462 METHODS AND FORMULAS DS 23.12 



23.12 METHODS FOE RICKETTSIAE 



The staining of Rickettsiae in sections is relatively easy, provided that an alkaline 

 solution be employed at some stage of the proceedings. The method of Castaneda 1930 

 and Macchiavello 1938, which involve the use of a phosphate buffer at a pH of 7.5, have 

 now almost replaced the other methods, which are given here largely for their historical 

 value, and to enable the research worker to check, if necessary, the results of the early 

 publishers in this field. Attention must therefore be drawn to the technique of Lupine 

 1932, in which a definitely acid solution is employed for staining. It must, however, in all 

 fairness, be pointed out that the technique of Lepine would equally well stain bacteria 

 or mitochondria were they present. Indeed, the general warning must be given that the 

 demonstration of Rickettsiae in cells is far more dependent upon the symptoms shown 

 by the animal from which they were taken, than on any demonstration which can be 

 made with an optical microscope. There are numerous methods for staining small par- 

 ticles occurring in cells, which have been given both under the cytological techniques 

 above and in various places in the previous divisions of the work. There is no evidence 

 of any kind that any of the techniques given in this section will stain Rickettsiae to the 

 exclusion of any other small particle or small body. 



23.12 Begg, Fulton, and van den Ende 1944 11431, 56:109 



REAGENTS REQUIRED: A. M/15 phosphatc buffer pH 7.6 10, 0.1% magenta 90; B. M/50 



citrate buffer pH 3.0; C. 1% methylene blue 

 method: [heat-fixed impression smears] — > A, 5 mins. — > wash —* B, }2 to V ^ mins. -^ 



wash — > C, 30 sees. — » dry 



23.12 Bohner test. 1928 Schmorl Schmorl 1928, 428 



REAGENTS REQUIRED: A. 1% methyl blue 35, 1% eosin 35, water 100; B. 0.005% sodium 



hydroxide in abs. ale; C. 0.1% acetic acid 

 method: [sections of ale. or acetone, fixed material] -^ water — * A, J^-5 mins. — > rinse 



-^50% ale, rinse — » B, 15-20 sees. — > abs. ale, rinse —>■ water, wash — ^ C, 1-2 mins. 



— ^ abs. ale, rinse — > abs. ale — > balsam, via usual reagents 



23.12 Bond test. 1938 Mallory Mallory 1938, 285 



REAGENTS REQUIRED: A. Water 100, eosin 0.1, methyl blue 0.1; B. xylene 30, aniline 60 

 method: [methanol-fixed smears] — * wash —* stain, 4-5 mins. — » wash -^ blot -^ dry -^ 

 B, till differentiated —* balsam, via xylene 



23.12 Castaneda 1930 11250, 47:416 



reagents required: A. phosphate buffer pH 7.5 100, 40% formaldehyde 5 DS 11.44 



Loffler 1890 1; B. 0.1% safranin 

 method: [very thin smears] -^ A, S mins. -^ drain -^ B, on slide, 2-3 sees. -^ water, till 

 no more color comes away -^ dry 



23.12 Clancy and Wolfe 1945 19938, 102 :483 



reagents required: A. water 100, methylene blue 0.02, magenta 0.02 

 method: [air-dried smears] -^ xylene, flooded on slide, few mins. -^ dry — > A, 5 mins. 

 -^ wash —)■ dry 



23.12 Cox 1939 see DS 23.12 Macchiavello 1938 (note) 



23.12 Darzins 1943 23684, 151:18 



reagents required: A. ADS 12.2 Lugol 50, water 50; B. 0.1% thionin in 10%, ale 

 method: [air-dried smears] -^ A, 30 sees. -^ rinse — > B, 5 sees. -^ wash -^ dry 



23.12 Goodpasture and Burnett 1919 see DS 23.12 Hertig and Wolbach 1924 (note) 



23.12 Gracian 1942 lest. 1943 von Brand 20540b, 18:150 



REAGENTS REQUIRED: A. 7% potassium dichromate; B. DS 13.13 Giemsa 1902 10, water 



90 

 method: [smear] -» xylene, 3 mins. -^96% ale, 1 min. -> water -^ A, 3 mins. -^ wash 



-^ B, 10-20 mins. -^ wash —> dry 



