Lecture 12

• Substrates


  • Starch
  • Lipid
  • Gelatin
  • Casein
• Differential Media


  • Tests are performed on microorganisms in order to determine characteristics about their metabolism
  • Enzyme production and activity can be observed on differential media
  • Differential media does not inhibit growth of organisms; its components/ingredients allow us to see metabolic differences between them
• Hydrolyzing enzymes


  Cleave substrates in the presence of H2O
• Starch Hydrolysis

  1. Starch is a large carbohydrate polysaccharide made up of Amylose (unbranched glucose polymer) and Amylopectin (large branched glucose polymer)

  2. Some organisms produce α-amylase to break down starch into short chains and monosaccharides (glucose)

  3. Nutrient agar with 1% starch is inoculated and incubated

  4. A zone of clearing next to the growth indicates the starch was broken down, organism is (+) for α-amylase production

  5. Iodine is used as the developer to enhance the zone of clearing and react with intact starch
• No zone of clearing


  • Organism is (-) for production of α-amylase and oligo-1,6-glucosidase
• Zone of clearing present


  • Organism is (+) for production of α-amylase and oligo-1,6-glucosidase
• Lipid Hydrolysis


  1. Lipids (fats) are high molecular weight hydrophobic molecules

  2. Triglycerides are hydrolyzed by lipases into glycerol and free fatty acid molecules

  3. Phospholipids are hydrolyzed by lipases, which is a factor in the spread of pathogenic bacteria

  4. Spirit blue plates (nutrient agar with spirit blue pH indicator) are used to test for lipase production

  5. Hydrolysis of lipids causes a decrease in pH, making the media darker blue
• No darker blue color change


  • Organism is (-) for production of lipase
• Darker blue color change


  • Organism is (+) for production of lipase
• Gelatin Hydrolysis


  1. Gelatin is a hydrolyzed form of collagen, the primary connective tissue protein

  2. Some organisms can break down gelatin to amino acids using gelatinase, which is a protease

  3. Nutrient agar with 1.5% gelatin is inoculated and incubated

  4. A zone of clearing next to the growth indicates the gelatin was broken down, organism is (+) for gelatinase production

  5. 15% TCA is used as the developer to enhance the zone of clearing and react with intact gelatin
• Zone of clearing present


  • Organism is (+) for production of gelatinase
• No zone of clearing


  • Organism is (-) for production of gelatinase
• Casein Hydrolysis


  1. Casein is a protein found in milk

  2. Some organisms can hydrolyze casein using caseinase, which is also a protease

  3. Nutrient agar with 1% skim milk is inoculated and incubated

  4. A zone of clearing next to the growth indicates the casein was broken down, organism is (+) for caseinase production

  5. 15% TCA is used as the developer to enhance the zone of clearing and react with intact casein
• Zone of clearing present


  • Organism is (+) for production of caseinase
• No zone of clearing


  • Organism is (-) for production of caseinase