The microbial analyses were done at five time points; before the trial and at the end of week 1, 2, 3 and
4. The fish were starved 24 h before sampling. The fish were sacrificed with a blow to the head, opened
aseptically and the whole intestine was removed. The intestine was dissected and the contents were collected
by carefully scraping with a rubber spatula and weighed. The microbial analyses were performed by
spreading appropriate dilutions in PBS from 101 to 1012 on a selective media for lactic acid bacteria,
MRS (de Man, Rogosa and Sharpe; MRS, Merck, Darmstadt). The plates were incubated anaerobically at
37 (C for two days.
The microbial analyses of skin mucus were done at the same time points as described above before the
fish were surface disinfected and dissected. The skin mucus was collected from the whole skin area of five
fish of each group by scraping with a rubber spatula. The microbial analyses of epidermal mucus were
performed as described above for intestinal Lactobacillus enumeration.
The presence of L. rhamnosus in the tank water was determined 24 h after LAB-feeding. To determine
the wash out of the LAB from the tank water, the samples were taken just before feeding on the 3rd week
of the trial. The samples were analysed by plate counting as described above.