318 THE TUBERCLE BACILLUS 



Boil for 15 minutes. Filter in the warm. Sterilize at 100 C. and pour 

 into Petri dishes. 



7. Fragments of tissue as culture media. A. and L. Lumiere obtain very 

 copious growths commencing in about 36 hours on fragments of liver or 

 spleen. 



Wash the liver and spleen of an adult bovine animal or calf in distilled water, 

 heat in the autoclave for three-quarters of an hour to shrink them and then cut 

 into rectangular pieces ; after soaking in a 6 per cent, aqueous solution of glycerin 

 for an hour the pieces are placed in potato tubes and sterilized in the autoclave for 

 15 minutes. It is best to sow from a potato culture. 



Gioelli uses pieces of human placenta immersed in broth, or, better, placenta- 

 broth containing O5 per cent, of sodium chloride and 6 per cent, glycerin 

 in place of liver. 



8. Bile. Calmette recommends pieces of potato soaked in a mixture of 

 95 parts fresh bile and 5 parts sterilized glycerin. The bile-glycerin mixture 

 ought to be kept for 3 weeks at the temperature of the laboratory before being 

 used. [On an ox-bile-glycerin-potato medium tubercle bacilli of the bovine 

 type grow more rapidly and more luxuriantly than on the usual media while 

 bacilli of the human type grow with difficulty on this medium and the avian 

 type not at all. On the other hand bacilli of the human type will grow 

 rapidly on an human-bile medium as will bacilli of the avian type on a fowl- 

 bile medium ; the cultivations of these two types on these media respectively 

 are similar in appearance to cultivations of the bovine type on ox-bile 

 (Calmette)]. 



9. Glycerin-broth. Glycerin-broth, or better, glucose-glycerin-broth is a 

 very useful medium for the growth of the tubercle bacillus. To sow a 

 broth culture of the tubercle bacillus, raise as large a piece of growth as 

 possible from the surface of an agar tube or other solid medium (it is even 

 better to lift the film of growth from the water of condensation) with a fairly 

 large platinum loop and float it very carefully on to the surface of the broth. 

 It is advisable to transfer three or four loopsful if a large flask is to be sown. 



Growth generally takes place in the form of a pellicle. After incubating 

 for about a fortnight a whitish area appears around the piece of growth 

 which was sown, this gradually extends and ultimately forms a thin delicate 

 film covering the whole of the surface of the medium. The film is at first 

 dry and fragile but becomes thicker in course of time : sometimes it remains 

 dry and scaly and sometimes becomes greasy, moist and wrinkled. Not 

 infrequently the film creeps up the sides of the vessel, sometimes to a height 

 of 1 cm. Rarely no film at all is formed and in this case the growth consists 

 of a flocculent deposit. Whatever the form of growth the broth remains 

 quite clear. 



[To obtain a successful growth on glycerin-broth requires considerable 

 attention to details. The material used for sowing the medium must be 

 young and actively growing perhaps the film growing on the water at 

 the bottom of a glycerin-potato culture gives the best results. If the culture 

 be sown at the right moment the growth will, in the case of the human type, 

 spread and cover the surface of the glycerin-broth in a Roux's bottle laid 

 on its flat side in a fortnight. At other times no change whatever is visible 

 in the material sown for weeks, then little white nodules appear which are 

 the precursors of a growth which once it starts to spread covers the surface 

 very rapidly. The flask must be carefully sealed. ] 



The tubercle bacillus can also be grown on ordinary broth containing no glycerin 

 (Gioelli [and others]). 



Pour a layer of vaseline oil about 1 mm. deep on the surface of the broth. Sterilize 



