Given the questions, I've pulled the  following description of our  assays 
that have been published and available on our websites since 1995.   Since I 
retired from UM, we're shifting  things over to our  new company web site, 
which is almost ready for launch.
 
As most of you know, from the early 1970s through the early 1990s, I did a  
considerable amount of work for EPA regarding the use of bees as 
environmental  monitors and for ecological assessments of potential hazards at EPA 
hazardous  waste sites (i.e., EPA authorized use of bees for this purpose in 
1989).   One  of our tasks was to identify measurement end points that could 
provide  data for assessments.  Here are two of the methods that we produced. 
  FYI - I realized after posting that the hygienic assay had been 
published, that  the description in the book had a major error - we do  NOT push the  
tube into the comb, in fact, we take care NOT to damage the cells.  Our  
tube for the liquid nitrogen makes a SLIGHT impression in the caps, but does 
not  break them.
 
For the following tests - the Brood Survival Test is one  of our go to  
methods for assessing  presence of materials that may poison the brood,  
whether a pollutant, natural product from plants bees are foraging, or  pesticides.
 
We will post these to our new web site, with photos.   We're  still 
checking that everything on the site works - I'll send the URL when we're  ready to 
go.  In the meantime, this will provide a summary of our  protocols.
 
 
Brood  Survival Test   
For  many years, we have used a simple yet effective method to assess brood 
survival.  We first find a comb where the queen has laid eggs in a patch 
that is at least  20 cells wide by 12 cells deep. We mark six rows with 
dressmaker pins. For each  row, a pin is inserted into a cell on the left side of 
the patch and another pin  is inserted into the 25th cell to the right of 
the first pin. We randomize the  colors of the pins marking each row and 
record the colors on a data sheet.   
The  test area consists of 120 cells (six rows of 20). We record the 
contents of each  cell-empty, honey, nectar, pollen, young larva, old larva, or 
pupa. The comb is  then returned to the center of the brood nest. Two weeks 
later we take the comb  out and again record the contents of each cell.  
Assuming  that a cell started with an egg or young larva, we should find a 
pupa in the  cell. If not, the pupa either died or was removed by the bees.  
 
Because  the bees often try to remove the pins, we mix the colors so that 
we can find the  original area, even if some of the pins have been pulled out 
of the comb. We use  a toothpick to remove the caps from any capped cells. 
We also record the age of  any pupae. We sometimes find pupae that are 
underage. In other words, the  original egg (or larva) did not survive, but the 
queen replaced it soon enough  for the replacement to reach the pupal stage.  
We  have learned never to assume that all of the initial eggs survived. 
Capped brood  does not guarantee survival. In many industrial areas, we find 
that brood losses  of up to 80% can occur, even though all of the cells may be 
capped.   
The  empty row (left) is a positive control. At the time of the initial 
inspection,  we swirl a round dowel in each of the 20 cells of the sixth 
(bottom) row. This  provides another mark to identify the test area, tests the 
ability of the queen  to replace lost eggs and serves as a reference for brood 
mortality. If no  mortality has occurred, all of the pupae should be healthy 
and similar in age  (right).  
Hygienic  Behavior   
The  hygienic behavior of a colony influences its susceptibility to 
microbial  pathogens, including natural pathogens such as chalk brood or foul brood 
and  presumably microbial insecticides. Several bee researchers employ a 
method  advocated by Steve Taber to rank colonies according to speed of 
uncapping and  removal of freeze-killed pupae. His method consists of cutting out 
a piece of  brood comb, freezing it overnight in a freezer, and carefully 
placing the brood  back into the comb.  
We  acknowledge the usefulness of this approach but found that it takes too 
much  time to assess large numbers of colonies. We also had problems 
getting  reproducible results. The act of cutting the comb seems to be sufficient 
to  stimulate our colonies to remove brood.  We’ve had the same problem 
using the pin prick  method. 
Therefore,  we developed a modification of the Taber technique. We use 
liquid nitrogen to  kill the pupae. 80 ml of liquid nitrogen is poured into a 
thin-walled metal tube  that is lightly pressed against the comb surface. A 
cloth wrapped around the  base of the tube prevents the nitrogen from leaking 
out and freezing additional  parts of the comb. We then have to wait about 
two minutes for the nitrogen to  evaporate and the metal tube to release from 
the comb. We then place an acetate  sheet over the comb and use a permanent 
marker to mark the positions of groups  of 20 undamaged cells (cells within 
the circle with intact caps). The comb is  then placed in the center of the 
brood nest. We mark the frame with a thumb tack  so we can quickly find it. 
Done correctly, this procedure leaves no visible  signs of having been 
applied other than the slight scoring of the caps caused by  the end of the 
metal tube.  
Twenty-four  hours later, we pull out the frame with the tack, index the 
acetate on the  frame, and count the number of uncapped as well as the number 
of cells from  which the bees have removed pupae have been removed by bees.  
 
Additional  tests conducted in spring 1996 established our recommendation 
in our technical  report to EPA to freeze several small patches, rather than 
one large patch of  capped brood to improve the accuracy of the assay.  We 
suggest a minimum of three small  frozen patches, each on a different frame 
face (requires 1-3 combs).  Hygienic  behavior is thought to be controlled by 
two recessive genes, one for uncapping,  the other for removal of dead or 
sick brood.  Colony populations are comprised of bees  from several 
sub-families – they all share the same mother, the queen; but may  have different 
fathers (drones).  Whether these sub-families are intermixed on the frames, or 
occur in  groups was unknown to us.

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