Measuring Bacterial Growth and Replication

Measuring Bacterial Growth and Replication

Measuring Bacterial Growth and Replication

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Binary Fission

  • Bacterial growth
    • Change in the total population rather than an increase in the size or mass of the individual organism
  • Bacteria divide by binary fission
    • increase of cell mass
    • duplication of genome
    • cell membrane and cell wall separation
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Binary Fission

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Growth Phases

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Growth Phases

  • Lag Phase
    • Population remains temporarily unchanged
    • Bacterial cells may be growing in volume or mass, synthesising enzymes, proteins, RNA etc. and increasing in metabolic activity
  • Exponential Growth Phase
    • Expressed as generation time, or double time, of the bacterial population
    • Generation time (G) is defined as the time (t) per generation (n = number of generations)
  • Stationary Phase: population growth is limited by:
    • exhaustion of available nutrients
    • accumulation of inhibitory metabolites or end products
    • exhaustion of space
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Exponential Growth Phases

  • One cell divides producing 2 daughter cells
  • Increase in population is by geometric progression
  • 'n' is the number of generations assuming no cell death
  • Total population, b, at the end of a given period starting with 1 cell: b = 1x2^n

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Generation Time

  • The generation time is the time interval required for the cells (or population) to divide

G = t/n

b = B x 2^n (expression of growth by binary fission)

G = t/(3.3logb/B)

  • G = generation time
  • t = time in minutes or hours
  • B = number of bacteria at beginning of time interval
  • b = number of bacteria at end of time interval
  • n = number of generations (number of times the cell population doubles during the time interval)
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Chemostat

  • Chemostat: bioreactor to which fresh medium is continuously added, while culture liquid is continuously removed to keep the culture volume constant
    • Control growth rate
    • Optimise production
  • Continuous fermentation
  • Batch fermentation
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Chemical Requirements for Growth

  • Energy source
    • Chemotrophs: chemical compounds
    • Phototrophs: radiant energy (light)
  • Source of electrons
    • Lithotrophs: reduced inorganic compounds
    • Organotrophs: useof organic compounds
  • Nitrogen source
    • Atmospheric nitrogen
    • Inorganic nitrogen (nitrate, nitrite or ammonium salts)
    • Organic compounds (amino acids)
  • Carbon source
    • Autotrophs: carbon dioxide as their major source of carbon
    • Heterotrophs: organic compounds as their carbon source
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Chemical Requirements for Growth

  • Oxygen, sulphur and phosphorus for cell components
    • Oxygen: water, nutrients or molecular oxygen
    • Sulphur: needed for the synthesis of certain amino acids: cysteine, cystine and methionine: use of organic and inorganic sulphur or elemental sulphur
    • Phosphorus: usually supplied in the form of phosphate nucleotides, nucleic acids, phospholipids, techoic acids and other compounds
  • Ions
    • metal ions such as K+, Ca2+, Mg2+, Fe2+ for normal growth
    • trace elements (low concentrations) in support of growth: Zn2+, Mn2+, Cu2+, Mo6+, Ni2+, B3+ and Co2+
    • enzyme cofactors: Zn2+, Mn2+, Cu2+, Mo6+, Mg2+, Fe2+
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Chemical Requirements for Growth

  • Oxygen and Carbon Dioxide
    • Aerobic bacteria: need oxygen for growth
    • Anaerobic bacteria: do not use oxygen to obtain energy
      • Strict (obligate) anaerobe: oxygen is toxic
      • Tolerant anaerobe: tolerate low levels of oxygen
    • Facultative anaerobe do not require oxygen for growth: they use oxygen if present
    • Microaerophilic bacteria tolerate low levels of oxygen for growth
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Chemical Requirements for Growth

  • Oxygen toxicity
    • inactivation of enzymes: oxidation of thiol (-SH) groups or enzymes
    • damage due to toxic derivatives of oxygen

formation of super oxide radical (O2-): O2 + e- --> O2-

formation of hydrogen peroxide (H2O2): 2O2- + 2H+ --> O2 + H2O2

formation of hydroxyl radicals (OH.): O2- + H2O2 --> O2 + OH. + OH-

  • Aerobic and facultative anaerobic organisms have developed various protective mechanisms against the toxic forms of oxygen
    • superoxide dismutase: elimination of superoxide radicals
    • catalase and peroxidase: elimination of hydrogen peroxide
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Physical Requirements: Temperature

  • Optimum growth temperature: allow maximum growth during a short period of time
    • Psychrophiles: optimum temperature 0-20 degrees
    • Mesophiles: optimum temperature 25-40 degrees
    • Thermophiles: >45 degrees
  • Bacteria that cause disease in humans grow best at 37-39 degrees
  • Control of temperature is essential for the storage of pharmaceutical products
    • deep freeze (-20 degrees): total parenteral nutrition, raw materials
    • 8-12 degrees: reconstituted syrups and multi-dose eye drops
    • 80 degrees: water for injection (regrowth of gram-negative and release of toxins)
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Physical Requirements: pH

  • Optimum pH for growth: 6.5-7.5
    • limits: 5-9
    • exception: Thiobacillus thiooxidans (0.5-6.0; optimum 2-3.5) and alkaline spring isolate (8-11.4; optimum 9-9.5)
  • Prevention of growth in pharmaceutical preparations
    • extreme pH prevent microbial growth
    • neutral pH: bacterial spoilage, e.g. in antacid mixtures, flavoured mouth washes, distilled and deionised water
    • pH>8: spoilage is rare (soap-based emulsion)
    • low pH: spoilage by moulds and yeast, e.g. fruit juice flavoured syrups
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Water Activity (Aw)

  • Estimates the proportion of uncomplexed water available in formulation
  • The greater the solute concentration, the lower the water activity
  • Most microorganisms grow best (except for halophiles) in dilute solutions (high Aw)
  • Limiting Aw values
    • Gram-negative rods: 0.95
    • Staphylococci, streptococci: 0.9
    • Most yeasts: 0.88 (down to 0.73)
  • Preparation of growth in pharmaceutical preparations
    • Water activity of aqueous formulations can be lowered to decrease microbial growth. Syrup BP (Aw = 0.86), tablets
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Enumeration

  • Sterile products
    • Preparations required to be sterile on the dosage form and other preparations labelled sterile
    • Need to comply with test for sterility
  • Preparations for topical use and for use in the respiratory tract except where required to be sterile and transdermal patches
    • Total viable aerobic count <10^2 microorganisms /g or /ml or /patch
    • Transdermal patches: absence of enterobacteria (on one patch) Other preparations: <10^1 enterobacteria /ml
    • Absence of Pseudomonas aeruginosa (/g or /ml or /patch)
    • Absence of Staphylococcus aureus (/g or /ml or /patch)
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Enumeration

  • Preparations for oral and rectal administration
    • Total viable aerobic count <10^3 aerobic bacteria and <10^2 fungi /g or /ml
    • Absence of Escherichia coli (/g or /ml)
  • Preparations for oral administration containing raw materials of natural origin (animal, vegetable or mineral) for which antimicrobial pre-treatment is not feasible and for which the competent authority accepts a microbial contamination of the raw material exceeding 10^3 viable microorganisms /g or /ml
    • Total viable aerobic count <10^4 aerobic bacteria and <10^2 fungi /g or /ml
    • <10^2 enterobacteria and absence of Staphylococcus aureus and Escherichia coli /g or /ml and Salmonella /10g or /10ml
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Enumeration

  • Herbal remedies consisting solely of one or more vegetable drugs (whole, reduced or powdered)
    • Herbal remedies to which boiling water is not added before use
      • Total viable aerobic count <10^5 aerobic bacteria and <10^4 fungi /g or /ml
      • <10^3 enterobacteria /g or /ml
      • Absence of Escherichia coli /g or /ml
      • Absence of Salmonella /10g or /10ml
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Colony Count

  • A single bacterium in the original culture being plated is assumed to give rise to a single viable colony
  • The viable count may be referred to as the number of colony-forming units (cfu) rather than as number of bacteria
  • The type of medium used and conditions of incubation might also alter bacterial growth and the formation of colonies
  • Plate count methods
    • Pour plate method
    • Spread plate method
    • Any other methods must be validated
  • Membrane filtration (0.45um membrane filters)
    • Cellulose nitrate filters for aqueous, oily and weak alcohol
    • Cellulose acetate filters for strongly alcoholic solutions
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Colony Count

  • Most probable number method
    • Reserved for the enumeration of bacteria in situations where no other method is available
      • Prepare a series of at least three subsequent tenfold dilutions of the product
      • From each level of dilution three aliquots of 1g or 1ml are used to inoculate three tubes with 9ml to 10ml of a suitable liquid medium
      • Incubate all tubes for five days at 30-35 degrees and record for each level of dilution the number of tubes showing microbial growth
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Enumeration

  • Cell count: directly by microscopy or electronic particle counter
  • Cell mass: directly my weighing, measurement of cell nitrogen; measurement of dry weight (oven)
  • Spectrophotometric measurement: measurements of tubidity, optical density
  • Cell activity: indirectly by relating the degree of biochemical activity to the size of the population
  • Rapid Microbiology Methods
    • Growth-based methods: a detectable signal is usually achieved by a period of subculture
    • Direct measurement: individual cells are differentiated and visualised
    • Cell component analysis: expression of specific cell components offers an indirect measure of microbial presence
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Rapid Microbiology Methods

  • Growth-based
    • Electrochemical methods (electric current)
    • Measurement of consumption or production of gas (carbon dioxide)
    • Bioluminescence (ATP)
    • Microcalorimetry (temperature)
    • Turbidimetry 
  • Direct measurement
    • Flow cytometry
    • Solid phase cytometry
    • Direct Epifluorescent Filtration (DEFT) - labelled fluorophore
  • Cell component analysis
    • Nucleic acid amplication techniques (NAAT)
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