STAINING OF SECTIONS 59 



in 25 per cent, sulphuric acid and in water until the film 

 is practically colourless after the last rinse in water. The 

 film is now well washed in water, and counter- stained 

 with Loftier' s methylene blue for one minute, again 

 washed in water, and allowed to dry. A drop of cedar 

 oil is added, and the film examined with the oil-immersion 

 objective direct. Any acid-fast organisms present will be 

 stained red. 



The method for staining the tubercle or leprosy bacillus 

 in section by this process is as follows: (1) Stain the 

 sections in warm carbol-fuchsin solution for ten minutes. 

 (2) Rinse in water. (3) Decolorise in 25 per cent, sulphuric 

 acid and in water, transferring from one to the other 

 alternately until almost decolorised; a faint pink does no 

 harm. (4) Rinse in water. (5) Counter-stain in Loftier' s 

 methylene blue solution for three minutes. (6) Dehydrate 

 in absolute alcohol for half to one minute. (7) Clear in 

 xylol or oil of cloves for five minutes, transfer to slide, 

 blot off excess of clearing agent, and mount with a drop 

 of balsam. 



Gramas Method. Grain's method can be applied 

 equally well to cover-glass preparations and to sections. 

 The process is as follows: (1) Stain the film in Ehrlich's 

 anilin gentian violet or carbol gentian violet for five 

 minutes, or a section for ten to thirty minutes, and 

 drain off the stain. (2) Immerse the preparation 

 without washing in iodine solution (1 gramme of iodine 

 and 2 grammes of potassium iodide dissolved in 300 c.c. 

 of water) for one to two minutes, when the colour changes 

 to a dirty brown. (3) Wash in alcohol until no more 

 colour comes away. (4) Counter-stain in eosin or Bis- 

 marck brown. Cover-glass specimens can now be washed, 

 dried, and mounted, while sections require to be further 

 treated as follows: (5) Dehydrate in alcohol. (6) Clear 

 in xylol or oil of cedar, transfer to the slide, if necessary, 

 with the section-lifter, lay out flat, blot off the excess of 

 clearing agent, add a drop of balsam, and mount. (Cover- 

 glass preparations of pure cultures do not require counter- 

 staining. After (3) rinse in water, dry, and mount.) All 

 bacteria do not retain their stain when thus treated. 

 Those which after the alcohol treatment, i.e. (3), are still 

 stained are referred to as ' Gram -positive,' while those 

 which are decolorised are known as ' Gram-negative.' 



