162 BIOLOGY AND TECHNIQUE 



One per cent Andradc Indicator. 



Acid to bring to neutral point of the indicator. 28 



One per cent Lactose. 29 



0.1 per cent Glucose. 



Brilliant Green in 0.1 per cent aqueous solution. 



Two dilutions of dye are used in routine plating, corresponding to 

 1-500,000 and 1-330,000 in terms of solid dye (0.2 c.c. and 0.3 c.c. of 0.1 

 per cent solution per 100 c.c. of agar). The sample of dye which Krumwiede 

 has used is from Bayer, but he has also tested and found equally satisfactory 

 samples from Griibler and Hochst. 0.1 gram of dye is accurately weighed 

 on a foil, washed with boiling H 2 into a 100 c.c. volumetric flask and made 

 up to the mark when cool. The flask should be clean and neutral (by test). 

 Fresh solutions vary in activity (see standardization tests) ; they keep about 

 a month. 



Each bottle is mixed and poured into six plates only (a thick layer of 

 agar gives the most characteristic colonies). Plates are left uncovered until 

 agar has "jellied"; porous tops are used; dry plates are essential to avoid 

 diffusion. 



Standardization: The agar must have proper "balance." The reaction is 

 important; sediment reduces the activity of the dye and light colored media 

 are better than darker ones. Different lots of agar with the same dye solution 

 act ununif ormly ; a new batch or a new solution must be tested. 



Any variation in the composition of the media necessitates a readjustment 

 of dye concentration; this statement cannot be over-emphasized. 



Brilliant green, in appropriate dilutions, not only inhibits all Gram- 

 positive and many Gram-negative bacteria, but exhibits differential action on 

 the colon-typhoid group. Paratyphoid and the B. lactis aerogenes are 

 untouched, typhoid is restrained only at low dilutions, while dysentery and 

 the other colon group are extremely susceptible. The typhoid colony on 

 this medium is characteristic. Looking through the plate against a dark 

 surface, in oblique light the colony has a snowflake appearance; the edge 

 delicately serrate. With artificial light and a hand lens, the texture is that 

 of a coarse woolen fabric. Acid production from the trace of glucose may 

 tinge the colony. The colony is large. 



Brilliant Green-Eosin Agar. 30 Meat infusion agar is prepared and 

 titrated to +1 to phenolphthalein. The following substances are added: 



28 An agar is neutral to Andradc when, hot, the color is a deep red, but fades 

 completely on cooling. This is determined by cooling 3 or 4 c.c. of acidified hot 

 agar in a serum tube under the tap and adjusting accordingly, 



29 These are conveniently added from one sterile solution containing 20% 

 lactose and 2% dextrose, 5 c.c. to 100 of agar gives the requisite concentration. 



30 Teague and Clurman, Jour, of Infcc. Dis., 18, 1916, 647. 



