Following infection the fungus becomes established in the developing seed embryo. Seeds of the crop being tested are placed in a beaker of sodium hydroxide water solution. The mixture is allowed to stand for an additional period of time. This will extract the embryos and they will rise to the surface of the container while the debris sinks to the bottom. Embryos are recovered from the surface of the container and prepared for clearing.
Clearing is obtained by placing the embryos in a petri dish containing lactic acid and trypan blue.
The dish is covered and placed in a dish overnight. The embryos vary from grayish blue through powder to turquoise, depending upon the cultivar and crop type. The 1000 embryos are carefully examined under a microscope using 10-25X magnification. During this examination infected embryos are counted and the total recorded. The standard for true loose smut in barley may be found in Table 2 of the Canada Seeds Act .