1. Agarose is frequently used instead of agar.

2. Cells are mixed in ~0.33% agarose/agar and layered on top of the base layer of 0.5% (or higher) agarose/agar.

3. Melt 1% agarose and cool in 40 °C water bath.

4. Warm 2X medium (e.g., 2X RPMI/20% FCS or 2X DMEM/20% FCS) to 40 °C.

5. Mix equal volumes of 1% agarose with 2X medium.

6. Pipet 1.5-2 ml to 35 mm dish or 6-well plate.

7. Allow to set without disturbing in hood for 30 minutes. Plates can be stored at 4 °C for 1 week.

8. If the plates are stored at 4 °C, warm to room temperature.

9. Melt 0.7% agarose and cool in 40 °C water bath.

10. Warm 2X medium (e.g., 2X RPMI/20% FCS or 2X DMEM/20% FCS) to 40 °C.

11. Count cells. 5,000-10,000 cells are plated for each 35 mm dish. Adjust cell concentration to 5,000-10,000 cells/0.1 ml.

12. 0.5-1.5 ml top layer agarose/medium is needed for each 35 mm dish.

13. Calculate the number of 0.7% agarose and 2X medium needed. Samples should be plated in triplicate. Mix equal volumes of 0.7% agarose with 2X medium.

14. Add 0.1 ml of cells for each 35 mm dish. Mix.

15. Carefully pipet 0.5-1.5 ml on top of the base layer.

16. Allow to set without disturbing in hood for 30 minutes.

17. Incubate at 37 °C in humidified incubator for 10 - 14 days. Longer incubation (up to 4 weeks) may be needed for slow growing cells.

18. Stain plates with 0.005% Crystal Violet or nitroblue tetrazolium.

19. Count colonies.