IN THE LARV^ OF ANISOPTERID DRAGONFLIES. 137 



larva must now be placed for 24 hours or more (according to tbe state o£ 

 hardness of the integument) in Soap Alcohol. This will extract all the fat, 

 and occasionally causes local damage or malformation of a particular block of 

 tissue ; but it is a safe softening agent to use, provided it is not left in for too 

 long a time. Hence the advice already given to soften only those larvae 

 which have recently undergone ecdvsis. Soap Alcohol seems to me, in any 

 case, preferable to Eau de Javelle. 



Certain very tough larvae, e. g. Austrdcordulia refracta, seem to resist 

 the softening treatment, even if immersed immediately atter ecdysis. The 

 integument of the larva is dark olive-green immediately after ecdysis, and 

 hardens almost at once to a thick dark brownish state, which resists all 

 attempts at softening within a reasonable time. In other larvas the 

 integument directly after ecdysis is very transparent and soft, and immersion 

 in the soap alcohol need not be prolonged beyond a few hours. 



The softening agent must be completely removed before dehydration by 

 placing the larva again in 70 per cent, alcohol, and washing it in several 

 changes extending over 24 hours or more. Before doing this, the abdomen 

 should be cut off at about the 4th or 5th segment to allow of greater 

 penetration on the part of the alcohol. 



Dehydrating and Clearing. — The object should now be run up in the usual 

 way through 90 per cent, alcohol (6 hours or less) to absolute alcohol (6 hours 

 or less). It is then placed in a mixture of absolute alcohol and cedar-oil in 

 «qual parts for 24 hours. Finally, it is removed to pure cedar-oil and left 

 there for 24 hours. 



Infiltration and Embedding. — After clearing in cedar-oil, some finely sliced 

 paraffin-wax of 36° M.P. (melting point) is introduced into the oil and 

 allowed to dissolve. Enough should be added to make the mixture just 

 pasty, so that, on warming ever so slightly, the fluidity returns again. The 

 object is left thus for 24 hours. It is then placed in pure melted paraffin- wax 

 of 36° M.P. for about 3 hours. From this it is passed into melted wax of 

 52° M.P. for a further 3 hours. Two or three changes may be given 

 during each three hours in order to ensure the removal of all the cedar-oil. 

 Finally, the object may be embedded in paraffin of 58° M.P. straight from 

 the melted 52° M.P. wax ; or, if the weather be warm, a further short period 

 in melted wax of 58° M.P. may be given before the final embedding. 



Sectioning. — As most of the gill-baskets are fairly large objects the 

 majority of sections should be cut at 12 or 15 fju, the thickness being reduced 

 to 5 or 7 yu, at any desired level through a number of sections, in order to 

 provide material for finer histological studies. 



In the case of the whole larva the cutting should begin at the anal end, so 

 that the rectum is reached as quickly as possible. The anal appendages can, 

 of course, be removed with scissors before the softening process is begun — or 

 after it, if preferred. 



