Microbiology Techniques Lecture 9

Lecture 9: Theory Behind Practical 2

• The lecture covers various microbiology techniques, including streaking bacteria, enumerating bacteria, and personal hygiene experiments.

• Laboratory Procedures:

  • Streaking bacteria onto selective media (Baird Parker and McConkey Agar) using the streak plate technique.
  • Enumerating bacteria using the serial dilution method followed by the spread plate technique.
  • A personal hygiene experiment.

• Online Resources:

  • The lecture mentions additional online material, including demonstrations related to:
    • Most probable number (MPN) test for water sampling.
    • Membrane filtration.
    • The dye reduction test.

Selective and Differential Media

• Selective Media:

  • Designed to encourage a specific group of bacteria while inhibiting the growth of others.
  • The choice is based on the desired organism's growth characteristics and the isolation source.
  • Example selective media: MacConkey agar and Baird-Parker agar.

• Differential Media:

  • Allows for the distinguishing of various bacterial species based on their colony morphology (size, color, or effect on the environment).
  • Example selective media that are also differential media: MacConkey agar and Baird-Parker agar.

McConkey Agar

• Description:

  • Routinely used for isolating and differentiating coliforms and other intestinal bacteria in diverse samples (e.g., water, dairy products, biological specimens).
  • Lactose is the carbon source; peptone is a nitrogen source; bile salts act as a selective agent.
  • Bacteria that ferment lactose develop a pH change. The pH indicator (neutral red) results in a localized dark red colouration.

• Composition: Specific ingredient amounts are provided in the slides (e.g. 17g Peptone, 10g Lactose, 1.5g Bile salts)

Baird-Parker Agar

• Description:

  • Primarily used for detecting and enumerating staphylococci, especially coagulase-positive species like S. aureus.
  • Widely used in food safety applications.
  • Contains lithium chloride and tellurite to inhibit other organisms, and additives (glycine and pyruvate) enhance Staphylococcus growth.
  • Coagulase-positive organisms often exhibit a clear halo around the colonies.

• Composition: Specific ingredient amounts are provided in the slides (e.g. 10g Casein Peptone, 5g Meat Extract, 1g Yeast Extract)

Streaking Bacteria

• Method for isolating two provided microorganisms
• Used on MacConkey agar and Baird-Parker agar
• Need to ensure no cross-contamination
• Incubate the plates at 37°C for 48 hours then photographs are to be submitted and documented

Enumeration of Bacteria - Serial Dilution

• Procedure:
  • Dilute the sample (e.g., milk) in a series of 10-fold dilutions; each dilution is added to a new tube
  • Spread the dilutions on nutrient agar plates.
  • Incubate to allow the bacteria to grow into isolated colonies on the agar plates.
  • Count the colonies on plates with appropriate colony counts
  • Calculate the original bacterial concentration

Spread Plate Technique

• Method for separating and isolating desired bacterial colonies and calculating bacterial concentrations.
• A sterile glass spreader is used to spread the sample homogenously on the agar.

Enumeration of Bacteria in Milk Sample

• Procedure:
  • Prepare a 10-fold dilution series of the milk
  • Spread diluted samples on nutrient agar plates
  • Incubate the plates to allow bacteria to grow into colonies
  • Count the bacterial colonies
  • Calculate the concentration per milliliter in the original milk sample.

Example Calculation

• Method: To determine bacterial concentrations using the number of colonies grown on plates after serial dilutions. This provides concentration in the original sample.

Personal Hygiene Experiment

• Goal: Assess the transfer potential of microorganisms from surfaces, particularly via tissue paper and hand-washing practices.

Dye Reduction Test

• Methylene blue as an indicator:
  • Color change indicates redox status—reduction changes the colour.
  • Used to evaluate milk quality.

Membrane Filtration

• Procedure:
  • Filter a sample through a membrane with appropriate microscopic pore size.
  • Bacteria are trapped on the filter.
  • Transfer the filter to a suitable growth medium.
  • The advantages include a rapid method, can be used in field settings, and produces reliable results

Most Probable Number (MPN) Test

• Description:
  • A statistically based method used for determining the concentration of coliform bacteria, indicators of faecal contamination, in water samples.
  • It involves incubating dilutions (10 ml, 1 ml, 0.1 ml) in a series of tubes containing a suitable growth medium.
  • An MPN index is calculated, yielding the concentration of bacteria in the sample.

This lecture delves into essential microbiology techniques such as streaking bacteria on selective media, enumerating bacteria, and performing personal hygiene experiments. It also discusses various methods like the Most Probable Number (MPN) test and membrane filtration. Perfect for students looking to enhance their practical microbiology skills.