DS 11.4-DS 11.40 



DYE STAINS OF GENERAL APPLICATION 



309 



for microorganisms. Indeed, it is very 

 nearly as specific as is the Fuelgen reaction 

 without any of tlie difficulties attached to 

 the latter. The Fuelgen reaction itself, 

 here cited from the paper of Fuelgen and 

 Rossenbeck 1924, is not now as widely em- 

 ployed as was formerly the case. It is 

 actually one of the oldest staining methods 

 known, having been introduced by Schiff 

 in ISGG (825, 93:140), as a reagent for al- 

 dehydes. Its nuclear reactions appear to 

 depend upon the selective staining of thy- 

 monucleic acid, but the stain itself is so 

 difficult and so temperamental that it is 

 doul)tful that it has occasioned any real 

 advance in microtomy. 



The next class of synthetic nuclear 

 stains, the thiazins, comprise methylene 

 blue and its oxidation homologs: Lauth's 

 violet and the azurs. These are used 

 mostly in admixtures of unknown compo- 

 sitions, commonly referred to as poly- 

 chrome methylene blues. These thiazins are 

 in general excellent and specific nuclear 

 stains and their use should not be confined 

 to blood, as is customarily the case. They 

 became unpopular at about the opening 

 of the present century because of the 

 difficulty at that time of securing mount- 

 ing media in which they could adequately 

 be preserved. They are hable to rapid fad- 

 ing when mounted in balsam, and also 

 tend to be extracted by alcohol in the 

 course of the dehydration period. If amyl 

 alcohol is used for dehydration, and one 

 of the neutral mounting media in place of 

 balsam, there is no reason why the thiazins 

 should not again become popular. The 

 formulas given are only those in which 

 methylene blue is employed alone. Many 

 more formulas will be found later in the 

 present chapter under the headings DS 

 13.1 and DS 13.2, with their subdivisions, 

 which deal with the common combinations 

 of thiazins and various eosins. 



The next class, the crystal violets and 

 gentian violets, are today almost confined 

 to botanical microtechniques. This is un- 

 fortunate since they are very specific for 

 nuclei and also do not stain albuminous 

 yolk material as does hematoxylin. It 

 is strongly recommended that those who 

 are handling heavily yolked embryos 

 should try the formula of Johansen 1932 



in preference to the more conventional 

 techniques. 



11.40 TYPICAL EXAMPLES 



Preparation of a strewn slide of pollen 



grains using the safranin stain 



of Johansen 1940 



It is not intended here to give an exam- 

 ple of the technique by which safranin is 

 used for general nuclear staining, but only 

 to give a method which may be employed 

 for the demonstration of the nuclei in pol- 

 len grains, a rather difficult subject. 



Pollen should be collected into a tube 

 containing 1% acetic acid in absolute al- 

 cohol. It is simple to do this by dipping the 

 anther under the surface of the fluid and 

 then resealing the tube. In this manner a 

 vast number of pollens can be collected in 

 a surprisingly short time. Pollen grains 

 ma}' remain in this fluid for a day of two, 

 but it is undesirable to leave them any 

 longer than this before allowing them to 

 settle. The supernatant liquid should then 

 be poured off and replaced with fresh ab- 

 solute alcohol. This : change should be 

 repeated several times or until the fluid 

 poured off no longer smells of acetic acid. 

 Wlieu the acetic acid has been washed out, 

 the absolute alcohol is replaced with a 

 mixture of equal parts of absolute alcohol 

 and ether, and the pollen grains are left 

 in this mixture overnight. 



A solution should then be prepared from 

 any nitrocellulose used for embedding, 

 such as celloidin (see Chapter 13). About 

 half of the alcohol-ether mixture should 

 then be poured from the top of the tube 

 containing the pollen and replaced with an 

 equal quantity of the weak celloidin solu- 

 tion. After this has been allowed to pene- 

 trate the pollen grains for a few hours — 

 (this will be definitelj^ established by the 

 grains falhng to the bottom) — about 

 three-quarters of the fluid is poured off 

 and the tube again refilled with a fresh 

 0.5% solution of celloidin. When this has 

 in its turn impregnated the pollen grains, 

 they may be left in the tube for an indefi- 

 nite period. 



When it is desired to make mounts, it is 

 necessary to clean as many co\-erslips as 

 the number of mounts required, taking 



