# Frozen brood test



## mike bispham (May 23, 2009)

In his successful experiment to raise resistant strains of bees [1] John Kefuss made good use of the frozen brood test to locate hygienic behaviours.

He also used plain 'bond' methods and selection by productivity, which we can all do, but I wondered if anyone here had any direct experience of frozen brood testing? 

Mike UK

[1] http://www.beesource.com/forums/sho...e-of-Resistance-Raising&p=1542537#post1542537


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## lharder (Mar 21, 2015)

Last year the UBC research team tested many of my bees using this method. What did you want to know?


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## mike bispham (May 23, 2009)

lharder said:


> Last year the UBC research team tested many of my bees using this method. What did you want to know?


Hi lharder,

Did they use nitrogen or other gas, or did they put combs in a freezer? What else; do they use sealed brood only? I'm just being lazy really, I'm sure I could look up instructions, but I thought it might be more fruitful to chat with somebody who has done it.

Mike


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## Juhani Lunden (Oct 3, 2013)

mike bispham said:


> Hi lharder,
> 
> Did they use nitrogen or other gas, or did they put combs in a freezer? What else; do they use sealed brood only? I'm just being lazy really, I'm sure I could look up instructions, but I thought it might be more fruitful to chat with somebody who has done it.
> 
> Mike


I have pdf -file of the work Paul Jungels is doing in Luxembourg. He is using liquid nitrogen. 

One Finnish friend of mine is using freezing. He has frozen (and melted)pieces of capped brood comb with him when going to yards. He makes the holes (rounds of capped brood) put these frozen (and again melted) pieces in these holes. The new pieces cut away go to the fridge in the end of the day.


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## mike bispham (May 23, 2009)

Juhani Lunden said:


> I have pdf -file of the work Paul Jungels is doing in Luxembourg. He is using liquid nitrogen.
> 
> One Finnish friend of mine is using freezing. He has frozen (and melted)pieces of capped brood comb with him when going to yards. He makes the holes (rounds of capped brood) put these frozen (and again melted) pieces in these holes. The new pieces cut away go to the fridge in the end of the day.


That makes good sense, thanks Juhani. Do you know what the advantage is to using gas at all? Your friend's system sounds fine.

Mike UK


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## Juhani Lunden (Oct 3, 2013)

mike bispham said:


> Do you know what the advantage is to using gas at all?


I don´t know.


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## Rader Sidetrack (Nov 30, 2011)

mike bispham said:


> Do you know what the advantage is to using gas at all?


The nitrogen used is a liquid, not a gas. The point of using liquid nitrogen is that it is so _cold_ that it is an _immediate_ kill, and can be done in the field, rather than waiting for a freezer to freeze-kill the sample. 

More on the technique: http://www.sare.org/Learning-Center...ting-Honey-Bee-Colonies-for-Hygienic-Behavior


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## Juhani Lunden (Oct 3, 2013)

Rader Sidetrack said:


> , and can be done in the field, rather than waiting for a freezer to freeze-kill the sample.


Like I just wrote, the freezed comb pieces( cut the previous day) are with you when you go to the yards. Doing it this way is easier on the field, you don´t need to carry the liquid nitrogen tanks with you.

And in your link they stated:
" Tests have shown that it does not matter if the frozen section comes from the same colony from which it was removed or from a different colony."


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## 1102009 (Jul 31, 2015)

Since I started to monitor I have some questions:



> For example, a colony may remove 95% of the frozen brood on the first test, but only 50% on the second. This colony is not hygienic!


I started to cut some brood cells from different frames and count mites.

- hygienic behavior is done by all colonies sooner or later. VSH is what we want. How to evaluate this with frozen brood test?

- the mite numbers change a lot from frame to frame. How do you estimate your infestation if you are not sure to have taken samples showing the average?

- the number of mites being phoretic change a lot from time to time. How do you estimate your infestation with only one alcohol shake at a time?

-What is more important as an evaluation, to check actively the hygienic behavior which means to watch for opened brood cells where mites are removed ( VSH) or to watch the seasonal fighting behavior of the bees which means to look for removed infested pupae early in season? ( all colonies do this IMO but the not resistant do it too late in season).

- is it important to watch how many mites are removed by grooming? Is it a sign of grooming if living mother mites are found on the board? Or must they be bitten?


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## beemandan (Dec 5, 2005)

mike bispham said:


> Did they use nitrogen or other gas, or did they put combs in a freezer? What else; do they use sealed brood only?


I was a grunt in a beelab about 10 or so years ago. We were doing liquid nitrogen hygienic testing. Putting an entire frame in a freezer isn't a good idea. The task for the house bees can be overwhelming. The population of nurse bees then becomes a factor. Doing a defined, smaller gives every hive a size for fair comparison. Sealed brood only.
Keep in mind this only tests for a general hygienic trait. To test for vsh is a bit more complicated. 
You should look up instructions for each.


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## mike bispham (May 23, 2009)

beemandan said:


> I was a grunt in a beelab about 10 or so years ago. We were doing liquid nitrogen hygienic testing. Putting an entire frame in a freezer isn't a good idea. The task for the house bees can be overwhelming. The population of nurse bees then becomes a factor. Doing a defined, smaller gives every hive a size for fair comparison. Sealed brood only.
> Keep in mind this only tests for a general hygienic trait. To test for vsh is a bit more complicated.
> You should look up instructions for each.


I think the idea is to take the samples - say a 2" circle - from a full capped frame, freeze them, then (when ready) thaw them and insert them in capped brood, taking away the removed circles to freeze ready for the next batch.

One of the things I like about this is it suits my detail-lite management. What is being tested is here and now. With my procutivity and age-only records I never really know if I'm looking at the same queen, or if she's new from supercedure, and will succumb top varroa come autumn. Freeze-brood seems to me to be a better assay, especially in combination with my records. I think I could take more of a chance on concentrating genetics with an intensive queen raising push. If I could bring vhs in too that would be better still.

Mike UK


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## JRG13 (May 11, 2012)

Freezing was pretty straightforward, more like a 4" diameter though.


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## lharder (Mar 21, 2015)

They inserted what looked to be a 2 inch plastic pipe over an area of purple eyed capped brood. Then liquid nitrogen was poured in, a couple of inches if memory serves. The pipe itself would freeze to the comb, and when it thawed, and could be removed, the number of empty cells were counted and the comb was labeled and placed back in the hive and the time recorded. The combs were removed 24 hours later and the number of cells still capped were counted.


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