Biochemical Reactions

• Determine the ability of an organism to ferment sugar incorporated in a medium producing acid or acid with gas.

  Sugar Fermentation
• Sugar Fermentation

  Positive: (Acidic) pinkish-red

  Negative: (Alkaline) yellow to colorless
• Demonstrates the ability of certain bacteria to decompose the amino acid tryptophane to indole, which accumulates in the medium.

  Indole Production
• Indole Production

  Positive: Red Ring

  Negative: Yellow Ring
• Detection of the production of sufficient acid during the fermentation of glucose so that pH of the medium falls, and it is maintained below 4.5.

  Methyl Red (MR TEST)
• Methyl Red (MR) Test

  Positive: Bright red color

  Negative: Yellow color
• Detects the presence of acetoin which is responsible to the conversion to diacetyl, and α napthol serves as a catalyst to form a red complex.

  Voges-Proskauer (VP) Test
• Voges-Proskauer (VP) Test

  Positive: Pink in 2-5 mins / Crimson Red in 30 mins

  Negative: Remains yellow or colorless for 30 mins
• Test for the ability of an organism to utilize citrate as the sole carbon and energy source for growth and an ammonium salts as the sole source of nitrogen with resulting alkalinity.

  Citrate Utilization
• Simmons' Citrate Medium (Citrate Utilization)

  Positive: Blue color and streak of growth

  Negative: Original green and no growth
• Koser's Citrate Medium (Citrate Utilization)

  Positive: Turbidity

  Negative: No turbidity
• Test for the presence of the enzyme nitrate reductase which causes the reduction of nitrate to nitrite which can be tested for by an appropriate colorimetric reagent. Almost all Enterobacteriaceae reduce nitrate.


  Nitrate Reduction Test
• Nitrate Reduction Test

  Positive: Red color (indicates the presence of nitrite)

  Negative: No color development
• Used to identify bacteria capable of hydrolyzing urea using the enzyme urease which splits urea to ammonia. Ammonia makes the medium alkaline and thus phenol red indicator changes to pink/red in color.

  Urease Test
• Urease Test


  Positive: Purple-pink color

  Negative: Pale yellow color
• Demonstrates the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide.

  Catalase Production
• Catalase Production


  Positive: Bubbling (oxygen is formed)

  Negative: No Bubbling
• Determine the presence of bacterial cytochrome oxidase using the oxidation of the substrate, a redox dye, tetramethyl-p-phenylene diamine dihydrochloride (oxidase reagent).

  Positive: purple color within 10 secs

  Negative: no reaction/color
• Determines whether the organism possesses the enzyme phenylalanine deaminase that deaminates phenylalanine to phenylpyruvic acid, which reacts with ferric salts to give a green color.

  Phenylalanine Deaminase Test
• Oxidase Reagent

  tetramethyl-p-phenylene-diamine dihydrochloride
• Phenylalanine Deaminase Test

  Positive: Green color

  Negative: No color change
• Some organisms decompose sulfur-containing amino acids producing hydrogen sulfide (H2S) among the products. It detects H2S liberated as a result of the degradation of sulfur containing amino acids.

  Hydrogen Sulfide Production
• Hydrogen Sulfide Production

  Positive: black color

  Negative: no color change
• Test the ability of an organism to grow in the presence of cyanide.

  Potassium Cyanide Test
• Potassium Cyanide Test

  Positive: turbidity due to growth

  Negative: Clear, no growth
• Used to determine whether a gram-negative rod utilizes glucose and lactose or sucrose fermentatively and forms hydrogen sulfide (H2S). TSI medium facilitates preliminary identification of gram-negative bacilli.

  Triple Sugar Iron (TSI) Agar
• Triple Sugar Iron (TSI) Agar

  Red color (alkaline): no fermentation

  Yellow color (acidic): fermentation of carbohydrate

  Bubbles in the butt: gas produce during fermentation of carbohydrate

  Blackening of the medium: H2S Production
• Triple Sugar Iron (TSI) Agar(Slant/Butt)


  Red/Red: no fermentation of glucose, lactose or sucrose

  Red/Yellow: Glucose fermented; lactose or sucrose not fermented

  Red/Yellow/H2S: Glucose fermented; lactose not fermented, H2S produced

  • Yellow/Yellow: Glucose and lactose/sucrose fermented