FORMULA. 



195 



NON-SYNTHETIC CULTURE MEDIA. 



Standard Peptonized Beef-Bouillon. 



Standard peptonized beef-bouillon is made as 

 follows : To 500 grams of finely minced lean 

 beef add 1,000 cc. of distilled water. The solu- 

 ble parts may be removed from the meat by al- 

 lowing the water to stand on it for 24 hours in 

 the ice-chest or for I hour in the water bath at 

 55 C. The writer prefers the second method. 

 Then boil for 60 minutes either in the steamer 

 or in a covered dish. Filter through a clean 

 cloth, using pressure (meat-press), cool, and 

 remove fat by filtering through S. & S. filter 

 paper; make up to 1,000 cc. by addition of more 

 water; then add I per cent Witte's peptonum 

 siccum and 0.5 per cent c. p. sodium chloride. 

 Steam one-half hour, filter, cool, titrate, add re- 

 quired alkali, steam again for one-half hour, 

 filter, pipette into tubes or flasks, and autoclave 

 or heat for a minimum time in the steamer. 

 Plugs should be well made and fit tightly; 

 glassware should be scrupulously clean. For 

 some purposes both the peptone and the salt 

 may be omitted. A greenish bouillon indicates 

 insufficient boiling, and will usually throw down 

 some additional vexatious precipitate when 

 heated in the test-tubes. Other meats may be 

 substituted for beef, and other peptones for 

 Witte's. Meat-extracts are not recommended. 

 Such extracts usually contain resistant spores. 

 Media which have been steamed, or boiled in an 

 open dish, are better for many bacteria than 

 those which have been sterilized in the autoclave. 



(For additional obse'rvations on proper steril- 

 ization see Culture Media, p. 29.) 



Dunham's Solution. 



Distilled water 1,000 



Witte's peptonum siccum 10 



C. P. sodium chloride 5 



First recommended by Dr. Ed. K. Dunham, of 

 New York. 



Standard Nutrient Agar. 



To 1,000 cc. of standard beef-bouillon add 10 

 grams of agar-flour, steam one-half hour, cool 

 to 58 C.; add whites of two eggs (beaten thor- 

 oughly and neutralized to litmus by dilute hy- 

 drochloric acid) and thoroughly mix with the 

 bouillon; steam I hour, filter hot through S. & S. 

 paper which has been thoroughly warmed with 

 boiling distilled water. Use two or three fun- 

 nels. That which remains unfiltered after a 

 reasonable time must be reheated and put 



through a fresh filter paper. Sometimes all can 

 be got through a second filter paper without re- 

 heating. Some advise filtering in the autoclave 

 or in the steamer, but the writer has not found 

 that necessary, and in recent years has also 

 abandoned the hot-water funnel. 



Clear agar may be obtained also by filtering 

 through absorbent cotton, and some prefer this 

 to filter paper. 



In preparing agar from the " slender kanten " 

 or the " square kanten," snip fine, soak in the 

 bouillon 15 minutes, and then heat on the sand 

 bath i hour at 110 C. or in the autoclave 45 

 minutes at 105 C. From this point proceed as 

 before. 



Long heatings in the autoclave at 110 C., or 

 shorter heatings at higher temperatures, are apt 

 to brown the agar, and should be avoided care- 

 fully, as this renders the medium less service- 

 able for the growth of bacteria. Agar which has 

 been properly superheated filters readily. One 

 per cent agar made from the agar-flour does not 

 require to be heated on the sand-bath or in the 

 autoclave, but filters satisfactorily after steam- 

 ing for an hour at 100 C. 



After the agar has been tubed it may be ster- 

 ilized, if it does not contain sugars, by one 

 steaming in the autoclave for 10 minutes at 110 

 C., or by short steamings in the steam sterilizer 

 at 100 C. on three successive days. 



To those who are dependent on the agar-strips 

 and do not have access to an autoclave, Schutz's 

 method may be recommended as very good. 

 The writer formerly made large use of this. It 

 consists in heating the agar very hot in a mini- 

 mum quantity of water or bouillon before add- 

 ing the bulk of the fluid. (See p. 34 and Bib- 

 liog., XVI.) 



Agar which has been soaked in 5 per cent 

 acetic acid for an hour or two before adding it 

 to the bouillon also enters into solution thor- 

 oughly and filters well after a short boiling. The 

 acid must first be removed completely by wash- 

 ing in running water for some hours under a 

 mosquito-net or a piece of gauze. 



Unfiltered agar does well enough for certain 

 fungi, and for lazy people, but the agar used for 

 delicate work in bacteriology should be as clear 

 as the bouillon from which it is made., i. e., per- 

 fectly free from cloudiness and precipitates. 

 Sufficient caustic soda is usually added to the 

 agar to render it + 15 of Fuller's scale. Other 

 degrees are useful, e. g., + 10, o, 10, etc. 



