides fragilis Facultative anaerobe Aerobe, can grow anaerobically Staphylococci, Escherichia coli Aerotolerant anaerobe Anaerobe, can tolerate O 2 Clostridium perfringens Micro-aerophilic Prefers reduced O 2 Campylobacter spp, Helicobacter spp Capnophilic Prefers increased CO 2 Neisseria spp
    26. 27. b. Growth temperature Descriptive term Property Example Growth temperature Psychrophilic Low temp <10 0 C Flavobacterium spp Thermophilic High temp >60 0 C B. stearothermophilus Mesophilic 20-40 0 C Most bacterial pathogens
    27. 28. 3. Bacterial metabolism <ul><li>Basic details of glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, ATP biosynthesis and AA metabolism are constant (core of their metabolism essentially similar to that of mammalian cells) </li></ul><ul><li>In addition to metabolic enzymes, many transport proteins are also closely related to the mammalian cells in structure and function </li></ul>
    28. 29. <ul><li>DNA sequencing: molecular families of proteins having common evolutionary origin </li></ul><ul><li>No mitochondria, so cytoplasmic membrane contains the components of electron transport chain </li></ul><ul><li>Bacteria can perform some energy-requiring processes at the cell surface, by directly exploiting the proton gradient rather than consuming ATP </li></ul><ul><ul><li>Flagellar rotation (motility) </li></ul></ul>
    29. 30. <ul><li>Advantages of bacterial metabolism to our environment </li></ul><ul><ul><li>Original development of O 2 in atmosphere </li></ul></ul><ul><ul><li>Availability of elemental Sulphur </li></ul></ul><ul><ul><li>Flow of Nitrogen </li></ul></ul><ul><li>Commercial exploitation of bacterial metabolism has given us </li></ul><ul><ul><li>Ethanol </li></ul></ul><ul><ul><li>Other alcohols </li></ul></ul><ul><ul><li>Acids resulting from fermentation </li></ul></ul>
    30. 31. Basic chemical reactions <ul><ul><li>Anabolism and Catabolism </li></ul></ul><ul><ul><li>Oxidation and Reduction reactions </li></ul></ul><ul><ul><li>ATP production and Energy storage </li></ul></ul>
    31. 32. Building and breaking down molecules <ul><li>Anabolic Reaction </li></ul><ul><li>(Anabolism) </li></ul><ul><li>The phase of metabolism in which simple substances are built into the complex materials of living tissue </li></ul><ul><li>Catabolic Reaction </li></ul><ul><li>(Catabolism) </li></ul><ul><li>The metabolic breakdown of complex molecules into simpler ones, often resulting in a release of energy </li></ul>
    32. 33. Anabolism <ul><li>Organisms catabolize carbohydrates as the primary energy source for anabolic reactions </li></ul>
    33. 34. Catabolism <ul><li>Glucose catabolized by </li></ul><ul><ul><li>Aerobic cellular respiration -> Results in complete breakdown of glucose to carbon dioxide, water and a lot of ATP </li></ul></ul><ul><ul><li>Anaerobic respiration and Fermentation -> Only partially breaks down glucose, into pyruvic acid and organic waste products and a little ATP </li></ul></ul>
    34. 35. Oxidation - Reduction reactions <ul><li>Redox reaction </li></ul><ul><ul><li>A chemical reaction in which electrons are gained, lost or shared </li></ul></ul><ul><li>Oxidation </li></ul><ul><ul><li>The loss of electrons by a molecule, atom or ion </li></ul></ul><ul><li>Reduction </li></ul><ul><ul><li>The gain of electrons by a molecule, atom or ion </li></ul></ul>
    35. 36. ATP production and Energy storage <ul><li>Phosphorylation An organic phosphate is added to substrate </li></ul><ul><li>Energy storing nucleotide </li></ul>
    36. 37. Bacterial respiration <ul><li>Used to denote involvement of a membrane associated electron transport chain in the process of oxidation </li></ul><ul><ul><li>Aerobic </li></ul></ul><ul><ul><ul><li>Final electron recipient in oxidation process is molecular oxygen </li></ul></ul></ul><ul><ul><li>Anaerobic </li></ul></ul><ul><ul><ul><li>Final electron recipient is an organic molecule in the absence of oxygen, this oxidative process is referred to as ‘Fermentation’ </li></ul></ul></ul><ul><ul><ul><li>All bacteria in evolution were anaerobes </li></ul></ul></ul>
    37. 38. Aerobic cellular respiration <ul><li>Utilizes glycolysis, synthesis of acetyl CoA, Kreb ’ s cycle, and electron transport chain; results in complete breakdown of glucose to carbon dioxide, water and ATP </li></ul><ul><li>A total of 38 molecules of ATP are formed from one molecule of glucose </li></ul>
    38. 39. Using oxygen in metabolism creates toxic waste <ul><li>Superoxide (O 2 - ), Hydroxyl (OH - ) and Hydrogen peroxide (H 2 O 2 ) </li></ul><ul><li>Microbes produce two enzymes to detoxify </li></ul><ul><ul><li>Catalase: H 2 O 2  H 2 O and O 2 </li></ul></ul><ul><ul><li>Superoxide dismutase (SOD): </li></ul></ul><ul><ul><li> Superoxide (O 2 - )  H 2 O and O 2 </li></ul></ul><ul><li>Microbes that don’t make these enzymes cannot exist in the presence of oxygen </li></ul><ul><li>Also protect the pathogenic organisms against Superoxide of the phagocytic cells </li></ul>
    39. 40. 4. Adaptive responses <ul><li>In response to environmental changes (stimuli), different sets of proteins are produced in the exponential and stationary phases of growth cycle </li></ul><ul><li>The effects of noxious stimuli on gene expression are the subject of intense current study </li></ul><ul><li>Each different stimulus leads to a specific adaptive ‘stress response’ </li></ul>
    40. 41. <ul><li>Different stresses: </li></ul><ul><ul><li>‘ Heat shock’ is the effect of raising temp to >45 0 C for a few minutes, has been most extensively studied </li></ul></ul><ul><ul><ul><li>‘ Heat shock proteins’ are the newly synthesized proteins in this response </li></ul></ul></ul><ul><ul><li>‘ Acid stress’ is provided by the stomach </li></ul></ul><ul><ul><li>‘ Oxidative’ and “pH stress’ are provided by the hostile environment of phagolysosomes </li></ul></ul><ul><ul><li>Osmotic stress </li></ul></ul><ul><ul><li>Cold shock </li></ul></ul><ul><ul><li>Nutrient limitation </li></ul></ul><ul><ul><li>Anaerobic environment </li></ul></ul>
    41. 42. <ul><li>There are ‘Global regulatory systems’ within bacteria, responsible for differential gene expression under different circumstances </li></ul><ul><li>Specific control mechanisms </li></ul><ul><ul><li>Stimulon </li></ul></ul><ul><ul><ul><li>All those genes whose expression is increased or decreased by a specific external stimulus </li></ul></ul></ul><ul><ul><li>Regulon </li></ul></ul><ul><ul><ul><li>All those genes under the influence of a specific regulatory protein. There may be several regulons in one stimulon </li></ul></ul></ul>
    42. 43. <ul><li>Specific control systems ensure that the organism synthesizes specific proteins as per the stress </li></ul><ul><ul><li>‘Virulence factors’, the proteins concerned with an organism’s progress in an infection </li></ul></ul><ul><ul><li>Chemicals secreted by an organism can themselves act as regulatory stimuli to individuals of the same species </li></ul></ul>
    43. 44. 5. Bacterial viability <ul><li>The ability of a cell to form a colony on an appropriate agar medium </li></ul><ul><ul><li>The colony count provides an accurate measure of viability </li></ul></ul><ul><li>The proportion of cells within a population which are capable of forming colonies </li></ul>
    44. 45. <ul><li>The ability of a particular cell to grow and undergo binary fission, and its progeny would have the same potential </li></ul><ul><li>VNC: (viable but non-culturable) </li></ul><ul><ul><li>The organisms which are viable, but in a (reversible) physiological state in which they cannot be induced to form colonies, e.g; in TB that have latent phases </li></ul></ul>

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