Litmus Milk Test Principle, Procedure, Result

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What is the Litmus Milk Test?

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

  • The Litmus Milk Test requires a litmus milk medium, which is a liquid medium that differentiates bacteria based on numerous metabolic events in litmus milk, such as fermentation, reduction, clot formation, digestion, and gas generation.
  • Based on the ability of enteric bacilli to decrease litmus, this medium is primarily used to distinguish between members of the genus Clostridium and other Gram-negative bacilli.
  • It is also used to establish and maintain lactobacilli cultures.
  • Litmus Milk is the most valuable medium in the dairy business because it is a dependable measure of bacterial activity in milk.
  • Litmus is an excellent indicator of acidity and alkalinity, and its oxidation-reduction potential is beneficial in milk media because it is less hazardous to bacteria than bromo cresol purple.
  • The addition of 1% w/v dextrose and/or 5% w/v yeast extract to Litmus Milk stimulates the growth of certain organisms that cannot grow in pure Litmus Milk. BIS recommends the current formulation for the detection of germs responsible for food poisoning.
  • In addition to the carbohydrate lactose, milk contains the proteins casein, lactalbumin, and lactoglobulin. Therefore, an organism may exhibit one or more of the following metabolic features in litmus milk, each of which is species-specific and aids in the identification of bacteria.
  • Lactose fermentation, litmus reduction, clot formation, peptonization (digestion), and gas generation are the various metabolic functions.
  • Litmus Milk is a medium used to distinguish between several metabolic activities.
  • Additionally, Litmus Milk is useful for maintaining and propagating lactic acid bacteria. To identify Clostridium perfringens in water, inoculate newly heated tubes of Litmus Milk with varying amounts of water and heat at 80 °C for 10 to 15 minutes to eliminate non-spore-forming organisms.
  • At 35°C, check for a positive Stormy Clot reaction every 24 hours for up to five days.
  • You can induce anaerobiosis in Litmus Milk by adding a small hot iron nail or 0.1 mg of reduced iron.
  • Substrate is skim milk, which is digested by several bacterial species in various ways. The bacterial actions can be classified as follows:

Acid Reaction

  • Pink to red colour of milk – Fermentation of lactose and/or dextrose in milk.
  • Acid coagulation – Lactic acid production, producing a casein curd in clear watery fluid.
  • Stormy clot – Gas formation in coagulated casein curd.

Alkaline Reaction

  • Blue color of the milk – Formation of basic amines or ammonia due to proteolysis.
  • Alkaline coagulation with a soft blue clot – Paracasein formation from casein by enzyme rennin.
  • Peptonization – Digestion of casein, evident by clearing of the medium and dissolution of the clot.

Redox Reaction

  • Decolorized medium (Similar to newly autoclaved Litmus milk) – Reaction of Litmus in the depths of the tube by reductase enzymes, resulting in oxygen removal to generate decolorized leucolitmus compound)
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Principle of Litmus Milk Test

  • Litmus is an indication of both the medium’s pH and its oxidation-reduction potential. Milk enables lactose, casein, lactalbumin, and lactoglobulin.
  • Under acidic conditions, litmus is red, but it is blue under alkaline conditions.
  • When an organism ferments lactose, lactic acid is generated, which imparts a pinkish-red hue to the medium.
  • Nitrogenous substrates used by bacteria in milk produce ammonia and a violet-blue hue.
  • The bacteria that decrease litmus take the oxygen, leaving behind a white base. Certain species produce proteolytic enzymes that hydrolyze milk proteins, resulting in clot formation.
  • Casease production results in peptonization, which leads to the digestion of the clot, as seen by the medium’s aqueous clearance.
  • Gas generation may be the consequence of lactose fermentation (CO2 and H2).
  • An acid clot is broken apart by an abundance of gas, resulting in a phenomenon known as stormy fermentation. This is possible with a few anaerobic Clostridium species.

Purpose of Litmus Milk Test

  • This test identifies bacteria on the basis of numerous metabolic responses in litmus milk, including fermentation, reduction, clot formation, digestion, and gas generation.
  • Lactic acid bacteria can also be grown in litmus milk.
  • This test measures an organism’s capacity to digest litmus milk.

Media Required

This test utilises Litmus Milk media. Litmus Milk is a medium for maintaining lactic acid bacteria and measuring the bacterial effect on milk.

Composition of Litmus Milk

IngredientsGms / Litre
Skim milk powder100 g
Sodium sulphite0.500
Final pH ( at 25°C)6.8±0.2

Preparation of Litmus Milk

  1. Suspend 101 grammes in 1 litre of distilled water while continually agitating.
  2. Dispense 10 ml portions into 15 x 150 mm tubes and autoclave at 15 pounds of pressure (121 degrees Celsius) for 5 minutes.

Procedure of Litmus Milk test

  1. Inoculate a 24-hour broth culture with four drops of litmus milk medium.
  2. 24-48 hours of incubation at 37°C in air at room temperature.
  3. Observe alkaline reaction (litmus goes blue), acid reaction (litmus turns pink), indicator reduction, acid clot, rennet clot, and peptonization daily for seven days.
  4. During the observation time, multiple changes may occur; therefore, it is necessary to document all changes.

Control strains

  • Escherichia coli ATCC 25922: Acid
  • Clostridium innocuum ATCC 1450: Alkaline, no clot, no digestion
  • Clostridium perfringens ATCC13124:  Acid, clotting, digestion
  • Pseudomonas aeruginosa ATCC27853— Peptonization (clearing)

Result Interpretation of Litmus Milk Test

Result Interpretation of Litmus Milk Test
Result Interpretation of Litmus Milk Test
  • Pinkish-red: Acid reaction, lactose fermentation
  • Blue:  Alkaline reaction, no fermentation, organisms attack nitrogenous substances in the medium
  • A clot or curd formation: Milk protein coagulation
  • Digestion (peptonization):  Milk protein digested, clearing of medium
  • CO2 and H2:  Bubbles in medium, a clot may be broken up
  • Purplish-blue:  No fermentation, no change of the indicator
  • White:  Reduction of litmus to a white  base by enzyme reductase
  • Stormy Fermentation:  Acid clot disrupted by an abundance of gas production
  • Negative test: color and consistency remain the same

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.

The appearance of Litmus Indicator

Pink or mauve colorAcid reaction (A)Escherichia coli
Blue medium or blue band at topAlkaline reaction (K)Alcaligenes faecalisPseudomonas aeruginosa
Purple (identical to uninoculated control)No change (NC)
White color (lower portion of medium)Reduction of litmus (R). It is recorded as decolorized
Result Interpretation of Litmus Milk Test
Result Interpretation of Litmus Milk Test – A, Acid reaction. B, Alkaline reaction.
C, No change. D, Reduction of indicator. E, Clot. (Note separation of
clear fluid from clot at arrow.) F, Peptonization.
Result Interpretation of Litmus Milk Test
Result Interpretation of Litmus Milk Test

Uses of Litmus Milk Test

  • Litmus Milk is a medium used to distinguish between several metabolic activities.
  • It is used for lactobacilli cultivation and maintenance.
  • It is used to differentiate bacteria on the basis of numerous metabolic events in litmus milk, such as fermentation, reduction, clot formation, digestion, and gas generation.
  • The most useful medium in the dairy business is the Litmus milk test, which is a reliable indicator of bacterial action on milk.
  • The Litmus milk test is an excellent diagnostic of acidity and alkalinity, and its oxidation-reduction potential is beneficial in milk media since it is less hazardous to bacteria than bromocresol purple.


  • The Litmus milk test is a complex medium that can yield diverse outcomes. Due to this, the findings of litmus milk testing might be extremely unreliable. Therefore, you should only use the litmus milk test as a confirming test and not as a decisive one.
  • A clot formation is reported simply as “clot” and cannot be distinguished from a curd formation in this medium.
  • Positive and negative control organisms can be employed to demonstrate the many possible reactions in Litmus Milk Medium.
  • For the conclusive identification of microorganisms, Litmus medium reactions are insufficient and should be supplemented with further assays.

Keynotes on Litmus Milk Test

  • For the quality control of Litmus Milk Medium Test, the following bacteria might be utilised: Clostridium perfringens ATCC13124 fermentation gas output, Clot formation and acid: Lactobacillus acidophilus ATCC11506 Pseudomonas aeruginosa ATCC27853 — cleared by peptonization.
  • Since the Litmus milk test is not specific, it must be supplemented by further tests for the definite identification of organisms.
  • If Clostridium is suspected or anaerobiosis is desired, immediately after inoculation, add sterile reduced iron (e.g., iron powder, nails, paper clips, metal filings) to the tube or pour a layer of sterile mineral oil over the medium surface.


  • Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition

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