FIXING AND STAINING MICROSCOPIC OBJECTS 243 



tone stage. The corrugation of the chromosomes of Lihum 

 at metaphase and first anaphase can be well seen with such 

 rapid fixation. Careful observation will show that the 

 zigzags do not form a regular spiral. The chiasmas at 

 diaphase and metaphase are also visible in well-fixed cells 

 after squeezing flat. 



Iron-acetocarmine is prepared by mixing 45 volumes 

 of glacial acetic acid with 55 volumes of distilled water, 

 heating to boiUng, and adding (about 1 gram to each 

 200 cubic centimeters, of) good carmine powder. The 

 mixture is to be cooled on ice, and decanted; and a drop 

 or two of solution of ferric salt (preferably the acetate) 

 added to every 100 cubic centimeters until the color on 

 standing is dark wine red. Too much iron precipitates the 

 carmine. The solution should be kept in a stoppered 

 bottle. Slides should be stored in the dark, in a cool place. 

 They keep best on ice. 



Iron-acetocarmine gives rapid fixation only if the pollen 

 mother cells are isolated in fresh undiluted solution, 

 unmixed with juice from anther walls or connective. 

 Hence the first washing in acetocarmine should not be 

 omitted. Also, as few anthers, or as small a piece of anther, 

 as can well be used, should be put on one slide. The 

 presence of the juice of the anther wall and connective, 

 and the lack of separation of masses of pollen mother cells, 

 are hindrances to rapid fixation. Large pollen mother 

 cells, like those of Canna or Lilium, are slower to fix than 

 the smaller cells of Nicotiana or Datura. In fact, much 

 care is needed to get good or even passable preparations of 

 Lilium pollen mother cells in iron-acetocarmine. 



Iron-acetocarmine easily gives good preparations with 

 the chromosomes of some Orthoptera, and of some 

 mammals. 



Smear Preparations Stained with Brazilin. — The writer 

 has spent some time investigating the best method of 

 making smear preparations of pollen mother cells. The 

 following method gives quick fixation and sharp staining, 

 if carefully adhered to in detail. 



