I invented the liquid nitrogen freeze method while working on a project for 
 EPA.  I found that neither the pin prick nor Steve Tabor's - cut and 
freeze  in freezer- produced consistent  results.  The problem is that  physical 
damage (pricking, cutting out bits of comb) can induce a repair  behavior.   
Hygenic behavior is supposedly  controlled by   two genes, each with a bit 
different behavior.    Also, removal  of paper is probably not a good test - 
its just part of a two step  process.
 
As per area of brood - the larger the area you  kill, the more certain  you 
will be to see bees take action - that's again not simply hygienic 
behavior,  but a response to a damage 'crisis'.   
 
What most have forgotten - our data showed that several small patches  over 
more than on brood frame provided the most reliable test.
 
Medhat followed up our work with his butter dish, and we did something  
similar with aluminum 'hockey' pucks.
 
Jerry
 
 
 

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