Litmus Milk Test: Principle, Procedure, and Results

Litmus milk is an undefined milk-based medium consisting of skim milk and the pH indicator azolitmin (litmus). Azolitmin is pink at pH 4.5 and blue at pH 8.3. Between these extremes, it is purple. In addition to being a pH indicator, litmus is an Eh (oxidation-reduction) indicator. Reduced litmus is white and oxidized litmus is purple.

Litmus milk medium differentiates microorganisms based on their ability to metabolize litmus milk. Four essential reactions in litmus milk are; lactose fermentation, reduction of litmus, casein coagulation, and casein hydrolysis. The combination of these reactions yields different results, each of which can be used to differentiate bacteria.

Litmus milk test
Litmus milk test
A, Acid reaction. B, Alkaline reaction. C, No change. D, Litmus reduction. E, Clot (note separation of clear fluid from clot at the arrow.) F, Peptonization. (Image source: Ref-1)

Litmus milk is mainly used to differentiate members within the genus Clostridium, and it also differentiates Enterobacteriaceae from other Gram-negative bacilli. Litmus milk also cultivates and maintains cultures of lactic acid bacteria.

Principle

The litmus milk test determines an organism’s ability to metabolize litmus milk. Four basic reactions occur in Litmus Milk: lactose fermentation, reduction of litmus, casein coagulation, and casein hydrolysis.

  1. Lactose fermentation: Fermentation of lactose is demonstrated when the litmus turns pink as a result of acid production. If sufficient acid is produced, casein in the milk is precipitated and forms an acid clot. Acid clots solidify the medium and can appear pink or white with a pink band at the top depending on the oxidation-reduction status of litmus. Acid clots can be dissolved in alkaline conditions. Fissures or cracks in the clot are evidence of gas production. Heavy gas production that breaks up the clot is called stormy fermentation. Stormy fermentation of litmus milk is the characteristic of Clostridium perfringens.
  2. Litmus reduction: Some microorganisms reduce litmus.  If litmus becomes reduced during lactose fermentation, it will turn the medium white in the lower tube portion where, the greater reduction rate is.
  3. Casein coagulation: Some bacteria produce proteolytic enzymes (caseases) such as rennin, pepsin, or chymotrypsin that coagulate casein and make a curd. A curd differs from an acid clot because it will not dissolve in alkaline conditions.  With some organisms, the curd shrinks, forming a straw-colored fluid called whey at the surface.
  4. Casein hydrolysis: Some bacteria produce caseases that hydrolyze casein, causing the milk to become straw-colored and resemble turbid serum. Bacteria that can only partially digest the casein produce ammonia (NH3), which raises the pH of the medium and turns the litmus blue. The formation of a blue or purple ring at the top of the clear fluid or bluing of the entire medium indicates an alkaline reaction.

Media:

Skim milk provides nutrients for growth, lactose for fermentation, and protein in the form of casein.

IngredientsAmount (Gram/Liter)
Powdered skim milk100 g
Litmus0.5 g
Sodium sulfite0.5 g
pH of the medium: 6.8

Quality Control

  • Fermentation: Clostridium perfringens (ATCC13124): gas production
  • Acid: Lactobacillus acidophilus (ATCC11506): clot formation
  • Peptonization: Pseudomonas aeruginosa (ATCC27853): Clearing

Procedure

  1. Take a litmus milk medium and inoculate with four drops of 24-hour broth culture.

  2. Incubate at 35°C to 37°C in ambient air.

  3. Observe daily for seven days and record all changes.

    Look for alkaline reaction (litmus turns blue), acid reaction (litmus turns pink), litmus reduction, acid clot, rennet clot, and peptonization. Multiple changes can occur over the observation period.

Litmus Milk Results and Interpretations

The appearance of Litmus Indicator

ColorInterpretationOrganism
Pink or mauve colorAcid reaction (A)Escherichia coli
Blue medium or blue band at topAlkaline reaction (K)Alcaligenes faecalis/ Pseudomonas aeruginosa
Purple (identical to uninoculated control)No change (NC)
White color (lower portion of medium)Reduction of litmus (R). It is recorded as decolorized
Litmus milk medium
Litmus milk medium
(From left to right: Alkaline reaction, uninoculated control, alkaline reaction (K), digestion of peptone (D), acid and gas production (G), acid (A), and acid clots (AC). The clear fluid on the surface of the D and G tubes is a mineral oil used to make the medium anaerobic. (Image source: Ref-2)

Appearance of Milk

Consistency of MilkInterpretationOrganism
Coagulation or clotFormation of clot
Semisolid and not pink; clear to gray fluid at the topCurd (C)
Pink and solid (white in the lower portion if the litmus is reduced); clot not movableAcid clot (AC)Lactobacillus lactis
Fissures in clotGas (G)Clostridium acetobutylicum
Clot is broken apartStormy fermentation (S)
Dissolution of the clot with transparent, grayish, watery fluid and a shrunken, insoluble pink clot.Digestion of peptone D (acid reaction)Clostridium sporogenes
Dissolution of the clot with grayish, watery fluid and a clear, shrunken, insoluble blue clotPeptonization* P (alkaline reaction)

*Peptonization: The conversion of a protein into peptones under the influence of the enzyme pepsin.

Limitation

Litmus media reactions are not specific and should be followed up with additional tests for the definitive identification of microorganisms.

References and further readings

  1. Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
  2. Leboffe MJ and Pierce BE. A Photographic Atlas for the Microbiology Laboratory, 5th Edition (2021). Morton Publishing Company.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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