Microbial Growth

1
Microbial Growth
Microorganisms can grow to extraordinary numbers
in a short time
2
Requirements for growth Chemical

• Water
• Carbon. Backbone of biomolecules
• Autotrophs can obtain carbon from the environment
  as CO2
• Heterotrophs obtain carbon from carbon-based
  molecules in food.
• Heterotrophs need at least basic carbon-based
  molecules (e.g.-sugars)
• Some species need certain specific additional
  carbon-based molecules
• Vitamins, amino acids

3

• Why do organisms need carbon?
• From where do autotrophs obtain carbon?
• From where do heterotrophs obtain carbon?

4
Requirements for growth Chemical

• Oxygen. Need varies
• Obligate aerobes Require oxygen.
• Example Mycobacterium tuberculosis
• Facultative anaerobes Oxygen optional
• Example Yeast
• Aerotolerant anaerobes Do not use oxygen but
  can tolerate it.
• Example Lactobacillus acidophilus.
• In digestive system, vagina.
• Used in yogurt making
• Microaerophiles Aerobes that require lower
  oxygen (2 to 10) than found in atmosphere (21)
• Example Helicobacter pylori. Causes stomach
  ulcers.
• Obligate aerobes Cannot tolerate oxygen.
• Examples Clostridium botulinum, Clostridium
  tetani.

• Can test for oxygen requirements by growing
  microbes in a liquid culture that has decreasing
  oxygen with depth

5

• Define each and give an example
• Obligate aerobe
• Facultative anaerobe
• Aerotolerant anaerobes
• Microaerophile
• Obligate anaerobe
• How can oxygen requirements for microbes be
  tested?

6
Requirements for growth Chemical

• Oxygen (continued)
• Oxygen is toxic. Reason
• O2 is converted into highly reactive forms that
  react with and damage cellular molecules
  including DNA and proteins.
• Organisms able to grow in oxygen (including many
  microbes and other organisms like humans) have
  enzymes that detoxify highly reactive forms of
  oxygen by converting them to water and/or O2.
• Examples of damaging forms of oxygen
• Superoxide radical. Detoxified by superoxide
  dismutase.
• Peroxide anion. Detoxified by catalase
• Catalase produces oxygen gas.
• Anaerobes lack enzymes for detoxifying damaging
  forms of oxygen.

7

• Why is oxygen toxic?
• How can organisms grow in oxygen if it is toxic?
• Give two examples of toxic forms of oxygen and
  the enzymes that detoxify them.

8
Requirements for growth Chemical

• Nitrogen. Needed to nucleotide, amino acids.
• Can be obtained from
• Nucleotides, amino acids in food. (Organic
  sources)
• Inorganic sources
• Nitrate. E.g.- in soil.
• Nitrogen gas in atmosphere.
• A limited number of bacterial species are the
  only organisms on Earth that can do this.
• This is essential for life on Earth since it
  deeps whole ecosystems supplied with nitrogen.
• Other substances
• Sulfur and phosphorus
• Trace elements Iron, copper, zinc, etc.
• Some photosynthetic microbes need only light,
  water, air and minerals to grow.

9

• Why do organisms need nitrogen?
• List two substances always needed for growth
  other than water, carbon, nitrogen and trace
  elements.
• Some photosynthetic microbes have minimal
  requirements for life. What are these
  requirements?

10
Requirements for growth Temperature
Growth - increase in numbers, not cell size

• Temperature. Species can be classified on basis
  of temperature range for growth

• Low range
• Psychrophiles.
• Optimal growth approximately 15oC. Can grow
  below 0oC.
• Found in polar, alpine, ocean depth environments.
• Includes substantial fraction of Earth
  environments.
• Some require low temperature, some can tolerate
  low temperature
• Includes both autotrophs and heterotrophs
• No growth of room temperature. No disease and
  little food impact.

11

• What is a psychrophile?
• Where are psychrophiles found?

12
Requirements for growth Temperature

• Mid range Mesophiles
• Optimal growth approximately 20oC to 40oC.
• Found in soil, animals, humans, food.
• Includes
• Pathogens
• Food spoilage microbes.
• Microbes involved in cheese making, alcoholic
  beverages.

13

• In what environments do mesophiles grow?
• What are three ways in which mesophiles are
  important to humans?

14
Requirements for growth Temperature

• High range Thermophiles
• Optimal growth approximately 45oC to 110oC.
• Enzymes can control high temperature.
• Found in hotsprings, deep sea hydrothermal vents,
  decaying materials (decay generates heat)
• Some require high temperature, some can tolerate
  high temperature.
• Example Thermus aquaticus

• PCR method for making many copies of a DNA
  fragment.
• Used in crime investigations and many other
  applications including clinical testing.
• Uses enzymes from thermophiles like Thermus
  aqauticus

15

• In what environments do thermophiles grow?
• Briefly explain the application for enzymes found
  in thermophiles.

16
Temperature Ranges for Growth
17
Requirements for growth pH
pH acid, neutral, base

• Most bacteria prefer near neutral

• Acidophiles Can tolerate acid conditions. In
  some cases extreme.
• Snottites Mucus-like biofilms of bacteria
  hanging from walls or ceilings of caves.
• Chemoautotrophs Obtain energy from volcanic
  sulfur compounds in warm water dripping from
  above.
• Biofilm can be as corrosive as battery acid.

18

• What is an acidophile?
• Briefly describe a snottite.

19
Requirements for growth Osmotic Pressure
Water moves toward area of more dissolved
material (salt, sugar, etc.). This osmosis.

• If there is more dissolved material outside cell,
  cell loses water.
• If there is less dissolved material outside cell,
  cell gains water.
• Hence, cell need right amount of dissolved
  material in surroundings.

• Most microorganisms are inhibited by high
  concentration of dissolved material Causes cell
  to lose water, dehydrate.
• Explains why salt preserves food and honey
  doesnt spoil.
• Note that honey can harbor a few inactive
  bacterial spores such as Clostridium botulinum
  spores. These are tolerated in adults but cause
  botulism (serious food poisoning) in infants.

• Halophiles Live in high salt
• Examples Great Salt Lake or smaller salt ponds.
• Skin microbes tolerate salt.
• Bacteriorhodopsin Protein from halophile.
• Used as computer memory

20

• Why would a cell lose water?
• Why does salt preserve food?
• Why doesnt honey spoil?
• What spore may be found in honey?
• What is a halophile?

21
Extremophiles
Bacteria adapted to extreme conditions (old,
heat, acid, salt, radiation)

• Deinococcus radiodurans
• A poly-extremophile. Withstands multiple extreme
  conditions Cold, heat, dehydration, vacuum,
  acid and radiation.
• Can withstand 1000 times (or more) the radiation
  that would kill a human.
• Can help with disposal of solvents and heavy
  metals.
• Can survive in outer space!

22
What is an extremophile?
23
Culturing Bacteria

• Microbes can grow
• In liquid Suspended in liquid containing needed
  materials.
• In solid On a solid (agar) containing needed
  materials.

• Terms
• Inoculum Sample of microbes introduced for
  growth.
• Environmental or clinical or stored samples
• Medium Nutrients (solid or liquid form)

24

• Briefly describe the growth of microbes in
• Liquid medium
• Solid medium
• Define each term
• Inoculum
• Medium

25
Culturing Bacteria

• Colonies
• Visible growth on surface of solid medium.
• Colony characteristics aid identification.

26
How are colony characteristics helpful?
27
Culturing Bacteria

• Pure cultures
• Must isolate a particular type of microbe so it
  can be identified
• Colonies are pure isolates.
• A colony on an agar plate is started by a single
  cell.
• Obtaining colonies by streaking
• Streak plate with a liquid inoculum using an
  inoculating loop so as to thin out the number of
  cells.
• Sterilize loop between each streak to reduce
  number of cells
• This will result in individual cells being
  deposited.
• Incubate for a suitable time, suitable
  temperature.
• Cells will divide forming colonies.
• Incubate for suitable time, suitable temperature.
• Can then lift a single colony with loop and grow
  cells from it as a pure culture.

28

• Why is a colony a pure isolate?
• Describe how to streak a plate to obtain isolated
  colonies.

29
Culturing Bacteria

• Obtaining colonies using pour plates
• Cell numbers are reduced by dilution rather than
  streaking.
• Serial dilution
• Dilute inoculum with liquid medium (e.g.- 1 mL
  inoculum with 9 mL medium). Mix.
• Dilute repeatedly as necessary.
• Dilutions are mixed with warm agar and poured
  onto plate.
• Colonies form after incubation.

30

• Describe how to perform a serial dilution.
• Describe how to make a pour plate.

31
Culture media

• Some microbes are more particular about culture
  conditions.
• Many microbes exist that have not been
  successfully cultured
• Media is prepared from powder. Examples
• Yeast extract, beef extract, peptone (amino acid
  mix).
• Process
• Dissolve powder in water.
• Sterilize
• If wish to make solid medium, mix with agar
  (gelling agent) before sterilizing. Pour into
  plate.

32
Culture media

• Types of media
• Defined media- Exact chemical composition of
  medium is known.
• Designed for particular organism being grown.
• May be used for experimental purposes in which it
  is necessary to control culture conditions.
• Complex media- Rich array of nutrients,
  composition not precisely known
• More widely used than defined media
• Easier to prepare
• Uses rich source sources of nutrients, sometimes
  pre-digested with enzymes. E.g.- yeast, beef,
  soy, milk
• Wide variety of nutrients so can support many
  kinds of microbes.
• Examples
• Nutrient agar
• Trypticase soy agar
• MacConkey agar
• May be enriched with blood to provide additional
  substances

33

• What are defined media?
• What are complex media?
• Give two examples of sources of nutrients used in
  complex media
• Why can complex media support many kinds of
  microbes?
• Give two examples of complex media.

34
Culture media

• Types of media (continued)
• Selective media
• Contain substances that favor/inhibit certain
  microbes. Examples
• Eosin, methylene blue, crystal violet, bile salts
  inhibit gram-positive but not gram-negative
  bacteria.
• High salt selects for halophiles or salt-tolerant
  species. Example Staphylococcus aureus lives
  on skin, is salt-tolerant.
• Sabourand dextrose agar- Slightly low pH favors
  fungi over bacteria

35

• Explain the concept of selective media.
• Give two examples of selective media.

36
Culture media

• Types of media (continued)
• Differential media Make it possible to see
  differences between kinds of microbes based on
  utilization of media ingredients. Examples
• Blood agar. Species differ in degree to which
  break down red blood cells.
• Streptococcus pyrogenes breaks down completely
• Streptococcus pneumoniae partially breaks down
• Enterococcus faecalis does not break down

• Carbohydrate utilization broth
• If bacteria can metabolize carbohydrate supplied,
  they release waste (acidic) and cause color
  change in pH indicator dye.

37

• Explain the concept of differential media.
• On what basis does blood agar differentiate
  between species?
• Explain how carbohydrate utilization broth
  differentiates species.

38
Culture media

• Types of media (continued)
• Selective AND differential media Example
• MacConkey agar
• Selective Crystal violet and bile salts inhibit
  gram positives
• Differential Neutral red is pH indicator
• Lactose is carbon source.
• If bacteria can metabolize lactose, will produce
  acid waste and indicator is red.
• If bacteria cannot metabolize lactose, will
  metabolize peptone (amino acid mix). This
  produces ammonia waste that is basic and
  indicator is colorless.

39
Culture media

• Types of media (continued)
• Others
• Anaerobic media Have chemicals that absorb
  oxygen
• Transport For clinical samples. Maintain ratio
  of microbes.
• Cell culture Viruses and some bacteria can only
  be cultured in live cells.
• Animal culture Some bacteria will not grow in
  culture and can only be grown in animals
• Mycobacterium leprae (leprosy) grown in
  armadillos.
• Treponema pallidum (syphilis) grown in rabbits.

40
Growth

• Populations of organisms increase exponentially
• Each generation doubles numbers.
• Therefore, the rate of increase gets greater each
  generation since a larger number is being
  doubled.
• 22 4
• 25 32
• 210 1,024
• 220 1,048,576
• 230 1,073,741,824

41
Growth

• Growth of microbial populations
• Generation time Time for cell to divide.
• Range from 20 minutes (E. coli) to 10 days
  (Mycobacterium)
• From one cell to over a billion in 30
  generations.
• For microbes that divide in 20 minutes Go from
  one to a billion in as little as 10 hours.
• Note Would take almost 32 years to count to 1
  billion at one number per second!
• At this rate, constant cell division for 26 hours
  could produce, from one bacterium, the weight of
  an aircraft carrier!

42
Living organisms increase exponentially. This
means that the rate of population increase gets
greater each generation. Why?
43
Growth

• Growth phases
• Lag phase. Little growth at start as inoculated
  cells adjust to new environment.
• Log phase. Period of constant rate of cell
  division so exponential (logarithmic) increase in
  number of cells.
• Stationary phase. Rate of death rate of cell
  division. Due to limiting factors (nutrients,
  waste accumulation)
• Death phase. Rate of death greater than rate of
  cell division. Nutrients exhausted, wastes
  buildup. Population declines.

44

• List and define each of the growth phases.
• Draw and label a growth curve

45
Counting Cells Methods of Counting

• Plate count Spread sample of liquid culture on
  plate and allow colonies to grow (overnight) and
  count colonies
• Since one cell forms one colonies, the number of
  colonies the number of cells originally put on
  the plate.

• Microscope Count cells in grid under microscope.

46
Counting Cells Methods of Counting

• Turbidity (cloudiness)
• More cells in liquid culture more cloudy
• Spectrophotometer is an instrument that measures
  light passage.
• The more cells present, the less light registers.

47
Counting Cells Dilution

• If there are too many cells to count as colonies
  or under microscope
• Serially dilute sample of liquid culture so can
  count more easily.
• Plate several dilutions, incubate, count
  colonies.
• Then multiply by the dilution to get actual count
  of original, undiluted sample.

48
Counting Cells Concentration

• If there are too few cells to count as colonies
  or under microscope
• Filter so cells can be made more concentrated
• Cells will be trapped on filter but liquid will
  pass through.
• Transfer filter to plate, incubate, count
  colonies.
• Can be done to count bacterial cells in stream,
  lake or drinking water.

49
Counting Cells Applications

• Clinical. i.e. Assessing urinary infection.
• Testing water Drinking, lake/stream
• Testing food

50

• What are the three methods for counting cells?
• Why does counting colonies produced by plating a
  liquid culture allow one to determine the number
  of cells in the liquid culture?
• Why might it be necessary to dilute or
  concentrate a liquid culture before plating?
• How might a cell culture be concentrated?
• Briefly explain how turbidity is used to
  determine numbers of cells.