Phage Cocktail for Shigella & Salmonella: Biofilm Control
- Okay, here's a breakdown of the methods described in the provided text, categorized for clarity.
- * Bacterial culture: Bacteria where grown in Tryptic Soy Broth (TSB) overnight at 37°C and 190 rpm.
- * Cell Lines: * RAW 264.7 macrophage cells * HT-29 human colorectal adenocarcinoma cells * Source: Obtained from the NIRBI Cell Culture Facility.
Okay, here’s a breakdown of the methods described in the provided text, categorized for clarity.
1. Biofilm Formation & Degradation Assay
* Bacterial culture: Bacteria where grown in Tryptic Soy Broth (TSB) overnight at 37°C and 190 rpm.
* Biofilm Formation:
* Bacterial culture was diluted.
* 20 µL of bacteria + 180 µL TSB were added to 96-well polystyrene microplates.
* Incubated for 24 hours at 37°C to allow biofilm to mature.
* Biofilm Degradation Assessment:
* Phages were added to the mature biofilm at a Multiplicity of Infection (MOI) of 10.
* Incubated for 8 hours at 37°C.
* Wells were rinsed three times with 1X PBS and air-dried.
* Biofilm Quantification:
* Biofilm was stained with 0.1% (w/v) crystal violet.
* Optical density was measured at 595 nm using a microplate reader (iMark Microplate Reader S/N 21673).
2. Mammalian Cell Culture
* Cell Lines:
* RAW 264.7 macrophage cells
* HT-29 human colorectal adenocarcinoma cells
* Source: Obtained from the NIRBI Cell Culture Facility.
* Culture Medium: Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with:
* 10% (v/v) fetal bovine serum (FBS)
* 5 mL/L antibiotic solution (containing 25 µg/mL Amphotericin B, 100 U/mL penicillin, and 100 µg/mL streptomycin).
* Culture conditions: 95% air, 5% CO2, 37°C.
* Passaging: Cells were grown to 80-90% confluence in T25 flasks and then used at a concentration of 5 x 105 cells/mL for experiments.
3. Cytotoxicity Assay (MTT Assay)
* Purpose: To assess cell viability of RAW 264.7 macrophage cells.
* Treatment: Macrophage cells (5 x 105 cells/mL) were co-cultured with:
* Single phages
* Phage cocktail (100 µL) at various dilutions
* Ceftriaxone (antibiotic)
* MTT Incubation: MTT was added to a final concentration of 0.5 mg/mL and incubated for 24 hours.
* DMSO treatment: 100 µL of DMSO was added to each well and incubated for 10 minutes to dissolve the formazan crystals formed by the MTT assay.
* Control: Cell culture without phage suspension.
Let me know if you’d like me to elaborate on any specific aspect of thes methods!