Oxidase Test Microbiology - Principle, Procedure, Limitations, Results, QC - in lab #Oxidase Test As the channel name suggests, our channel will be a perfect lounge for the malayali medicos..we wil be covering videos which will be like lecture classes related to the subjects biochemistry and microbiology in which we are specialised.. It will be a better learning experience for the students especially for those who are not able to understand and follow the normal classes in college..we assure the students that you will get a basic idea regarding the topic and extra reading can be done from the reference textbooks... If you like my video #like #comment #subscribe my channel don't forget to subscribe my channel Qualification Maneesha M Joseph MSc MLT (Microbiology) Assistant Professor Baby memorial college of allied Health science Kozhikode Our Partner Channel Health & Voyage channel link - https://youtu.be/nzKqRVjlwc0 #Oxidase Test #Medical #Microbiology # malayalam lecturer #Mallu Medicos Lounge #MalluMedicosLounge #MLT

1. By,
Maneesha M Joseph
Assistant Professor
Baby Memorial College
Kozhikode
Video Class uploaded in YouTube Channel “Mallu Medicos Lounge”

2. PRINCIPLE OF OXIDASE TEST
v Cytochromes are iron containing hemoproteins, they are involved in respiration
and electron transport chain.
v Cytochrome oxidase are enzymes that participates in the electron transport chain
by transferring electrons from a donor molecules (NADH) to oxygen.
v Oxidase test identifies organisms that produce enzyme cytochrome oxidase
v This enzyme has the property that it can take electrons from external synthetic
compounds such as TEMED
v The test reagent, N, N, N’, N’-tetramethyl-p-phenylenediamine dihydrochloride
is colorless in reduced state and acts as an artificial electron donor for the
enzyme oxidase,thus the dye become oxidised
v The oxidised reagent forms the coloured compound indophenol blue.
v The cytochrome system is usually only present in aerobic organisms which are
capable of utilising oxygen as the final hydrogen receptor.
OXIDASE TEST

3. A number of reagents can be used for this test.
•Kovacs Oxidase Reagent:
1% tetra-methyl-p-phenylenediamine dihydrochloride, in water
•Gordon and McLeod’s Reagent:
1% dimethyl-p-phenylenediamine dihydrochloride, in water
•Gaby and Hadley (indophenol oxidase) Reagent:
1% α-naphthol in 95% ethanol
1% p-aminodimethylaniline HCL

4. Since the oxidase reagent is unstable and has to be freshly prepared for use, this method is
convenient.
1.Strip of Whatman’s No. 1 filter paper are soaked in a freshly prepared 1% solution of
tertramethyl-p-phenylene-diamine dihydrochloride.
2.After draining for about 30 seconds, the strips are freeze dried and stored in a dark bottle
tightly sealed with a screw cap.
3.For use, a strip is removed, laid in a petri dish and moistened with distilled water.
4.The colony to be tested is picked up with a platinum loop and smeared over the moist
area.
5.A positive reaction is indicated by an intense deep-purple hue, appearing within 5-10
seconds, a “delayed positive” reaction by colouration in 10-60 seconds, and a negative
reaction by absence of colouration or by colouration later than 60 seconds
DRY FILTER PAPER METHOD

6. 1.A strip of filter paper is soaked with a little freshly made 1% solution of the
reagent.
2.A speck of culture is rubbed on it with a platinum loop.
3.A positive reaction is indicated by an intense deep-purple hue, appearing
within 5-10 seconds, a “delayed positive” reaction by colouration in 10-60
seconds, and a negative reaction by absence of colouration or by colouration
later than 60 seconds.
WET FILTER PAPER
METHOD

7. DIRECT PLATE METHOD
1.Add 2 -3 drops of reagent directly to suspect colonies on an agar plate. Do not
flood the plate with the reagent.
2.Observe for colour change within 10 seconds.
Note: The Direct Plate method should be carried out on a non-selective agar
plate.
3.When using Kovac’s oxidase reagent, microorganisms are oxidase positive
when the color changes to dark purple within 5 to 10 seconds. Microorganisms
are delayed oxidase positive when the color changes to purple within 60 to 90
seconds. Microorganisms are oxidase negative if the color does not change or it
takes longer than 2 minutes.
4.When using Gordon and McLeod reagent, microorganisms are oxidase
positive when the color changes to red within 10 to 30 minutes or to black
within 60 minutes. Microorganisms are oxidase negative if the color does not
change.

8. TEST TUBE METHOD
1.Grow a fresh culture (18 to 24 hours) of bacteria in 4.5 ml of nutrient broth
(or standard media that does not contain a high concentration of sugar).
2.Add 0.2 ml of 1% α-naphthol, then add 0.3 ml of 1% p-
aminodimethylaniline oxalate (Gaby and Hadley reagents).
3.Shake vigorously to ensure mixing and thorough oxygenation of the culture.
4.Observe for color changes.
5.Microorganisms are oxidase positive when the color changes to blue within
15 to 30 seconds. Microorganisms are delayed oxidase positive when the
color changes to purple within 2 to 3 minutes. Microorganisms are oxidase
negative if the color does not change

9. 1.Dip swab into reagent and then touch
an isolated suspect colony
2.Observe for colour change within 10
seconds.
SWAB METHOD

10. Result Interpretation of Oxidase Test
Positive Result
Development of a deep purple-blue/blue colour indicates oxidase
production within 5-10 seconds.
Negative Result
No purple-blue colour/No colour change.

11. Examples
Oxidase Positive Organisms: Pseudomonas, Neisseria, Alcaligens,
Aeromonas, Campylobacter, Vibrio, Brucella, Pasteurella, Moraxella,
Helicobacter pylori, Legionella pneumophila, etc.
Oxidase Negative Organisms: Enterobacteriaceae (e.g. E. coli)
QUALITY CONTROL FOR OXIDASE TEST
Positive Control: Pseudomonas aeruginosa ATCC 27853
Negative Control: Escherichia coli ATCC 25922