Lactobacillus fermentum

The growth inhibition effect of the chitosan–metal composites was tested against a range of microorganisms that acted as model organisms for gut microflora of livestock gastrointestinal tract. E. coli (ATCC 8739) and Staphylococcus aureus (ATCC 25923) were grown in tryptic soy media (Sigma-Aldrich). Enterococcus faecalis (ATCC 29212) was grown in brain heart infusion (BHI) media (BD Biosciences). Lactobacillus fermentum (ATCC 9338) was grown in MRS medium (BD Biosciences). All incubations were done at 37°C.

The PP mixture comprised various Lactobacillus strains supplied by the American Type Culture Collection (ATCC) (Manassas, VA, USA). The strains included Lactobacillus gasseri (ATCC 33323), Lactobacillus plantarum (ATCC BAA-793), Lactobacillus reuteri (ATCC 23272), Lactobacillus helveticus (ATCC BAA-2840), Lactobacillus fermentum (ATCC 23271), Lactobacillus rhamnosus (ATCC BAA-2836), and Lactobacillus casei (ATCC BAA-2843). Following the supplier’s protocol, we cultured the strains in MRS broth (Beckton Dickinson, Sparks, MD), and preserved them as glycerol stocks at −80°C. To initiate culture growth, we prepared 5 mL of pre-warmed MRS broth with the glycerol stocks and then incubated these starter cultures at 37°C in a 5% CO₂ environment for 2 h. The bacteria were cultured overnight in 1 L of MRS broth under identical conditions until they reached the logarithmic growth phase, confirmed by measuring optical density at 600 nm (OD600). After growth, the bacterial cells were collected through several centrifugation steps at 3000×g and 4°C, followed by a wash in cold PBS. We then resuspended the bacterial pellets in 10% glycerol in PBS and promptly froze them in 1 mL aliquots for future administration to mice. The viability and concentration of the bacteria were verified by serial dilution and colony-forming unit (CFU) enumeration on agar plates.

Data collector was trained for two days on data collection tools and how the sample collection procedures will be performed, by the principal investigator before the actual sample collection. The quality of data collection pretested at Jugal general hospital among 29 (10% of sample size) adult dental caries patients. Quality control measures were implemented throughout the whole process of the bacteriology laboratory work. Staining reagents, culture media, and antibiotic discs were checked for their normal shelf-life before use. All culture media was prepared according to the manufacturer’s instructions and sterility of the prepared culture media was checked by incubating 3–5% of the batch at 37°C overnight and observed for growth. Culture media, which showed any growth, were rejected and replaced by a new sterile batch, and all culture plates and antibiotic discs were stored at recommended refrigeration temperature (2–8°C) after preparation. Reference strains like Streptococcus mutans (ATCC 25175) Lactobacilli uscasei (ATCC 393), Lactobacillus fermentum (ATCC 9338), S. aureus (ATCC 25923), E.coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and K. pneumoniae (ATCC 700603) were used [17 ].