Nitrate Reductase & Catalase

Materials and Equipment for Laboratory Tests

  • Live Organisms:

    • Catalase Test:

    • Enterococcus faecalis

    • Staphylococcus saprophyticus

    • Micrococcus luteus

    • Bacillus spp. (any species of Bacillus)

    • Hydrogen peroxide (H₂O₂)

    • Nitrate Reductase Test:

    • Pseudomonas stutzeri

    • E. coli

    • Micrococcus luteus

    • Nitrate broth (3 tubes per group of 3 students)

    • Reagents:

      • Nitrate A (sulfanilic acid)

      • Nitrate B (alpha-naphthylamine)

      • Zinc dust

Procedure for Catalase Test

  1. Slide Preparation:

    • Obtain a glass slide.

    • Use a sterile loop to obtain an inoculum of bacteria (utilize slant cultures) and place it on the slide.

    • Alternatively, you may first place a drop of hydrogen peroxide on the slide, then add the bacteria.

  2. Adding Hydrogen Peroxide:

    • Add a drop of hydrogen peroxide onto the bacteria.

  3. Observation:

    • Observe for the presence of bubbles within 10 seconds.

    • Important Note:

      • Do not apply hydrogen peroxide directly on the slant!

  4. Specific Observation for Bacillus spp.:

    • Bacillus spp. are sometimes weak catalase producers.

    • Use a heavy inoculum of this organism and observe for bubble production under a microscope.

Procedure for Nitrate Reductase Test

Day 1

  1. Inoculate the nitrate broth with the bacteria.

  2. Incubate at 37°C.

Day 2

  1. Remove the caps from all 3 cultures.

  2. Add 10 drops of Nitrate A (sulfanilic acid) into each of the 3 tubes.

  3. Add 10 drops of Nitrate B (alpha-naphthylamine) into each of the 3 tubes.

  4. Observation:

    • If the solution does not turn red, proceed to the next step.

  5. Add a small amount of zinc dust using a spatula to the respective tube.

Safety and Disposal Procedures

  • Day 1:

    • Discard slides used for the catalase test into the contaminated slide dish containing disinfectant.

  • Day 2:

    • After completing the nitrate reductase test and recording results, remove the tapes from the tubes and place them in the wire basket for appropriate disposal.

Important Definitions

  • Catalase Test:

    • A biochemical test utilized to identify organisms that produce the enzyme catalase, which catalyzes the decomposition of hydrogen peroxide into water and oxygen, resulting in observable bubble formation.

  • Nitrate Reductase Test:

    • A test to assess the capability of an organism to reduce nitrate (NO₃⁻) to nitrite (NO₂⁻) or further to nitrogen gas (N₂), utilizing specific reagents to detect the presence of nitrites in a culture after incubation.

Notes on Bacterial Organisms

  • Enterococcus faecalis: A Gram-positive bacterium commonly found in the gastrointestinal tract.

  • Staphylococcus saprophyticus: A Gram-positive, coagulase-negative staphylococcus associated with urinary tract infections.

  • Micrococcus luteus: A non-pathogenic, Gram-positive bacterium found in various environments, often used in laboratory settings.

  • Pseudomonas stutzeri: A Gram-negative, motile bacterium known for its ability to reduce nitrate.

  • E. coli: A Gram-negative bacterium commonly found in the intestines of humans and animals, with strains relevant to foodborne illnesses.

  • Bacillus spp.: A diverse group of Gram-positive rod-shaped bacteria, some of which can produce catalase and are used in various applications, including biotechnological processes.