Exercise 14: Enzyme Assays

Introduction

Enzyme assays are rapid tests used to determine the presence or absence of an enzyme by directly applying a reagent to a small amount of culture. These tests are useful for screening primary cultures to help determine the appropriate course of testing for identification. Two of the most commonly used enzyme assays are the catalase test and the oxidase test. Both are enzymes that are associated with the bacterial cell membrane. Catalase is involved in removing toxic oxygen free radicals from the cell. Catalase is useful in distinguishing Micrococcus and Staphylococcus (+) from Streptococcus and Enterococcus (–). Cytochrome c oxidase is a component of the electron transport chain found in some bacteria. Oxidase is used to distinguish Enterobacteriaceae (–) from other Gram negative bacilli such as Pseudomonas & Vibrio (+).

Catalase Test: Catalase is an enzyme that converts hydrogen peroxide (H₂O₂) into water and gaseous oxygen. The ability to synthesize these protective enzymes accounts for an organism’s ability to live in the presence of oxygen. Bacteria that produce catalase can be detected easily using 3% hydrogen peroxide. When hydrogen peroxide is added to a catalase-positive culture. Oxygen gas bubbles form immediately. If no bubbles appear, the organism is catalase-negative.

Oxidase Test: The function of oxidase is to transfer electrons to oxygen converting it to water. The oxidase test uses a special oxidase reagent (tetramethyl phenylenediamine dihydrochloride) that reacts with the enzyme. When the reagent is oxidized it turns from colorless to a bright purple color. To perform the test students will add 1 – 2 drops of reagent to a small strip of filter paper. A loop is used to transfer and smear a well isolated colony onto the filter paper. If the bacteria produces oxidase, it will turn a bright purple color within 20 – 30 seconds. Do not count any color change after 30 seconds. Oxidase reagent will eventually turn purple from exposure to air.

Course Intended Outcomes

Describe and perform at least 3 biochemical tests and describe their roles in microbial identification. Interpret results obtained and draw conclusions from biochemical tests performed.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Bunsen burner
• Inoculation loop
• Glass slides, cleaned
• Filter paper
• Test tube rack
• Additional materials and cultures will be assigned by your instructor

Procedure

Catalase Technique:

1. Refer to Exercise 2 for proper use of instruments and aseptic technique.
2. Using a sterile loop, transfer a small amount of one bacteria onto a clean glass slide.
3. Add 1 drop of 3% hydrogen peroxide (H₂O₂) over sample.
4. Observe for the production of bubbles.

Oxidase Technique:

1. Refer to Exercise 2 for proper use of instruments and aseptic technique.
2. Place a strip of filter paper over a clean slide.
3. Add 1 or 2 drops of oxidase reagent to the filter paper
4. Using a sterile loop, transfer a visible amount of one bacteria onto the filter paper. If the colonies are small, you may need to pick several.
5. Observe for the production of a blue/purple color within 30 seconds.
6. When testing is completed dispose of the used filter paper in the biohazard trash.

Sampling/Inoculation:

1. Your instructor will assign the cultures to be used for inoculation by your group.
2. Collect the required materials for the lab.
   • Place all tubes in a tube rack.
   • Never hold a culture tube by its cap.
   • Bacterial culture tubes are always to be shared with your table group.
3. Use slides from your slide box for enzyme tests.
4. When testing is completed apply bleach to the slides for one minute before cleaning.

Lab Clean-up

Wash slides with detergent, rinse and dry. Return slides to your slide box and store in the bin labeled for your section.

Return bacteria cultures to the front bench after inoculations are complete.

Remove labels from tubes and place in the tube rack ABOVE the biohazard container.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

Catalase Test

Oxidase Test

Critical Thinking Questions

What is a cytochrome and where is it found?

What causes oxidase reagent to turn blue after 30 seconds?

What is it called if your negative control for catalase shows bubbles? What could cause this?