To start a culture put 4 rounded tablespoons of the dry mixture into the bottle. Put in a couple of pinches of baker’s yeast. Shake it up for a bit to mix the yeast with the medium. Add 3 tablespoons of warm water. Finally, plug the top with a cotton ball or something similar to keep wild fruit flies and other insects out.
It will look like you did not put in enough water, and it will take a while for the water to sink it. Don’t try to stir it. Just put it aside for a couple of days in a fairly warm spot to let the yeast to get started. I just set them on top of an aquarium.
After a couple of days remove the plug and you will smell the yeast. The next step is to place something for the flies to crawl on. I use strips of plastic canvas about 1” wide. Plastic canvas is sold in craft shops.
Put a funnel in the bottle and pour about a dozen flies from another culture into it. Remove the funnel and replace the plug. Depending on the temperature, in a few days you will see the small maggots crawling around if you look closely. Eventually they will crawl up the sides and form pupae. Then they will hatch as adults. The first generation adults will then appear and can be shaken into the tank for feeding.
Although most bottles will be good for a couple of generations I try to start a new culture when the first new adults appear. The adults are reported to be most fertile shortly after emerging. At any given time I try to have 3-4 bottles at different stages. It is easy for a culture to get too old if you are not starting new ones to replace them. I know one aquarist who marks the date on his cultures and starts another every 9 days.