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Growth and multiplication

Microbial physiology. Microbial metabolism . Enzymes. Nutrition. Bioenergetics. Bacterial growth and multiplication. mode: Binary fission. Growth and multiplication. Bacterial Cell Division. 1. Replication of chromosome 2. Cell wall extension 3. Septum formation

emmanuel-maddox
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Growth and multiplication

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  1. Microbial physiology. Microbial metabolism. Enzymes. Nutrition. Bioenergetics. Bacterial growth and multiplication.

  2. mode: Binary fission Growth and multiplication

  3. Bacterial Cell Division • 1. Replication of chromosome • 2. Cell wall extension • 3. Septum formation • 4. Membrane attachment of • DNA pulls into a new cell.

  4. Growth • It is an increase in all the cell components, which ends in multiplication of cell leading to an increase in population. • It involves - an increase in the size of the cell & an increase in the number of individual cells. • Bacteria divide by binary fission.

  5. Generation time • Interval of time between two cell divisions OR • The time required for a bacterium to give rise to 2 daughter cells under optimum conditions • Also called population doubling time.

  6. Generation time • Coliform bacilli like E.coli & other medically important bacteria – 20 mins • Staphylococcus aureus- 27-30 mins • Mycobacterium tuberculosis - 792-932 mins • Treponema pallidum -1980 mins

  7. Growth form in Laboratory • Colony – formed by bacteria growing on solid media. (20-30 cell divisions) • Each bacterial colony represents a clone of cells derived from a single parent cell. • Turbidity – liquid media - 107-109 cells/ml • Biofilm formation – thin spread over an inert surface.

  8. Solid medium Colony

  9. Liquid medium

  10. Bacterial counts • Cell Counts ... many ways • 2 methods – Total cell count - Viable cell count

  11. Total Count • Total number of cells in the sample = living + dead. Can be obtained by : • Direct counting under microscope using counting chambers. • Counting in an electronic device – Coulter counter.

  12. Counting chambers

  13. Over method • Direct counting using stained smears - by spreading a known volume of culture over a measured area of slide. • Opacity measurements using an absorptiometer/ nephalometer. • Chemical assays of cell components.

  14. Turbidity- a spectrophotometer measures how much light gets through

  15. Viable Cell Count • Measures the number of living cells. • Methods – Surface colony count • Dilution method • Plating method • Number of colonies that develop after incubation gives an estimate of the viable count.

  16. Plate counts

  17. Bacterial Growth Curve • When a bacterium is added to a suitable liquid medium and incubated, its growth follows a definite course. • If bacteria counts are made at intervals after inoculation & plotted in relation to time, a growth curve is obtained. • Shows 4 phases : • Lag, • Log or Exponential, • Stationary • Decline.

  18. Phases of Growth Curve • 1. Lag phase – No increase in number but there may be an increase in the size of the cell. • 2. Log OR Exponential phase – cells start dividing and their number increases exponentially.

  19. Phases of Growth Curve • 3. Stationary phase – cell division stops due to depletion of nutrients & accumulation of toxic products. - equilibrium exists between dying cells and the newly formed cells, so viable count remains stationary • 4. Phase of Decline – population decreases due to the death of cells – autolytic enzymes.

  20. Morphological & Physiological alterations during growth • Lag phase – maximum cell size towards the end of lag phase. • Log phase – smaller cells, stain uniformly • Stationary phase – irregular staining, sporulation and production of exotoxins & antibiotics • Phase of Decline –involution forms(with ageing)

  21. Factors Affecting Bacterial Growth • Availability of Nutrients & H2O • Temperature • Atmosphere – O2 & CO2 • H-ion concentration • Moisture & drying • Osmotic effects • Radiation • Mechanical & sonic stress.

  22. Bacterial Nutrition • Water constitutes 80% of the total weight of bacterial cells. • Proteins, polysaccharides, lipids, nucleic acids, mucopeptides & low molecular weight compounds make up the remaining 20%.

  23. Moisture & Drying • Water – essential ingredient of bacterial protoplasm. Hence drying is lethal to cells. • Effect of drying varies : • T. pallidum – highly sensitive • Staphylococci sp– stand for months • Spores – resistant to desiccation, may survive for several decades.

  24. Nutrients Functions • Generation of energy • Synthesis of cellular materials • Essential nutrients (basic bioelements needed for bacterial cell growth) • H2O: universal solvent; hydrolyzing agent • Carbon: food & E* source; in form of prot., sugar, lipid • Nitrogen: for prot. syn; nucleic acid syn (purines & pyrimidines) • Sulfur (sulfate): AA syn (i.e., Cystine) • Phosphate: key component of DNA & RNA, ATP, and inner & outer membrane phospholipids • Minerals: assoc’d w/ PRO (i.e., Fe:PRO); common component of enzymes.

  25. Nutrients 2 types • Macronutrients– needed in large quantities for cellular metabolism & basic cell structure • C, H, O, N • Micronutrients – needed in small quantities; more specialized (enzyme & pigment structure & function) • Mn, Zn • Fastidious Bacteria: microbes that require other complex - nutrients/growth factors ( i.e., Vitamins or AAs)

  26. Temperature • Vary in the temperature requirements. • Temperature range – growth does not occur above the maximum or below the minimum. • Optimum Temperature – growth occurs best, 37ºC for most pathogenic bacteria.

  27. Uptake of nutrients by bacteria • Passive diffusion • simple diffusion • facilitated diffusion • Active transport

  28. Microbes differ in their temperature optimum Psychrophiles: -10 to 20C Psychrotrophs: 0 to 30 C Mesophiles: 10 to 48C e.g. most bacterial pathogens Thermophiles: 40 to 72C Hyperthermophile: 65 to 110C 29

  29. How does optimal growth temperature relate to your daily live? Some pathogens can multiply in the refrigerator: Listeria monocytogenes 30

  30. H-ion Concentration • Neutral or slightly alkaline pH (7.2 – 7.6) – majority of pathogenic bacteria grow best. • acidic pH – Lactobacilli • alkaline pH -Vibrio cholerae

  31. Osmotic Pressure or Osmolarity • Most bacteria require an isotonic environmentor a hypotonic environment for optimum growth. • Osmotolerant - organisms that can grow at relatively high salt concentration (up tp 10%). • Halophiles - bacteria that require relatively high salt concentrations for growth, like some of the Archea that require sodium chloride concentrations of 20 % or higher.

  32. Effect of salt on cells Similar effect with sugars 34

  33. Radiation, stress • Radiation • X rays & gamma rays exposure – lethal • Mechanical & Sonic Stress • May be ruptured by mechanical stress.

  34. Growth Factors • Some bacteria require certain organic compounds in minute quantities – Growth Factors OR Bacterial Vitamins. It can be : • Essential – when growth does not occur in their absence. • Accessory – when they enhance growth, without being absolutely necessary for it.

  35. Growth Factors Identical with eukaryotic nutrition • Vitamin B complex – • thiamine • riboflavine • nicotinic acid • pyridoxine • folic acid & • Vit.B 12

  36. Presence or Absence of Gases • Primary gases = O2, N2, & CO2 • O2 - greatest impact on microbial growth (even if the microorganism does not require it) • Aerobic respiration – terminal electron acceptor is oxygen. • Anaerobic respiration – terminal electron acceptor is an inorganic molecule other than oxygen (e.g. nitrogen).

  37. Depending on the O2 requirement • Strict (Obligate) Aerobes – O2 present, require O2 for growth e.g. Pseudomonas aeruginosa • Obligate aerobe – 20% O2: only grows with O2 • Microaerophile – 4% O2: best growth with small amount O2 • e.g. Campylobacter spp, Helicobacter spp • Strict (Obligate) Anaerobes – O2 depleted, grow in the absence of O2 & may even die on exposure to O2 e.g. Bacteroides fragilis • Obligate anaerobe: only grows in absence of O2 • Aerotolerant anaerobe: anaerobes that “tolerate” +/or survive in O2, but do NOT utilize O2 during E* metabolism • e.g. Clostridium perfringens • Facultative Anaerobe – grows both in presence & absence of O2; but grows BEST under Aerobic conditions; considered to be aerobic organism; O2 present – aerobic respiration for E*; O2 absent – anaerobic pathways (fermentation) • e.g. Staphylococcus spps • Capnophilic organism – requires high CO2 levels eg Neisseria spps

  38. Oxygen-related growth zones in a standing test tube

  39. Oxygen is toxic Oxygen is readily converted into radicals (singlet oxygen, superoxide, hydrogen peroxide, hydroxyl radical) Most important detoxifying enzymes are superoxide dismutase and catalase Cells differ in their content of detoxifying enzymes and hence, ability to grow in the presence of oxygen 42

  40. Catalase is an important diagnostic enzyme Classification of gram-positive cocci Staphylococci are catalase + Streptococci are catalase - Staphylococci Streptococci 43

  41. pH • Majority of bacteria grow BEST at neutral or slightly alkaline pH • pH 7.0 – 7.4 => this is near most normal body fluids • Acidophiles: grow BEST at low pH (acid: pH 0 – 1.0) • T.B. - pH 6.5-6.8 • Alkalophiles: grow BEST at high pH (alkaline: pH 10.0) • V. cholerae - pH 8.4-9.2

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