Bee-Quick gave a good explanation.  Personally, "everyone uses" isn't  a 
good justification  for freezing LARGE areas.   Three inches is  a tad on the 
large size - don't go bigger.
 
Our  initial data demonstrated that smaller areas, at least 2 or 3  over 
more than 1 side of one frame provided a MORE  ACCURATE  test.  Freeze too 
large an area, and any hive will uncap and remove.   Keep the frozen patches 
small.
 
I prefer keeping the freeze area down to no more than 2 inches in  
diameter, and you can reduce the amount of liquid nitrogen per tube  proportionally.
 
I use thin-wall aluminum  tubing from the hardware store.  Medhat  used a 
butter dish.
 
As per dry ice, CO2 from fire extinguisher, any number of gases that freeze 
 when they hit the air (like the ones used to freeze off warts) - I agree, 
the  liquid nitrogen is hazardous.  We developed this method for EPA as a 
test  to be used in assessments of brood survival in colonies placed on 
hazardous  waste sites.  The data had to be able to hold up in court.
 
We did NOT  anticipate the average beekeeper would be using  this.  Pat 
Heitkham was one of the first, and he borrowed a dewer from his  local cattle 
AI guy.  Marla said he couldn't use my test, and he  decided and proved that 
he could.
 
So, I'm all in favor of a SAFER alternative.  Cold is cold.  For  accuracy 
of the test, you need to minimize damage to the comb AND you  need  to 
thoroughly freeze the brood.  Frost-bite isn't sufficient.  We  found dry ice to 
be slow and CO2 will put bees to sleep and even kill  them.  Not a problem 
in open air, but don't drop into  a hive - we use  it to kill off colonies at 
the end of experiments where we need to gather  bees.
 
When we were developing  a way to be sure the bees were frozen (DEAD),  our 
criteria was simple.  Pick an area of the comb that has pupae  on  both 
sides.  Freeze the test area with your  method of choice.   When you go in to 
complete the test, flip the comb over.  If you completely  froze the test 
area, you should have frozen at least some of the pupae in the  same place on 
the reverse side of the comb.
 

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