Enumeration of Microorganisms in Foods

Enumeration of Microorganisms in Foods

• Microorganisms in raw, processed, and processed food ingredients are examined to determine their presence, types, number, and products.
• Knowing this information helps estimate shelf life, identify spoilage, or determine causation of foodborne illness.

Methods of Enumeration

• Direct Enumeration Methods: Directly count microorganisms.

  • Direct Counting Methods:
    • Direct microscopic count (DMC): Observe and count cells under a microscope. Useful for high cell counts (10⁶/ml) like in dairy products
    • Direct counting on membrane filters: Use filters with small pores (typically 0.45µm) to retain bacteria for counting.
  • Culture-Based Methods: Grow microorganisms in media to form colonies
    • Plate count method: Count colonies grown on solid media, using serial dilutions to obtain appropriate counts (30-300 colonies). Preferred for heat-sensitive bacteria.
    • Pour plate method: Mix sample with molten agar, pour the mixture onto plates, and let it solidify for incubation.
    • Spread plate method: Spread diluted sample over solid agar.
    • Most probable number (MPN) technique: Estimate organisms in low numbers. Repeated inoculation into media (3 or 5 tubes) to evaluate probable number from absence or presence of growth.

• Indirect Enumeration Methods: Measure products produced from microorganisms.

  • Alternative methods (chemical and physical):
    • Dye reduction test
    • Electrical methods
    • ATP determination
    • Rapid Methods
      • Immunological methods
      • DNA/RNA methodology

Study of Microorganisms in Foods by Conventional Methods

• Enumeration of microbes in foods typically involves direct counting of all organisms or allowing them to grow into colonies and counting those.

Direct Counting Methods (DMC)

• Direct microscopic count (DMC):
  • A simple method for observing microorganism directly in the food/culture.
  • Smear of food specimen on slides, staining, and microscopic observation (oil immersion objective).
  • Only useful for high-microbial counts
  • Can assess cell morphology.
  • Can be aided by fluorescent probes for improved efficiency.
• Direct counting on membrane filters:
• Concentrate bacteria on polycarbonate filter (0.45µm).
• Stain, count the retained bacteria.
• More efficient for samples with lower bacterial counts as less sample is required.
  • Can use florescent dyes (like acridine orange) to increase efficiency (DEFT)

Culture Based Methods (Plate Count Method)

• Principle: Viable bacteria multiply and form visible colonies. Counting colonies provides information on bacterial cell numbers.
• Procedure:
  • Homogenize, dilute, plat the sample on the chosen media, incubate at appropriate temperature.
  • Count colonies for colony-forming units (CFUs)
• Factors affecting SPC:
  • Sample method
  • Distribution of microorganisms in food
  • Nutritional adequacy of plating media
  • Incubation temperature/time
  • Presence of other microorganisms
• Types of methods
  • Pour plate method
  • Spread plate method

Most Probable Number (MPN) Technique

• MPN technique useful for estimating microbial numbers when low counts are anticipated.
• Involves inoculating multiple tubes w/ different dilutions, then incubating to identify growth.
• Consult MPN tables to determine probable number from growth.
• Relatively easy and comparable results.
• Used for detection of low numbers of bacteria.