134 CULTURE MEDIA AND THEIR STERILIZATION 



Hemoglobin Agar. Prepare agar tubes by the usual method and 

 keep them in the incubator for some time to permit the condensed 

 water to evaporate. Prepare hemoglobin as follows : Allow aseptically 

 obtained blood to run into a flask containing sterile physiologic salt 

 solution. Shake and leave in the refrigerator until the red blood 

 corpuscles have settled. Pipette off the clear fluid and replace by 

 fresh sterile salt solution. Repeat this operation once. (The pro- 

 cedure has been fully described above as the washing of red blood cor- 

 puscles). If only a small amount of hemoglobin is needed the washing 

 may be done in the tubes of the centrifuge in fifteen minutes. Remove 

 the hemoglobin from the corpuscles by shaking with ether; evaporate 

 the latter on a water bath at a low temperature or in the incubator, 

 and filter the watery solution of hemoglobin through a Pasteur filter. 

 Add the clear hemoglobin filtrate to dry sterile agar tubes. 



Milk. Milk is sometimes used as a culture medium. It is first 

 boiled, the cream is removed after it has separated out, and 10 c.c. 

 of the fat-free milk is placed into test-tubes. These are then sterilized 

 as usual. Before the tubes are prepared the reaction of the milk must 

 be tested; it should be + 10 to + 20 acid to phenolphthalein, but 

 neutral or faintly alkaline to litmus. 



Litmus Milk. Prepare the milk as above and before distributing 

 it to tubes add enough sterile litmus solution to give it a deep lavender 

 color. If the milk is too acid to produce this color effect, add enough 

 -$ solution of caustic soda to produce the desired color. 



Beer Wort. -Wort gelatin and wort agar are frequently used for 

 the cultivation of yeast cells, saccharomyces, or blastomyces. Beer 

 wort, before hops have been added, may be obtained from a brewery. 

 It can also be prepared in a laboratory as follows: 



1. Take 250 grams of crushed malt and place it in a 2 liter flask. 



2. Add 1000 c.c. distilled water heated to 70 C. and close the flask 

 with a rubber stopper. 



3. Place in a water bath kept at 60 C. for one hour. 



4. Strain through muslin into another flask and heat in the steam 

 sterilizer for one-half hour. 



5. Filter through a dense paper filter and sterilize in the steam 

 sterilizer. If the beer wort is to be used as such it should be dis- 

 tributed to the test-tubes before the sterilization. 



Wort Gelatin and Wort Agar. These are prepared like ordinary 

 nutrient gelatin or agar, except that the nutrient bouillon is replaced 

 by beer wort. The reaction of the latter is not interfered with; 

 however, if gelatin is used the strongly acid reaction of the latter has 

 to be corrected. 



Potato Culture Media. For ordinary work the preparation is as 

 follows: Select good potatoes which have not yet germinated in the 

 cellar; potatoes which have been frozen cannot be used. Their out- 

 side must be thoroughly cleansed by scrubbing with soap and water, 

 then with pure water, and finally with 1 to 1000 bichloride solution. 



