Rugai Modified with Indole and Lysine

Goal:

• Differential culture medium for the presumptive identification of negative Enterobacteria oxidase.

Important details:

• Colonies must be freshly obtained (maximum of 24 hours) from Gram negative bacilli in suitable isolation media, such as MacConkey Agar, CLED etc.;
• Colonies should be analyzed whether they are lactose fermenters or not;
• If the strain is lactose negative, it must be subjected to the oxidase test. If it is positive oxidase, another identification methodology must be verified;
• The rugai medium is indicated only for fermenting bacteria;
• The colonies to be identified are sown by deep pricking and surface streaking in a tube;
• The procedure is followed by incubation at 35ºC for 18-24h.
• After incubation, the Modified Rugai medium is read, and the data can be checked directly online using the “Rugai test” tool on the RenyLab website: renylab.ind.br

Evidences:

Apex medium

• L-Tryptophan deamination: positive test when there is development of green bottle coloring at the apex of the tube.
• Sucrose fermentation: positive test when yellow color develops at the apex of the tube.
• Glucose fermentation: the appearance of yellow color in the lower portion of medium.
• Gas production from glucose: if the bacteria under study produces gas from glucose.
• Production of hydrogen sulphide (H2S): the production of hydrogen sulphide is observed by the appearance of black coloring.
• Urea hydrolysis: the test is considered positive when there is the formation of a bluish color in the lower portion of the medium.

Lysine medium

• Lysine decarboxylation: glucose fermentation reduces the pH changing the lysine medium to yellow. When the activity of the lysine decarboxylase enzyme occurs, the pH will increase, due to the transformation of lysine into a primary amine: cadaverine, and the medium will turn to purple again.
• Motility: when bacterial growth is restricted to the inoculation point, negative motility is considered, for positive motility there is diffuse growth with partial or complete turbidity of the medium.

Cotton plug

• Indole: the appearance of red color characterizes the positive test.

OBS: The Rugai medium with lysine does not allow a definitive and precise identification of the Enterobacteria species, being the use of a more specific methodology required.