136 BIOLOGY AND TECHNIQUE 



habit-acquired skill which the individual worker attains. None of these stool 

 isolation media are ordinarily successful at once in the hands of anyone, and a 

 certain amount of practice must be attained before one can judge of the use- 

 fulness or uselessness of a medium. 



Brilliant Green Agarfor Typhoid Isolation. Krumwiede has recently 

 devised a brilliant green agar with which he has had excellent results. 1 

 The basis is an extract agar like that used for Endo's medium: 



Beef Extract 0.3% 



Salt 0.5% 



Peptone 1.0% 



Agar 1.5% 



(Domestic peptones are satisfactory.) 



Dissolve in autoclave; clear and filter. A clear agar is essential. The final 

 reaction of the medium is to be neutral to 2 Andrade's indicator, which in terms 

 of phenolphthalein is 0.6-0.7% acid (normal HC1). It is more convenient to 

 i have the reaction set slightly alkaline to litmus at the time of preparation and 

 to acidify each bottle as used. The agar is bottled in 100 c.c. amounts and auto- 

 claved. When needed, the bottles are melted and the volume of each cor- 

 rected (if necessary) to an approximate 100 c.c. Add to each bottle: 



One per cent Andrade's Indicator. 



Acid to bring to neutral point of the indicator. 3 



One per cent Lactose. 4 



0.1 per cent Glucose. 4 



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 18 accurately weighed on a foil, washed 

 with boiling H 2 into a 100 c.c. volumetric Iflask 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. 



1 We are indebted to Dr. Krumwiede for a preliminary account of this method. 



2 Andrade's Indicator: 0.5 per cent aqueous acid fuchsin 100 c.c. 



Normal NaOH 16 c.c. 



The dye is slowly (2 hours) alkalinized to the color-base; the red tint is restored by 

 acids. 



3 An agar is neutral to Andrade when, hot, the color is a deep red, but fades com- 

 pletely 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. 



4 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. 



