Litmus Milk Test: Introduction, Principle, Composition, Preparation, Procedure, Result Interprettation, Uses and Keynotes

Litmus Milk Test: Introduction, Principle, Composition, Preparation, Procedure, Result Interprettation, Uses and Keynotes

Introduction of Litmus Milk Test

Litmus Milk Test needs a litmus milk medium and it is a liquid medium that helps to differentiate bacteria based on various metabolic reactions in litmus milk, including fermentation, reduction, clot formation, digestion, and the formation of gas. This medium is used primarily to differentiate members within the genus Clostridium and it also differentiates Enterobacteriaceae from other Gram-negative bacilli based on the ability of enteric bacilli to reduce litmus. It is also applied to cultivate and maintain cultures of lactobacilli.

Principle of Litmus Milk Test

Litmus acts as both an indicator of pH and of the oxidation-reduction potential of the medium. Milk facilitates lactose, casein, lactalbumin, and lactoglobulin. In an acid condition litmus is red while under alkaline conditions it is blue.  When an organism ferments lactose, lactic acid is produced that changes the medium to a pink-red color. Nitrogenous substrates using bacteria in the milk release ammonia and yields a purplish-blue color. Bacteria that reduce litmus cause the oxygen to be removed, leaving a white base. Proteolytic enzymes produced by certain organisms hydrolyze milk proteins that result in clot formation. Casease formation results in peptonization which causes digestion of the clot, exhibited by a watery clearing of the medium. The end result of lactose fermentation may be gas formation (CO2  and H2). Plenty of gas breaks up an acid clot causing a reaction referred to as stormy fermentation. This may happen with certain anaerobic Clostridium species.

Composition of Litmus Milk Test

Ingredients- Gms / 100 ml

Skim milk powder:  10.0

Litmus: 0.05

Sodium sulfite:  0.05

Final pH (at 25°C) 6.8±0.2

 Preparation of Litmus Milk Test

Suspend 10.1 grams in 100 ml distilled water.

Agitate the mixture continuously.

Dispense 10 ml amounts into 15 x 150 mm tubes.

Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 5 minutes.

Avoid overheating.

 

Storage and Shelf life of Litmus Milk Test

  • Store at 2-8ºC  and away from direct light.
  • Media should not be used if there are any signs of deterioration  (discoloration), contamination.
  • The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Test Requirements for Litmus Milk Test

Procedure of Litmus Milk Test,

  1. Inoculate test organisms (4 drops of a 24-hour broth culture or pure culture of 18-24 hours well-isolated colony of the test isolate) into a litmus milk medium and incubate in the appropriate atmosphere at 37°C in ambient air for 24-48 hours.
  2. In the case of Clostridium, anaerobiosis is desired, add sterile reduced iron like iron powder or nails or paper clips or metal filings to the tube or pour a layer of sterile mineral oil over the medium surface immediately after inoculation.
  3. Observe daily for 7 days for alkaline reaction (litmus turns blue), acid reaction (litmus turns pink), indicator reduction, acid clot, rennet clot, and peptonization (clearing).
  4. Multiple changes can happen over the observation period and record all these changes.

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

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)

Uses of Litmus Milk Test

  1. Litmus Milk is a differential medium used to determine different metabolic Functions.
  2. It is used for the cultivation and maintenance of lactobacilli.
  3. It is applied to differentiate bacteria based on various metabolic reactions in litmus milk, including fermentation, reduction, clot formation, digestion, and the formation of gas

Keynotes on Litmus Milk Test

  • For quality control of Litmus Milk Medium Test following bacteria can be used for the following purposes- Fermentation: Clostridium perfringens ATCC13124 — gas production, Acid: Lactobacillus acidophilus ATCC11506— clot formation and Peptonization: Pseudomonas aeruginosa ATCC27853 — clearing.
  • Litmus milk test is not specific and thus it should be followed up with additional tests for definitive identification of organisms.
  • If Clostridium is suspected or anaerobiosis is desired, 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 immediately after inoculation.

Further Readings on Litmus Milk Test

  • https://assets.fishersci.com/TFS-Assets/MBD/Instructions/IFU61274.pdf
  • https://www.himedialabs.com/TD/M609.pdf
  • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092029/
  • https://catalog.hardydiagnostics.com/cp_prod/product/CatNav.aspx?oid=6939&prodoid=K34
  • https://www.sigmaaldrich.com/Litmus Milk
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