Oxidase Test

Many organisms that respire have the cytochrome C oxidase enzymes in their electron transport chain that transfers electrons to oxygen, reducing oxygen to either water or hydrogen peroxide.  (If the toxic hydrogen peroxide is generated, catalase will convert the hydrogen peroxide to water and oxygen.)  The tests described here use Kovac's  oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride) that changes color when it becomes oxidized by cytochrome C oxidase.  As this enzyme requires oxygen, it is present only in aerobes, a few microaerophiles, and facultative anaerobes.  Because all known strains that are oxidase positive are catalase positive (either +i or +s), but not all catalase positive strains are oxidase positive, these two tests together may really help narrow down the identification of an unknown.

This test can be performed using one of two similar procedures, your instructor will inform you of which.

Purpose
to test for the presence of cytochrome oxidase.  This test is helpful in identifying members of Neisseria, Enterobacteriaceae, Pseudomonadaceae, to aid in differentiating Gram negative nonenterics from Enterobacteriaceae, and in identifying a few others.

Colonial Growth Procedure:
  1. Have freshly grown growth in streaks, lines, or colonies on a solid media, such as Nutrient Agar or TSA.  Do not use blood agar plates as blood cells contain oxidase and may give a false positive reading.
  2. Watch the time closely.  Directly place two or three drops of the reagent onto some growth.
  3. Within 20 seconds, observe if the growth under the reagent turns blue.
  4. If the color changes blue within 20 seconds, record the test as positive for the presence of cytochrome C oxidase.
  5. Keep watching for a color change to blue within 30 seconds.  If the color changes to blue within 30 seconds, record the test as potentially positive or inconclusive.  If the color does not change to blue within 30 seconds, record your results as negative for the presence of cytochrome C oxidase.  Important, the reagent may oxidize on its own and turn blue, so a color change after 30 seconds is negative.
  6. Compare your results with both a positive and a negative control.

Oxidase Slide Procedure:

  1. This procedure requires BBL's DrySlide TM from Becton Dickinson and Company.  These slides already have the reagent on them.
  2. Use freshly grown growth or colonies from a solid media that is not blood agar, such as Nutrient Agar or TSA.  Old growth is not reliable.
  3. Transfer a large colony or equivalent amount of cells to the reagent slides using a tool that is not metal. Sterile pipettes and the wooden handles of swab sticks are suggested.  Some use cotton swabs to transfer the cells, but too many cells often stay with the swab, so this is not recommended.  If you are unable to pick up a large colony of cells, an agar plug may be cut out of a spare plate of cells with cleaned instruments, remove and invert the plug with cleaned forceps, and place the cells on the plug directly on the BBL's DrySlide TM.  Do not allow the forceps to touch the slide.  (This procedure works well but will likely contaminate the plate, so make sure it is a spare plate.)
  4. Observe the slides for a color change to blue watching the time closely.
  5. If the color changes to blue within 20 seconds, record the test as positive for the presence of cytochrome C oxidase.  If the color becomes blue within 30 seconds, record the test as probably positive.
  6. If the color does not change to blue within 30 seconds, record the test as negative for the presence of cytochrome C oxidase.
  7. Compare your results with both a positive and a negative control.  Note, many specimens will turn blue after 30 seconds from a side reaction, a few as early as 45 seconds, so watch the time closely and record these as negative.

References:

BD BBL's DrySlide TM Oxidase Product Insert.  2013.  Becton, Dickinson and Company, Sparks, MD, USA.

Cappuccino J.G. and Sherman N.  2008.  Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA.

Collin C.H., Lyne P.M., and Grange J.M.  1989.  Collins and Lyne's Microbiological Methods, 6th ed. Butterworth & Co, Ltd., London, UK.

Harley J.P.  2008.  Laboratory Exercises in Microbiology, 7th ed. McGraw-Hill Companies, New York, NY, USA.

Leboffe M.J. and Pierce B.E.  2011. Exercises for the Microbiology Laboratory, 4th ed. Morton Publishing Company, Englewood, CO, USA.

MacFaddin J.F. 2000. Biochemical Tests for the Identification of Medical Bacteria, 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA, USA.

Instructor Notes:

The reagent can oxidize (go bad) and should be tested with a known positive control.  If the reagent looks bad, do not use unless known controls test correctly.  Escherichia coli is oxidase negative and Bacillus subtilis, Alcaligenes faecalis, and the Pseudomonas species are all positive.  With the DrySlides, some known positive controls may not turn blue within 20 seconds (BD BBL's DrySlide TM Oxidase Product Insert, 2013) but usually do within 30 seconds.  The Colonial Growth procedure does seem to work better than the Slide procedure if fresh reagents and fresh cultures are used.  Not only is the Colonial Growth procedure easier to visibly observe than the Slide procedure, but the color change also occurs earlier and further away from the cutoff time.  Collin et al., 1989, say that a clean platinum wire may be used, but determining a clean from a mostly clean wire and a platinum from non-platinum wire may be too difficult for most students so that metal instruments are not recommended.  A trace of iron in a metal instrument that touches the reagent slide may result in a false positive result.  The oxidase test should not be performed on cells that have been grown in a medium containing glucose as glucose fermentation inhibits oxidase.  There are also other oxidase tests that use other reagents, but they are less sensitive than the test and reagent discussed here.