How to Calculate Viability

Calculating seed viability can be done in a number of ways. The easiest and most reliable method of seed viability testing is using 2,3,5-triphenyltetrazolium chloride, a stain commonly abbreviated as TTC or TZ. This stain reacts in an oxidation-reduction reaction with respirating (living) tissue in seeds, turning living seed embryos red, in contrast to no color change in non-living seed embryos. This test can also be used in the animal kingdom under different conditions to examine brain or other living tissue.

2,3,5-triphenyltetrazolium chloride (powder)

Distilled water

3 containers

Scissors or needle

Gloves

Measuring spoon

Scale (0.01 gram specific)

Weighing boat

Graduated cylinder

Calculate the volume of TTC stain you need. A standard concentration of TTC/water commonly used is 1 gram per liter of solution. For a smaller sample, you can mix 0.1 gram of TTC with 100 mL of distilled water.

Wearing gloves, weigh out the appropriate amount of TTC powder (0.1 g, 0.5 g, 1 g) and add to the appropriate amount of distilled water (100 mL, 500 mL, or 1 L, respectively).

Store the stain in a dark, cool place until it is ready to be used. Do not prepare the stain more than 24 hours in advance of when it is needed, as it can react in adverse ways with light, water, or heat.

Soak the seeds that you are going to use for at least 24 hours in distilled water. This process allows the seeds to swell--making them easier to cut and handle as well as allowing the stain to permeate the seed membranes.

Dissect or pierce the seeds with a needle or scissors. This is dependent on seed size, etc. This is done to increase stain permeation throughout the seed during the staining process. Try not to destroy the embryo during your dissection process

Remove the seeds from the distilled water and place them into the TTC stain previously prepared. If you're examining for specific embryo characteristics, remove from the stain as soon as red is seen in the seed tissue. If you're examining merely for viability (presence/absence of red in the embryos), you can leave the seeds in the stain longer (up to 24 hours). Remove the seeds from the stain and wash with distilled water to prevent further staining.

Examine the seeds for red color. To calculate viability, count the total number of seeds used as well as the number of seeds stained red and as a result, determined viable. Divide the number of "viable seeds" by the total number of seeds and multiply this by 100 to get a percentage. This is your overall percent viability.

Please handle TTC powder with care, as it can have adverse effects on your skin. Call a doctor immediately if it's ingested.