130 LABORATORY MANUAL OF MICROBIOLOGY 



Exercise 47 

 Crude Cultures of Higher Sulphur Bacteria 



Place some mud from ditches or other bodies of water in a large 

 flask or cylinder and cover to a depth of 3 to 6 inches with fresh 

 or salt water. 



Add 5 grams of magnesium sulphate and 2 grams of calcium 

 carbonate per liter of water. This furnishes a supply of H2S 

 due to the action of sulphate-reducing bacteria present in the 

 mud. 



Inoculate the solution with material that contains the sulphur 

 bacteria, namely pond scum, submerged plant material, organic 

 matter from sulphur springs, etc. H2S may also be used in the 

 form of a gas. 



Incubate in the dark for colorless sulphur bacteria, while, for 

 colored forms, the cultures are exposed to transmitted light. 

 Growth may appear on surface of liquid, along walls of container, 

 or on surface of mud. 



Concentration of H2S, amount of mud added to flasks, purity 

 of water used, amount and nature of inoculum will all influence 

 the type of organism developing. The red forms develop better 

 under higher partial pressures of H2S than the colorless forms. 



Exercise 48 

 Isolation of Pure Cultures of Higher Sulphur Bacteria 



100-cubic centimeter portions of Medium 70 are placed in 

 250-cubic centimeter Erlenmeyer flasks and steriUzed, the 

 ammonium sulphate being steriUzed separately in stock solutions, 

 then added to the rest of the medium, after sterilization. 



Inoculate flasks from enrichment cultures obtained in previous 

 experiment, using as little foreign material as possible. 



The flasks are then placed under jar (e) into which hydrogen 

 sulphide is introduced, as shown in Fig. 1 (p. 30). 



The sulphide is generated by the action of HCl upon FeS in 

 (a). 



Hydrogen, CO2, oxygen, or air may be introduced from com- 

 pressed gases (5) in regulated amounts controlled by manometer 

 attachment (/). 



