448 LEON S. MEDALIA 



b. Indicator watery solutions. The strength of the indicator 

 solutions found most useful to prepare the " standard colors" as 

 well as for use in measuring the H. I. C. of fluids were 0.02 per 

 cent of the respective indicators in sterile distilled water, (ex- 

 cepting phenol red in which case 0.04 per cent was found neces- 

 sary). These solutions were prepared from the "stock alcoholic 

 solutions" by placing 45 cc. (40 cc. for phenol red) of distilled 

 water in each of eight amber colored bottles of proper size stop- 

 pered with cotton and sterilized in the autoclave at 10 pounds 

 for one-half hour (rubber stoppers were sterilized alongside). 

 To each of the 45 cc. bottles of sterile distilled water, 5 cc. of the 

 "stock" alcoholic solutions of the indicators were added with 

 sterile pipets in an aseptic way making a 0.02 per cent solution, 

 except in the case of phenol red, in which case 10 cc. of the alco- 

 holic stock solutions were added (to the 40 cc. of water) making a 

 0.04 per cent solution. These water solutions were kept in the 

 dark, well stoppered with the sterilized rubber stoppers. 



The flocculent precipitate of the methyl red, formed when the 

 alcoholic solution was added to the distilled water, was cleared 

 away by the addition of 0.5 cc. of sterile & NaOH. This has 

 not interfered with the production of red in the acid, and yellow 

 in the alkaline solutions. 



Preparation of "color standards" 



For the preparation of "color standards" for standardizing cul- 

 ture media, the regular B. B. H. test tubes were used (thick 

 walled, without lips, 130 by 16 mm. outside diameter) 14 or at 

 most 28 such tubes being necessary. They should be of clear 

 glass and as nearly alike in diameter as possible. The impor- 

 tance of having the tubes alike in diameter cannot be emphasized 

 too strongly. 



Two sets of test tubes, seven pairs in each, are made use of, one 

 set for brom thymol blue, and the other for phenol red. Seven tubes 

 of each set are filled with approximately & NaOH, and the other 

 seven with 0.1 per cent HC1 (made from concentrated HC1 0.1 

 cc. and distilled water 100 cc). The seven pairs of tubes are 



