Bacterial Differentiation Techniques: Blood Agar, Catalase, Oxidase

Posted on Jan 9, 2025 in Biology

Blood Agar

  • Blood agar differentiates bacteria based on what reaction?

    • Blood agar is differential. The medium contains 5% sheep red blood cells that are added to trypticase soy agar. It is a rich medium that allows many organisms to grow, and it is differential for the action of bacterial enzymes on red blood cells.

    • Shows different types of hemolysis.

      • Alpha, beta, gamma

        • Beta-hemolytic bacteria produce enzymes that are able to completely lyse red blood cells, which causes a clear transparent zone in the media where the bacteria are growing.

        • Alpha-hemolytic bacteria are able to chemically reduce the hemoglobin in red blood cells. This causes a green/brown color in the media where the bacteria are growing.

        • Gamma-hemolytic bacteria do not lyse red blood cells or reduce hemoglobin, so the bacteria grow on the media but have no effect on the blood cells (see Figure 14.2). A pathogen like Streptococcus pyogenes is beta-hemolytic, whereas the less pathogenic Streptococcus faecalis is gamma-hemolytic.

  • If the organism is Streptococcus faecalis, what would you see on your blood agar plate?

    • No effect but has bacterial growth

Catalase

  • What is the substrate in the catalase reaction? What is the enzyme? What are the products?

    • Catalase: An exoenzyme present in some bacteria that hydrolyzes peroxide into water and oxygen.

Catalase

  • Staphylococcus and Streptococcus are both Gram-positive cocci. What test would you perform to differentiate them?

    • Catalase test

  • Catalase is produced by which bacteria?

    • Aerobic and facultative bacteria

  • Is it positive or negative when bubbles are formed?

    • Positive

  • Why are bubbles produced?

    • Break down of hydrogen peroxide into water and oxygen

Oxidase

  • What test would you perform to differentiate Pseudomonas from other Gram-negative rods?

  • The presence of what enzyme is being tested for in an oxidase assay?

    • Cytochrome oxidase, and it oxidizes cytochrome c and uses the electrons to reduce oxygen to water; therefore, cytochrome c acts as an electron donor. An oxidase test uses an alternate molecule as an electron donor, and when this molecule loses electrons, it turns a dark blue color

  • What color does the test strip turn if a bacteria is positive for the enzyme? What color does the test strip turn if the bacteria is negative?

    • Negative: No color change

    • Positive: A dark color develops after a specific reagent is added

Oxidase Test

  • What enzyme does it test for?

    • Oxidase

  • What organism was oxidase-positive?

    • Pseudomonas fluorescens

  • What is indicative of a positive result? Explain.

    • Dark blue color

Rapid Membrane Test for Esculin Hydrolysis

  • Which Gram-positive cocci can hydrolyze esculin into esculetin?

    • Group D streptococci

  • What color does the membrane turn if a bacteria is positive for esculin hydrolysis?

  • Describe how the test is performed?

Bile Esculin Test

  • Explain the purpose of the test.

    • To check to see if the Gram-negative rod-shaped bacteria cause UTI

  • What organism produces a positive result?

    • Streptococcus faecalis

    • E. coli

    • Enterobacter aerogenes

    • Serratia marcescens