NOTES ON THE CYTOLOGICAL TECHNIQ.UE 



probably not indefinitely. After lo years I have sometimes had to discard an old stock 

 owing to the formation of a chocolate-coloured precipitate when it was mixed with 

 water. In old stocks it is also sometimes better to dilute down to 1% instead of |% for 

 the working strength, though at this dilution the stain becomes exhausted more rapidly; 

 it may, however, give very bright and clear preparations while it lasts. 



Alum. The correct strength of iron alum varies somewhat with the material, but 

 usually the Pteridophyta require much weaker alum than the Flowering Plants. A 

 2 % solution for mordanting and an 8 % solution for differentiating is often satisfactory. 

 (In Flowering Plants I have generally used the strengths in the reverse order.) 



Bismark brown. A 2% solution in 90-95% alcohol is convenient. The counterstain can 

 then be introduced, after haematoxylin, last thing before finishing the dehydration. 

 Alternatively, the stain may be dissolved in a weaker alcohol (50 or 70%) and intro- 

 duced during the dehydration, a procedure which is sometimes preferred as leading to 

 better washing off of surplus stain. The length of time required in the stain varies with 

 the age of the solution but should be of the order of 2 min. 



Acetocarmine. The only secret in this stain is to have one's solution strong enough. 

 Heat a 45% aqueous solution of glacial acetic acid to boiling-point with excess of 

 carmine. Cool and filter. Use distilled water and keep the vessel covered while 

 heating. 



Schiff's reagent {Leuco-basic fuclisin) for Feulgen technique. Pour 100 c.c. boihng, distilled 

 water on to 0-5 g. of Gurr's basic fuchsin. Agitate thoroughly and cool to 50° C. Filter 

 and add 10 c.c. n/i -hydrochloric acid and 0-5 g. potassium metabisulphite. Shake well 

 and keep in the dark in a glass-stoppered bottle for 1 2- 1 8 hr . During this time the solution 

 bleaches to a pale straw colour and is then ready for use. Stored as above, the reagent 

 will keep in good condition for up to 3 months. 



SQUASH METHODS 



I. Simple acetocarmine. With large sporangia the very simplest of the squash methods may 

 be satisfactory. Thus in all the Osmundaceae 1 2 hr. fixing in acetic-alcohol followed by 

 breaking of a group of sporangia with a flat-ended needle into a drop of acetocarmine 

 will provide ideal material for squash preparations, without the need for any manual 

 pressure, provided that all the empty sporangia and other solid bodies are removed. It is 

 then only necessary to put on a cover-slip, conveniently a no. i thickness, | in. square, 

 and boil the preparation violently under one corner for a second or two, during which 

 process the cover-slip itself provides all the pressure which is needed. The initial size 

 of the drop of liquid should be such that after boihng the cover-slip clings closely to the 

 slide without air bubbles. In this condition it may be ringed with wax for further study 

 in the fresh condition or it may at once be made permanent. If this is to be done it 

 should be left for as long as possible, i.e. half an hour or until air begins to enter, before 

 lifting the cover-shp, to ensure the maximum adhesion of the squashed cells, and it may 

 be beneficial to the intensity of colour to heat gently several times, care being taken not 

 to reach boihng-point again or the cover-slip will merely blow off explosively and the 

 preparation be lost. 



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