DS 23.10 



DYE STAINS OF SPECIAL APPLICATION 



459 



cause it appears to be the most usually 

 employed in laboratories. The guinea pig 

 may be killed by any method; it is then 

 strapped down on a board in the custom- 

 ary manner. Rickettsiae are most readily 

 obtainable to the beginner from the inner 

 surface of the scrotal sac of a male, which 

 has the advantage of being free from hair 

 because it hes entirely within the coelomic 

 cavity and is free from dermal adhesions. 

 The guinea pig is opened by a median in- 

 cision and the skin stretched back. This 

 will disclose perfectly enormous vesicula 

 seminales, and a very large prostate gland. 

 These are pushed to one side to disclose 

 the testes lying within a peritoneal fold. 

 This is washed free of extravasated blood 

 with a jet of normal saline, and a piece 

 about 5 mm. square is cut from the an- 

 terior surface of the sac. This piece is now 

 removed and laid with the inner surface 

 ui)wards in the center of a 3" X 1" glass 

 slide, where it may remain while the body 

 of the guinea pig is being disposed of with 

 the customary precautions. 



It is now necessary to cut, not to scrape, 

 a few cells from the exposed inner surface 

 of the material. This is best done with a 

 freshly sharpened razor or with a change- 

 able-blade scalpel, using a new blade, cut- 

 ting parallel to the surface of the glass 

 with short, jerky strokes. One should be 

 careful to draw the knife as much as push 

 it. The debris accumulated on the blade 

 of the knife is then spread over the surface 

 of a second chemically clean slide, which 

 is put aside to dry. More sUdes are now 

 prepared in the same manner until a dozen 

 or two smears have been accumulated. As 

 soon as the smears are dry, the slides are 

 placed in a large jar of absolute alcohol, 

 and if care is taken that the entire surface 

 of the slide is covered, the jar may now be 

 lemoved from the operating room and 

 turned over to a technician. If the slides 

 are, moreover, picked one at a time from 

 the jar with sterile forceps and placed in 

 another jar outside the operating room, it 

 is now safe to proceed with the staining 

 without any further aseptic precautions. 



The preparation of the stain presents 

 few difficulties, for it is a three-step proc- 

 ess involving first, gross overstaining in a 

 magenta solution buffered to a pH of 7.5, 



followed by differentiation and counter- 

 staining in an acid thionin solution, with 

 a final clearing of the thionin with weak 

 citric acid. 



Each slide is removed from the absolute 

 alcohol and waved backward and forward 

 in the air until it is dry. The slides are 

 then examined unstained under the low 

 power of the microscope, and those which 

 show more than one or two cells lying on 

 top of each other are rejected. Normally, 

 in a batch of about a dozen slides, one or 

 two will be found in which the smear is so 

 thin that numerous isolated, undamaged 

 cells will be seen in the field of the low 

 power. These slides alone are worth stain- 

 ing, and each must be treated individu- 

 ally. The sUde is placed on a rack, or held 

 in the hand, and flooded with the ma- 

 genta-phosphate buffer stain for three 

 minutes. The magenta is then drained off 

 without washing, and the citric acid-thio- 

 nin solution is flooded onto the surface 

 and off again immediately. The simplest 

 technique is to hold the slide in the left 

 hand, to take a pipetful of the stain in the 

 right hand, allow this to fall over the slide, 

 moving the pipet backward and forward 

 to insure an even distribution, and then 

 to tip the slide immediately onto a thick 

 layer of blotting paper so that the stain 

 runs off. While the stain is being run 

 from the slide in the left hand, the right 

 hand picks up a pipetful of the citric acid 

 solution, and squirts this against the in- 

 clined slide in such a manner as to wash off 

 the blue dye. The slide is then passed into 

 tap water, where it may remain while suc- 

 cessive shdes from the same batch are 

 treated with the stain. 



The slides should remain in tap water 

 until no further color comes away, and 

 should then be washed in distilled water 

 and air-dried. It is usually not worth while 

 to mount the slides under a coverslip, but 

 to accumulate them dry and to observe 

 them with an oil immersion objective. 

 After each observation the immersion oil 

 may be washed off with xylene. 



A successful preparation shows deep red 

 Rickettsiae in light blue cells. The usual 

 error is t(^«Riove too much of the blue 

 which, unoerlfc^ very high powers neces- 

 sary, is a desinR(8B background for picking 



