Microbial growth
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Microbial Growth . Factors that influence growth: Physical/Environmental Chemical/Nutritional. Microbial adaptations are remarkable Extremeophiles May adapt to a point of no return . Temperature. Microbes within different ranges ( - 20 0 C – 120 0 C) Min and max typically ~ 30°C apart

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Microbial Growth

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Microbial growth

Microbial Growth

  • Factors that influence growth:

    • Physical/Environmental

    • Chemical/Nutritional


Microbial growth

  • Microbial adaptations are remarkable

    • Extremeophiles

    • May adapt to a point of no return


Temperature

Temperature

  • Microbes within different ranges (-200C – 1200C)

    • Min and max typically ~ 30°C apart

    • Optimum temp closer to max than min


Microbial growth

  • Psychrophiles:

    • Optimum temperature ~15°C

  • Psychrotrophs (moderate psychrophiles)

    • Optimum temperature ~25°C

  • Mesophiles:

    • Optimum temperature ~37°C


Microbial growth

  • Thermophiles:

    • Optimum temperature ~60°C

  • Hyperthermophiles:

    • Optimum temperature above 70°C

    • Usually Archaea


Microbial growth

pH

  • Most microbes are neutrophiles (6.5 – 7.5)

    • Optimum pH of most bacteria is 7

  • Some bacteria are considered acid tolerant

    • Helicobacter pylori

  • Typically fungi grow over a wider pH range

    • Usually responsible for spoilage of acidic foods


Microbial growth

  • Acidophiles optimum is lower (below 5.5)

    • Sulfolobus - Archaea from acidic hot springs

    • Lactobacillus – Bacteriaproduces lactic acid

    • Thiobacillus - Bacteria produces sulfuric acid

  • Alkalophiles have optimum above 8.0

    • Bacillus alcalophilus ~ 10.5

    • Vibriocholerae prefers pH of 9.0 outside host


  • Water activity

    Water Activity

    • Osmotic Pressure

    • Adaptations

      • inclusion bodies, compatible solutes, stretch receptors


    Microbial growth

    • Facultative halophiles

      • Osmotolerant/Halotolerant

      • Staphylococcus aureus

      • Fungi tend to be more tolerant than other microbes

    • Obligate halophiles

      • Most marine microbes

    • Extreme halophiles

      • Salt flats of Utah and Dead Sea


    Microbial growth

    • All organisms need:

      • Macroelements

        • CHNOPS

      • Microelements

        • K, Ca, Cl, Na

        • Trace elements

      • Growth factors


    Carbon

    Carbon

    • One of the most important growth requirements

      • ½ the dry weight of a bacteria cell is carbon

    • Carbon skeleton base of organic compounds

      • Hydrocarbons


    Metabolic diversity

    Metabolic Diversity

    • Organisms grouped by energy, carbon and electron source

    • Energy Source

      • Phototroph or Chemotroph

    • Carbon Source

      • Autotroph or Heterotroph

    • Electron Source

      • Lithotroph or Organotroph


    Microbial growth

    • Photolithoautotroph

      • CO2, Light and inorganic e- donor

    • Chemoorganoheterotroph

      • Organic carbon, organic chemicals and organic e- donor

    • Chemolithoautotroph

      • CO2, inorganic chemicals and inorganic e- donor

    • Photoorganoheterotroph

      • Organic carbon, light and organic e- donor

    • Chemolithoheterotroph

      • Organic carbon, inorganic chemicals and inorganic e- donor


    Oxygen requirements

    Oxygen Requirements

    • Oxygen has many toxic forms

      • Organisms require enzyme systems to protect them

    • Superoxide (O2-) radical

      • Neutralized by superoxide dismutase (SOD)

      • 2 O2- + 2 H+ → H2O2 + O2


    Microbial growth

    • Peroxide

      • Detoxified by catalase or peroxidase

      • Catalase: H2O2 → H2O + O2

      • Peroxidase: H2O2 + 2 H+ → 2 H2O


    Microbial growth

    • Obligate (Strict) Aerobes

      • only aerobic metabolism

      • Have SOD and catalase or peroxidase

    • Obligate (Strict) Anaerobes

      • Destroyed by oxygen

      • Do not have SOD, catalase or peroxidase

      • only anaerobic metabolism


    Microbial growth

    • Facultative Anaerobes

      • Have SOD and catalase or peroxidase

      • Grow with or without oxygen

      • aerobic or anaerobic metabolism

      • Grow faster in the presence of O2

    • Aerotolerant Anaerobes

      • Have SOD

      • Grow with or without oxygen

      • only anaerobic metabolism

      • Grow faster in the absence of O2


    Microbial growth

    • Microaerophiles

      • Grow only in low levels O2

      • small amounts of SOD and catalase

      • Produce toxic levels of superoxide free radicals and peroxide at high levels of O2

      • only aerobic metabolism


    Growth in liquid media

    Growth in liquid media

    Obligate

    Aerobes

    Facultative

    Anaerobes

    Obligate

    Anaerobes


    Microbial growth

    Aerotolerant

    Anaerobes

    Microaerophiles


    Nitrogen

    Nitrogen

    • Needed for amino acids, nucleic acids, and ATP

    • Amino acids from protein degradation

    • Nitrogen reduction

      • Reduce nitrate to ammonia then utilize the ammonia

    • Nitrogen fixation

      • assimilate gaseous nitrogen ( N2)


    Sulfur

    Sulfur

    • Needed for building some amino acids (cysteine and methionine), vitamins (thiamine and biotin) and some carbohydrates

    • Sulfur containing amino acids from protein degradation

    • Reduce sulfates (SO42-) or sulfides (H2S)


    Phosphorus

    Phosphorus

    • Tends to be a limiting growth requirement

    • Phosphorus is needed for building nucleic acids, phospholipids, and ATP

      • Phosphate ion (PO43-)is an important source


    Microelements

    Microelements

    • Several are essential for proper cell function

      • Signal molecules, membrane potential, enzyme cofactors

    • Trace Elements

      • Minerals required in very small amounts

      • Iron, copper and zinc


    Growth factors

    Growth Factors

    • Organic compounds essential growth

    • Cannot be synthesized by microorganisms

      • Vitamins

      • Amino acids

      • Nucleic acid bases

    • Fastidious

      • Neisseria


    Cultivation of microorganisms

    Cultivation Of Microorganisms

    • culture medium

      • nutrient material prepared for growing microorganisms

    • inoculation

      • introduction of a microorganism into medium

    • culture

      • growth of a microorganism observed on/in a medium


    Types of culture media

    Types Of Culture Media

    • Chemically defined media:

      • Exact composition known

    • Complex media:

      • Exact composition varies


    Microbial growth

    • Selective media:

      • Favors the growth of desired microorganisms

      • Inhibits the growth of unwanted ones

    • Differential media:

      • Distinguishes between groups of microorganisms


    Macconkey s agar

    MacConkey’s Agar

    • Selective medium:

      • Inhibits Gram-positive bacteria growth

      • Encourages Gram-negative bacteria growth

    • Differential medium:

      • Lactose fermenters produce acid and form pink colonies

      • Non-lactose fermenters form colorless colonies


    Macconkey s agar1

    MacConkey’s Agar

    Escherichia

    Salmonella


    Microbial growth

    • Differential media

      • Blood agar

        • Alpha Hemolysis

        • Beta Hemolysis

        • Gamma Hemolysis


    Microbial growth1

    Microbial Growth

    • Refers to increase in number of cells not size of individual cells

    • Bacteria typically reproduce by binary fission

    • Generation time

      • time required for a bacterial population to double

        • Typically 1-3 hours


    Microbial growth

    • Generation number is expressed as a power of 2

      • Original cell is 20, 2nd generation (after one cell division) would be 21

    • 20= 1 cell

    • 21= 2 cells

    • 22= 4 cells

    • 23= 8 cells


    Microbial growth

    Phases of Bacterial Growth Curve

    • In closed system or batch culture

      • Lag phase

      • Log phase

      • Stationary phase

      • Decline phase


    Microbial growth

    • Phases of the growth curve can be observed in liquid media

      • Solid media, different colonies in different phases

    • Continuous cultures

      • Open system


    Microbial growth

    Measuring Bacterial Growth

    • Direct Methods

      • Viable plate count, Membrane filtration, Microscopic count, Most Probable Number (MPN), Electronic Counters

    • Indirect Methods

      • Turbidity, Metabolic activity, Weight


    Microbial growth

    Direct Methods

    • Viable Plate Count

      • Important to limit colonies to a countable number

        • 30-300 colonies (CFUs)

        • Serial dilutions ensure colony counts within range

      • Advantage: only living cells

      • Disadvantage: incubation time, growth requirements, may underestimate count


    Microbial growth

    • Plate count methods

      • pour-plates

      • Spread-plates methods


    Microbial growth

    2. Membrane Filtration

    • Sample (liquid) passed through filter

    • Filter placed on surface of solid medium

    • Organisms retained on filter will grow

  • Advantage: only living cells, can be used to count low cell concentrations

  • Disadvantage: must have at least 100 ml of media, requires incubation time, may underestimate count


  • Microbial growth

    3. Microscopic Count

    • Known volume of sample placed in counting chamber

    • Viewed under microscope

    • Cells counted

  • Advantage: no incubation time is required

  • Disadvantage: dead cells may be counted, tedious, requires a high concentration of cell (10 million per ml)


  • Microbial growth

    4. Most probable numbers (MPN)

    • Multi-tube statistical assay

    • Advantage: measures only living cells, useful for culturing cells that wont grow on solid media

    • Disadvantage: incubation time, expensive & time consuming


    Microbial growth

    • Series of dilution sets

      • Each set inoculated with 10X less sample than previous set

      • Incubated and results compared to MPN table

        • gives statistical estimation of cell concentration


    Microbial growth

    5. Electronic Counter

    • Coulter Counter – electrical current

    • Flow Cytometry – light transmission

    • Advantage: No incubation time

    • Disadvantage: dead cells may be counted, not very sensitive due to clumping and debris in media


    Microbial growth

    Indirect methods

    1.Turbidity

    • Uses spectrophotometer

    • Advantage: no incubation time

    • Disadvantage: must have high concentration of cells, may count dead cells


    Microbial growth

    • Measures light transmitted through sample

      • Measurement is inversely proportional to cell concentration


    Microbial growth

    2. Weight

    • Wet weight

      • Cells centrifuged and packed cells weighed

    • Dry weight

      • Packed cells dried at 100°C for 8 to 12 hours then weighed

    • Disadvantage: Tedious and time consuming

    • Advantage: Useful in measuring filamentous organisms


    Microbial growth

    3.Metabolic Activity

    • Based on enzyme activity

    • Advantage: once metabolic rate is established provides reliable estimate of cell number

    • Disadvantage: requires incubation time, requires metabolic rate be established in advance


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