Gordon-McLeod Reagent (Oxidase reagent) R026

Intended use
Gordon-McLeod Reagent (Oxidase reagent) is used to carry out the oxidase test, to determine the presence of
oxidase enzymes.
Composition**
Ingredients
N,N-Dimethyl p-phenylenediaminehydrochloride 0.15 gm
Distilled water 10.0 ml
**Formula adjusted, standardized to suit performance parameters
Directions
1. Biochemical identification was carried out by using Gordon-McLeod Reagent (R026) and Soyabean Casein
Digest Agar Plate (M290) containing 24-48 hours old culture.
2. Place 2 to 3 drops of Gordon Mcleod Reagent (R026) on to a filter paper in a petridish. With a platinum wire
loop (not nichrome), plastic loop or glass rod, smear some of the growth from Soyabean Casein Digest Agar Plate
(M290) on the prepared filter papers.
3. Observe for appearance of deep blue purple colour within 10 seconds.

Principle And Interpretation

The oxidase test is based on bacterial production of an intracellular oxidase enzyme. This oxidase reaction is due to
a cytochrome oxidase system that activates oxidation of reduced cytochrome by molecular oxygen, which in turn
acts as an electron acceptor in the terminal stage of the electron transfer system. All Pseudomonas and Neisseria
spp., produce an oxidase enzyme which, in the presence of atmospheric oxygen, cytochrome c, and an oxidase
reagent, oxidize the reagent to a colored compound, indophenol.

Type of specimen
The specimen is any isolated colony on primary or subculture plates.

Specimen Collection and Handling

1. For clinical samples follow appropriate techniques for handling specimens as per established guidelines.
2. For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines.
3. For water samples, follow appropriate techniques for sample collection, processing as per guidelines and local
standards.
After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
In Vitro diagnostic use only. Read the label before opening the container. Wear protective gloves/protective
clothing/ eye protection/face protection. Follow good microbiological lab practices while handling specimens and

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culture. Standard precautions as per established guidelines should be followed while handling clinical specimens.
Safety guidelines may be referred in individual safety data sheets.

Limitations
1. The reagents used in the oxidase test have been shown to auto-oxidize, so it is very important to use fresh
reagents, no older than 1 week.
2. Both bacteria and yeast grown on media containing high concentrations of glucose show inhibited oxidase
activity, so it is recommended to test colonies grown on media without excess sugar, such as nutrient agar. Tryptic
soy agar is also an excellent media.
3. Bacteria grown on media containing dyes may give aberrant results.
4. The test reagents will effectively kill the microorganisms, so sub-culturing should be done prior to adding any
reagent to an active culture.
5. Use of a nichrome or other iron containing loop may yield false-positive reactions. Platinum loops are
recommended.
6. Most Haemophilus are oxidase-positive. Less sensitive strips or reagents may yield false-negative results.
7. The oxidase test can be used in the presumptive identification of Neisseria and in the differentiation and
identification of gram-negative bacilli. Oxidase-positive organisms should be examined by gram stain to determine
morphology and gram reaction. Additional biochemical tests are recommended for complete identification.
8. Oxidase reactions of gram-negative bacilli should be determined on non-selective and non-differential media to
ensure valid results. Also, colonies taken from media containing high levels of glucose may give false-negative
reactions.
9. It is recommended to use colonies that are 18-24 hours old. Older colonies will produce weaker reactions.
10. Any color changes appearing after 20 seconds should be disregarded.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when
stored at recommended temperature

Quality Control
Appearance : Black coloured solution.
Clarity : Clear without any precipitate.
Cultural Response

Organism Oxidase Reaction

Cultural Response 24-48 hours old culture grown on Soyabean Casein Digest Agar Plate
(M290). Place 2 to 3 drops of Gordon Mcleod Reagent (R026) on to a
filter paper in a petridish. smear some of the growth from Soyabean
Casein Digest Agar Plate (M290) on the prepared filter paper. Observe for
appearance of deep blue purple colour within 10 seconds.

Neisseria gonorrhoeae ATCC 19424 Positive (development of purple-blue colour)

Pseudomonas aeruginosa ATCC 27853 Positive (development of purple-blue colour)
(WDCM 00025)

Staphylococcus aureus subsp. aureus Negative (No change in colour)

ATCC 25923 (WDCM 00034)

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Storage and Shelf Life

Store between 10-30°C in tightly closed container and away from bright light. Use before expiry date on label. On
opening, product should be properly stored in dry ventilated area protected from extremes of temperature and
sources of ignition. Seal the container tightly after use.

Disposal
User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product.
Follow established laboratory procedures in disposing of infectious materials and material that comes into contact
with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques.

Reference
1. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition. Vol. 2.
2. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015)
3. Downes F. P. and Ito K. (Ed.), 2001, Compendium of Methods for the Microbiological Examination of Foods,
4th ed., APHA, Washington, D.C.
4. Rice E.W., Baird, R.B., Eaton A. D., Clesceri L. S. (Eds.), 2012, Standard Methods for the Examination of Water
and Wastewater, 22nd ed., APHA, Washington, D.C.
5. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products,
17th Ed., APHA Inc., Washington, D.C.
6. Lapage S., Shelton J. and Mitchell T., 1970, Methods in Microbiology', Norris J. and Ribbons D., (Eds.), Vol.
3A, Academic Press, London.
7. MacFaddin J. F., 2000, Biochemical Tests for Identification of Medical Bacteria, 3rd Ed., Lippincott, Williams
and Wilkins, Baltimore
8. Jean F. Macfaddin, 2000, Biochemical Tests for Identification of Medical Bacteria,3rd edition, Williams &
Wilkins, Baltimore.
9. Godkar B. P., 1996, Textbook of medical laboratory technology: 25(340)

Storage temperature Do not use if package is damaged

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Revision : 03/2022

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User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in
this and other related HiMedia™ publications.The information contained in this publication is based on our research and development
work and is to the best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to
specifications and information related to the products at any time. Products are not intended for human or animal or therapeutic use but
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