Lesson 7 - Microbial Growth + Culture

Why are microorganisms important to food webs?

• Microorganisms form the hidden foundation of food webs.

• Essential nutrients such as carbon are limited and must be recycled.

• Microorganisms help decompose organic matter and recycle nutrients, supporting other organisms.

How do microorganisms contribute to the carbon cycle?

• Autotrophs fix inorganic CO₂ into organic carbon.

• Heterotrophs obtain carbon from organic compounds.

• Decomposition and respiration help return carbon to the environment.

• These processes recycle carbon through ecosystems.

What is nitrogen fixation, and what are diazotrophs?

• Atmospheric N₂ makes up approximately 79% of the atmosphere but cannot be directly used by most organisms.

• Bacteria play key roles in the global nitrogen cycle

• Diazotrophs are microorganisms capable of nitrogen fixation.

• They use nitrogenase (enzyme) to convert atmospheric N₂ into ammonia/ammonium (NH₄⁺).

• Nitrogen fixation supplies biologically available nitrogen.

• Only a few bacteria can fix N2 ...so all life depends on them!

What is the relationship between Rhizobium bacteria and plants?

• Rhizobium species are nitrogen-fixing root endosymbionts.

• They inhabit root nodules of certain plants, especially legumes.

• The bacteria receive organic nutrients from the plant.

• The plant receives biologically available nitrogen produced by the bacteria.

What is the difference between facilitated diffusion and active transport?

• Facilitated diffusion:

  • Passive transport through membrane proteins.

  • Moves substances down their concentration gradient.

  • Does not require metabolic energy.

• Active transport:

  • Uses energy to transport substances.

  • Can move substances against their concentration gradient.

What is binary fission, and what are its main stages?

• Binary fission is asexual reproduction in which a bacterial cell increases in size/cell volume and biomass then splits into two equal daughter cells.

• Population doubles at each division (generation time)

• Stages:

  • Cell growth

  • DNA replication

  • Formation of division septum

  • Cell separation

• Some bacteria and yeasts reproduce through asymmetrical budding instead.

What is generation time, and how is bacterial population growth calculated?

• Generation time (doubling time) is the time required for a bacterial population to double under favourable conditions.

• In a favourable/optimal environment, with unlimited & appropriate resources and environmental conditions, bacteria divide at a constant interval

• Nt = N0 × 2^n

• Nt = final cell number

• N0 = initial cell number

• n = number of generations

What are examples of fast and slow bacterial generation times?

• Clostridium perfringens: Approximately 10 minutes

• Mycobacterium leprae: Approximately 14 days

• Generation time varies between bacterial species.

What is the difference between liquid and solid culture media?

• Liquid media (broth):

  • Cells grow in suspension.

  • Used to study pure culture growth and produce large quantities of cells or extracellular products.

  • Often used as a closed system (batch culture).

• Solid media (agar):

  • Cells grow as visible colonies.

  • Separate bacteria in samples

  • Isolation of a pure culture (bacterial population grown from a single isolated ‘pure’ colony

  • Studying diversity in a sample, specific traits e.g., colony morphologies, physiology, growth, genetics, antibiotic production etc.

What are the five major types of microbial culture media?

• Complex/rich: Nutrient-rich; exact composition poorly defined.

  • E.g. contain general extracts of yeast cell, plant and animal tissues

• Minimal defined: Contains only essential nutrients needed for growth of a specific microbe; exact composition and concentrations known.

• Enriched: Complex media to which specific factors are added; the microbe is not capable of making them but needs them to grow.

  • E.g., blood proteins, nucleotides, vitamins etc.

• Selective: Favours growth of some microorganisms over others.

• Differential: Distinguishes microorganisms based on biochemical or physiological activities.

What is the difference between selective and differential media? Give an example.

• Selective media favour the growth of certain microorganisms while inhibiting others.

  • E.g., high or low pH, plus antimicrobials, specific nutrients

• Differential media distinguish microorganisms based on biochemical or physiological differences,

  • E.g., contain a dye that changes in colour when bacteria produce specific byproducts; differentiate microbes based on their metabolic activities

• MacConkey agar is both:

  • Selective for Gram-negative bacteria.

  • Differential for lactose fermentation: fermenters produce pink/red colonies, while non-fermenters appear colourless/white.

How does an autoclave sterilize culture media?

• Uses high-temperature and high pressurized steam (moist heat) to kill microorganisms, including resistant spores.

• Moist heat is most effective at killing microbes

• Moist heat damages essential cellular components.

• Sterilization effectiveness can be assessed using biological indicators containing resistant bacterial spores.

What is the difference between complex and synthetic (defined) media? Give examples

• Complex media:

  • Contain biological extracts with incompletely known chemical compositions.

  • Example: Lysogeny broth (LB), containing tryptone and yeast extract.

• Synthetic/defined media:

  • Exact chemical ingredients and concentrations are known.

  • Examples: M9 medium and sulfur oxidizer medium.

What are dilution streaking and spread plating used for?

• To obtain well isolated single colonies which can be used to establish pure cultures or estimate the total number of bacteria in a sample

• Spread plating can also estimate viable bacterial numbers.

• Assumption: One cell = one colony! (not always true, some bacteria naturally live/occur in pairs or multiples and cannot be separated by these simple techniques)

How does dilution streaking isolate pure bacterial cultures?

• Bacteria are progressively diluted across an agar surface.

• Dilution of the sample separation a single bacterium

• Separated bacteria multiply to form visible isolated colonies.

• An isolated colony can be used to establish a pure culture.

What are the steps and goals of serial dilution and viable plate counting?

• Set up 10-fold serial dilutions in liquid culture

• A small amount (e.g. 0.1 ml) of each dilution is then plated on agar medium.

Goal:

• Obtain a dilution that gives nicely separated colonies

• Select plates with ~30-300 Colony Forming Units (CFU)

• Calculate number of bacteria in original sample

Why is counting microorganisms important?

• Government regulations: Food safety and health applications.

• Quality control: Water safety, contamination, and soil health.

• Research: Measuring microbial populations and studying growth.

What are the three main methods used to count or estimate microbial populations?

• Direct microscopic counts using a haemocytometer.

• Plate counts using dilution plating.

• Optical density using a spectrophotometer.

• Microbes must be suspended in liquid for these methods.

• Results may be expressed as bacteria/mL or bacteria/g.

What is optical density (OD₆₀₀), and how does it relate to microbial growth?

• Microbial cells in liquid suspension increases the turbidity (cloudiness) of the liquid; the higher the concentration of the cells the more turbid the suspension

• Cells scatter light, reducing transmitted light measured by a spectrophotometer.

• Usually measured at 600 nm (OD₆₀₀). • Higher cell concentration → greater turbidity → higher OD₆₀₀.

Describe the Microbial Growth Cycle

• Growth cycle is analysed as cells density (OD600nm or cell counts) as a function of time

• When grown in batch liquid culture (i.e. in a test tube or flask, in a closed system, where media and nutrients are not refreshed) there are four phases of growth:

  • lag, exponential/logarithmic (log), stationary, death

What occurs during the lag phase of bacterial growth?

• Cells need time to sense and adjust to their environments, during lag phase, bacteria adapt themselves to growth conditions

• Bacteria are maturing and not yet able to divide

• Synthesis of RNA, enzymes and other molecules occurs

What occurs during the log/exponential phase of bacterial growth?

• Characterized by cell doubling

  • Vegetative growth

  • Planktonic cells – single cells in suspension

• If not limited, doubling will continue at constant (exponential) rate

• Slope of (log scale) line = growth rate: number cell divisions/unit time

• Rate of this growth depends upon the growth conditions

• Log growth cannot continue indefinitely as medium is depleted of nutrients and enriched with wastes in batch cultures

What occurs during the stationary phase of bacterial growth?

• In stationary phase overall population growth plateaus

• Due to a growth-limiting factor such as the depletion of essential nutrient(s), and/or formation of an inhibitory product

• The result is a horizontal linear part of the curve

What occurs during the death phase of bacterial growth?

Without any new nutrients (and/or production of toxic byproducts by dying cells), all cells will eventually die off

What is a chemostat, and why is it useful?

• The chemostat ensures continuous logarithmic growth by constantly adding and removing equal amounts of culture medium

• Chemostat allows growth and growth conditions/environment to be highly controlled

• Conditions approximate that of the native environment allowing detailed study of microbes and microbial communities in more natural conditions (e.g. study of human microbiome of the human large intestines/colon)

Why are many microorganisms difficult to culture, and how does the iChip help?

• 85–99% of bacterial species from many environments do not grow on conventional agar plates.

• Many require specific nutrients or growth factors.

• The iChip helps cultivate microbes using conditions closer to their natural environment.

• This technology contributed to new antibiotic discovery.