The Preparation of Whole Mounts and Smears 107 



be tested until the optimum fixation is obtained for a given species or 

 stage. Plant materials can be stored in these fluids for months at 0°C., 

 depending on specific response. Most workers transfer the tissues 

 after 1 or 2 days of fixation to 70% alcohol, in which tissues can be 

 stored for many months in a refrigerator. Make the smears as with 

 fresh material. 



Root tip smears are being used extensively for chromosome counts 

 and for the study of the morphology of metaphase chromosomes. The 

 principal problem is to obtain separation and flattening of cells. 

 Some large root tips, such as onion, may be squashed flat easily, 

 whereas the small root tips of sweet clover resist crushing. 



Fix the root tips in Farmer's or Carnoy's fluid for at least one day. 

 Smear directly from the fixing fluid, or transfer to 70% alcohol for 

 prolonged storage. The responses of a species to the fixing formula, 

 the fixing interval and the permissible storage period in fixing fluid 

 or 70% alcohol, must be determined by experimentation. 



The piece of root tip that is fixed is usually at least three times 

 as long as the most actively meristematic region. Cut off: this active 

 end into a drop of acetocarmine, and macerate into linear strips with 

 needles. Some materials are benefited by some heating at this stage. 

 Cover and heat carefully. Press or smear the cover until the cells are 

 sufficiently separated and flattened. 



Chromosome groups tend to become clumped with the above 

 simple treatment. This defeats the main goal of the smear method, 

 the rapid and positive counting of chromosome complements. Clump- 

 ing can be prevented, and some morphological details accentuated 

 by pre-treatment of the living root tips. Soak the tips in a saturated 

 aqueous solution of paradichlorobenzene for 1 to 4 hours and fix in 

 the preferred formula. Methyl alcohol, in concentrations of 1% to 

 .3% in water has also been used for pre-treatment and found to yield 

 spread-out chromosome complements (Dr. J. G. O'Mara) . There are 

 unlimited opportunities for testing other benzene derivati\es. and 

 unrelated compounds in the smear method. 



The separation of cells can be improved by hydrolyzing the 

 middle lamella with 5 to 10%, HCl. The acid may be in water, or 

 70% alcohol, or in Farmer's fluid. After immersion in the acid for 

 5 to 30 min., return the roots to fixative, which should be changed 

 at least once. Enzymes also have been suggested for digesting the 

 middle lamella. 



Several readily available plants, especially Rhoeo and Trades- 

 cant ia. yield excellent sporocyte smears that show the coiled chromo- 



