W6 Botanical Microtechnique 



ACETOCARMINE SMEARS 



The aceLocarmine method has become so commonplace that it 

 may well be included in an elementary manual. This rapid method 

 combines killing, fixing, and staining. The freshly-made preparations 

 can be used in nonpermanent form for counting chromosomes, de- 

 termining their association, and studying intimate details of structure. 

 71ie slides can be made permanent if desired. 



The stain is prepared by dissohing 1 g. carmine in 100 cc. of 

 boiling 455( acetic acid (or propionic acid) . Cool and decant. Add 

 2 drops of a saturated aqueous solution of ferric acetate, and allow 

 to stand for 12 hr. Filter and store the main stock in a refrigerator, 

 keeping a small cjuantity in a dropper bottle in the laboratory for 

 immediate use. Some workers omit the iron salt and incorporate the 

 correct amount of iron from the reaction between steel dissecting 

 needles and the acetic acid in tlie dye. This requires considerable 

 experience. Excessive iron prevents clear differentiation and may 

 produce a dark grantdar precipitate. This can be minimized by clean- 

 ing the steel needles in -ib'/i acetic acid periodically during dissection. 

 If iron is added to the dye formula, use nickel or chromium plated 

 needles or pyrex needles drawn to an abrupt, fine point. 



The simplest method of using acetocarmine consists of macerating 

 or smearing a fresh anther in a drop of the stain. Small anthers may 

 be dropped into the dye entire, then dissected under a binocular, 

 discarding pieces of anther wall and leaving only the masses of sporo- 

 cytes. Large anthers shoidd be sliced into the thiiniest possible slices 

 on a sheet of wet l^lotiing paper, the fragments transferred to a drop 

 of dve and macerated as above. 



Lower a cover glass over the drop of dye containing the sporocvtes, 

 and press or tap gently. A carefid sliding motion on the (()\er glass 

 sometimes aids in smearing the cells into a thin film. Pass tlie slide 

 qincklv o\er an alcohol lamp llame several times. Ihe amount of 

 heatinu iiuisi be determined bv trial, l)ut do not luai lo the boilin" 

 j)oini. Diain oil excess stain, and seal tlu' edges ol the coxer glass 

 will paraffin. Examine the slides with a microscope, and stcire satis- 

 factory ones in a refrigerator. I'lic coloi impioxcs in a lew clays, 

 readies maxiimmi intensiix. then begins to dctc i ioi ate. 



Eresh anthers are not available at all times, and it is often neces- 

 sary to make extensive collections in the field lot subsecjuent study. 



Kill entire anthers, or grass spikelets, or e\cii large portions of 

 an inlloicscciuc in Farmer's oi- (".arno\"s lluid. Mai/e cytc^logists 

 prefer an alccjhol-acid latio ol j:l, but ratios ol 1:1, 2:1 etc. may 



