Identifying Bacteria based on Enzymes and multiple test media Lab # 9 Medgar Evers College Prof. Victor Santos

Aim To identify bacteria based on the enzymes they have that allow them to carry out certain specific reactions! You will detect the presence of Amylase, proteases, lipases, tryptophanase, urease, and phenylalanine deaminase.

Enzyme Medium used Color change By product detected Amylase (B. subtilis) Starch agar plate Add iodine look for clear zone due to starch degradation We look for clear zone

Proteases (B.subtilis) Skim milk plate Look for clear zone due to degradation of casein protein Clear zone of degradation

Lipases (S.aureus) Spirit blue agar We look for a dark blue precipitation due to lowering of pH as a result of fatty acids being released Blue precipitation or sometimes just depletion of fat droplets on the agar.

Tryptophanase (E.coli) Tryptone broth We add kovac’s reagent to detect the indole ring produced. Tryptophan is broken down into indole, pyruvate and ammonia. We detect the indole.

Urease (P.vulgaris) Urea agar slant When urea is broken down it releases ammonia and carbon dioxide. The ammonia raises the pH and the phenol red indicator causes a color change from yellow to bright pink We detect the raise in pH due to the ammonia being released.

Phenylalanine deaminase   (P.vulgaris) Phenylalanine agar We look for a greenish precipitate after the addition of 10% ferric chloride Phenylalanine is broken down by the enzyme into ammonia and phenylpyruvic acid. The ferric chloride reacts with the phenylpyruvic acid to yield a greenish color.

Multiple testing media These media are important because they save us time and money by allowing us to test for several parameters at once. Examples; Kliger’s Iron Agar, SIM, and Litmus Milk

Kliger’s Iron Agar The Kliger’s medium contains 1.0 % lactose, 0.1 % glucose and the amino acid cysteine, peptones, ferrous salts and the pH indicator phenol red. This medium allows you to detect glucose fermentation, lactose fermentation and hydrogen sulfide production. Inoculate by streaking and stabbing one tube with your unknown and one with P. vulgaris, the control.

SIM This medium is 0.7% agar so its considered semi-solid to allow motile organisms to be detected. This medium allows you to detect motility, hydrogen sulfide production from the breakdown of cysteine, and detect the breakdown of tryptophan. Inoculate one tube with unknown, and three controls; one with S. aureus, and one with P. vulgaris and the last one with E. coli. YOU MUST STAB THESE!

Litmus milk Allows you to detect several properties! A) Fermentation of sugar will lead to a pink color change due to acid formation. B) The breakdown of proteins leads to ammonia formation which raises the pH and leads to a blue color change. C) Litmus reduction leads to a white color change. This happens due to a drop in oxygen and the dye is then used as an electron acceptor becoming reduced and changing the medium to a white color. D) Coagulation of proteins can lead to curd formation. E) Peptonization or the medium becomes translucent or sometimes brown due to the breakdown of milk proteins. F) Ropiness or the formation of thick slime due to the accumulation of waste products and cells.

Inoculate a tube of litmus milk with your unknown only!