Journal of 



Applied Microscopy. 



Volume I. 



JUNE, 1898. 



Number 6 



Fixing and Imbedding Lichens. 



For many years lichens have been 

 studied by descriptive and systematic 

 botanists mainly. Although a few mor- 

 phologists and physiologists have made 

 important contributions to our knowl- 

 edge of these interesting associations of 

 organisms, the great number of prob- 

 lems in plant morphology and physiology 

 to which the study of more rapidly 

 growing, larger, and simpler structures 

 promised a speedier solution, have 

 caused the lichens to be more or less 

 ignored by investigators. When any 

 attempts to study them have been made, 

 various technical and other difficulties 

 have tended to discourage the investi- 

 gator. The excessively slo'v growth of 

 lichens not only tries one's patience, but 

 it also very greatly increases the danger 

 of culture experiments becoming failures 

 through the invasion of the more 

 prolific bacteria and moulds. 



Even the study of their microscopic 

 anatomy is attended with unusual dif- 

 ficulty, and comparatively seldom, if at 

 all, have modern histological methods 

 been applied to them. Yet the careful 

 study of their minute structure would 

 not only settle the disputed points as to 

 the nature of the lichen association and 

 the reproductive parts, but would sug- 

 gest and prepare the way for physiologi- 

 cal studies of no slight importance. 

 Since De Bary and Schwendener con- 

 vinced botanists (and even the "lichen- 

 ologists") finally that lichens are com- 

 posed of two very different kinds of 

 plants, green or blue-green algae, capa- 

 ble of manufacturing their own food 

 from inorganic materials, and fungi, 

 absolutely dependent upon already pre- 

 pared food, there has been no agreement 

 as to whether, as Reinke claims, these 

 associations are sufficiently intimate and 

 independent to justify their being called 

 autonomous, or whether, as many be- 

 lieve, they are composed of two kinds 

 of plants mutually benefited by being 



associated; or, again, whether the asso- 

 ciation is simply that of host and para- 

 site. Careful study of the structure of 

 these associations, and culture experi- 

 ments upon the component organisms, 

 will settle the question. 



For the study of lichen structure, I 

 have found proper fixing and careful 

 imbedding to be indispensable. I have 

 been most successful with a concentrated 

 (or saturated) solution of corrosive sub- 

 limate in thirty-five per cent, alcohol. 

 This I use hot, which secures very rapid 

 penetration, killing, and fixing, during 

 and after which no contraction need 

 take place. If the lichen material is air 

 dry, but still alive, it should be thor- 

 oughly wetted and kept moist for two 

 or three days, and then small pieces only 

 should be put into the hot fixing agent, 

 in which they will promptly sink to the 

 bottom, where they may be left for a 

 suitable length of time — from five min- 

 utes to half an hour. Slow dehydration 

 in the alcohols, using five or six grades 

 and comparatively large volumes in 

 proportion to the size of the objects, will 

 secure the removal of the last traces of 

 corrosive sublimate. 



After absolute alcohol, alcohol-xylol 

 (equal parts), and pure xylol, imbedding 

 may be begun. For many lichens a harder 

 grade of paraffin must be used than for 

 most vegetable structures. A mixture 

 of hard and soft paraffin which melts 

 at about 60°C. is to be recommended. To 

 the pure xylol in which the material has 

 already become clear, small pieces of 

 paraffin may be added, keeping the dish 

 warm at the same time, both to increase 

 the solvent power of the xylol and also 

 gradually and finally to evaporate it all. 

 By this means the material is slowly 

 warmed and penetrated with paraffin. 

 After remaining in melted paraffin 

 absolutely free from xylol for three 

 hours, the objects may be imbedded. The 

 sections should be very thin, and before 

 cutting, the block should be chilled to 



