To improve signal detection we decided to encapsulate our bacteria inside beads made of gellan gum and set about developing our own procedure. Research yielded a number possible options and having experimented with several, we eventually settled modifying a technique developed by Carol Nolan (Nolan, Carol L. (Blue Bell, PA). 1992. Method for microbial immobilization by entrapment in gellan gum. United States Monsanto Company (St. Louis, MO). http://www.freepatentsonline.com/5093253.html)
For more detail into how the beads work, gellan gum and other methods we tried, go to the bead page
Bead Making Procedure
It is first necessary to set up a 50mL LB overnight inoculated with a colony of cells you wish to encapsulate, and leave this overnight at 37°C. The next day, follow these steps:
- Add 0.24g of Gellan Gum to 19.6mL of ddH2O. Heat in a waterbath to 90°C, stirring occasionally, then cool to 54°C.
- Meanwhile centrifuge 2 x 25mL overnight at 4500 x g for 8 minutes. Discard the supernatant.
- Wash the pellet in 2mL of ddH2O, re-suspend and centrifuge at 4500 x g for 5 minutes. Repeat once.
- Slurry the cells in 0.25mL ddH2O and add to the cooled gum, mix gently by shaking.
- Pipette dropwise into 0.5mL of CaCl2 using the p1000 pipette from approximately 1.5 inches above the surface solution.
- Gently agitate the beads for at least 1 minute then pour off the CaCl2.
This will result in beads approximately 1 - 2mm in diameter.
Note: It is essential to pipette the beads into CaCl2 as soon as possible once the bacteria have been added to the gellan gum. The cooler the gum-bacteria mix gets, the larger and less uniform the beads formed will be.