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Litmus Milk Test Procedure

In this litmus milk test procedure post we have briefly explained about litmus milk test principle, objectives, requirements, litmus milk test procedure, uses and limitations of litmus milk test.

Remel Litmus Milk Medium is a liquid medium for determining the activity of bacteria on milk and maintaining lactic acid bacteria in qualitative investigations.

Lactose fermentation, casein hydrolysis and coagulation, gas production, and litmus reduction are all utilised to distinguish bacteria in Litmus Milk Medium.

It’s very good for separating Clostridium species and distinguishing Streptococcus bovis (no growth) from Streptococcus equinus (growth). Because litmus is a reliable indicator of effect upon milk, it is also used to cultivate lactic acid bacteria associated with dairy products.


Litmus is a pH indicator as well as an indicator of the medium’s oxidation-reduction (Eh) potential. Lactose, casein, lactalbumin, and lactoglobulin are all found in milk. Litmus is red in an acid solution and blue in an alkaline solution. When an organism ferments lactose, lactic acid is generated, and the medium turns pink-red.

Some bacteria in the milk react with the nitrogenous substrates, producing ammonia and giving the milk a purplish-blue colour. The oxygen is eliminated by organisms that decrease litmus, leaving a leuco (white) base. Proteolytic enzymes, which are produced by certain organisms, hydrolyze milk proteins, causing them to coagulate.

Peptonization occurs as a result of casease synthesis, causing the clot to be digested, as seen by a watery clearing of the medium. Lactose fermentation may result in the creation of gas (CO2 and H2). Stormy fermentation occurs when an acid clot is broken apart by a large amount of gas. Certain anaerobic Clostridium species may cause this.



1. Incubator


1. Skim Milk

2. Demineralized Water

3. Sodium sulphite


1. Skim milk powder: 100.000, Litmus: 0.500, Sodium sulphite: 0.500, Final pH ( at 25°C) 6.8±0.2

Litmus Milk Test Procedure

1. 4 drops of a 24-hour broth culture inoculated into litmus milk medium, Incubate for 24-48 hours at 37°C in room temperature.

2. Alkaline reaction (litmus goes blue), acid reaction (litmus turns pink), indicator reduction, acid clot, rennet clot, and peptonization should all be observed every day for 7 days.

3. Multiple changes may occur during the observation period, and all changes should be recorded.


litmus milk

Control , pink=acid , white=reduction , stormy fermentation , blue=alkaline

litmus milk

Positive Reaction/Test

litmus milk

Acid pH – Pink to red color, Alkaline pH – Purplish- blue color, Reduction – White, Acid curd -Hard curd with clear supernatant, Digestion – Dissolution of clot with clear, grayish, watery fluid, Rennet curd – Soft curd followed by peptonization , Gas production – Bubbles in coagulated milk, Negative test  – Color and consistency remain same.


1. Litmus milk test is the most useful medium in dairy industry as it is a reliable indicator of bacterial action on milk.

2. Litmus milk test is a good indicator of acidity, alkalinity and its oxidation-reduction potential is useful in milk media with lower toxicity to microorganisms than bromocresol purple.


1. Litmus milk test is a complex medium that can produce a diversity of results. Because of this, litmus milk test can give quite unreliable results. Thus, you would be advised to use litmus milk test as a confirmatory test but not a definitive test.

2. A clot formation is simply recorded as “clot” and cannot clearly differentiate between a clot and curd formation in this medium.


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