Tryptic soy agar

Streptococcus pneumoniae from frozen glycerol stocks was inoculated onto tryptic soy agar (Millipore Sigma, Billerica, MA) plates containing 20 μg/mL neomycin and 3% defibrinated sheep blood and then incubated overnight at 37°C with 5% CO₂. Because S. pneumoniae is naturally resistant to neomycin, it was added to prevent contamination of other bacterial species. Liquid cultures in semi-defined media C + Y or Todd-Hewitt Broth and 2% yeast extract were inoculated with the bacteria grown on the blood agar plate.^{50 (link)}

On day 18 of incubation, one egg from each flat was set aside for embryo staging. Each flat of developing eggs was injected at a time. Eggs were injected on their large end, into the amniotic sac. The needle punctured each egg at a depth of 2.49 cm to deliver each 50-μL concentration automatically. The different concentrations of the probiotic culture were injected in ascending concentration of bacteria to ensure the correct dosage was applied according to each treatment. However, between each treatment applied, a sanitization cycle was conducted to eliminate any contamination in the Inovoject equipment. After each cycle, 50 μL were collected and spread onto tryptic soy agar (Millipore Sigma, St. Louis, MO) plates to confirm that no bacterial contamination occurred between treatments. After all treatment inoculations, the eggs removed from each flat were in ovo inoculated with 50 μL of a Coomassie blue dye and immediately euthanized via CO₂ asphyxiation. Each embryo was analyzed to confirm that the injected eggs were in the appropriate stage of development for 18 d of incubation. Also, the presence of the dye surrounding the embryo's body through the amniotic fluid confirmed that the inoculation was correctly delivered in the amniotic fluid and did not puncture the embryo's tissue.