MS U.O-MS 11.1 



METAL STAINS 



527 



acid is seen to be cloudy, or if there is a 

 black precipitate on the bottom of the 

 bottle, the ovary should be removed to 

 another bottle containing a fresh supply 

 of osmic-acid solution. It is a waste of 

 time to add fresh osinic to the existing 

 bottle, wliich has demonstrated, through 

 the reduction of the reagent, that it is al- 

 ready contaminated. 



When the ovary is sufficiently impreg- 

 nated, the bottle containing it should be 

 filled to the brim with triple-distilled 

 water and tilted backward and forward 

 once or twice to mix the contents. The 

 ovary is then allowed to settle to the bot- 

 tom, and the surplus water is poured off. 

 This should be repeated once or twice, and 

 then only half the water removed, leaving 

 the ovarj^ immersed in about ten milli- 

 liters of water. The bottle with the water 

 in it should then be placed in an oven, or 

 on a water bath, which will maintain a 

 temperature of about 38°C. for one or two 

 days. The ovary is then examined under 

 the surface of water with a binocular mi- 

 croscope. It should present a very dark 

 brown, but not absolutely dead-black, 

 appearance. If it is covered with a dead- 

 black, amorphous deposit, it is evident 

 that it has been too long in osmic acid. If it 

 is light brown, it has not been in osmic long 

 enough. In either circumstance it had 

 better be thrown away. If, however, it has 

 been properly impregnated, it should now 

 be thoroughly washed, either in at least 

 ten changes of triple-distilled water with 

 not less than one hour in each, or over- 

 night in slowly running, triple-distilled 

 water. 



The ovary is then embedded in paraffin 

 by the methods described in Chapter 12, 

 and sections are cut longitudinally at a 

 thickness of from one to two microns. 

 The sections are dried on a shde, the wax 



melted, and the slide dropped into xylene 

 to remove the wax. Meanwhile, set up two 

 tubes, or coplin jars, one containing an- 

 hydrous turpentine and the other oil of 

 cedar. As soon as the wax has been dis- 

 solved, pass the sHde into the cedar oil 

 until the xylene has been removed, with- 

 draw the slide, wipe the surplus oil from 

 the bottom, and examine it under an oil- 

 immersion lens. If impregnation has been 

 successful — that is, if the Golgi apparatus 

 and yolk granules are shown as dead-black 

 spots against a pale brown background 

 — return the sUde to xylene until the cedar 

 oil has been removed, and then mount it 

 in balsam under a coverslip. If no signs of 

 Golgi apparatus are present, the shde may 

 be thrown away, and it must be presumed 

 that some mistake has been made in the 

 technique. If, however, as frequently hap- 

 pens, there is some evidence of Golgi 

 apparatus as black dots, but the back- 

 ground is obscured by other scattered 

 black granules, place the sUde in turpen- 

 tine for about two minutes, rinse it rapidly 

 in xylene, return to cedar oil, and re-exam- 

 ine. Repeat these operations until the 

 section has been properly differentiated. 

 Some people recommend that all sections 

 should be treated with turpentine, but this 

 is not usually necessary in the case of so 

 simple an object as the earthworm ovary. 



If turpentine has been used, wash the 

 slide in a jar of clean xylene in order that 

 all turpentine may be removed from the 

 slide before it is finally mounted. 



The majority of formulas from this sec- 

 tion have been transferred to the appro- 

 priate section of Chapter 18. Those re- 

 tained were originally suggested as stains 

 and have never been recommended as fix- 

 atives. Simple solutions of osmic acid are 

 not listed separately, but are given with 

 individual techniques. 



11.1 van Gehuchten 1927 

 11.1 Hamilton 1897 



11.1 Staining Solutions 

 see F 1700.0000 van Gehuchten 1927 



3464, 20:180 

 STOCK solutions: I. Brain tissue hardened in F 7000.0000 Muller 1859 for 3 weeks 50, 



F 7000.0000 Muller 1859 100. (Grind to a paste and filter.) II. 1% osmic acid. 

 WORKING solution: stock I 100; stock II 0.5. 



11.1 Kolossow 1892 23632,9:39 



formula: water 50, 05% ale. 50, nitric acid 2, osmic acid 1 



