548 Trow. — On Fertilization in the Saprolegnieae. 
years — a point of no small importance. The best and most convenient 
culture vessels are Petri dishes, and these too should be sterilized. Sterilized 
tap water is suitable where the water is sufficiently free from lime. If lime 
is precipitated on sterilization, distilled water or rain water should be used. 
On no account should cultures be attempted on white of egg, chopped beef, 
or other similar substrata, unless the inoculating material has been entirely 
freed from bacteria and other micro-organisms. The flies, having been 
transferred to Petri dishes half filled with water, are inoculated by cuttings 
of hyphae from healthy cultures, a single filament being transferred by 
means of a sterilized needle. When this process is repeated with the 
requisite care for a few generations, micro-organisms are practically 
eliminated, and if an even temperature be maintained the sexual organs 
appear on the cultures in great abundance with clock-work regularity a few 
days (generally about five) after inoculation. I have kept such cultures 
going for twenty to thirty generations in perfect health and vigour, and 
such as these were alone used by me for fixing. The first essential in 
cytological work is thoroughly healthy and vigorous material. The time 
and labour spent in the preliminary study of the living plant meets with its 
due reward in the greater constancy and accuracy of the final results. All 
sorts of anomalies occur when the conditions of life are unfavourable, as 
the Saprolegnieae are exceptionally susceptible to changes in their environ- 
ment. Both the species of Achlya collected for me by Mr. Pole-Evans 
were found in the hard waters which everywhere flow from the lias in the 
Vale of Glamorgan. They frequently occur in a curious stunted, coral-like 
form which is almost, if not quite, sterile. Such cultures, if transferred to 
distilled water, develop normal growths at once. 
The best fixative is a solution of chromic and acetic acids in water in 
such proportions that the percentages of the two acids are respectively 0-7 
and 0-3. The much weaker solution used by Davis was tried, but the 
results in A. De Baryana were distinctly bad. The solution should be 
allowed to act for twenty-four hours and the material should then be 
washed in running water for another twenty-four hours. The transference 
to absolute alcohol is effected very readily and safely by the use of the 
diffusion shells of Schleicher and Schiill. All danger of shrinkage is 
avoided by this method and it has the merits of being easy to use and 
certain in its effect. The transition from absolute alcohol to paraffin may 
be successfully made by means of xylol, chloroform, or cedar-wood oil, but 
care must be taken to avoid sudden transitions at every stage of the process. 
The material itself should never be handled either with lifters, forceps, or 
other tools, at any time, but rather floated gently from one vessel to another 
when changes are necessary. The two essentials for success are undoubtedly 
fine healthy cultures free from micro-organisms and an appropriate fixative. 
The method of applying the fixative is important. Two cultures grown 
