472 



METHODS AND FORMULAS 



DS 23.20 



tory investigations, making it unnecessary 

 to go to the trouble of infecting a rabbit 

 for the purpose of securing the necessary 

 demonstration material. 



If, then, in the course of routine dissec- 

 tions, a sacrificed rabbit is found to have 

 a pneumococcal infection of the liver, 

 which may easily be seen as yellow lesions 

 on the surface, it is only necessary to cut 

 the lesion and some surrounding tissue, 

 and to place it in a suitable fixative. Mal- 

 lory himself recommends fixation in 

 Zenker 1894 (Chapter 18, F 3700.0010) 

 for about 24 hours. As Zenker contains 

 mercuric chloride it is undesirable that the 

 specimen should remain in it for more than 

 three or four days, but the actual time of 

 fixation is not critical. As always, when 

 deahng with dichromate fixatives, a large 

 quantity of fixative should be used, and 

 the object should be suspended in the 

 fixative solution in a loose cloth bag. When 

 fixation is complete the pieces are re- 

 moved from the fixative and washed in 

 running water overnight. They are then 

 embedded in paraffin and sectioned in the 

 ordinary manner. Sections of from five to 

 eight microns are desirable if it is intended 

 to demonstrate the bacteria, though these 

 are quite readily seen in the ten-micron 

 sections customarily employed for histo- 

 logical examinations. When the sections 

 have been fixed on the shde, they may, if 

 desired, be freed from the last traces of 

 mercuric chloride by treating them with 

 iodine and bleaching with sodium thio- 

 sulfate. The writer does not usually do 

 this, but the treatment is insisted upon by 

 Mallory in the description cited. 



The staining solutions used in this tech- 

 nique are simple to prepare and relatively 

 stable. The first solution is a 2.5 % solution 

 of phloxine in water. Two stock solutions 

 are also required: one of 1% each of 

 methylene blue and borax in distilled 

 water, and the other a 1% solution of 

 azur II in distilled water. Five milUhters 

 of each are added to 90 milUhters of dis- 

 tilled water for use. Differentiation is in 

 Wolbach's resin-alcohol (ADS 21.2 Wol- 

 bach 1911). 



It is difficult to secure a sufficiently 



heavy stain in phloxine to withstand the 

 alkaline tliiazine solutions used for couu- 

 terstaining. Mallory recommends that the 

 sections be placed in a coplin jar of the 

 solution in a 55°C. oven and that they 

 remain there for at least an hour. The 

 coplin jar is then removed from the oven 

 and cooled before the sections are re- 

 moved; they should be stained a dense 

 orange. If they have not yet acquired this 

 color, they should be returned to a 

 paraffin oven for a further period. If the 

 sections are satisfactorily stained, the 

 solution may be poured off or the slides 

 removed from it and briefly rinsed in 

 water. The purpose of this rinse is not to 

 differentiate the eosin in the section but to 

 remove it from the glass. The slides bear- 

 ing the sections are then placed in the 

 methylene blue-azur solution in another 

 coplin jar for 5 to 20 minutes; the exact 

 time varies according to the specimen 

 which one is staining and can only be 

 determined by experiment. Mallory rec- 

 ommends that the solution be freshly 

 filtered onto each slide, rather than that 

 the solution be used in a coplin jar; but 

 the writer has not found this nearly as 

 convenient, nor does it appear to be in any 

 way obligatory. After the slides have taken 

 up sufficient methylene blue solution to 

 appear bluish rather than pinkish, they 

 may be accumulated in water, before 

 being differentiated in the resin alcohol 

 one at a time. This differentiation is best 

 conducted in a dish which is large enough 

 to admit the slide in a flat position. The 

 slide is taken in a pair of angle forceps, 

 dipped under the surface of the solution, 

 and waved gently backward and forward 

 for about a minute. As it is being moved 

 backward and forward in the differentiat- 

 ing solution, the blue color will come off in 

 clouds; it is much easier to overdifferenti- 

 ate than to underdifferentiate. 



The differentiation may be readily con- 

 trolled by inspection under the microscope, 

 though it is not necessary to observe the 

 bacteria, since the nuclei have exactly 

 the same staining reaction. Differentiation 

 should be stopped when the nuclei can be 

 seen, under the low power of the micro- 



