THE PREPARATION OF CULTURE MEDIA 143 



Methods of Clearing Media. Clearing with Eggs. When culture 

 media" are prepared from substances containing no coagulable pro- 

 tein, it is often necessary, for purposes of clearing, to add the whites 

 of eggs, and then to heat for forty-five minutes in the Arnold 

 sterilizer. In the following detailed descriptions, the direction 

 "clear with egg" has been given whenever such a step is deemed 

 necessary. The exact technique of such a procedure is as follows: 



In a small pot or pan, the whites of several eggs (one or two 

 eggs to each liter of medium) are beaten up thoroughly with a little 

 water (20 c.c.). This egg white is then poured into the medium, 

 which, if hot, as in the case of melted agar or gelatin, must first 

 be cooled to about 50 to 55 C. The mixture is then thoroughly 

 shaken and steamed in the Arnold sterilizer for thirty minutes. At 

 the end of this time, the flask containing the medium is removed from 

 the sterilizer and thoroughly shaken so as completely to break up 

 the coagulum which has formed. It is then replaced and allowed 

 to steam for another fifteen minutes. At the end of this time the 

 medium between the coagula should be clear. It is now ready for 

 filtration through cotton. 



Filtering Media through Cotton. The filtration of media after 

 clearing, either by the addition of eggs or by the coagulation of the 

 proteins originally contained in it, is best done through absorbent 

 cotton. A small spiral, improvised of copper wire is placed as a 

 support in the bottom of a large glass funnel. A square piece of 

 absorbent cotton is then split horizontally giving two squares of 

 equal size. Ragged edges and incisures should be avoided. These 

 two layers of cotton are then placed in the funnel, one piece above 

 the other in such a way that the direction of the fibers of the two 

 layers is at right angles one to the other. They are then gently 

 depressed "into the filter with the closed fist. The edges of the 

 cotton are made to adhere to the sides of the funnel by allowing 

 a thin stream of tap water to run over them, while smoothing them 

 against the glass with the hand. 



The medium, when poured into such a filter, should be poured 

 along a glass rod at first, to avoid running down the sides or burst- 

 ing the filter. After filtration has begun, the filter should be kept 

 as full as possible. The first liter or so which comes through may 

 not be clear, but the filter gains in efficiency as the coagulum settles 

 into the fibers of the cotton, and the first yield may be sent through 

 a second time. Filtration of agar or gelatin is best done in a warm 



