Paired nasal and axillary skin swabs were collected from each patient and were immediately transferred to Mannitol Salt broth (HiMedia Laboratories, Mumbai, India) for overnight selective enrichment. The following day, aliquots were plated onto Blood Agar (BA) media. Single colonies from plates which showed suspected S.aureus colony growth, were further processed by Gram stain, catalase and coagulase test, and checked for the ability to hydrolyse DNA. Disc diffusion tests with cefoxitin (10 μg) were used to identify suspected MRSA according to British Society for Antimicrobial Chemotherapy guidelines. All media were locally prepared and batch controlled using appropriate internal quality control strains (S.aureus ATCC 25923, S.epidermidis ATCC 12228, E.coli ATCC 25922). These initial screening tests were performed in Thika Hospital, Kenya. Isolates were initially kept at -20°C and then transferred to -80°C for storage. Isolates were later shipped to the University Medical Centre Groningen, the Netherlands for further investigations.
Mannitol salt broth
Manufactured by HiMedia
Sourced in India
Mannitol Salt broth is a growth medium used in microbiology laboratories. It is designed to selectively isolate and differentiate Staphylococcus species. The broth contains mannitol, a carbohydrate, and sodium chloride, which inhibits the growth of most other bacteria, allowing for the cultivation of Staphylococcus.
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2 protocols using mannitol salt broth
1
Isolation and Characterization of MRSA
2
Isolation and Identification of Staphylococcus
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To accumulate Staphylococcus microorganisms, an aliquot of milk (1 ml) was added to 9 ml of mannitol salt broth (HiMedia Laboratories Pvt. Ltd., India). Reinoculation of this culture into Baird Parker Agar (HiMedia Laboratories Pvt. Ltd., India) and Azide Blood Agar Pronadisa (Conda, Spain) differential-diagnostic media yielded 30–100 colonies. For further study, isolated colonies with characteristic features (causing β-hemolysis and creating a zone of lecithinase activity) were selected. To interpret biochemical features, a system for identification of staphylococci, micrococci and related genera (bioMérieux, Australia) was used. Attribution of the strains to coagulase-positive staphylococci was done using a preparation of freeze-dried plasma (freeze-dried citrate rabbit plasma; ECOlab, Russia).
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