Lab 2 - Indicator Microorganisms

What are indicator microorganisms?

• Present in HIGHER numbers than pathogenic bacteria; their concentrations are correlated with the presence of pathogenic bacteria

• Some are directly associated with product safety and some with product quality

What are coliforms?

A group of bacteria that are:

• Gram -

• Aerobic or facultative anaerobic

• Non-spore forming

• Rods

• Ferment lactose and produce acid and gas at 32-37℃

What is violet red bile agar?

• Peptone and yeast extract provide carbon, nitrogen, and vitamins to support and stimulate the growth of bacteria

• Bile salts and crystal violet inhibit most gram-positive microorganisms

  • Bile acid conjugate and precipitate when pH is lower

• Lactose will be fermented by coliforms and reduce the pH

• For facultative anaerobes (but grow more efficiently in aerobic conditions)

• REQUIRES: over-layer method

How do you calculate the LOD?

LOD = (1 CFU*Dilution factor)/volume plated

What is the most probable number (MPN)?

• MPN technique is a serial dilution test to estimate the concentrations of microbes of interest when their concentrations are too low for plating and direct enumeration

• It’s an estimated number

  • Estimation based on positive tube numbers for each dilution factor

  • Many dilution factors can be made, but only choose three dilution factors for the final calculation

  • Durham tubes positive result - turbid and gas production

• EXAMPLE: 3/3 of the 10 mL, 3/3 of the 1 mL, 2/3 of the 0.1 mL, 3/3 of the 0.01 mL, 0/3 of the 0.001 mL, 0/3 of the 0.0001 mL

  • Use numbers 3,2,3

How do you calculate the dilution correction factor?

If no MPN table fits the needs based on the number of samples tested, you can convert the MPN number identified to DCF

DCF = lowest dilution from the table (or most concentrated sample)/lowest dilution used in your series (or most concentrated sample)

• EX: 0.1/10 = 0.01 * MPN/100 mL

  • 0.8 cell/100 mL —> 8 cell/1000 mL (present like this)

Why use slants over agar?

Slants provide both aerobic and anaerobic conditions by doing a streak and stab inoculation

MacConkey agar

Changes red at a pH below 6.8

What are the Four main genera of coliform?

Escherichia, Enterobacter, Klebsiella, and Citrobacter

How does plating without agar work?

Use Petri Film - dehydrated media, use liquid suspension or rehydrate media, no prep ahead, no need for refrigeration, very small amount of sample needed, shows gas production