Counting Microorganisms

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Counting Micro-organisms

• Counting elephants is easy counting bacteria is
  not quite as easy!

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Counting Micro-organisms

• A single tiny drop of nutrient broth incubated
  overnight may contain 5 000 000 cells this
  is a lot to count.
• 1cm3 may contain 108 cells.
• In order to estimate numbers it is necessary to
  dilute the sample.

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Counting Micro-organisms
SERIAL DILUTION 1ml into 9ml 110 dilution
conc. 10-1 10-2
10-3 10-4 10-5
10-6
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Counting Micro-organisms

• It is possible to dilute a cell suspension in
  known steps in order to plate a specific range of
  cells. This set of plates contain a 1 to 10
  dilution series.

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TOTAL COUNTS

• Direct counts
• The haemocytometer is a specialized microscope
  slide used to count cells.
• The centre portion of the slide has etched grids
  with precisely spaced lines.

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TOTAL COUNTS

• To get an accurate count there should be between
  40 and 70 cells in a 1 mm square.
• If not you dilute or concentrate the cell
  suspension as necessary .

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TOTAL COUNTS
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TOTAL COUNTS

• Indirect counts
• These depend on the effects of the organisms to
  estimate their numbers.
• As organisms grow they make the nutrient broth
  turbid.
• This turbidity can be measured with a
  colorimeter

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TOTAL COUNTS

• The more organisms the greater the optical
  density of the solution.

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TOTAL COUNTS

• Indirect count
• The Coulter counter is a probe which measures the
  change in conductivity of a solution as a
  bacteria passes through a narrow gap.

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TOTAL COUNTS

• Problem with INDIRECT COUNTS and DIRECT COUNTS is
  that they can not tell living cells apart from
  dead cells
• Advantage of INDIRECT COUNT is that the process
  can be automated

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VIABLE COUNTS

• Count only those cells capable of growing
• Viable counts can be accomplished by such
  techniques as pour plating.
• Assumption each viable cell gives rise to a
  colony.

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VIABLE COUNTS

• Advantages
• The method can be made to be very sensitive.
• One can count subsets of the population
• Disadvantages
• Colony-forming units may underestimate cell
  numbers because of clumping or chains of cells.
• Counts require at least a few hours, usually
  overnight, for incubation.