Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Development of a method for in-situ characterisation of biofilms grown on meat muscle  (#404)

Nirmani Wickramasinghe , Josh T Ravensdale , Ranil Cooray , Gary A Dykes , Scott P Chandry

Psychrotrophic Pseudomonas species are the dominant bacteria on aerobically stored chilled meat. Biofilm formation during chilled storage and transportation results in slime formation which is a major spoilage characteristic. Few studies have investigated biofilm formation on meat. In this work, a method was developed to investigate biofilm growth on meat using evenly sectioned sterile meat slices placed in six well plates permitting modest throughput and replication. Pseudomonas fragi (n=5) and Pseudomonas lundensis (n=5) were selected from a previously characterised collection based on their volatile production during spoilage on sterile beef. Meat samples were inoculated with cell numbers similar to those on retail meat and incubated at 4 ⁰C and 10 ⁰C for three, five and seven days to form mono species biofilms. At each time point, biofilms were stained with Syto 9 and Propidium Iodide to image live/dead proportions of bacteria. To observe the structure of eDNA network, TOTO 1 and Syto 60 were used on the seventh day. Two independent experimental rounds were carried out and confocal microscopy was used to obtain Z stacks which were quantified for mean biomass using biofilm analysing software COMSTAT. The structural and cellular organisation of the biofilms on muscle tissue were analysed using 3D visualization software AVIZO. The cell count data on biofilms correlated well with microscopic observations and the results were reproducible. Pseudomonas biofilms on sterilised beef were structurally similar to those of adventitious microorganisms on non-sterile beef slices incubated under identical conditions. Based on mean bio volume data, some P. fragi strains produced biofilms more rapidly than P. lundensis at 4⁰C than at 10⁰C. Highly dense, thick biofilms are a characteristic of P. fragi strains. The bacterial cells in P. fragi biofilm appeared to be vertically oriented whereas this characteristic was not observed in P. lundensis biofilms.