368 Special Procedures I (chap. 22) 



procedure: 



Temporary Mounts 



1. Place material, fixed or fresh, on slide with small mount of either 

 stain, just enough to reach edge of cover glass. Apply pressure. 

 Sometimes it is advantageous to allow material to remain in stain 

 5-10 minutes, then remove to fresh batch before squashing. Frag- 

 ments of animal tissue (ovaries, testes, biopsy bits) can be put in 

 a tube with a large excess of stain for 2-7 days. Remove to fresh 

 solution on slide and squash. 



Methods of applying pressure: 



(rt) Press with ball of thumb. 



(b) Tap or press with blunt or flat instrument. 



(c) Roll with glass rod or a vial. 



All methods are subject to ease and amount of spreading, some 

 materials spread more easily than others. Avoid actual crushing. 



Alternate squashing method: Use a large amount of stain and press out 

 excess with a blotter, flattening cells at same time. Staining may 

 be improved by passing slide 3-4 times through a small flame. Do 

 not alloAv solution to boil. 



2. Seal edges with paraffin or vaseline and allow to stand 1 day. 



results: 



acetocarmine — red 



aceto-orcein — dark purple 



iron acetocarmine — deep bluish red 



comments: 



Rothfels and Simonovitch (1938) use an air-drying technique for 

 spreading chromosomes in cells gTown in tissue culture, and recom- 

 mend the method for material which is lumpy and contains debris 

 making squashing difficult. The material growing on slides or cover 

 glasses is fixed and at completion of fixation is placed horizontally to 

 dry at room temperature with no airstream or humidity control. This 

 produces uniform flattening of division stages and can be followed 

 by acetocarmine or aceto-orcein staining. 



Puck {1958), Ruddle et al. {1958), Harnden {1959), and others first 

 treat tissue cultures gTowing on cover-slip with hypotonic solutions 

 (page 409), 37 °C, 15 minutes to swell the cells, followed by fixation 

 and then air drying. The combination of swelling and air drying- 

 helps to spread and flatten the chromosomes. 



