MORPHOLOGY 507 



Sections. Sections through influenzal broncho-pneumonic patches contain 

 numerous bacilli. Pfeiffer recommends the following method for staining the 

 bacilli in sections : 



Fix in alcohol, embed in celloidin (paraffin is better, Besson) stain for 

 half an hour in dilute carbol-fuchsin, differentiate for a few seconds in 

 absolute alcohol slightly acidified with acetic acid the deep red colour of 

 the sections is now changed to a uniform pinkish- violet tint clear in clove 

 oil and xylol and mount in balsam. The bacilli are stained deep red and the 

 ground work a pale pink. 



2. Cultural characteristics. 



Conditions of growth. Pfeiffer's bacillus does not grow on the ordinary 

 media : for its cultivation outside the body media containing blood, serum, 

 or haemoglobin must be used. It is strictly aerobic and grows at temperatures 

 between 26 and 42 C., the optimum being 37 C. 



When an emulsion of sputum containing Pfeiffer's bacillus is sown on agar (p. 192) 

 a scanty growth of the organism is generally obtained but sub-cultures on the same 

 medium fail. The sputum apparently supplies the substances necessary for the 

 growth of the primary culture ; but for sub-cultures blood-agar must be used. 

 It is important that the agar should be neutral or only just alkaline, for even a 

 slight excess of alkali may give a negative result. 



Characters of growth. Blood-agar. Agar to which blood has been added 

 is the best medium for growing Pfeiffer's bacillus. To prepare the medium, 

 liquefy a tube of agar and pour the contents into a Petri dish : when set 

 spread a large drop of blood in as thin a layer as possible 

 over the surface. Agar slopes may be treated in the 

 same way. Human blood or the blood of rabbits or 

 pigeons, collected of course aseptically, gives the best 

 results. Blood-agar prepared according to Bezan9on's 

 directions (p. 53) is equally suitable. 



Surface cultures on blood-agar sown with an emulsion 

 of sputum and incubated at 37 C. for 24-48 hours give 

 a copious growth of very small, delicate colonies like 245 _ Pfeiffer s 



minute dew drops, visible only with a lens : the colonies bacillus. Single colonies 

 never become confluent. A colony here and there may at 3^c d ) aga x handYeS 8 

 attain the size of a pin's head. 



Hsemoglobin-agar. Huber relying on Pfeiffer's observation that haemo- 

 globin is the constituent in blood essential for the growth of the bacillus 

 prepared an haemoglobin medium. 



Huber used Hommel's commercial haemoglobin : this liquid, deep red in colour, 

 was made strongly alkaline with potash solution to prevent coagulation during 

 heating and then sterilized at 100 C. The product was added to sterile melted 

 agar cooled to 50 or 60 C. in sufficient quantity to make the agar deep red in 

 colour. The tubes were then sloped and allowed to set. The medium is not very 

 satisfactory, and the same remark is applicable to Nastikow's method of adding 

 yolk of egg to agar. 



It is better to use a 1 per cent, aqueous solution of commercial haemoglobin and 

 to sterilize by filtration through a porcelain bougie (Achalme, Rosenthal). 



Liquid media. In broth containing pigeons' blood Pfeiffer's bacillus give& 

 rise to delicate whitish flakes but the growth is not particularly charac- 

 teristic. A better cultivation is obtained by using rabbit serum into which 

 a certain amount of haemoglobin has been allowed to diffuse by leaving the 

 serum in contact with the clot (Rosenthal). 



Pfeiffer's bacillus does not grow on glycerin-agar. The cultures which were 



