CLEAVAGE. 53 



made unnecessary, since the attachment of the ovum to the walls of the uterus 

 provides for direct parental nutrition. In the separation of the cells of the 

 morula into an inner cell mass and an outer covering layer is seen the earliest 

 differentiation into cells (inner cell mass), which are destined to form the 

 embryo proper, and cells (outer cells covering layer) which are to engage in 

 the development of certain accessory structures. 



PRACTICAL SUGGESTIONS. 



The ova of the star-fish or sea-urchin afford excellent material for the study of total 

 (holoblastic) equal cleavage up to and including the blastula and gastrula stages. During the 

 breeding season for these animals (late in June or early in July in the latitude of New York) 

 the ova are readily obtained and easily fertilized under artificial conditions. The ova are 

 removed and placed in shallow vessels containing sea-water. The seminal fluid is mixed 

 with a small quantity of sea-water and some of the mixture is added to the water containing 

 the ova. Gentle agitation will serve to disseminate the spermatozoa. Fertilization occurs 

 within half an hour and cleavage follows very shortly. A few of the ova are placed on a 

 slide and watched under the microscope until the first cleavage is nearly complete and then 

 any desired quantity may be treated as follows: Remove the ova from the vessel by means 

 of a pipette, care being taken not to take up any more water than is absolutely necessary. 

 Eject them into 5 per cent, formalin contained in a slender, cylindrical bottle. After a 

 few minutes (when the ova have settled to the bottom) it is best to change the formalin. The 

 ova may remain in the formalin indefinitely. 



The later stages in cleavage may be determined and secured in the same manner. The 

 process goes on rapidly and blastulae will appear in a few hours, gastrulae in a little longer 

 time. 



To prepare permanent mounts, proceed as follows: 



Alcohol, 30%, 50%, 70%, an hour in each grade. 



Borax-carmin, few hours. 



Alcohol, 70%, slightly acidulated with HC1, few hours. 



Alcohol, 70%, several changes, few hours. 



Alcohol, 95%, one hour. 



Alcohol, absolute, one hour. 



Xylol, one hour. 



Thin xylol-damar, indefinitely. 



Remove some of the ova in a pipette, place on a slide and apply a cover-glass. In order 

 to prevent crushing, it is best to place a few bits of broken cover-glass in the damar on the 

 slide before applying the cover-glass. 



Eggs of the common frog are good examples of total unequal cleavage. The ova in 

 various stages of cleavage can be procured in ponds during the spring and preserved indefi- 

 nitely in 5 per cent, formalin. A hand lens or even the naked eye will be sufficient to deter- 

 mine the earlier stages (two, four, eight and sixteen cells). For closer examination a few 

 of the ova may be placed in the formalin or in water in a watch-glass. The cleavage furrows 

 on the surface are remarkably clear. 



Permanent preparations of a number of stages for use in a large class may be made by 

 removing the individual ova, each with its gelatinous capsule still intact, from the general 

 mass and placing in formalin in slender test-tubes. The tubes should have a diameter just 



