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Litmus Milk Test (WINTER 2014 version) CAUTION: Litmus milk is a complex medium that can produce a variety of results. Because of this, litmus milk can give quite unreliable results (i.e., the results often give negative results when something positive should happen). Thus, you would be advised to use litmus milk as a confirmatory test, but not a definitive test (except as a last resort). Introduction Litmus milk is a complex medium that can potentially distinguish among many species of bacteria. Litmus milk has several components that can be metabolized: lactose (milk sugar); casein (milk protein); and litmus (a pH indicator that is purple to blue at neutral to alkaline pH and pink under acidic conditions). If lactose is fermented, the solution should turn pink. If gas is produced during fermentation, you may be able to see bubbles or cracks in the milky medium (but this is often difficult to observe). If lactose is not fermented and proteins are metabolized instead, the solution will become alkaline and turn blue in color. If casein is digested, the milk will coagulate to form a curd (a solid). Casein may be metabolized all the way down to individual amino acids. This process, called peptonization, results in a clear (not milky) liquid that is usually brown in color. Finally, the litmus may be reduced and become colorless. The culture will then look milk white. Results Possible Results Description No Change Alk Appearance of 7-Day Cultures Purple Alkaline Blue A Acid Pink Ac Acid with Curd Acid, somewhat solid Acid with Curd, Litmus reduction Pink Top, White Bottom, somewhat solid Acid and Gas with curd, Litmus reduction Pink Top, White Bottom, broken or cracked solid Alkaline with Peptonization Clear brown top, brown bottom Alkaline with Peptonization, Litmus Reduction Clear brown top, brown bottom Ac-R AcG-R AlkP AlkP-R Methods 1. Obtain two Litmus Milk broths from the back shelf. 2. Inoculate one broth with cells from your stock unknown culture. Leave the other broth uninoculated (this will act as a negative control). 3. Incubate both broths at an appropriate temperature (whichever is a better growth temperature for your organism). 4. Observe your cultures over the next several days. Changes should be visible in five to seven days. Photograph Figure 1. Several different results in Litmus milk Figure 2. A solid curd. Notice that the culture does not flow when the test tube is tilted.