TITLE: Isolation and identification of potential probiotic bacteria from the soil of Saint Martin&#39;s island Bangladesh
AUTHORS: Md. Mahbubur Rahman, Sulav Indra  Paul

[DESCRIPTION]
Soil bacteria of Saint Martin’s Island modulates gut microbiota and prevents Enterococcus faecalis infection in tilapia (Oreochromis niloticus)

[STEPS]
SECTION: Collection of soil sample.
1. the marine soil samples were collected from the shore of Saint Martin’s Island (20°37'40.3"N’ 92°19'22.3"E) of the Bay of Bengal, Bangladesh
SECTION: Collection of soil sample.
2. the samples were collected from 6-15 inch depth using an auger
SECTION: Collection of soil sample.
3. immediately transferred to a 50 mL sterile falcon tube
SECTION: Isolation and maintenance of bacteria.
4. 1 g of each soil sample was suspended in 9 mL of autoclaved seawater in an individual test tube 
SECTION: Isolation and maintenance of bacteria.
5. 100 µL suspension from each diluted stock (10-2 and 10-3) was aseptically inoculated on individual Starch Casein Agar (SCA) 
SECTION: Isolation and maintenance of bacteria.
6. After incubation, different colonies were picked up based on their colony characteristics
SECTION: Phenotypic identification of bacterial isolates.
7. Individual colonies grown on SCA plates were carefully observed and colony characteristics viz, colony size, shape, color, type and elevation were recorded
SECTION: Phenotypic identification of bacterial isolates.
8. Biochemical tests such as oxidase, catalase, motility, oxidative-fermentative (O-F) tests were accomplished
SECTION: Screening of antagonistic activity of marine soil isolates.
9. Bacteria were grown in Marine Broth (MB) 2216 (Merck, USA) for 7 days at 28°C
SECTION: Screening of antagonistic activity of marine soil isolates.
10. The broth culture was centrifuged at 10,000 ×g for 15 min and the culture supernatant was passed through a 0.22 µm millipore membrane filter 
SECTION: Screening of antagonistic activity of marine soil isolates.
11. The inhibitory activity of the culture supernatant was determined by agar well diffusion assay
SECTION: Molecular identification of marine soil isolates.
12. Genomic DNA of the selected isolates was extracted by using a commercial GenJET genomic DNA purification kit (Thermo Fisher Scientific, USA)  #K0721
SECTION: Molecular identification of marine soil isolates.
13. DNA was amplified by using universal primer 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGATACCTTGTTACGACTT-3'). 
SECTION: Molecular identification of marine soil isolates.
14. The PCR amplification condition was done by an initial denaturation at 94 °C for 5 min; 35 cycles of a denaturation at 94 °C for 1 min, an annealing at 57 °C for 40 sec and an extension at 72 °C for 1 min and a final extension step at 72 °C for 10 min. 
SECTION: Molecular identification of marine soil isolates.
15. Then the PCR amplicons were purified by using a commercial kit (Thermo Fisher Scientific, USA) 
SECTION: Molecular identification of marine soil isolates.
16. 16S rRNA gene sequencing was done  by Sanger Sequencing
SECTION: Evaluation of digestive enzymes activity of the marine soil bacteria.
17. Enzymatic activity such as, protease, lipase, amylase, and cellulose activity of the B. haynesii strain CD223 and A. mimigardefordensis strain SM421were assessed to evaluate their probiotic effects.
SECTION: Evaluation of the viability of bacteria in different pH and bile esculin.
18. The pH of SCA broth was adjusted from pH 3–9. Then, B. haynesii strain CD223 and A. mimigardefordensis strain SM421 were inoculated in this broth and kept for 24 h incubation at 28°C. 
SECTION: Evaluation of the viability of bacteria in different pH and bile esculin.
19. The viability of the cells was confirmed by inoculating them onto SCA agar plates (pH 7) by the spread plate method
SECTION: Preparation of bacterial extracellular products (ECPs).
20. Bacteria were enriched in MB at 28oC for 10 days
SECTION: Preparation of bacterial extracellular products (ECPs).
21. The ECPs were harvested and mixed with an equal volume of ethyl acetate in a separatory funnel
SECTION: Preparation of bacterial extracellular products (ECPs).
22. The air-dried extract was weighted and dissolved in methanol for further use
SECTION: Minimum inhibition concentration (MIC).
23. MIC was measured with different concentrations of ECPs extracts (1000, 500, 250, 125, 62.5, and 31.25 μg mL-1) 
SECTION: Measurement of Hematological Parameters
24. Fish from each treatment were anesthetized with the clove oil (0.05 mL per 500 mL of water) for hematological analysis
SECTION: Measurement of Hematological Parameters
25. Blood was collected from fish using a 3 cc syringe containing 10% blood anti-coagulant (EDTA) inserted into the caudal peduncle region to drag out blood.
SECTION: Measurement of Hematological Parameters
26. The blood was transferred to a test tube coated with EDTA, and stored at -30 oC until use.
SECTION: Measurement of Hematological Parameters
27. Red blood cells (RBCs) and white blood cells (WBCs) were counted using an improved Neubauer hemocytometer (MarienFeld Company Germany) under the light microscope (DM 100; Leica, Wetzlar, Germany)
SECTION: Measurement of Hematological Parameters
28. To measure hemoglobin, fresh blood was collected from fish from each treatment and was poured in the edge of a strip of hemoglobin meter before the coagulation of blood. 
SECTION: Measurement of Hematological Parameters
29. Estimation of immunoglobulin (IgM) was carried out by using Humalyzer-3000 analyzer.
SECTION: Metagenomics study
30. Gut microbiome DNA was extracted using a commercial kit
SECTION: Metagenomics study
31. V3 and V4 primer were used for sequencing