Monitoring growth

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Monitoring growth

• Learning objective
• To be able to describe ways of growing bacteria
  and ways of monitoring their growth

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Questions
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Similarities

• Lag phase.
• Little increase in cell number
• Individual bacteria may be increasing in size
• Enzymes may be being synthesised to utilise the
  nutrient mediummax.2
• Log phase.
• The population shows exponential phase
• Nutrient supply is not a limiting factor
• Stationary phase.
• The population growth slows down
• The number of new cells formed is balanced by the
  number of cells dying
• Limiting factors, such a nutrient supply and
  metabolic wastes have started to influence
  further increase in population size max 2

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Differences

• Lag phase for lactose alone longer nutrient less
  readily available
• Growth of population less than with glucose
  fewer cells and longer to increasemax.2
• Growth with lactose and glucose gives a greater
  population
• Has a second lag around 100-120 minute and then
  increases again
• Uses glucose first as growth curve similar to
  glucose alone and then uses lactosemax 3
• Death phase is occurring for glucose alone as all
  the energy source/glucose has run out.1

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Growing Microbes

• Microbes for experiments can be obtained from
  natural sources (e.g. soil, food, skin, water,
  air), or bought from suppliers in agar slopes.
• You don't need much, since a dot may contain
  millions of viable cells, each of which could
  grow into a whole colony.

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Medium

• The mixture that the microbes are grown
  (cultured) on.
• The medium must contain all the nutrients
  (sugars, minerals, proteins) needed for the
  microbes to grow.
• By adjusting these nutrients, the medium can be
  made selective for one type of microbe.
• Culture media can be made up by mixing together
  known amounts of specific chemicals (a defined or
  synthetic medium), or they can be made from a
  natural source such as boiled meat or yeast
  extract, which generally contains the nutrients
  required by most microbes (an undefined or
  complex medium).

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Nutrient medium

• A cheap general-purpose complex medium used for
  most school experiments.

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Broth

• A liquid medium (i.e. without agar).

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Agar

• Agar is mixed with a liquid medium to make a
  solid medium, which is very useful to observe,
  separate and store bacteria cultures.
• A solid medium in a petri dish is known as an
  agar plate, while a solid medium in a Universal
  bottle is called an agar slope.
• Agar is actually a polysaccharide extracted from
  seaweed.
• It melts at 41C (so can be incubated at 37C
  without melting), is reasonably transparent, and
  is not broken down by microbes, so it remains
  solid.

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Aseptic transfer

• Also called aseptic technique.
• The transfer of a sample of bacteria from one
  vessel to another.
• This is the most common and basic technique and
  is used in almost all micro-biological
  experiments.
• The bacteria are usually transferred using a wire
  or glass inoculating loop, which can carry a tiny
  volume of culture (10 µl) or a scraping of cells
  from an agar plate.
• Larger volumes are transferred using a sterile
  syringe or pipette.

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Key words

• Inoculate -
• Incubate -
• Culture -
• Colony -
• Streak Plate
• Lawn -

• To add few cells to a medium, so that they may
  grow.
• To leave a culture to grow under defined
  conditions.
• A growth of microbes in a medium. The culture can
  be pure (one species of microbe) or mixed (many
  species).
• A visible growth of bacteria on an agar plate
  containing many millions of cells.
• A method of inoculating an agar plate with
  bacteria so that the bacteria are gradually
  diluted.
• A layer of bacteria growing on the surface of an
  agar plate.

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Measuring the Growth of Microbes

• Growth of cells in a liquid culture is generally
  measured by simply counting the number of cells.
• There are various techniques for doing this. Some
  give total cell counts, which include both living
  and dead cells, while others give viable cell
  counts, which only include living cells.

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Haemocytometer

• This counts the total cells by observing the
  individual cells under the microscope.
• This is reasonably easy for large cells like
  yeast, but is more difficult for bacterial cells,
  since they are so small.
• The cell counter (or haemocytometer) is a large
  microscope slide with a very accurate grid drawn
  in the centre.
• The grid marks out squares with 1 mm, 0.2 mm and
  0.05 mm sides.
• There is an accurate gap of 0.1 mm between the
  grid and the thick coverslip, so the volume of
  liquid above the grid is known.
• The number of cells in a known small volume can
  thus be counted, and so scaled up. The units are
  cells per cm3 .

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For example

• A 0.2 mm square has an average of 80 cells in a
  1000x dilution
• Volume above square 0.2 x 0.2 x 0.1 0.004 mm³
• 80 cells in 0.004 mm³ 20 000 cells per mm³ in
  the diluted suspension
• which is 20 000 x 1000 2 x 107 cells per mm³ of
  undiluted suspension
• or 2 x 1010 cells per cm³

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Turbidometry

• This technique also counts the total cells.
• It is quicker than using a haemocytometer, but
  less accurate.
• A sample of the liquid culture is placed in a
  cuvette in a colorimeter, and the absorbance of
  light is measured.
• The greater the concentration of the cells, the
  more cloudy or turbid the liquid is, so the more
  light it scatters, so the less light is
  transmitted to the detector.
• A wavelength of 600nm is normally used.
• Although the absorbance scale of the colorimeter
  is used, light is not actually absorbed by the
  cells (as it is by pigment molecules), but
  scattered.
• If the same sample is counted in a haemocytometer
  and its absorbance measured, than a calibration
  curve can be plotted.
• From this calibration curve the concentration of
  cells can be read off for any absorbance.

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Dilution Plating

• This technique counts viable cells.
• A sequence of ten-fold dilutions is taken from
  the original culture flask, using sterile medium.
  
• This is called a serial dilution, and allows
  large dilutions to be made using small volumes.
• From each dilution a 1 cm³ sample is taken and
  spread evenly onto an agar plate.
• Each viable cell in the sample will multiply and
  grow into a colony.
• In most of the samples there will be too many
  colonies to count, but in one of the dilutions
  there will be a good number (20-200) of
  individual colonies.
• From this we can calculate the concentration of
  viable cells in the original culture.

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For example

• Suppose there were 83 colonies in the x10 000
  dilution agar plate.
• How many viable cells would there have been per
  cm³ in the original culture?
• There were 83 viable cells in the 1 cm³ sample of
  the x10 000 dilution
• So there were 83 x 10 000 8.3 x 105 cells per
  cm³ in the original culture

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Questions