-Use sterile technique and sterile solutions throughout this method.-

1. Grow a starter culture at 30 C with shaking (250 rpm) until it reaches saturation.

***It is important to make sure all strains to be tested are completely saturated, since cell density can affect the size of the halo.

2. Microwave a sterilized solution of 0.5 % agar until melted and place in a 55 C water bath.

***Don't leave unattended when microwaving since the agar tends to boil over suddenly.

***Make sure the agar equilibrates to 55 C to 60 C before starting the assay; otherwise it will kill the cells.

3. Distribute several paper disks (Difco, #1599-33-6) into a sterile petri dish, and spot either 5 or 15 of synthetic alpha-factor (1 - 5 mg/ml) onto each disk.

***In some instances, it may be beneficial to use more disks per plate to obtain a wider range of alpha-factor concentrations.

***This should be done no more than 1 to 2 hrs before use.

4. Aliquot 4 ml of 0.5 % agar into a 5 or 14 ml plastic tube with a pop-off cap (e.g. Falcon, #35-2063).

5. Immediately before performing the assay, transfer a small volume of the saturated starter culture into the tube containing agar. 100 ul of starter culture should be used for strains grown in SCD; 10 ul should be used for strains grown in YPD.

6. Invert a few times, and pour onto a pre-warmed plate containing the appropriate solid media. Swirl to cover the plate evenly. This step should be done quickly to prevent the agar from solidifying.

***It is convenient to do two tubes at a time, one in each hand.

***Bubbles can usually be eliminated by poking with flamed sterile forceps.

7. On the plate, place one paper disk containing 5 ul alpha-factor, and one disk containing 15 ul alpha-factor.

***When doing several assays, it is helpful to use a paper template for consistent placement of the disks on each plate.

8. Place the plate at 30 C until a lawn of cells appears. The halo assay will result in a zone of growth inhibition surrounding the filters soaked with pheromone, as seen in the example below.  Halos can usually be seen clearly after 24 hrs for most strains.

***It is often desirable to let the cells grow longer (up to several days) to get a denser lawn and to observe any changes that might occur over an extended period of time.

9. Differences in halo size are normally big enough to be detected by eye.

***It is also possible to measure halo size and plot this value vs. log [alpha-factor].