After reading this article you will learn about the requirements and procedure of litmus milk reactions.
Milk sugar lactose, the milk proteins casein, lacto-albumin and lacto-globulin are the milk substrates that can be transformed by microorganisms, bringing about changes in pH. In order to distinguish the metabolic changes, a pH indicator, the oxidation reduction indicator litmus is incorporated in the medium. The various biochemical changes that occur are lactose fermentation, gas production, litmus reduction, curd formation, proteolysis and alkaline reaction.
1. Broth cultures of E.coli, Streptococcus lactis, Alcaligenes faecalis and Pseu- domonas aeruginosa.
2. Litmus milk broth:
Skimmed milk powder – 100.0 g
Litmus – 0.075g
Water – 1000.0 ml
Autoclave at 15 lbs (121°C) for 15 minutes
3. Inoculation loop.
4. Glass marking Pencil.
5. Test tubes.
6. Bunsen flame.
Inoculate different organisms in litmus milk broth and incubate at 37°C for 24 to 48 hours.
Organisms that can utilise lactose as carbon and energy source will degrade it by producing β-galactosidase releasing lactic acid making the purple litmus turn pink due to a fall in pH to pH4.
When lactose is fermented, C02 and H2 are evolved which can be observed as separation of curd or as tracks or fissures in the curd from which gas, bubbles out to the top.
This anaerobic process involves bio-oxidations that occur in the absence of molecular oxygen. This oxidation of lactose produces lactic acid, butyric acid, C02 and H2. The H2 that is removed is accepted by litmus which turns from purple to white or milk coloured.
Two distinct types of curds are formed when organisms grow in litmus milk. Based on the biochemical mechanism, curds are either acid or rennet. Acid curd is formed due to lactic or other organic acids which cause milk protein, casein precipitate as calcium caseinate forming an insoluble clot.
This clot is hard and will not retract from the walls of the tube. Invert the tube, the clot remains immobile. Rennet curd is formed when organisms produce an enzyme renin that acts on casein to form paracasein which is converted to calcium paracaseinate in presence of calcium. This insoluble curd is soft semi-solid and will flow out slowly when the tube is tilted.
Organisms which cannot utilise lactose in milk produce proteolytic enzymes, hydrolyse casein in milk and utilise it. This leads to evolution of large amounts of NH3 leading to an alkaline pH which turns litmus deep purple in the upper region of the tube. The medium produces a translucent brown whey like appearance, due to hydrolysis of proteins to amino acid.
When casein is partially degraded, short polypeptide chains with simultaneous release of alkaline end products result. This alkaline reaction makes the colour of litmus unchanged.