ASPERGILLUS FUMIGATUS 



697 



examination. The surface of the culture is at first velvety and white then 

 bluish-green, blackish-green and finally, after 5 or 6 days, blackish-brown. 



On gelatin. Very small flakes slowly make their appearance along the 

 line of sowing : a few spores may appear about the fourth week : in the end 

 there is a very slight liquefaction of the medium. 



On agar. After incubating for 2 days at 37 C. a white film is seen along 

 the line of sowing : little by little the growth acquires a green tint which 

 gradually deepens in colour. (Agar made with Raulin's liquid is the best.) 



Grijns recommends the following medium : 



Water, - - - - 100 c.c. 



Extract of malt, - 1 gram. 



Saccharose, 2 grams. 



Agar, 1-75 grams. 



On this medium asci are formed. 



On potato. An abundant growth rapidly appears along the line of sowing 

 which afterwards becomes blackish-green in colour. 



3. Detection and isolation of the fungus. A. Sputum. In suspected 

 cases of Aspergillosis the sputum should 

 be examined for the parasite both by 

 microscopical examination and by cul- 

 tures. 



Microscopical examination. Renon 

 recommends the following method : Pre- 

 pare films with the green-coloured part 

 of the sputum and stain for 10 minutes 

 in an aqueous solution of safranm. The 

 mycelium and spores are stained pale 

 orange. Carbol-thionin may also be used. 



Cultures. Pick up some small frag- 

 ments from the centre of the sputum and 

 sow in tubes of Raulin's medium. In- 

 cubate at 37 C. and after 2 days the 

 mycelium will have formed a whitish, 

 velvety layer on the surface of the medium, 

 and this soon becomes covered with green 

 spores. On inoculating an emulsion of these spores into the veins of a rabbit 

 or pigeon a fatal pseudo-tuberculous aspergillosis is set up in a few days, and 

 if a small piece of the kidney of the experimental animal be sown in Raulin's 

 fluid a pure culture of the fungus can be obtained. 



B. Sections. Harden the tissue in alcohol, embed in paraffin and stain 

 either by Gram's method or by the following modification of Weigert's method. 



1. Stain with Orth's picro-carmine. 



2. Stain for 20 minutes in carbol-gentian-violet. 



3. Wash rapidly in 0'7 per cent, normal saline solution and blot with a 

 piece of filter paper. 



4- Treat for 1 minute with Gram's solution and soak up the excess with 

 filter paper. 



5. Transfer for a few moments to aniline oil. 



6. Replace the oil with xylol, blot up the excess of fluid. Mount in balsam. 

 One or other of the methods described on p. 694 may also be used. 



4. Toxin. According to Kotliar, Aspergillus fumigatus forms neither 

 toxins nor immunizing substances in culture media. Cecci and Besta have 

 however extracted from the spores a toxic substance of unknown composition 

 which is unaltered by boiling and can be preserved in alcohol. In rabbits, 



FIG. 332. Mycelium of Aspergillus fumi- 

 tus in the sputum of an hair-comber. (After 



