FORMUL^K. 



195 



Non-vSynthetic Culture Media. 



Slandard Feplnnicrd Bccf-Bonilloii. 



Standard peptonized becf-liouillnn is made as 

 follows To 5(X) grams of finely minced lean 

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

 l>lc parts may be removed from the meat by al- 

 lowing the water to stand on it tor 24 hours in 

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

 55'^ C. The writer prefers the second method. 

 Then lioil for 60 nnnutes either in the steamer 

 or in a covered dish. Filter through a clean 

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

 remove fat by liltering through S. & S. filt'_'r 

 papcr; 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- 

 ciuired alkali, steam again for one-half hour, 

 filter, pipette into tubes or llasks, and autoclave 

 ("■r 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. 

 i\[edia which have been steamed, or boiled in an 

 ri|)en dish, are better for many bacteria than 

 thnse wdiich have been sterilized in the autoclave. 



(For additional observations on proper steril- 

 ization see Culture Media, p. 29.) 



DiinJiain's Solution. 



Distilled water 



Witte's peptonum siccum 



C. P. sodium chloride 



1,000 

 10 



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 wdiich has been thoroughly warmed with 

 boiling distilled water. Use two or three fun- 

 nels. That which remains unfiltered after a 

 reasonable time must he reheated and put 



through a frch filter paper. Sometimes all can 

 be ,got through a second filter paper without re- 

 heating. Some advise filterin.g in the autoclave 

 or in the steamer, but the writer has nut found 

 that necessary, and in recent years has also 

 abandoned the hot-water funnel. 



Clear agar may be obtained aKi' ]'>y filterin.g 

 through absorbent cotton, and ^(une prefer this 

 til filter paper. 



In preparing agar from the " slender kanten " 

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

 bouillon 75 minutes, and then heat on the sand 

 bath I hour at no'' 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 bi.gber 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 readil)'. One 

 per cent agar made from the agar-llonr does not 

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

 autoclave, but filters satisfactorily after steam- 

 ing for an lioiir at T00° C. 



After the agar has been tubed it may he ster- 

 ilized, if it does not contain su.L'ars, 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 fonnerI_\- made large use of this. It 

 CI insists in heating the agar very hot in a mini- 

 nmm quantity of water or bouillon before add- 

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

 liog., XVI.) 



Agar wdiich 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 he 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 bacteriolo.gy should be as clear 

 as the bouillon from which it is made., /. 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. 



