BIO 215
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LAB PROTOCOL: LITMUS MILK TEST
INTRODUCTION AND PURPOSE: Litmus milk is an interesting diagnostic medium that consists of skim milk,
peptone, and a color indicator dye (litmus). The color indicator is PINK when acidic, BLUE when alkaline, and
PURPLE when neutral. Do you remember litmus paper from when you were in elementary school? Same thing!
It is important to remember that milk contains protein (mostly casein) and sugar (mostly lactose). In order to
grow in litmus milk medium, an organism must first be able to break down protein or lactose, with exoenzymes.
Then, other things can happen. Milk may separate, forming a solid portion (CURD) and liquid portion (WHEY).
Here is a summary of all that go on and be observed. It is easiest to think of each reaction separately
when making observations:
1.LACTOSE FERMENTATION: Lactose is broken into glucose and galactose using beta-galactosidase.
Glucose is then FERMENTED and a major end product is lactic acid. In a lactic acid solution, pH is close to 4.
2. GAS FORMATION: If gas is a product of the fermentation, you will see small fissures in the solidified milk, or
a STORMY appearance if large amounts are produced and curd is completely disrupted.
3. CURD (OR CLOT) FORMATION: There are two distinct types of curd that may form as milk separates.
a) ACID CLOT - acid formation causes milk to separate. The clot is solid and sticks to sides of glass.
b) RENNET CURD - some organism make enzymes like rennin that breaks the protein into smaller
pieces. A solid curd is formed, one that separates from the liquid greyish 'whey.'
4. ALKALINE REACTION: If mainly PROTEIN is being consumed and broken down into shorter peptide chains,
alkaline products (ammonia) will be made.
5. PROTEOLYSIS: Complete breakdown of peptides into individual amino acids: this is called proteolysis. It
frees up amino acids which can then be utilized. Ammonia is also produced and solution becomes alkaline
(deep blue in upper portion of tube). As protein continues to be broken down, Remaining material liquifies and
may turn yellowish or brownish, producing very dark whey.
6. LITMUS REDUCTION: Finally - some organisms can 'reduce' the litmus itself. This causes the litmus to turn
WHITE. It happens, is characteristic of each species, but is not metabolically significant for the organism, it is
just a side reaction in the medium that occurs.
PROTOCOL: Inoculate each litmus milk tube with loopful of organism. Incubate 48 hours with loosened caps.
Examine results. Examine again in 5-7 days.
ATTACHED SHEET DIAGRAMS POSSIBLE REACTIONS, AND SHORTHAND NOTATION FOR RECORDING
THEM.
Biology 215, Ruth A. Gyure
Western CT State University