Bcrazy
Drone Bee
- Joined
- Nov 14, 2008
- Messages
- 1,460
- Reaction score
- 5
- Location
- Warboys, CAMBS
- Hive Type
- None
- Number of Hives
- nil bees given away all colonies
I have been asked by Tim my method of maceration of bee body parts for the BBKA Microscopy exam. The method i use works extreamly well for me and it is as follows;
My method is:-
Kill and fix in alcohol
Remove wings and return them to alcohol. Caustic will destroy the membrane.
Place bee in cold 20% caustic (soda or potash)
Heat until the alcohol inside the bee boils - this will happen at about 75 deg. C - well before the caustic itself boils. You can just usually see the bubbles forming inside the bee, and sometimes the bee moves quickly about as the alcohol vapour escapes from it. Leave it "boiling" for 5secs - do not boil the caustic at any time as this will over soften the bee.
Remove heat for 5mins. During this time the caustic will seep into the bee to replace the alcohol that boiled off during the heating.
Repeat heating/cooling at least twice more. During the second heating the liquid expelled from the bee will be brown due to dissolved body contents. This will get even darker on the third heating. If this doesn't happen try one more heating cooling, but still never boiling the caustic.
Remove bee from caustic and place in water for about 12 hours to get rid of most of the caustic. This water may also turn a little brown as more contents come out.
Remove bee from water and place in 20% acetic for several hours. This will neutralise any caustic that is left and remove the small amount of calcium salts that are in the chitin.
After this the bee will be very soft, and easily dissected. This should be done as far as possible under water - certainly without letting the parts dry out, as any air bubbles will be very difficult or impossible to remove.
Place the dissected limbs etc onto small squares of microslides, arrange exactly as you want them to appear when finished, cover with another square, and clip together. This was the last chance to move any of the limbs as from now on they will harden, and probably break if movement is attempted.
Place the clipped slides into 100% alcohol for 3 hours.
Remove and place in fresh alcohol for 3 hours.
Remove and place in xylene for 3 hours
Remove and place in fresh xylene for 3 hours
Remove clipped slides to petri dish with a little xylene in it, unclip part slides, a very carefully remove limb into xylene. From there move onto final slide, position carefully, apply balsam and cover slip.
The bee is fixed in alcohol, which initially stays inside the bee when it is placed into the caustic soda. When the caustic soda and bee are heated the alcohol in the bee boils long before the caustic soda does. After about the third boiling the alcohol is mostly boiled off, and the contents are dissolved, and turning the caustic brownish. At this point the caustic itself can be brought just to the boil, but the heat removed immediately it boils, and then allowed to cool a little before proceeding to the water, then acetic etc. The bee is not left boiling for any length of time as it would be over softened.
The bee only stays in the caustic during the 3 or 4 heating cycles - probably over in 30mins.
The mixture for boiling is 20% caustic soda (or caustic potash). 20% means that there are 20 grams of caustic dissolved in 100mls of water, or pro rata.
Regards,
Mo
Best of luck Tim and to all others who intend to take the microscopy exam just follow what is says in Modul 9 and you cant go wrong.
Best wishes
Mo.
My method is:-
Kill and fix in alcohol
Remove wings and return them to alcohol. Caustic will destroy the membrane.
Place bee in cold 20% caustic (soda or potash)
Heat until the alcohol inside the bee boils - this will happen at about 75 deg. C - well before the caustic itself boils. You can just usually see the bubbles forming inside the bee, and sometimes the bee moves quickly about as the alcohol vapour escapes from it. Leave it "boiling" for 5secs - do not boil the caustic at any time as this will over soften the bee.
Remove heat for 5mins. During this time the caustic will seep into the bee to replace the alcohol that boiled off during the heating.
Repeat heating/cooling at least twice more. During the second heating the liquid expelled from the bee will be brown due to dissolved body contents. This will get even darker on the third heating. If this doesn't happen try one more heating cooling, but still never boiling the caustic.
Remove bee from caustic and place in water for about 12 hours to get rid of most of the caustic. This water may also turn a little brown as more contents come out.
Remove bee from water and place in 20% acetic for several hours. This will neutralise any caustic that is left and remove the small amount of calcium salts that are in the chitin.
After this the bee will be very soft, and easily dissected. This should be done as far as possible under water - certainly without letting the parts dry out, as any air bubbles will be very difficult or impossible to remove.
Place the dissected limbs etc onto small squares of microslides, arrange exactly as you want them to appear when finished, cover with another square, and clip together. This was the last chance to move any of the limbs as from now on they will harden, and probably break if movement is attempted.
Place the clipped slides into 100% alcohol for 3 hours.
Remove and place in fresh alcohol for 3 hours.
Remove and place in xylene for 3 hours
Remove and place in fresh xylene for 3 hours
Remove clipped slides to petri dish with a little xylene in it, unclip part slides, a very carefully remove limb into xylene. From there move onto final slide, position carefully, apply balsam and cover slip.
The bee is fixed in alcohol, which initially stays inside the bee when it is placed into the caustic soda. When the caustic soda and bee are heated the alcohol in the bee boils long before the caustic soda does. After about the third boiling the alcohol is mostly boiled off, and the contents are dissolved, and turning the caustic brownish. At this point the caustic itself can be brought just to the boil, but the heat removed immediately it boils, and then allowed to cool a little before proceeding to the water, then acetic etc. The bee is not left boiling for any length of time as it would be over softened.
The bee only stays in the caustic during the 3 or 4 heating cycles - probably over in 30mins.
The mixture for boiling is 20% caustic soda (or caustic potash). 20% means that there are 20 grams of caustic dissolved in 100mls of water, or pro rata.
Regards,
Mo
Best of luck Tim and to all others who intend to take the microscopy exam just follow what is says in Modul 9 and you cant go wrong.
Best wishes
Mo.
