A critical analysis of microbiota associated with the production of shellfish in Saldanha Bay, South Africa

Ethical Clearance: 199021384/05/2019

Mussels and oysters samples will be collected by the principal investigator and two research assistants. Ten mussels and ten oysters will be placed in sterile Whirlpack bags and transported to the laboratory at a temperature of less than 4°C. Upon arrival at the laboratory, the mussels and oysters will be scrubbed under running tap water to remove shell debris and attached algae. Mussels and oysters will be homogenised to obtain a stock suspension. Thereafter, various analytical methods will be used for the detection of bacterial (Salmonella, Escherichia coli, Vibrio cholera, Vibrio parahaemolyticus and Vibrio vulnificus) and viral (Hepatitis A, Norovirus and Male-specific bacteriophages) presence.

Shellfish sample analysis: The Most Probable Number (MPN) of E. coli in samples will be used. The multiple tube method with the 5-tubes-5 dilutions test according to the norm ISO/TS 16649 -3 (2005) will be employed. A 1:3 dilution mixture containing Seventy-five grams (75g) of shellfish and intervalvar liquid (FIL) will be prepared using tryptone salt water (Biokar Diagnostics). The mixture will be homogenised using a food homogenizer for 2 minutes to obtain a stock suspension. Thirty millilitres (30ml) of this mixture will be added to 70 ml tryptone salt water (Biokar Diagnostics) and will be homogenised to reach a 1:10 dilution. Ten millilitres (10ml) of this homogenate will be inoculated into five tubes of double concentrate of Minerals Modified Glutamate Broth (MMGB). Five tubes of normal concentration MMGB will be inoculated with 1ml of the 1:10 homogenate, while the other five tubes will be inoculated with 1ml of a 1:100 dilution mixture per tube. After homogenization, all tubes will be incubated at 37°C for 24h. Escherichia coli confirmation will be determined by culturing 1uL of positive tubes (tubes showing acid production) on the Tryptone Bile X-Glucuronide agar (TBX) (Oxoid, Wesel, Germany), followed by incubation at 44°C for 24h. The growth of blue colonies would indicate the presence of E. coli. The number of E. coli per 100g will be determined using MPN tables (ISO/TS16649-3, (2005) according to the number of positive results in the three dilutions.