CYTOGENETIC ANALYSIS 491 



slide in the upper one-third of the pad. Reexamine and repeat this step, if necessary. 

 The more favorably squashed cells will appear (in the microscope field) along the upper 

 edge in the center region. Just why the lower edge of the cover glass responds so 

 favorably to the squashing is not fully understood. In the event this area of the cover 

 glass is noted to have overlapping chromosomes and unfiattened cells, added pressure 

 is sorely needed. 



8. Seal the edges of the cover glass with Kronig's cement (see Addendum). 



9. Store for several days to several weeks. 



10. Make slides permanent after 48 hours or more of storage, following the freezing 

 technique of Conger and Fairchild. 221 



The above schedule is primarily for light-medium phase-contrast microscopy, 

 using a light green filter (Zeiss Opton, Model W). In the event dark contrast objec- 

 tives are employed, such as Neofluars, the mordanting step may be eliminated or 

 destaining may be more effectively conducted with 45 per cent acetic acid in place of 

 0.5 per cent acetocarmine for the final rinse. On the other hand, bright-field micro- 

 scopy may require excess mordanting and minimal destaining to enhance the differen- 

 tial affinity of the mordanted stain for chromosomes. The ferric acetate mordant 

 prepared with razor blades has eliminated the troublesome precipitate generally 

 witnessed in the past with botanic specimens. Excess staining of the cytoplasm may 

 result when slides are stored for a long period. This can be alleviated at the time the 

 slides are to be made permanent, simply by rinsing once with 45 per cent acetic acid, 

 or dilute stain, following separation of the cover glass and prior to initiating dehydration. 



Unsquashed preparations for general morphology 



This procedure permits observations immediately after fixation and in the per- 

 manent state. All pretreatments (hypotonic and the like) essential for chromosomal 

 preparations must be avoided. 



1 . Fix by adding 1 ml. of 3 : 1 fixative directly to the medium. Store until 

 necessary, as mentioned above, or proceed to complete the schedule. 



2. After 10-15 minutes of fixation, remove the cover glass and place atop the 

 pedestal. 



3. Rinse with several drops of fresh 3: 1 fixative. Decant. 



4. For phase-contrast microscopy, add 3 drops of 1-1^ per cent acetocarmine (no 

 mordant) and wait for 20 seconds before rinsing with 3 : 1 solution. For bright-field 

 microscopy, add a touch of mordant, blend into stain, and wait for 60-90 seconds. 

 Rinse rapidly with several drops of 0.5 per cent acetocarmine and halt further destain- 

 ing by adding a few drops of 3 : 1 fixative. 



5. Dehydrate by placing the cover glass in a vial containing 95 per cent ethyl 

 alcohol and mount within 10 seconds, using Euparal (see Addendum) sparingly. Pass 

 over a low flame. Press very gently under many layers of the bibulous pad. Observe 

 under low power. Store flat until dry. 



Hematoxylin-and-eosin preparations are most satisfactory for viewing general 



