Food Microbiology - Litmus milk test

Food Microbiology

1. WELCOME YOU ALL

2. Vivekanandha arts and science college for women Veerachipalayam – 637 303, sankagiri , Salem dt. , tamilnadu India. Affiliated to Periyar University - Salem

3. Department of microbiology Subject : Food microbiology Subject incharge : Dr. R.Dineshkumar Assistant professor, Department of Microbiology, VIASS , Sankagiri . Submitted by : K.Geetha III – Bcs.Microbiology , Department of Microbiology, VIASS , Sankagiri .

4. Topic Litmus Milk Test

5. Overview : Introduction Objective Principle Media Procedure Result interpretation Positive test Negative test Limitation Uses

6. Introduction : Milk is an excellent medium for the growth of microorganisms because it contains the milk protein, casein, the sugar lactose, vitamins, minerals and water. Litmus milk is a Milk – based medium used to distinguish between different species of bacteria The lactose (milk sugar), litmus (pH indicator) and casein (milk protein) contained within the medium can all be metabolized by different types of bacteria.

7. Objective : To differentiate microorganisms based on the various metabolic reaction in litmus Milk, including fermentation, reduction, clot formation, digestion, and the formation of gas.
Litmus Milk is used primarily to differentiate members within the genus Clostridium .
It differentiates Enterobacteriaceae from other Gram – negative Bacilli based on the ability of enterics to reduce litmus.
Litmus Milk also is used to cultivate and maintain cultures of lactic acid bacteria.

8. Principle : Litmus, a pH indicator is incorporated in the medium for the detection of production of acid or alkali and oxidation – reduction activities. Fermentation of lactose in milk with the production of lactic acid is demonstrated when the litmus turns from blue to pink as a result of acid production.The accumulation of acid acts on casein, resulting in the formation of clots due to the precipitation of casein as calcium caseinate . With some organisms, the curd shrinks, and whey is formed at the surface.

9. Principle : Acid curd is identified by the clot which remains immobile when the tube is inverted. Some bacteria produce rennin enzymes that act on casein and in presence of calcium ions form paracaseinate that is insoluble and is called rennet curd.Unlike acid curd, it is a semisolid clot that will flow slowly on tiltins the tube. Some bacteria possess proteolytic enzyme caseinase and hydrolyzed casein. Casein hydrolysis results in the release of large quantities of ammonia that makes the medium alkaline with a foul smell, the litmus turns into a purplish to blue with further incubation, an opaque cleaning of milk occurs as the casein is hydrolyzed to peptides and amino acids. The opaque liquid supernatant which is a whey – like appearance turns brown in colour . Additionally, some organisms reduce litmus, in which case the medium becomes colourless in the bottom of the tube.

10. Media : Powdered skim milk (100g) Litmus (0.5g) Sodium sulphite (0.5g) Per 1000ml pH – 6.8

11. Procedure : Inoculate litmus milk medium with 4 drops of a 24 – hours broth culture. Incubate at 37 degree C in ambient air for 24 – 48 hours. Observe daily for 7 days for alkaline reaction (litmus turns blue), acid reaction ( litmus turns pink), indicator reduction, acid clot, rennet clot, and peptonization . Multiple changes can occur over the observation period and record all changes.

12. Result Interpretation Pink colour Acid reaction Pink and solid; clot not movable Acid clot Fissures in clot Gas Clot broken apart Stormy fermentation White colour Reduction of litmus Semisolid & not pink; clear to gray fluid at top Curd Clarification of medium; loss of “body” Digestion of peptone; peptonization Blue medium or blue band at top Alkaline reaction No change None of the above reaction

13. Results: Positive test : Acid pH : pink to reduce colour Alkaline pH : purplish – blue colour Reduction : white Acid curd : hard curd with clear supernatant (whey) Digestion : dissolution of the clot with clear, grayish, watery, fluid and a shrunken, insoluble pink Clot Rennet curd : soft curd followed by peptonization (alkaline pH, supernatant brown) Gas production : bubbles in coagulated milk

14. Positive test :

15. Positive test :

16. Negative test : Colour and consistency remain the same.

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

18. Uses : The litmus milk test differentiates members of the Enterobacteriaceae from other Gram – negative Bacilli based on the enterics ability to reduce litmus It is commonly used to differentiate members within the genus Clostridium . It mainly aids in the identification and differentiation of Enterococcus , and Lacticacid bacteria . The media may also be used to grow lacticacid bacteria .

19. Reference https://microbenotes.com Https://microBiologyinfo.com