546 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(n) Savage. 



(1) Peptone 10.0 g., Liebig's extract of 

 beef, 10.0 g., sodium chlorid 5.0 g., 

 are boiled up in an enamalled dish 

 with one liter of distilled water. 

 The mixture is then poured into a 

 flask, 30.0 g. of powdered agar 

 added, and the whole heated in the 

 autoclave at 115°C. for one hour. 

 The flask is removed, and after 

 cooling to about GO'^C, the white 

 of one egg mixed with a little 

 distilled water is added. The 

 contents are coagulated by heating 

 in current steam in the usual way, 

 filtered, and the filtrate made up to 

 1 liter. The mixture is made 

 neutral, litmus paper being used 

 as the indicator. Then 19.0 cc. 

 of normal sodium carbonate solu- 

 tion and 10.0 g. c.p. lactose are 

 added. The flask is replaced for 

 30 minutes in the steam sterilizer. 

 Almost invariably there is a con- 

 siderable precipitate and the mix- 

 ture has to be again filtered. 



(2) Seven cc. of the fuchsin solution 

 (see below) are added, followed by 

 25.0 cc. of a quite freshly prepared 

 10,0% sodium sulphite solution. 

 The mixture becomes much less 

 red, but is not immediately 

 decolorized. It is then distributed 

 conveniently into small flasks, 

 each containing 50.0 to 60.0 cc. of 

 media, and sterilized in current 

 steam for 2 days, 30 minutes each 

 day. 



(3) The fuchsin solution is made as 

 follows: 3.0 g. powdered crystal- 

 line fuchsin are placed in a dry 

 flask, and 60.0 cc. of absolute 

 alcohol are added. The contents 

 are thoroly mixed, and the flask 

 tightly stoppered, allowed to stand 

 for exactly 24 hours at 20° to 22°C. 

 The alcoholic extract is then 

 decanted and preserved in a clean 

 glass-stoppered bottle. Made in 

 this way a uniform fuchsin extract 

 is obtained which keeps well, 

 and the same quantity of fuchsin 

 is added each time a fresh batch of 

 medium is prepared; a matter of 

 much importance. 



(4) The medium must be stored in the 

 dark, since light gradually turns 

 it red. When solidified it is almost 

 free from color, 

 (o) U. S. A. Med. Dept. 



(1) Into a container put 1 liter of tap 

 water, marking the level of the 

 fluid. Add 30.0 g. thread agar, 

 10.0 g. peptone, 5.0 g. NaCl, 5.0 

 g. beef extract. Cook until dis- 

 solved—it is best to autoclave 30 

 minutes at 15 pounds; filter thru 

 sterile gauze or cotton. If neces- 

 sary clear with egg. For this 

 purpose, for each liter beat up the 

 white of one egg with 10.0 cc. of 

 warm water until the egg is well 

 mi.xed. Add this to agar cooled to 

 55°C., mix thoroly, heat for 30 

 minutes or autoclave and filter 

 thru cotton. 



(2) This stock agar is kept on hand in 

 quarter-liter flasks or bottles. 

 Agar is standardized just before 

 use and reaction adjusted to 0.2% 

 acid to phenolphthalein. Before 

 use, fuchsin and sodium sulphite 

 are added. A filtered, saturated 

 solution of basic fuchsin in 95% 

 alcohol is kept on hand. A 10.0% 

 solution of dry sodium sulphite 

 crystals in sterile water is freshly 

 made. 



(3) Teague has shown that a 10.0% 

 solution of crystalline sodium 

 sulphite can be heated for 20 

 minutes at 15 pounds pressure with 

 practically no change, and that the 

 10.0% sodium sulphite solution 

 covered with a layer of liquid 

 petroleum about one cm. thick 

 and sterilized in the autoclave 

 can be kept at room temperature 

 for 3 weeks and probably much 

 longer with but very slight change. 



(4) One and eight-tenths cc. of fuch- 

 sin solution is added per liter to 

 the agar. After this has been 

 done the sodium sulphite solution 

 is added gradually until the hot 

 agar is almost decolorized— usually 

 about 25.0 cc. to the liter. A pale 

 rose color should be present in the 

 hot agar which fades to a very 

 faint pink on cooling; 10.0 g. of 



