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BIOLOGICAL PROPERTIES 279 



extending throughout. During the next few days a white wrinkled pellicle 

 forms on the surface, which as growth proceeds becomes thicker, dry and 

 brown and falls to the bottom of the tube where it forms a dirty white 

 deposit, the broth at the same time becoming deep green 

 in colour and afterwards brownish. The culture is viscous 

 and ropy and has a peculiar odour. 



(ii) Gelatin. Stab culture. After incubating for 2 days 

 at 20 C. small colonies appear along the line of the stab : 

 these coalesce and form a white streak : liquefaction 

 commences about the third day (champagne glass lique- 

 faction) and rapidly extends to the walls of the tube. The 

 medium is coloured green. 



Isolated colonies. Small, yellowish, granular colonies 

 appear on the plates after incubating for 2 days. Lique- 

 faction occurs round them and gradually extends through- 

 out the plate. The gelatin assumes a green tint. 



(iii) Agar. After incubating for 24 hours at 37 C. 

 a greenish streak appears on the agar which rapidly 

 spreads over the surface, the agar taking a fluorescent 

 green colour. 



(iv) Potato. Along the line of sowing a thick brown 

 layer is formed, and if this be removed the surface of 

 the potato beneath becomes green on exposure to air. 



SECTION III. BIOLOGICAL PROPERTIES. 



1. Pigments (Gessard). FIQ 



When a broth culture of the bacillus pyocyaneus is cuiture^on agar-3 days 

 shaken up with a little chloroform and allowed to stand 

 for a moment the chloroform separates at the bottom of the tube and is 

 coloured pure blue, while a beautiful fluorescent green watery liquid floats 

 to the surface. 



The bacillus pyocyaneus secretes three pigments, one blue (pyocyanine) ; 

 another fluorescent and green and identical with the pigment produced by 

 saprophytic fluorescent bacilli ; the third is greenish and non-fluorescent 

 and of little importance. 



In contact with air pyocyanine oxydizes and forms a brown substance, 

 pyoxanthose. 



Pyocyanine is easily obtained by extracting a broth or agar culture with chloro- 

 form. In the case of agar it is only necessary to leave the chloroform on the culture 

 for a few hours without shaking. The chloroform acquires a blue colour, and if 

 evaporated long blue needles of pyocyanine crystallize out. Solutions of pyo- 

 cyanine are turned red by dilute acids but the blue colour is restored on the addition 

 of an alkali. Cultures in broth or peptone solution retain their colour after filtration 

 through a Chamberland bougie. Pyocyanine is not toxic. 



The formation of pyocyanine and of the green pigment may be varied 

 at will and even suppressed by growing the organism on different culture 

 media. 



In a solution of peptone, Gessard was able to suppress the formation of the green 

 pigment and the culture then had a very pretty blue colour (this phenomenon cannot 

 be obtained with all peptones). In the same way, on glycerin-peptone-agar (the 

 test medium of Gessard) the amount of pyocyanine produced is considerably increased. 

 Pyocanine is the only pigment formed when the organism is sown in a 10 per cent, 

 gelatin medium containing a little glycerin and incubated at 35 C. 



On the other hand, the green pigment is formed to the exclusion of the others when 



