112 STAINING METHODS 



and dissolve in 



Distilled water 1000 c.c. 



Filter. 

 METHOD. 



1. Prepare and fix films in the usual way. 



2. Pour stain I on the film and allow it to act for 

 two minutes. 



3. Wash thoroughly in water. 



4. Treat with Lugol's iodine for ten seconds. 



5. Wash thoroughly in water. 



6. Pour stain II on to the film and allow it to act for 

 thirty seconds. 



7. Wash thoroughly in water. 



8. Dry and mount. 



NOTE. The cultivation from which the films are prepared 

 must be upon blood-serum which has been incubated at 37 C. 

 for from nine to eighteen hours. 



The bacilli are stained a light red by the neutral red, 

 which contrasts well with the two or three black spots, 

 situated at the poles and occasionally one in the centre 

 representing protoplasmic aggregations (? meta-chro- 

 matic granules) stained by the acid methylene-blue. 



Wheal and Chown (Oxford) Method. (To demonstrate actino- 

 myces.) 



1. Stain briefly with Ehrlich's hsematoxylin (until nuclei are 

 faint blue after washing with tap water) . 



2. Wash in tap water. 



3. Stain in hot carbol-fuchsin (as for tubercle bacilli) for five 

 to ten minutes. 



4. Wash in tap water. 



5. Decolourise with Spengler's picric acid alcohol. This is 

 prepared by mixing: 



Alcohol, absolute 20 c.c. 



Picric acid, saturated aqueous solution 10 c.c. 

 Distilled water 10 c.c. 



During the progress of steps 1-5 the preparation must be re- 

 peatedly examined microscopically with the -inch objective. 



