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No. 3.] ORIGIN OF THE CLEAVAGE CENTROSOMES. 811 
5). The sperm attraction sphere is present until the head of 
the spermatozoon begins to develop into the male pronucleus, 
when it also totally disappears (Fig. 6). Both spheres are 
absent during a relatively long period (z.e., while the young 
pronuclei are developing) ; and when the pronuclei have at- 
tained their maximum size and are in contact, two attraction 
spheres appear again in the cytoplasm, and the cleavage spindle 
is formed. 
Method.— The middlepiece of the spermatozoén was first 
differentiated from the centrosomes by iron haematoxylin fol- 
lowed by erythrosin. The same results were obtained by 
staining with a mixture of orange and methyl green. (Satu- 
rated aqueous solution of orange two parts, in eight parts 
water. Saturated aqueous solution of methyl green one part, 
in four parts water. Wash in absolute alcohol.) This stain 
was the outcome of a series of experiments with the anilins, 
undertaken with the aim of supporting the method of double 
staining (Figs. 8-10) by one containing fewer possibilities 
of error. This method demands relatively no technical manip- 
ulation, and practically the same results were obtained whether 
the sections remained in the stain fifteen minutes or twenty- 
four hours. The stain was used after several fixatives, with the 
same result, but all the figures for this paper were drawn from 
chromo-acetic preparations. 
An examination of the figures will show that a variety of 
structures select the methyl green, — vzz., the nucleoli, micro- 
somes, centrosomes, chromosomes, and sperm granules, —and 
that the orange is selected by the chromatin of the vesicles and 
resting nucleus, the cytoplasmic network, and the archoplasm 
(which is present in the attraction spheres, fertilization cone, 
spindle, and throughout the cytoplasm). 
This method of staining sharply differentiates the very young 
nucleoli of the vesicles found at the telophase of the second 
maturation spindle, and destined to become the female pro- 
nucleus. These vesicles when first formed are eleven in num- 
ber, thus corresponding with the chromosomes. As _ they 
progress towards the formation of the female pronucleus, their 
walls break and fuse with each other, thus forming the chro- 
