CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



323 



2. Plasmon (sodium caseinate). 500.0 g. 



3. KH2PO4 5.0 g. 



4. Magnesium phosphate 0.3 g. 



5. Ammonium lactate 

 Preparation : 



(1) Mix 500.0 g. of "plasmon" (sodium 

 caseinate) with 2^ liters of lukewarm 

 2.0% Na2C03 solution. 



(2) Add 25 liters of distilled water. 



(3) Allow to soak and place in a large 

 flask. 



(4) Soak 10.0 g. of trypsin in a little 

 water and add to (3). 



(5) Place a layer of toluol about 1 cm. 

 high to keep down bacterial growth. 



(6) Place at 37°C. for several days shak- 

 ing occasionally. 



(7) From time to time test for trypto- 

 phane with acetic acid and bromine 

 water. The maximum amount of 

 tryptophane is reached after about 

 10 to 20 days. 



(8) Remove the toluol and heat to 80 °C. 

 to coagulate any undigested al- 

 bumin. 



(9) Add 100.0 g. talcum and shake 

 thoroly. 



(10) Allow to stand several hours until 

 it is rather clear. 



(11) Tyrosin and some cystin will settle 

 out during the cooling. 



(12) Filter. 



(13) Add H2SO4 to the clear filtrate until 

 it is 5.0% H2SO4. 



(14) Add a 10.0% solution of HgS04 in 

 5%, H2SO4 to (13). A lemon colored 

 precipitate of a union of HgS04 and 

 tryptophane is formed. 



(15) Allow to stand for 24 hours and 

 filter. Test the filtrate to see that it 

 is free from tryptophane by adding 

 more HgS04 to it. 



(16) Wash the precipitate with 5.0% 

 H2SO4 until the wash water no longer 

 gives a red coloration with Millon's 

 reagent. 



(17) Add the damp precipitate to 500.0 g. 

 of distilled water. 



(18) Decompose the precipitate by heat- 

 ing and pass in H2S. 



(19) Pass in CO2 to drive out the excess 



of H2S. 



(20) The liquid contains tryptophane and 

 cystin while the precipitate con- 

 tains HgS. 



(21) Filter and add H2SO4 to the filtrate 

 so that it is 5.0% H2SO4. 



(22) To separate the cystin, precipitate 

 fractionally with HgS04 in that one 

 adds first HgS04, carefully, until 

 just a conglomerate precipitate of 

 cystin mercury sulphate is formed. 



(23) Allow to stand a few hours and filter, 



(24) Precipitate again with HgS04. 



(25) Allow to stand, filter, wash with 

 5.0% H2SO4 as in (16) and add the 

 moist precipitate to distilled water. 



(26) Decompose the precipitate with H2S 

 as in (18) and (19). 



(27) Filter and evaporate to dryness. 

 (2S) This brown colored mass may be 



crystallized in hot water several 

 times or pure tryptophane may be 

 obtained. 



(29) To obtain pure tryptophane add lead 

 carbonate (about 10.0% of the used 

 albumin, therefore about 50.0 g.) to 

 the filtrate (27) and heat for about 

 30 minutes in a water bath. Am- 

 monia is given off. 



(30) After cooling precipitate the lead 

 by passing thru H2S. 



(31) Filter and evaporate the filtrate to 

 dryness. 



(32) After several crystallizations with 

 dilute alcohol one obtains pure 

 silvery rectangular platelets of tryp- 

 tophane. 



(33) Dissolve 0.3 g. of (32), 5.0 g. of 

 secondary potassium phosphate, 0.3 

 g. of Magnesium phosphate and 

 ammonium lactate (amount not 

 given) in 1 liter of distilled water. 



(34) Distribute in 10.0 cc. lots in tubes. 

 Sterilization: Sterilize on 2 successive days 



in a steam sterilizer. 

 Use: Indol production. Preparation of 



pure tryptophane. 

 Variants: The author added glucose and 



glycerol, amounts not given. 

 Reference: Zippel (1912-13 p. 572). 



1127. Bacto Tryptophane Broth 

 (Dehydrated) 



Constituents : 



1. Distilled water 1000.0 cc. 



2. Hydrolyzed casein 1.0% 10.0 g. 



(Bacto tryptophane broth dehydrated) 



Preparation : 



(1) Prepare a 1.0% solution of Bacto 



