	Laboratory Investigations in Microbiology
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Chapter 28: Milk microbiology

One of nature's most elegant and nutritious food sources is milk. Produced by mammals as the sole source of nutrition for the newborn, milk contains crucial nutrients such as protein, sugar, and fat; vitamins; minerals such as calcium; and even antibodies! Not surprisingly, this nutritious fluid also contains many bacteria, including Lactobacillus and Lactococcus. Bacteria in milk may be beneficial for intestinal health (e.g Bifidobacterium and Lactobacillus), contribute to the eventual spoilage of milk through fermentation, and may cause disease (e.g. Salmonella) if present. To prevent disease and slow down spoilage, milk must be pasteurized before being sold. Pasteurization heats milk to temperatures of ~ 63 or 72C, effectively killing Gram-negative bacteria. However, depending on the initial bacterial count, spoilage organisms may still be present in varying numbers. To test the quality of milk, one can measure the rate at which oxygen is being consumed in a milk sample - the more rapidly oxygen is used, the poorer the quality of milk. It is also possible to determine bacterial numbers in milk samples using the same methods we used for food testing.

Lastly, we will also examine some of the different effects bacteria have on milk by examining their reaction in litmus milk (photo on the right) - a complex differential medium containing milk sugar and proteins. Some bacteria ferment lactose (acid), which may also produce a curd. Others do not ferment and thus produce a basic reaction. Some even hydrolyze casein (proteolysis), producing a brownish clear fluid full of amino acids. This photo shows (L to R) Streptococcus lactis (Acid + Curd), Bacillus subtilis (Proteolysis), Alcaligenes faecalis (Base) and E. coli (Acid).

Materials per lab group

4 Litmus milk broths
2 sterile test tubes
Milk samples
Methylene blue dye
4 sterile Petri plates
2 melted VRBA deeps
2 melted TSA deeps

Procedures

Litmus milk test

Inoculate 4 Litmus milk broths with 100 ul of the cultures provided (EC, AF, SL, PV)
After incubation, examine the Litmus milk tubes for the production of acid, base, curd, and proteolysis. Record your data.

Bacterial counts

Obtain 15 ml of milk samples (A and B) in the sterile test tubes by gently pouring milk from the sample flask into your test tube (1/2 full).
Pipette 1 ml of a milk sample into a tube of melted VRBA; mix. Pour into sterile Petri plate and allow to harden. Repeat for melted TSA.
Repeat for your second milk sample
Incubate for 48 hrs
After incubation, count the number of colonies for each milk sample on the VRBA and TSA plates. Record your data.

Methylene blue test

Add 1 - 2 drops of methylene blue to the remaining ilk samples in each test tube. Mix and incubate.
Check on the methylene blue samples occasionally (once in 8-12 hours, and again after 24 hours) during the next 24 hours. Record approximately how many hours it took for each sample (A and B) to turn white.