>  Medhat added some other refinements that made this a bit easier for the
>average beekeeper.


What refinements?

When  we tested thousands of colonies when I was in Ontario, we found the 
following to get reliable results you need to:
1. Pick a frame of capped brood that contains pupae with light pink eyes.
2. use a smaller ring that isolates 30-50 cells to be treated with Liquid 
nitrogen.
3. Use 4 replicates /hive that means 4 rings placed on a single capped 
brood frame Then apply your liquid nitrogen . The total cells treated is 
120-200 capped cells in each hive.
4. Apply 1-2 scopes  (30ml) of Liquid nitrogen first to all 4 rings then 
repeat second treatment after 1-2 minutes. This will ensure that pupae 
were killed. Think of it is freeze / thaw out system to kill the pupae. In 
some cases when we applied one time liquid nitrogen, pupae survived and 
emerged as adults without any problem. 
5. The frame will be placed back in the hive in the same position as it 
was before applying the liquid nitrogen.
6. After 24 h count all uncapped and removed pupae. Calculate % uncapped 
and removed pupae. All removed cells or partially removed should be 
considered uncapped, too. In fact you can compare uncapping vs removal 
activities for each hive. Keep in mind these are two different traits.
7. You need to take the average of the 4 readings for each hive to present 
the hive activities for hygienic. We used to consider only removal of 
pupae is the final criterion for selection of the hygienic trait.
8. Test should be done away from the honey flow time. Honey flow will 
encourage bees to remove and clean cells faster. Your figures will be over 
estimating the trait  if you test during the honey flow.


Medhat

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