NOTES ON THE CYTOLOGICAL TECHNIQUE 



II. Making the preparation permanent. The method used has been almost exactly that 

 originally devised by McChntock, though for ease of reference the details may be given 

 here as follows : 



(i) Dissolve off the cover-slip by immersing the slide face upwards in 45 % acetic acid. 

 It is essential not to attempt to hurry this process by poking the cover-slip with a needle 

 or the more tightly attached cells will wash off. 



(ii) Dehydrate by passing slide and cover-slip through graded mixtures of acetic acid 

 and absolute alcohol of the following strengths i : i, i : 3, i : 9, absolute. 



(iii) Partially clear in i : i absolute and xylol from which it is safe to mount in 

 balsam. It is essential in mounting that the cover-shp should be replaced exactly over 

 the area from which it came. This may be clearly marked by the lines of dried carmine 

 formed round the edges of the cover-slip after the original boihng and which should on 

 no account be cleaned off until the preparation is finished, but if this is not sufficient it 

 may be convenient to mark the position of two corners of the cover-slip with a diamond 

 on the back of the shde before immersing it in the acetic acid. 



III. Acetocarmine squash with manual pressure. With small sporangia in which it is im- 

 possible to remove the empty cases entirely additional pressure is needed. This applies 

 particularly to the Polypodiaceae with mixed sori, in which ripe spores which resist 

 pressure are always liable to be present on the same slide as the softer mother cells. The 

 spore mother cells are squashed out of their sporangia as thoroughly as possible with a 

 flat-ended needle into the stain and the larger lumps of tissue such as the indusia re- 

 moved. Not too many sori should be dealt with at one time, six or so on a slide are 

 generally enough. A cover-shp, which may be large or small according to taste, is then 

 put on and the preparation heated gently. Whether it should be boiled or not depends 

 on the intensity of colour and the softness of the material. If staining is faint, boiling 

 enhances it; on the other hand, if the cells are soft they may blow to pieces on boiling. 

 In either case the pressure which produces flattening of the cells is apphed by hand. The 

 warm preparation is put down on the bench, momentarily covered with a piece of 

 blotting paper and a finger passed rapidly over it with the precise degree of pressure 

 which can only be learned by experience. The field should then be searched at once, 

 preferably without ringing, and ruthlessly discarded if it does not show any specially 

 favourable cells. This is essential, because in the Polypodiaceae, where only 16 or some- 

 times 8 mother cells may be the entire contents of a sporangium, successfully squashed 

 and usable cells may occur singly on a slide and if not found at once be looked for in vain 

 afterwards. 



As a routine procedure in deahng with this type of material every instance of an excep- 

 tionally perfect cell was photographed at once and perhaps also drawn while the pre- 

 paration was in the first wet condition, as a precaution against its loss on transfer to 

 balsam. A few such photographs have been included in this book (e.g. Figs. 183/^, 2i8(^). 

 In every case, however, the transfer to balsam by the method fisted in paragraph II 

 above was attempted, the only additional precaution needed being slow dehydration, a 

 delay of a quarter of an hour or longer in each of the alcohols being not too much. In 

 most cases a cell could be rephotographed in balsam and the greater number of the 

 figures in this book are of these ; occasionally the critical ceU became detached and lost 



296 



