Laboratory Exercise #20 Litmus Milk Reactions1 Purpose: To examine the various ways microorganisms can hydrolyze the

he sugar and proteins in milk There are many nutrients in milk that can serve as substrates for bacterial growth. Milk contains the proteins casein, lactoalbumin, and lactoglobulin. The sugar that is present is lactose. There are also abundant vitamins present. By analyzing the end products formed, it is possible to determine which components of the litmus milk are being used by bacteria as an energy source. To assist in differentiating the metabolic changes that occur, the pH indicator litmus is added to the medium. The following reactions may be observed: Lactose fermentation Litmus reduction Gas formation Proteolysis Alkaline reaction Organisms that produce the enzyme -galactosidase (i.e. have the Lac Operon) are capable of fermenting lactose to lactic acid. The lactic acid can be detected as the litmus indicator turns pink when the pH of the medium falls to about 4. Litmus also serves as an artificial electron acceptor; excess hydrogen ions produced during bio-oxidation reactions are accepted by the litmus present in the medium. In the oxidized state, litmus is purple; in the reduced state, it turns white or the color of the milk. Carbon dioxide and hydrogen can be produced during the fermentation of lactose. These gases may be visualized as cracks, fissures or bubbles in any curd present. Two types of curds can be formed in the litmus milk tubes: acid or rennet curds. An acid curd is an insoluble clot formed when the acids precipitate the casein in the milk. To detect whether the curd is an acid curd, invert the tube and see if the clot remains in place. A rennet curd is a softer curd, which has a semi-slid consistency such as that of yogurt. The rennet curd will flow freely if the tube is tilted. Some microorganisms may attack the protein in the milk. These organisms use proteolytic enzymes to hydrolyze the casein in the milk to its constituent amino acids. The tube will take on the appearance of a brownish straw-colored fluid. Large quantities of ammonia may also be released, causing the litmus to take on a deep purple color near the top of the tube.

This lab was adapted from: Investigating Microbiology: A Laboratory Manual for General Microbiology. P.E. Stukus. Saunders College Publishing. 1997. Microbiology: Laboratory Theory and Application. M.J. Leboffe and B.E. Pierce. Morton Publishing Company. 2002. 1

An alkaline reaction occurs when there is no change in the litmus or the change is to a deeper color of blue. This represents a partial decarboxylation or deamination of the amino acids and the release of alkaline end products. Litmus milk is a very complex test to read, some organisms may be capable of a number of the possible reactions while other organisms may only perform one reaction. To help you understand what these different reactions look like, you may want to consult the photographic atlas available in the laboratory. Materials: Litmus milk tubes Cultures o Unknown environmental isolates o Escherichia coli o Pseudomonas aeruginosa Procedure: 1. Using a sterile inoculating loop, inoculate a litmus milk tube with your environmental unknown culture. 2. The instructor will inoculate control organisms for this experiment. 3. Incubate at room temperature (25 C) until next week. Next week 4. Observe tubes and record all reactions. You may want to review the color atlas when examining the litmus milk tubes to aid in the analysis.

Laboratory Report #20 Name__________________________________________ Date _________ Reactions: Organism S. lactis E. coli P. aeruginosa Environmental Unknown Curd Formation Acid Alkaline Proteolysis Litmus Reduction Section ___

Review Questions: 1. How can you differentiate curd formation and proteolysis?

2. Is litmus milk a selective or differential medium or both? Why?

3. Compare and contrast the pH indicators that are used in the (a) carbohydrate fermentation, (b) methyl red test, (c) citrate test, (d) urease test, and (e) litmus milk test.