26 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(1) is distributed (100 cc. in 250 cc. 

 flasks usually). 



Sterilization : Sterilize in flowing steam for 

 30 minutes on 3 consecutive days. 



Use: Isolation and cultivation of Thio- 

 bacillus thiooxidans. The organisms can 

 grow at a pH = 1.0 with a maximum at 

 pH = 3.0 to 4.0. 



Variants: The author added a trace of 

 FeS04 and the following combinations of 

 potassium salts of phosphoric acid to the 

 above solution containing no potassium 

 phosphate, 

 (a) KH2PO4 3.0 g. pH = 4.2 



'KH2PO4 1.5 g.\ ^TT _ 5 4 



(c) K2HPO4 3.0 g. pH = 6.0 

 . ,. /K2HPO4 3.0 g.l ^„ _.. 

 ^^) icaCOa 10.0 g./ P^ - ^-^ 



At the various pH values the author 

 found starting with acid medium some 

 Th. thiooxidans give final pH = 1.2. Soil 

 bacteria produce no acid at pH = 4.2 

 but change pH = 8 to pH = 7.6. 



Reference: Waksman (1922 pp. 606, 609 

 to 613). 



75. Waksman's Sulphide Solution 



Constituents : 



1. Water 1000.0 cc. 



2. K2S 5.0 g. 



3. NH4CI 0.1 g. 



4. MgClj 0.1 g. 



5. Na2HP04 or K2HPO4 0.2 g. 



6. NaHCOs 1.0 g. 



7. CaC03 10.0 g. 



or CaClj 0.25 g. 



Preparation : 



(1) Dissolve 2, 3, 4 and 5 in part of 1. 



(2) Dissolve 6 and 7 in rest of 1. 



(3) Add (1) to (2) by means of sterile 

 pipettes after sterilization. 



(4) pH = 7.5. 



Sterilization: Autoclave at 15 pounds pres- 

 sure for 15 minutes. 



Use: To show oxidation of K2S by Thio- 

 bacillus thiooxidans . 



Reference: Waksman (1922 p. 609-15). 



76. Beijerinck's Thiosulphate Solution 



Constituents : 



1. Water 100.0 cc. 



2. Na2S203-5H20 0.5 g. 



3. NaHCOa 0.1 g. 



4. K2HPO4 0.02 g. 



5. NH4CL 0.01 g. 



6. MgCl2 0.01 g. 



Preparation: 



(1) Dissolve 2, 3, 4, 5 and 6 in 1. 



(2) Adjustment of reaction not specified. 



(3) Inoculate with a little ditch or canal 

 water or ditch mud. Incubate at 

 28 to 30 °C. 



Sterilization: None required. 



Use: Enrichment and isolation of organ- 

 isms utilizing carbonic acid, Thiobacillus 

 thioparus. Two or three days after 

 inoculation the surface is covered with a 

 layer of free sulphur filled with bacteria. 



Variants : 



(a) When Beijerinck used an inoculum 

 from sea water, 3.0% NaCl was added 

 to the solution. 



(b) Waksman used the above solution 

 with either 0.02 g. Na2HP04 or K2- 

 HPO4 and added one of the following 

 combinations: 



(1) KH2PO4 3.0 g. pH = 5.4 



(2) NaHCOa 1.0 g. pH = 8.8 

 fNaHCOalLOg. ^3 = 9 4 



^'^' \CaCO3 jlO.Og. P^ ^-^ 

 He found that Thiobacillus thio- 

 oxidans produced a pH = 1.4 in 

 variant (b) (1). 



(c) Trautwein substituted Na2HP04 for 

 K2HPO4 in the original solution and 

 used the solution to grow Thionic acid 

 bacteria (Omeliansky). 



Reference: Beijerinck (1903 p. 595), Waks- 

 man (1922 p. 609), Trautwein (1921 p. 

 518). 



77. Kaserer's Hydrogen Ammonium Chlor- 

 ide Solution 



Constituents: 



1. Water 1000.0 cc. 



2. K2HPO4 (0.05%) 0.5 g. 



3. MgS04 (0.02%) 0.2 g. 



4. NH4CI (0.1%) 1.0 g. 



5. NaHCOs (0.05%) 0.5 g. 



6. Iron chloride trace 



7. Hydrogen 

 Preparation : 



(1) Dissolve 2, 3, 4, 5 and 6 in 1. 



(2) Flask. 



(3) Following sterilization, if the solu- 

 tion is sterilized, inoculate the solu- 

 tion with soil. 



