iii„-8 MANUAL OF METHODS FOR PURE CULTURE STUDY 



phosphorus used does not ignite spontaneously but merely gives off 

 a grey smoke, ignite it before the jar is sealed by a match held with 

 the forceps. Since considerable heat is developed, place beaker, 

 unless resistant glass is used, three inches from the top of the con- 

 tainer and put a "blank" plate under the beaker rather than an inocu- 

 lated plate. After the phosphorus ignites, and the jar is tightly 

 sealed, place it directly in the incubator. At the time the container 

 is opened, have available a crock or pan filled with water. As soon 

 as the lid is taken from the jar, remove the beaker containing the 

 phosphorus with the tongs and submerge under the water in the pan 

 and save for later use. After this, remove the cultures from the jar. 



Advantages: Quick method of obtaining anaerobiosis. It is relatively inexpensive 

 since the only materials are phosphorus and a container which may be sealed. Dis- 

 advantages: Care must be exercised to prevent accidental burns which are very pain- 

 ful. Inexperienced technicians should be cautioned concerning the dangers. 



ALKALINE PYROGALLOL METHODS 



Another chemical method for removing oxygen in order to promote 

 anaerobic growth is to utilize the oxygen absorptive capacity of the 

 reaction between alkali and pyrogallic acid. Of the technics and 

 devices reported which make use of this reaction, two may be recom- 

 mended as being especially useful. One of these concerns a technic 

 applied to individual plate culture and the other relates to a system 

 for individual tube cultures. 



Spray (or Bray) Plate Cultures 



Materials: (1) Spray (1930) anaerobic dish''; (2) plasticene 

 (see footnote 3) or tape for sealing; (3) 20% aqueous NaOH. (4) 

 40% aqueous pyrogallic acid. 



Note: The Spray dish consists of an ordinary glass petri dish top and a special 

 bottom which is deep and which has a raised ridge across the center. The top of the 

 bottom dish has a lip into which the top section of the dish fits. Although constructed 

 of heat resistant glass, in practice considerable breakage during sterilization and hand- 

 ling of the Spray dish may be encountered. This is eliminated in the Bray^ dish, 

 which is Pyrex, and which is essentially the same in design as the Spray dish. In the 

 Bray dish, however, the need for the lip is eliminated since the top of the bottom 

 section is slightly smaller in diameter than the remainder of the bottom section. 

 This allows the top to fit down over the rim of the bottom section. 



Method: Pour anaerobic medium in the top half of the dish, 

 and after solidification, streak from sample or culture, or pour 

 seeded plate. After inverting dish, place 10 ml. of 20% aqueous 

 NaOH solution in one section of the bottom dish and 4 ml. of 40% 

 aqueous pyrogallic acid in the other. Seal dish with plasticene or 

 tape. Tilt dish to mix solutions and place in incubator. 



''Spray anaerobic dish. Fisher Scientific Company, Pittsburgh, Pennsylvania, or 

 E. H. Sargent Company, Chicago, Illinois. 



^Bray anaerobic dish. Corning Glass Works, Corning, New York, Pyrex No. 3155, 

 or dealer. 



