Detection of Microorganisms II

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Detection of Microorganisms II By Ms. Atittaya
T. 452-8203 Mr. Monthien S. 442-2411
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Detection

• 4 Basic methods used for total numbers are
• Standard Plate Counts (SPC) for viable cells
• The most-probable numbers (MPN) for viable cells
  (statistical determination)
• Dye-reduction techniques for viable cells that
  posses reducing capacities
• Direct Microscopic Counts (DMC) for both viable
  and nonviable cells

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Alternative Plating Techniques

• Spiral Plating
• ? Using Spiral plater
• - Continuously distributes the liquid inoculum
  on the surface of a rotating agar plate
• - Results in depositing the sample in an
  Archimedes spiral
• - After inoculation colonies will grow
  intensively near the center of the plate while
  fewer grow toward the edge
• - Approved by Association of Official Analytical
  Chemists (AOAC)

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Spiral Plating
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Spiral Plating

• Advantages
• - Less agar is used
• - Fewer plates and pipettes are required
• - Little training is required for its operation

• Disadvantages
• - Food particles may block the dispensing stylus
• - It is more suitable for use of liquid food
• - It is expensive

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Membrane Filters

• Membranes with a pore size will retain bacteria
  (generally 0.45 ?m)
• Then the membrane is placed on an agar plate
• Collected microorganisms are viewed and counted
• Suitable for samples that contain low numbers of
  bacteria
• Example of membrane filters is Hydrophobic Grid
  Membrane Filter (HGMF)

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Membrane Filter

• Advantage
• More than 100ml samples can be tested.
• Effective and acceptable technique. Used to
  monitor drinking water in government
  laboratories.
• one of a few methods that will allow the
  isolation and enumeration of microorganisms.

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HGMF

• Advanced by Sharpe and Michaud
• Widely used to enumerate microorganisms from a
  variety of food products
• 1,600 wax grids on a single membrane filter
  (hydrophobic wall to prevent the spread of
  colony)
• Can detect as few as 10 cells/g
• Can be used to enumerate all cfus including
  indicator organisms
• Approved by AOAC for total coliforms, fecal
  coliforms, and salmonellae

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The Most-Probable Number

• Presumptive test
• Determine by gas produced
• Confirmed test
• Streak on EMB agar plate
• -Completed test
• Tranferring to lactose broth and NA slant

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The Most-Probable Number
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The Most-Probable Number

• Advantages
• It is relatively simple
• Results from one laboratory are more likely than
  SPC results to agree with those from another
  laboratory
• Specific groups of microbes can be determined by
  use of appropriate selective and differential
  media
• It is the method of choice for determining fecal
  coliform densities

• Disadvantages
• Large amount of glassware is required
• The lack of opportunity to observe the colonial
  morphology of the organisms
• Its lack of precision

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Dye Reduction

• Estimate the number of viable organisms in
  suitable products
• Methylene blue and resazurin
• Add prepared supernatants of food to standard
  solution
• Methylene blue blue ? white
• Resazurin blue ? pink or white
• Time used is inversely proportional to the number
  of organisms in the sample
• A long history of use in the dairy industry,
  especially to measure microbial quality from raw
  milk

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Dye Reduction

• Advantages
• Simple, rapid, and inexpensive
• Only viable cells actively reduce the dyes

• Disadvantages
• Not all organisms reduce the dyes equally
• Not applicable to food specimens that contain
  reductive enzymes

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Direct Microscopic Count

• Smears of food specimens or cultures onto a
  microscope slide
• Stain with an appropriate dyes
• View and count cells with the aid of microscope
• Widely used in dairy industry

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Direct Microscopic Count

• Advantages
• It is rapid and simple
• Cell morphology can be determined
• Fluorescent probes for improved efficiency

• Disadvantages
• Results depends on each analyst
• Both viable and nonviable are counted
• Food particles are not always distinguishable
  from microorganisms
• Some cell may not take the stain well

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Questions ??
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Questions ??

• What is the method used for determining both
  viable and nonviable cells?
• - Direct microscopic count
• Which method is popular in testing the presence
  of coliforms?
• - The most-probable number method
• What is the relationship between time used and
  number of microorganisms in dye reduction method?
• - Inversely proportional

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THANK YOU