Bacterial Enumeration

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Bacterial Enumeration
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Enumeration

• Goal
• To determine the number of bacteria per
  milliliter of sample
• Cells/ml
• Methods
• Direct
• Staining and counting under microscope
• Heterotrophic plate count
• Count colonies and assume each colony represents
  1 cell in original culture
• Indirect estimate based on another property
• MPN
• Statistical estimate based on growth patterns in
  media
• Spectroscopy
• Estimate based on turbidity (or transmittance of
  light)

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Enumeration of Bacteria

• Methods
• Viable Live cells only
• Heterotrophic plate count
• MPN
• Total Live and dead
• Spectroscopy
• Staining w/ microscopy

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Direct Count Indirect Count

• Advantage
• More accurate
• Disadvantage
• More time
• Uses
• To get accurate counts of cells in clinical or
  environmental samples

• Advantage
• Quicker to do
• Disadvantage
• Less accurate
• Uses
• To get quick and dirty count in controlled
  circumstances

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Viable Total

• When you are concerned about only living and
  metabolically active organisms
• EX) CLINICAL SAMPLES

• When you need to know a number of all organisms
• EX) Microbial Ecology

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Spectrophotometer

• Measuring turbidity or optical density of a
  solution by measuring transmittance of light
• OD mathematical way of expressing turbidity
• As OD increases turbidity increases and
  Transmittance decreases
• As transmittance increases
• Incident light into culture at specific
  wavelength
• 540, 600 or 660 nm
• Some light scatters more scatter with more cells
• The non-scattered light exits and is read by a
  photocell
• T

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OD

• Optical Density
• OD2-(log T)
• For our E. coli culture
• 1 OD 1.25 x 106 cells/ml
• This conversion is different depending on
  species!

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Guidelines for Spec 20

• Use clean cuvettes
• Wipe with KimWipes before inserting
• Zero your spec with a blank before you measure.
• Solutions should be diluted first
• Readings are not accurate when the culture is too
  turbid

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Heterotrophic Plate Count

• Spread a sample evenly across a plate, incubate
  24 hours, and count colonies
• Each colony represents 1 single cell that was in
  original sample
• These require dilutions prior to plating
• The plate should have between 30 and 300 colonies
• lt30 will give statistically inaccurate count
• gt300 TNTC (colonies too clumped)

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Once you count the plate you can calculate number
in original