56 



THE ART OF MAKING MICROSCOrE SLIDES 



Clearing 



quantity of anhydrous copper sulfate at 

 the bottom of the aljsolute alcohol bottle 

 and cease to regard the alcohol as absolute 

 when the salt starts turning from white to 

 blue. More wholemounts are ruined by 

 being imperfectly dehydrated than by any 

 other method, and even the smallest speci- 

 men should have at least 24 hours in abso- 

 lute alcohol before being cleared. 



If the specimen is to be mounted in 

 Canada balsam, or one of the substitutes 

 for it, it must next be cleared, but if it is 

 to be passed directly to Venice turpentine 

 the reader should turn to end of the chap- 

 ter for a detailed description of this 

 technique. 



The Choice of a Clearing Agent 



A clearing agent must be some sub- 

 stance which is miscible both with abso- 

 lute alcohol and with the resinous medium 

 which has been selected for mounting. 

 The ideal substances for tliis purpose are 

 essential oils for they impart just as much 

 transparency to the specimen as does the 

 resin used for mounting, so that one has, 

 as it were, a preview of the finished 

 specimen. The use of xylene or benzene, 

 which is so widespread in the preparation 

 of paraffin sections (see Chapter 12) has 

 tended to spread into the preparation of 

 wholemounts, for which purpose, in the 

 author's opinion, they are worthless. They 

 have a relatively low index of refraction ; 

 hence one cannot tell whether or not the 

 slight cloudiness of the specimen is due to 

 imperfect dehydration until after they 

 have been mounted in balsam. 



The writer's first choice is terpineol 

 (synthetic oil of hlac) which has advan- 

 tages possessed by no other oil. It is read- 

 ily miscible with 90% alcohol, so that it 

 will remove from the specimen any traces 

 of water which may remain in it through 

 faulty dehydration, and it has also the 

 property of not making specimens brittle. 

 The odor is very shght and rather pleas- 

 ant. Oil of cloves is the most widely 

 recommended essential oil for the prepa- 

 ration of wholemounts and it has only two 

 disadvantages: its violent odor and the 

 fact that objects placed in it are rendered 

 brittle. If a small arthropod be cleared in 

 oil of cloves, it is almost impossible to get 



it into a wholemount without breaking off 

 some appendages. Oil of cloves is, how- 

 ever, miscible with 90% alcohol. Oil of 

 cedar (more correctly oil of cedar wood) 

 has been recommended in the literature 

 and has the advantage of having a pleas- 

 ant odor and of not rendering ol:)jects 

 brittle. Unfortunately it is very sensitive 

 to water so that perfect dehydration in 

 absolute alcohol is necessary before en- 

 deavoring to clear with it. 



Two clearing agents, wliich are excellent 

 for unstained specimens, are very little 

 known. These are turpentine and acetic 

 acid. The acid cannot be used with stains 

 for obvious reasons, while the turpentine 

 is a strong oxidizing agent and cannot, 

 therefore, be used after hematoxyhn, 

 though it is perfectly safe with carmine. 

 Absolute (glacial) acetic acid is miscible at 

 all proportions both with water and with 

 Canada balsam. If small arthropods are 

 to be mounted in balsam, rather than in 

 the manner described in Chapter 4, they 

 may be dropped into acetic acid, left there 

 until they are completely dehydrated, and 

 then transferred directly to balsam. This 

 little-known technique is strongly to be 

 recommended. 



Mounting Specimens in Balsam 



Nothing is easier than to mount a speci- 

 men in a resinous medium, provided that 

 it has been perfectly dehydrated and 

 cleared. A properly made wholemount 

 should be glass-clear, but it will not be 

 clear in balsam unless it is clear in ter- 

 pineol or clove oil. Not more than one in a 

 thousand wholemounts has this vitreous 

 appearance, and the Avorker who is ac- 

 customed to looking at rather cloudy 

 wholemounts should take the trouble to 

 dehydrate a specimen thoroughly, then to 

 remove the whole of the dehydrating 

 agent with a clearing agent, and then to 

 mount properly in balsam. 



The first step, therefore, in making a 

 mount in, say, Canada balsam is to make 

 quite certain that the specimen in its es- 

 sential oil is glass-clear; the second step 

 is to make certain that one has "natural" 

 Canada balsam and not "dried" balsam 

 which has been dissolved in xylene. Solu- 

 tions of dried balsam in hydrocarbons are 



