312 



METHODS AND FORMULAS 



DS 11.40 



But the results obtained on insect material 

 with magenta are so much better than 

 those obtainable with any other stain that 

 it has in the present work been confined 

 to this dye. The only two solutions re- 

 quired are a 1 % solution of potassium per- 

 manganate, which must be freshly pre- 

 pared before each use, and a 1 % solution 

 of magenta in distilled Avater. Each of 

 these can most conveniently be handled in 

 a standard coplin jar. 



The slides bearing the smears are re- 

 moved from the distilled water and placed 

 in 1 % potassium permanganate for about 

 five minutes. A word of warning is neces- 

 sary at this point. The water from wliich 

 the slides are passed to the permanganate 

 must be distilled, for the least trace of a 

 reducing agent in it will cause the potas- 

 sium permanganate to be precipitated on 

 the surface of the smear, from wliich it 

 cannot subsequently be removed without 

 great trouble. If, however, the distilled 

 water is pure and the potassium per- 

 manganate is freshly made, the sections 

 as they are removed from the potassium 

 permanganate will be of a medium purple 

 color with only a trace of brown. They 

 must be passed very rapidly into another 

 jar of distilled water, without giving them 

 an opportunity to oxidize, or they will be 

 ruined. After the briefest rinse in this sec- 

 ond dish of distilled water, the sections 

 are dropped directly into the 1 % magenta. 

 As the time in which they should remain 

 in this is variable, it is desirable to take a 

 single smear from the batch and run this 

 through ahead of the remainder in order 

 to estabhsh the timing before running the 

 remainder through in a single operation. 

 In general, about five minutes should be 

 sufficient, but the first slide should be re- 

 moved from the magenta and rinsed 

 briefly in water after about two minutes 

 and examined with the naked eye against 

 a white background. It should be a deep 

 purple color: between the light purple 

 (which it is hkely to show after three min- 

 utes immersion) and a dense black purple 

 (which will be almost impossible subse- 

 quently to differentiate). If it is already 

 sufficiently stained at three minutes, an- 

 other sfide should be tested at one and two 

 minutes; if it is not sufficiently stained 



after three minutes, it should be returned 

 and examined at one-minute intervals un- 

 til it shows the desired coloration. It may 

 then be transferred to a jar of distilled 

 water and allowed to remain there until 

 the rest of the batch have been put 

 through, on the timing already estab- 

 lished, and placed in the same jar. 



Each sfide will have to be differentiated 

 separately, because alcohol removes the 

 stain rapidly while clove oil removes it 

 slowly. Each section is therefore placed in 

 95% alcohol when color clouds will im- 

 mediately be seen to come from it. After 

 a few minutes of rinsing in the alcohol, the 

 slide is then removed to the stage of the 

 microscope and examined under an eight- 

 millimeter objective, if such is available, 

 or under a 16-milliraeter objective with a 

 20-X eyepiece if the eight-millimeter one 

 cannot be used, to see if the nuclei are yet 

 clearly differentiated from the cytoplasmic 

 background. If the slides are left to differ- 

 entiate in alcohol until the nuclei alone 

 remain colored, it will be impossible to 

 stop the process in time. They should 

 therefore be carefully watched to deter- 

 mine the time at which the nuclei can be 

 clearly distinguished against a lightly 

 stained background. Each slide is then 

 placed in clove oil in which it may be left 

 to differentiate until the background, 

 which now may be examined under a 

 high-power objective, is seen to be entirely 

 free from stain, leaving the chromosomes 

 briUiantly colored. It must be emphasized 

 that the differentiation in clove oil is very 

 slow, and if insufficient differentiation in 

 alcohol be given, it may be necessary to 

 wait some weeks until the clove oil has 

 completed the job. The process sounds 

 complicated to describe, but is, as a matter 

 of fact, very easy to learn and yields re- 

 sults with great certainty. 



As soon as the differentiation in clove 

 oil has proceeded to the desired degree, 

 the slides are washed in xylene to re- 

 move the whole of the clove oil and then 

 mounted in balsam in the ordinary man- 

 ner. These preparations are remarkably 

 permanent, and the stain is, if anything, 

 more specific to chromosomes than is the 

 iron hematoxylin which is more usually 

 employed. 



