570 S. HENRY AYERS AND COURTLAND S. MUDGE 



The filtrate, which is of a yellowish-white color, will appear 

 cloudy and can not be filtered clear in the concentration used. 

 This makes no difference, because it is clear in the dilution of 

 the final medium. 



Make up the filtered milk-powder solution to 250 cc. with 

 distilled water. This completes the milk-powder solution "a." 



PEPTONE-EXTRACT SOLUTION 



To make the peptone-extract solution "b" dissolve 5 grams 

 peptone and 3 grams Liebig extract in 100 cc. distilled water by 

 steaming in the Arnold sterilizer or by boiling over flame for 

 twenty minutes. Filter until clear and make solution up to 

 250 cc. with distilled water. This completes solution "b." 

 "Difco" peptone has been used in our experiments because of 

 its hydrogen-ion concentration, which is near the neutral point, 

 and because, with the extract in the proportion of 5 grams 

 peptone to 3 grams extract, a precipitate is usually formed which 

 permits filtration with a resulting clear solution. For a standard 

 medium, whatever makes of peptone and extract are selected 

 should be universally used. 



The milk-powder solution "a" is now mixed with the peptone 

 extract solution "b" which gives a total volume of 500 cc. To 

 this mixture 500 cc. of double strength (3 per cent) washed-agar 

 solution is added. This completes the medium, which is now 

 ready for sterilization. 



THE USE OF DISTILLED WATER 



We specifically mention distilled water. Tap water may or 

 may not contain dissolved substances, the effect of which on 

 media making and bacterial growth is unknown. By the use 

 of distilled water this uncertainty is obviated and one is assured 

 of a constant definite solvent, "standard" everywhere. 



WASHED-AGAR SOLUTION 



A stock solution of double strength (3 per cent) washed agar 

 is prepared and put up in flasks and sterilized. This agar is then 



