- The oxidase enzyme is a key factor in distinguishing Staphylococcus from Micrococcus, and the Microdase Disk is a reagent-impregnated disc recommended for use in qualitative procedures.
- Kovacs first described the oxidase method for distinguishing gram-negative bacilli in 1956.
- Tetramethyl-p- phenylenediamine (TMPD) in dimethyl sulfoxide was used by Faller and Schleifer to create a new oxidase reagent in 1981, improving upon Kovacs’ original formula (DMSO).
- When compared to Kovacs’ oxidase reagent, modified Kovacs performed significantly better in tests conducted by Tarrand and Groschel.
- Cytochrome c is present in every type of microorganism, however only a minority of staphylococci do.
- To confirm the existence of cytochrome c type, an oxidase reagent is used.
Objective
- This test is used to distinguish between gram-positive, catalase-positive cocci (micrococci from staphylococci).
- To find out the difference between Micrococcus and Staphylococcus spp. by figuring out where the enzyme oxidase is.
Principle of Microdase Test
- By looking for the enzyme oxidase, the microdase test is a quick way to tell the difference between Staphylococcus and Micrococcus spp.
- For detecting the oxidase enzyme, tetramethyl-p-phenylenediamine dihydrochloride (oxidase reagent) is mixed with dimethyl sulfoxide (DMSO) and put on filter paper discs in the shape of a circle.
- The modified oxidase reagent is made by putting 1% (w/v) tetramethyl-p-phenylenediamine in dimethyl sulfoxide that has been tested and is of a good quality.
- DMSO makes the reagent more soluble and stable against self-oxidation. It also helps the reagent get into the cells.
- In the presence of oxygen in the air, the oxidase enzyme reacts with the oxidase reagent and cytochrome C to make the coloured compound indophenol, which shows up as a blue or purple-blue colour on the disc after a bacterial colony has been placed on it.
- All micrococci have cytochrome c, but with a few exceptions, most staphylococci don’t have this type of cytochrome. The oxidase reagent proves that type c cytochrome is there.
Requirement
- Microdase Disk (25 disks/vial) Reactive Ingredients: Tetramethyl-p-phenylenediamine Dimethyl Sulfoxide
- Loop sterilization device
- Inoculating loop swabs, collection containers
- Incubators, alternative environmental systems
- Supplemental media
- Quality control organisms
- Forceps
- Microscope slide or petri dish
- Wooden applicator stick.
Procedure of Microdase Test
Analyses must be conducted on gram-positive, catalase-positive, aerobic cocci. Colonies should be harvested from a plate of Blood Agar (TSA with 5% Sheep Blood) at less than 24 hours of age, ideally between 15 and 18 hours.
- Put the disc on a clean glass slide or in a petri dish using forceps. The disc should not be rehydrated.
- Do not use a mixed culture; instead, use a wooden applicator stick to inoculate the disc with many isolated colonies (a visible inoculum).
- You should look for a change to a blue hue for at least two minutes.
Interpretation And Result Of The Test
- Positive Test – A positive result would be a blue or purple-blue colour within 2 minutes.
- Negative Test – No colour change (from white to grey) within 2 minutes
Quality Control
Microdase Disk has passed tests using the following quality control organisms. Establish quality control protocols for the laboratory before beginning testing on control organisms. It is not acceptable practise to publish patent results that have been flagged as having failed quality control.
- Positive: Micrococcus luteus (ATCC10240)
- Negative: Staphylococcus aureus (ATCC25923)
Limitations
- S. lentus, S. sciuri, and S. vitulus are the only species of Staphylococcus that are known to react positively to Microdase. If these species are suspected, it is suggested that a Lysostaphin Test be done as a way to tell them apart. These species live in some animals all the time and are not usually separated from human samples. Most of the time, they are not thought to be dangerous pathogens.
- It is also known that Macrococcus species and Kocuria kristnae have cytochrome c. Macrococci have not yet been found in clinical samples and identified. K. Kristnae can be told apart from micrococci by its ability to make acid aerobically from glycerol and D-mannose, make acetoin, and break down esculin.
- Microdase is not made to test oxidase activity in organisms other than Staphylococcus and Micrococcus on a regular basis.
Uses
- The modified oxidase test is used to tell the difference between Staphylococcus and Micrococcus. The result for micrococci should be good. Except for Staphylococcus sciuri, S. sciuri, the results for staphylococci should be negative. lentus, while S. vitulinus
References
- Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
- https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU21132.pdf
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC273944/
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC272470/
