HiMRSA™ Confirmation Agar Base

Intended Use

Recommended for selective isolation of Methicillin Resistant Staphylococcus aureus (MRSA) from other Methicillin resistant Staphylococci species on the basis of coagulase activity from clinical samples.

Composition**

Final pH ( at 25°C) 7.4±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 73.05 grams in 900 ml purified/ distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Aseptically add rehydrated contents of 1 vial of ACC Selective Supplement (FD363) along with 10 vials of Coagulase Supplement (FD362). Mix well and pour into sterile Petri plates.

Principle And Interpretation

Staphylococci can cause many forms of infection (1) and also causes food poisoning by releasing enterotoxins into food (2). MRSA is a resistant variation of the common bacterium Staphylococcus aureus. It is an invasive pathogen that can cause disease in almost any tissue or organ in the human body, primarily in compromised individuals (3). Staphylococcal infections were earlier treated using Penicillin. But over the years resistance to this drug developed. Methicillin was the next drug of choice. While methicillin is very effective in treating most Staphylococcus infections some strains have developed resistance to methicillin and can no longer be killed by this antibiotic. These resistant bacteria are called Methicillin Resistant Staphylococcus aureus (MRSA) (4). Resistance to methicillin was obsereved in other species of Staphylococcus. This necessitates the differentiation of Methicillin Resistant Staphylococcus aureus from other species of Methicillin Resistant Staphylococcus.

Coagulase test is a biochemical test that is used to differentiate Staphylococcus aureus from other Staphylococci species like S.epidermidis and S.saprophyticus on the basis of the ability to produce the coagulase enzyme. Coagulase-positive S.aureus is among the most ubiquitous and dangerous human pathogens, for both its virulence and its ability to develop antibioticresistance.

On this medium mannitol acts as carbohydrate source, the fermentation of which is indicated by indicator mixture. Mannitol positive staphylococci appears as yellow coloured colonies and negative appears as light mauve to purple coloured colonies. Coagulase supplement (FD362) aids identification of Staphylococcus aureus by coagulase reaction (5). Coagulase positive organisms are surrounded by an opaque zone while the absence of opaque zone indicates coagulase negative Staphylococcus.

Tryptone serves as a source of nitrogen, carbon, sulphur and vitamins. Growth factors and amino acids helps in better recovery of the organisms. Selective agent inhibit most of the contaminating microflora except Staphylococcus species. The selective supplement ACC Selective Supplement (FD363) helps to select the Methicillin Resistant Staphylococcus species and inhibits the other accompanying flora.

Type of specimen

Clinical samples : Pus, wounds

Specimen Collection and Handling

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (6,7). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use only. For professional 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 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 medium is recommended for detection of coagulase positive S. aureus, but other bacteria may grow.
2. It should be noted, however, that bovine strains, in particular, do not always produce this zone and confirmatory testing is needed.
3. Each lot of the medium has been tested with the standard strains, slight variation in growth may be observed depending on the source from where the organism has been isolated.

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 Beige to purple homogeneous free flowing powder

Gelling Firm, comparable with 2.0% agar gel.

Colour and Clarity of prepared medium Purple to reddish purple coloured clear to slightly opalescent gel forms in Petri plates.

Reaction Reaction of 7.3% w/v aqueous solution at 25°C. pH : 7.40±0.2

pH 7.20-7.60

Cultural Response Cultural response was observed after an incubation at 35-37°C for 24-48 hours. Recovery rate is considered as 100% for bacteria growth on Soyabean Casein Digest Agar.

Key : (*) Corresponding WDCM numbers.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 2 - 8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance is best if used within stated expiry period.

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 (6,7).

Reference

1. Bhakdi S, Tranum-Jensen J. Alpha-toxin of Staphylococcus aureus. Microbiol Rev. 1991;55:733.
2. Lyon BR, Skurray R. Antimicrobial resistance in Staphylococcus aureus: genetic basis. Microbiol Reviews. 1987;51:88. Methicillin Resistant Staphylococcus aureus Copyright ã 1997-2005 Canadian Centre for Occupational Health and Safety, Sept 19th, 2005.
3. DWorkin M et. al 2006. The Prokaryotes (a Handbook on the Biology of Bacteria) 3rd ed, Vol. 2, page 345.
4. Salfinger Y., and Tortorello M.L., 2015, Compendium of Methods for the Microbiological Examination of Foods,5th Ed., American Public Health Association, Washington, D.C.
5. Beckers N. J. et al, 1984, Can. J. Microbiol., 30:470.
6. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
7. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.