ch4- Dynamics of Microbial Growth

bacterial growth

it is an increase in number

bacteria divide by binary fission

• 1 cell → 2 cells, 2 → 4, 4 → 8, 8 → 16, etc…

• growth is exponential: population doubles with each division

generation time

time it takes for a cell to divide or population to double

• varies among species and with growth conditions

• environmental conditions

  • different media, temperature

pure culture and how to obtain it

bacteria are study in pure culture

• pure culture: population of cells derived from a single cell (clones, genetically known)

pure culture maintained using aseptic technique

• minimizes potential contamination

bacteria are grown in/on culture medium

• contains nutrients dissolved in water

• can be broth (liquid) or solid gel

growing microorganisms on solid medium - solid media/bacteria

solid media - broth media with the addition of agar

• agar - marine algae extract

• liquefies at >95 *C

• solidifies at 45 *C

  • remains solid at RT and body temperature

Bacteria grow in colonies on solid media surface

• all cells in colony descend from single cell

• approx. 1 mil cells per visible colony

growing microorganisms on solid medium - streak plate method

simplest and most commonly used for isolation

spreads out cells to separate

• obtain single cells → individual colonies

bacterial growth under laboratory conditions

bacteria grown on agar plates or in tubes or flasks of broth are closed systems

• nutrients not renewed; wastes not removed

• termed batch cultures

  • yield characteristic growth curve

The Growth Curve

characterized by 5 distinct stages:

Lag phase:

• number of cells does not increase

• cells prepare for growth

Log/exponential phase:

• exponential growth

• cell most sensitive to antibiotics

Stationary phase:

• overall population stabilizes

• cells exhausted nutrients, waste build-up

Death phase:

• total number of viable cells decrease

• death is exponential

Phase of prolonged decline:

• 99% of cells died

• very gradual decrease in viable cell population

• most adapted cells survive

environments & microbial growth

prokaryotes inhabit nearly all environments

• some live in comfortable habitats favored by humans

• some live in harsh environments

  • termed extremophiles: most are archaea

environmental factors that influence growth

• temp, oxygen, pH, water availability

temperature requirements

each species has a well-defined temperature range

• optimum growth temperature

prokaryotes divided into 5 categories

• psychrophiles (-5C to 15C)

  • found in arctic and Antarctic regions

• psychrotrophs (20C to 30C)

  • important in food spoilage

• mesophiles (25C to 40C)

  • more common

  • disease causing

    • human pathogens

• thermophiles (45C to 70C)

  • common in hot springs

• hyperthermophiles (70C to 110C)

  • usually members of archaea

  • found in hydrothermal vents

oxygen requirements

prokaryotes divided based on oxygen requirements

pH requirements

bacteria survive within a narrow pH range

• have an optimum pH

• maintain constant internal pH (near neutral)

neutrophiles

• multiply between pH of 5-8, optimum near neutral

acidophiles

• thrive at pH below 5.5

  • picrophilus oshimae has optimum pH of <11

alkaliphiles

• grow at pH above 8.5

water availability

all microorganisms require water for growth

water not available in all environments

• In high salt environments, a cell may undergo plasmolysis

some microbes can withstand or even require high salt

• halotolerant: withstand up to 10% (ex. Staphylococcus )

• halophiles: require high salt concentrations

  • marine bacteria ~3%

  • extreme halophiles >= 9%

    • dead sea, utah’s salt flats

nutritional facts that influence microbial growth

growth of prokaryotes depends on nutritional factors as well as physical environment

required elements - essential components for macromolecules

organisms classified based on

• carbon sources

  • heterotrophs

    • use organic compounds as nutrient source

  • autotrophs

    • use inorganic carbon CO2 as carbon source

• energy sources

  • phototrophs

    • derive energy from sunlight

  • chemotrophs

    • derive energy from chemical compounds

• trace elements (minerals)

  • required in minute amounts

  • cobalt, zinc, copper, molybdenum and manganese

• growth factors

  • must be added to growth media

  • bacteria can’t synthesize

    • certain amino acids, vitamins, purines, pyrimidines

  • organisms with many requirements → fastidious

cultivating microorganisms in the laboratory

microorganisms grown on culture media

general categories of culture media

• complex media contain a variety of ingredients

  • exact composition highly variable

  • often a digest of proteins

  • NA, blood agar, chocolate agar

• chemically defined media

  • contain precise amounts of pure chemicals

  • not practical for routine laboratory use

    • invaluable in research

      • each batch is chemically identical

        • does not introduce experimental variable

cultivating prokaryotes in the laboratory

special types of culture media

• useful for isolating and identifying a specific species

• selective media

• differential media

selective media

inhibit growth of unwanted organisms

• allow only sought after organisms to grow

ex.

• Thayer-Martin agar

  • for isolation of Neisseria gonorrhoeae

• MacConkey agar

  • for isolation of Gram-negative bacteria

differential media

contain substances that are changed by bacteria in recognizable ways

example:

• blood agar

  • identifies bacteria that produce hemolysin (break down RBCs)

    • hemolysis

• MacConkey agar

  • contains lactose and pH indicator → identify bacteria that produce acid from lactose

providing appropriate atmospheric conditions

anaerobes

• die in the presence of oxygen

  • growing anaerobic bacteria using

    • anaerobic jar

    • reducing agents (ex. sodium thioglycolate)

    • anaerobic chamber

methods to detect and measure microbial growth - direct microscopic count

• liquid dispensed in specialized slide

• viewed under microscope

• cells counted

• does not distinguish between living and dead cells

methods to detect and measure microbial growth - viable cell count

quantify living cells on solid culture media

assumption: single cell gives rise to colony

• plate out dilution series: 30-300 colonies ideal

• count colony forming units (CFU)

methods to detect and measure microbial growth - membrane filtration

concentrates microbes by filtration - trapped on surface

filter is incubated on appropriate agar medium → colonies counted

membrane filtration - turbidometric method

measures biomass

• turbidity (cloudiness)

  • measured with spectrophotometer (absorbance)

  • proportional to concentration of cells