820 
MR. W. GARDINER ON THE CONTINUITY OF THE 
small vacuoles, or no vacuoles at all, than with large full-grown cells when large 
vacuoles are present. 
In the latter case there is every opportunity for contraction, and there is moreover 
always a tendency to a dilution of the fixing fluid by the cell-sap. This being the 
case, any successful results with full-grown cells may be regarded as very favourable 
evidence for the efficacy of the reagent employed. In order to eliminate any doubt 
as to whether the salt solution influenced the result, analogous experiments were made 
with fresh tissue. The great drawback to the thorough efficiency of picric acid is that 
it wets the tissue with some difficulty and only penetrates after some time. This fact 
becomes very apparent when large pieces of tissue are used at any time of difficult 
permeability attendant on peculiar histological structure. A saturated solution of 
picric acid in absolute alcohol to some extent obviates this difficulty, but it is not 
so successful as a saturated watery solution, although it appears to be a valuable 
reagent for ordinary work. 
With regard to other manipulative details, it is, as mentioned above, important to 
cut up the material into small pieces, and also to place it at once upon cutting in the 
preservative medium. My usual plan, in fact, was to cut off the pulvini and allow 
them to drop, then and there, into picric acid, in order to avoid any loss of water due to 
evaporation, which as far as delicate investigations are concerned will soon very gravely 
affect the whole cell-equilibrium. After treatment with picric acid for about 24 hours 
the material is removed, rapidly washed with water, and placed in alcohol, the latter 
being changed until the yellow coloration of the picric acid is no longer obtained. 
Any method of preservation is, however, very imperfect. Not only is appreciable 
contraction produced, but a great amount of rigidity of the protoplasm occurs due to 
coagulation and death. These considerations and results determined me to use fresh 
material, which I employed afterwards all through the investigation.* 
* In connexion with the experiments upon fresh material the results obtained with Spirogyra are of 
ome interest. They confirm those alluded to in the text. Absolute alcohol was shown to be an utter 
ailure. Watery picric acid was the best reagent, preserving the lenticular form of the nucleus, and 
demostrating the threads going to the chlorophyll bands with great success. A saturated solution of 
picric acid in absolute alcohol is to be preferred next, but it causes definite shrinking. The great point, 
however, that these experiments made evident was that throughout the entire process of preservation 
and staining it is necessary to keep all the solutions as nearly as possible of the same density and to 
avoid any rapid diffusion. Thus if it be required to put up a preparation of Spirogyra, one caji first fix 
the cell with saturated watery picric acid. Then wash in dilute alcohol and stain with either dilute 
ammonia-liEematoxylin or a dilute alcoholic solution of one of the aniline dyes. Any dense staining 
solution will at once cause shrinking. But after this point comes the difficulty. Dilute or strong 
glycerine will at once cause great shrinking, whatever be the precautions employed, and the only way 
which is apparently left open to adopt is to mount in such a medium as camphor water, which will cause 
swelling, in a dilute solution of potassium acetate or calpium chloride, or, still better, in dilute alcohol. 
To the latter there is the obvious objection that it will act upon most of the varnishes that are used to 
surround the cover glass and so work its way out, I should suggest as a varnish in this case, a strong 
solution of gelatine in glacial acetic acid, but hitherto J have not been able to try whether it would work. 
These results are, however, worth consideration. 
