Identify Enterococcus by Using Litmus Milk Decolorization Test

The litmus milk decolorization test is a rapid inexpensive technique to assist in the identification of enterococci. It is based on the ability of most strains of Enterococcus species to reduce litmus milk by enzyme action as shown by decolorization of the litmus.

Note: Enterococci can also be identified using an Aesculin Hydrolysis Test 

Principle Behind the Litmus Milk Decolorization Test

A heavy inoculum of the test organism is incubated for up to 4 hours in a tube containing litmus milk. Reduction of the litmus milk is indicated by a change in colour of the medium from mauve to white or pale yellow.

Requirement

Litmus Milk Reduction-   No. 50

Method

1. Using a sterile loop, inoculate 0.5 ml of sterile litmus milk medium with the test organism.

Important: A heavy inoculums of the test organism must be used. Scraping the loop three times across an area of heavy growth is recommended.

2. Incubate at 35 – 37oC for up to 4 hours, examining at half hour intervals for a reduction reaction as shown by a change in colour from mauve to white or pale yellow (compare with the positive control). 

Note: The incubation time should not be more than 4 hours because some strains of viridans streptococci will reduce litmus milk with prolonged incubation.

Results

White or pale yellow-pink colour …….   Suggestive of Enteroccocus

No change or a pink colour …………..  Probably not Enterococcus

Controls

Positive control:           Enterococci species

Negative control:                   Viridans streptococci

Note: The work of Schierl and Blazevic* demonstrated that up to 83% of Enterococcus could be identified by the rapid litmus milk reduction test. A negative result can be checked by culturing the organism in aesculin broth and examining daily for up to 7days of aesculin hydrolysis as shown by a blackening in the medium, Enterococci hydrolyze aesculin.

Litmus milk decolorization test

Aesculin Hydrolysis Test to Identify Enterococci

This test can be economically performed using a Rosco bile aesculin tablet (Bile esculin 404 – 21). The tablets are available from Rosco Diagnostica. They have good stability (3 – 4y).

The test can be performed by placing a tablet on a blood agar plate inoculated with the test organism and incubating is at 35 – 37oC overnight. A positive test is indicated by the tablet and colonies around it turning black/grey. A negative test is shown by the tablet remaining white and no change in colour of the colonies. A zone of inhibition may appear around the tablet.

Alternatively, the test can be performed by making a dense suspension of the test organism in 0.25 ml of physiologically saline in a small tube, adding a tablet, and incubating at 35 – 37oC for 4 hours (for overnight). A positive reaction is shown by a black/grey colour in the medium.

Note: An aesculin hydrolysis can also be performed by incubating the test organism on bile aesculin agar but this medium is expensive and only available in 500g pack size.

Litmus milk is a medium that differentiates bacteria based on multiple metabolic reactions. The medium contains skim milk, pH indicator azolitmin (which is red at acidic conditions and blue at alkaline conditions), lactose and casein. Bacterial fermentation of lactose yields lactic acid which changes the color of the indicator to pink. Nitrogenous substrates used by some bacteria decrease litmus by taking oxygen, leaving a white base.

Litmus milk decolorization test

Inoculation

Litmus milk is a differential medium used to distinguish microorganisms that metabolize various milk substrates into varied metabolic end products. It contains the milk sugar lactose, the milk protein casein and a pH indicator litmus. Different organisms transform these substrates by a variety of reactions, such as fermentation, reduction of litmus, clot formation and digestion. It is primarily used to differentiate members of the genus Clostridium and other Gram-negative bacilli, such as Enterobacteriaceae and lactic acid bacteria. It is also used to cultivate and maintain cultures of these bacilli.

The medium is prepared by adding powdered skim milk and litmus to a volume of 1000 ml sterile water and mixing well. It is then dispensed into 15 x 150 mm tubes and sterilized by autoclaving at 121 °C for five minutes. The tubes should be stored at 2 °C to 8 °C and protected from light, excessive heat and moisture.

To check for the presence of an acidic or alkaline substance, a piece of red and blue litmus paper is torn into thirds and dipped into the sample. If the red litmus paper turns pink, the material is acidic; if it remains blue, it is alkaline.

Skim milk provides nutrients for growth, the lactose for fermentation and the protein in the form of casein. If the test organism grows in the medium, it can cause different reactions. When organisms ferment the lactose, they produce lactic acid that changes the medium color to a pink-red. Bacteria that reduce the litmus release ammonia and change it to a blue or purple color. Organisms that can digest casein clot the sample, leaving a straw-colored ring at the top of the medium and a clear fluid below. Organisms that cannot digest the casein produce proteolytic enzymes that hydrolyze the protein, resulting in the formation of an insoluble curd (rennet curd) or peptonization.

Observe the medium daily for seven days and record all changes. Look for an alkaline reaction (litmus turns blue), an acid reaction (litmus turns pink), indicator reduction, acid clot, rennet clot and peptonization. It is important to note that litmus milk reactions are not specific and should be followed up by additional tests for the definitive identification of the microorganism.

Incubation

Milk is a natural medium for the growth of microorganisms. It contains the milk protein casein, the sugar lactose, vitamins, minerals and water. To distinguish between different types of bacteria, a pH indicator, the oxidation-reduction indicator litmus, is added to the medium. Microorganisms metabolize the milk substrates in the medium to produce varied metabolic responses. The reactions include lactose fermentation, litmus reduction, clot formation, peptonization (digestion) and gas production.

Powdered skim milk, litmus and sodium sulphite are mixed to prepare the medium. The medium is inoculated with 4 drops of a 24-hour broth culture and incubated at 35deg-37degC for 7 days. Observe the medium every day for alkaline reaction (litmus turns red), acid reaction (litmus turns blue), indicator reduction, clot formation, casein coagulation and peptonization. Many changes can occur over the observation period, but only one or two of the most probable reactions will aid in the identification of the organism.

Certain bacteria such as Clostridium perfringens ferment the milk sugar lactose, producing acids like CO2 and H2. This results in the precipitation of a curd that solidifies the medium. The acid clot may also develop tracks or fissures in it, indicating heavy gas production. A heavy gas production that breaks apart the clot is called stormy fermentation, and it is characteristic of Clostridium perfringens.

Other organisms decompose sulphur containing amino acids in the medium to produce H2S and other volatile compounds. The medium may appear black or brown if this occurs. Some organisms also produce indole from tryptophan, causing it to become a dark green color in the medium. This reaction can be detected by adding a drop of Kovac’s reagent to the medium. If the reagent appears red, there is a positive reaction.

Some bacteria reduce the amount of acid that they produce by reducing the activity of their enzymatic complement. This causes the medium to become straw colored and resemble turbid serum. Other bacteria produce proteolytic enzymes that hydrolyze the casein in the milk, resulting in the formation of an acid clot or rennet clot that is observed as a watery clearing of the medium.

Litmus Milk Decolorization Test

The Litmus milk decolonization test is a quick inexpensive technique to assist in the identification of enterococci. The test relies on the ability of most strains of Enterococcus to reduce a milk-based medium by enzyme action. This decolorization can be observed by a change in the color of the litmus milk medium from mauve to white or yellow. This change in the color of the medium can be used to differentiate between the different species of Enterococcus and also between these and other lactic acid bacteria.

The medium used in this test is a special type of milk-based medium that contains the pH indicator azolitmin. The azolitmin turns pink when the medium is at pH 4.5 and blue when the medium is at pH 8.3. Litmus is also an Eh (oxidation-reduction) indicator; it turns white or milk colored when it accepts hydrogen from a reducing agent such as a protein.

To perform the litmus milk decolorization test, a tube of litmus milk is inoculated with a single drop of an overnight Todd Hewitt broth culture or a sterile suspension from growth on a plate. The medium is then incubated at 35C for seven days. The tubes are examined daily for several metabolic reactions including lactose fermentation without and with gas, reduction of litmus, clot formation, acid reaction, and peptonization.

Certain lactic acid bacteria produce proteolytic enzymes that hydrolyze the protein casein in the milk. This leads to a coagulation of the milk and a watery clearing of the medium, similar to the appearance of milk in an egg wash. In some cases, the bacterium may also ferment other carbohydrates such as sucrose and agar.

Litmus milk also contains arginine, which if reduced by an organism causes the medium to turn alkaline. This is a common feature of many enterococci strains as they have the enzymes to oxidize arginine. Another way that Enterococcus can be identified is by the fact that the organisms hydrolyze the bile pigment aesculin. The presence of this pigment can be confirmed by examining a smear on a slide fixed with crystal violet and then a smear made on agar that has been stained with Gram’s iodine.

Results of the Litmus milk Decolorization Test

The Litmus milk decolorization test differentiates bacteria based on numerous metabolic responses in the medium, including lactose fermentation, reduction, acid production, clot formation, digestion and gas generation. The test is primarily used to distinguish members of the Clostridium genus, but can also be used to cultivate and maintain Lactobacilli cultures.

The medium contains skim milk, the pH indicator azolitmin and the oxidation-reduction indicator litmus. The medium turns pink when the pH is above 4.5, and blue when the pH is below 5.0. The medium also contains the major milk substrates lactose and the milk proteins casein, lactalbumin and lactoglobulin. Bacteria metabolize these substrates in a variety of ways, depending on their enzymatic complement.

Four essential reactions in the Litmus milk medium are; lactose fermentation, reduction of litmus, casein coagulation and casein hydrolysis. Observe for changes such as a blue color change in the coagulated casein curd, acid production with a shrunken, watery clot or fissures in the clot that indicate heavy gas generation (stormy fermentation).

When lactose is fermented by bacteria, it produces carbon dioxide and hydrogen, which cause a pink to red coloration of the medium. In addition, some anaerobic species of Clostridium produce acids that can break apart the clot, resulting in the formation of a stormy fermentation appearance.

Clostridium perfringens is a common organism that causes this reaction. Likewise, the presence of ammonia in the clot or a dark violet-blue hue in the medium indicates proteolytic activity. The presence of peptonization demonstrates the ability of certain bacteria to hydrolyze milk protein into soluble components such as amino acids and peptides.

The bile aesculin hydrolysis test is a reliable indicator of the presence of enteric bacterivores. The test is simple to perform and can be done with an aesculin tablet, available from Rosco Diagnostica. The test can be performed daily for up to seven days, and if the result is positive, the presence of enteric bacterivores can be confirmed by using other tests.