Microbiology Practical 2 Theory

Lecture 9: Theory Behind Practical 2

• The practical sessions include:
  • 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.

Extra Online Material

• Most Probable Number (MPN) test for water sampling
• Membrane filtration
• The Dye Reduction test

Selective and Differential Media

• Many media are developed to select or differentiate between organisms.
• Selective media: favour the growth of certain organisms while suppressing the growth of others.
  • The choice depends on the organism and the sample's nature.
• Differential media: show differences between bacterial species through colony morphology like size, colour, or influence on the surrounding environment.
  • Baird-Parker agar and MacConkey agar are selective and differential examples.

McConkey Agar

• A common selective and differential media for isolating and differentiating coliforms and other intestinal bacteria in water, dairy, and biological specimens.
• Key ingredients: Lactose, peptone, bile salts.
• Lactose fermenters produce a localized drop in pH, which, with neutral red as an indicator, creates dark red colonies.
• This agar allows the isolation and differentiation of lactose-fermenting, bile salt-tolerant bacteria.
• Composition:
  • Peptone: 17 g/L
  • Polypeptone: 3 g/L
  • Lactose: 10 g/L
  • Bile salts: 1.5 g/L
  • Sodium Chloride: 5 g/L
  • Agar: 13.5 g/L
  • Neutral red: 0.03 g/L
  • Crystal violet: 0.001 g/L
  • Distilled water: 1 L
  • Final pH = 7.1

Baird-Parker Agar

• Used for detecting and enumerating staphylococci, particularly coagulase-positive organisms like S. aureus.
• Widely used in food safety applications.
• Ingredients include: lithium chloride and tellurite to inhibit other organisms, glycine, and pyruvate to enhance staphylococcal growth.
• Tellurite reduction produces a grey-to-black colour and coagulase-positive organisms show a clear halo around the colonies.
• Composition:
  • Casein Peptone: 10 g/L
  • Meat Extract: 5 g/L
  • Yeast Extract: 1 g/L
  • Lithium Chloride: 5 g/L
  • Glycine: 12 g/L
  • Sodium Pyruvate: 10 g/L
  • Agar: 15 g/L
  • Final pH (at 25°C): 6.8 ± 0.2

Streaking Bacteria

• Use the streak plate method to inoculate MacConkey and Baird Parker agar, with provided organisms, while avoiding cross-contamination.
• Incubate at 37°C.
• Take photos and note observations after 48 hours for practical report.
• Each organism is streaked on half of each plate. Example organisms: Staphylococcus aureus and Escherichia coli.

Enumeration of Bacteria (Serial Dilution Method)

• Add sample (milk or otherwise) to an appropriate diluent (diluting solution - often water).
• Repeat n dilution steps adding fresh diluent following each step.
• Spread different dilutions across the agar plates.
• Incubate the inoculated plates.
• Count the colonies on the plates.
• Calculate the concentration of bacteria in the original sample by taking the count, the plating factor, and the dilution factor into account.

Spread Plate Technique

• Isolate colonies of interest visually.
• Count bacteria colonies.
• Use a sterile glass spreader to spread bacteria evenly on the surface of the agar plates to make colonies visible.

Enumerating Bacteria in Milk

• Create a 10-fold dilution series of milk samples.
• Use Nutrient Agar plates to identify bacteria in 10⁻², 10⁻³, and 10⁻⁴ dilutions.
• Calculate the number of bacteria per millilitre in the original sample.

Personal Hygiene Experiment

• The experiment involves the transfer of microorganisms through tissue paper.
• It demonstrates the concept of hand washing preventing microorganism spread.
• Different plates are handled by gloved and ungloved hands to observe differences.

Dye Reduction Test

• Methylene blue is used as a redox indicator for Streptococcus lactis.
• If Streptococcus lactis is present, the blue color will reduce indicating presence and quality of milk.
• Poor quality milk will decolorise within two hours; high-quality milk retains color.

Membrane Filtration

• Uses cellulose acetate or cellulose nitrate filters with pore sizes of 0.45 - 0.2 µm to separate bacteria.
• Trapped bacteria can be viewed after transfer to an appropriate medium.
• Quick, useful for field testing and higher reproducibility of results.

Most Probable Number (MPN) Method

• Coliform bacteria in faeces/water can be determined via MPN.
• MPN method involves diluting samples to different concentrations.
• Inoculating these dilutions into different tubes.
• Incubating at appropriate temperature.
• Counting positive samples in each dilution series
• Using a table to assess the total amount of coliform bacteria in 100 mL of sample.

This quiz covers the theoretical principles behind Practical 2 in microbiology, including methods for streaking bacteria and enumerating them through various techniques. It also delves into selective and differential media, particularly Baird Parker and McConkey agar, for studying bacterial growth. Explore the concepts of personal hygiene tests and additional water sampling methods as well.