The first difficulty of the experiment was to manufacture labeled arylphorin that had a high specific activity level (high ratio of radioactive molecules to nonradioactive molecules). First this process was attempted "in vivo" by injecting insect larvae at the fifth instar with radiolabeled phenylalanine. The idea was to allow the larvae to manufacture the arylophorin (using the labeled phenylalanine). This procedure, however, did not result in a high enough specific activity of arylophorin. Additional arylophorin was injected when even high amounts of radiolabeled phenylalanine failed to produce the desired specific activity level. The additional arylophorin, however, resulted in a high mortality rate among the insects.
Another method of producing labeled arylphorin was decided upon--incubating the insect fat body, which produces the arylophorin, from the the fifth instar stage under certain conditions to achieve the desired specific activity level. This proved the second (and perhaps greatest) difficulty of the experiment--finding the right conditions from which the highest (or most acceptable) specific activity could be achieved.
After much experimentation it was determined that incubating one-half fat body in 1 ml of medium (0.15 mM of labeled phenylalanine and amino acids at 25% normal level in Grace's medium) for 15-24 hours, chasing labeled phenylalanine after one hour with unlabeled phenylalanine, yielded most desirable results. Temperature was shown to have little or no affect on the results, so room temperature was used in further experimentation and final implementation in achieving high specific activity (more convenient).