Experiment 2: Standard Plate Count of Milk

What is SPC?

• agar plate method of estimating bacterial populations

• Official method for determining sanitary quality of milk, widely used.

How does SPC work?

• Serial dilutions of milk prepared

• Aliquots are spread over the agar surface (spread plate) or mixed in the molten agar (pour plate)

What does each colony that arose mean?

Means they came from multiplying during incubation of one single bacterial cell that was fixed in the media when it was plated.

What is the total number of colonies equivalent to?

Number of viable microorganisms in the diluted sample/

How can we determine the number of bacteria in the original sample (undiluted)?

Counting number of colonies formed and accounting for the appropriate dilution factor.

Why are serial dilutions prepared?

To reduce the microbial population sufficiently so the sample that we are to obtain will produce separate and distinct colonies

What are some advantages of SPC?

• Only counts viable organisms

• accurate use with milk having low bacterial content

What are some disadvantages of SPC?

• only detects organisms that are able to use particular nutrients supplied and under certain incubation conditions

What is TTC (2,3,5-triphenyltetrazolium)?

Dye used to count microbial colonies in solid culture media

Characteristics of SPC

• Colourless when oxidized

• Red when reduced

• Live orgs reduce TTC by enzymatic actions

What is formazan?

Live orgs reduce TTC by enzymatic actions and form formazon

• bact cells turn red

• Only viable bacteria can make formazan and be red when against food product.

Where is formazan kept?

In granules in bacterial cells

Why is TTC added to SPC agar?

To make living bacteria more visible for counting (we count the red cells)

What does a high CFU count in pasteurized milk indicate?

• poor milk quality

• low CFU = longer shelf-life

Why do we need to mix the milk and agar well before allowing it to solidify?

To evenly disperse colonies on the surface and agar.