THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA in„-5 



boiled (heat reduction) immediately prior to its being placed in the container. A some- 

 what less sensitive system can, in an emergency, be prepared by adding a tinge of color 

 from Loeffler's alkaline methylene blue to a tube of glucose broth. 



Tiie procedure recommended (Fildes, 1931) is: Prepare three stock solutions: (1) 

 CO ml. N/10 NaOH diluted to 100 ml. with distilled water; (2) 3.0 ml. 0.5% aqueous 

 methylene blue diluted to 100 ml. with distilled water; (3) 6.0 g. of glucose in 100 ml. 

 distilled water to which has been added a small crystal of thymol. 



Each time the indicator solution is needed, mi.x equal parts of the three solutions 

 in a test tube and boil in a cup of water until the color disappears. Place tube in 

 anaerobic container immediately and begin process of securing anaerobic conditions. 

 If the container is satisfactorily deoxygenated, the color in the solution should not 

 reappear. If the blue color does return it is a sign that the container leaks or has not 

 been satisfactorily exhausted of oxygen. (In the vegetable tissue jar, to be described, 

 the color may appear but will disappear with the development of anaerobiosis during 

 the incubation period). 



Biological Methods for Oxygen Removal 

 vegetable tissue jar 



Materials for method of McClung, McCoy and Fred (1935): 

 (1) Jar, or other container which may be sealed air tight {Recom- 

 mended: 6" X 18" or 6" X 12" Pyrex cyhnder^); (2) square (7" X 7") 

 of plate glass or a glazed plate; (3) plasticene^, 3^ pound; (4) glass 

 tumbler; (5) supply of oats or other grain (other tissues, particularly 

 chopped Irish potatoes, may be used, but are less conveniently 

 stored for occasional use, and in some cases produce objectionable 

 odors which are evident when the jar is opened) ; (6) tap water. 



Method: Place inverted tumbler (if plates are to be used), or 

 other support, in bottom of cylinder. Add oats to fill at least one 

 tenth of the capacity of the cylinder. Add sufficient tap water to 

 moisten the oats. Stack plates (or other cultures) on support. 

 Add tube of methylene blue solution (see above). Place layer 

 of plasticene (previously softened by placing in incubator) on rim 

 of cylinder. Push plate glass square firmly against plasticene; 

 using fingers, press the clay against both the square and the cylinder 

 until a satisfactory seal is obtained. Place jar in incubator immedi- 

 ately. (A 40-48-hour incubation period is recommended). 



If plate cultures are employed, use unglazed porcelain ("clay") 

 tops'* to replace the ordinary petri dish cover to absorb the moisture 

 which collects within the cylinder. If porcelain tops are unavailable, 

 add a petri dish lid containing CaCU to absorb the moisture. 



^Pyrex cylinder. Corning Glass Works, Corning, New York or supply house. 

 Pyrex Catalogue No. 850. 



^Plasticene The most satisfactory product of this type seems to be the English 

 clay called "Plasticene" (gray or green colored). This is obtainable in this country 

 from J. L. Hammet Company, Cambridge, Massachusetts, and perhaps other supply 

 houses. Other types may be found which are satisfactory but these must be tested 

 individually for suitability as some have been encountered which dry to a hard cake 

 upon incubation. 



^Unglazed porcelain ("clay") tops for Petri dishes. The Coors porcelain dish, sold 

 by Arthur H. Thomas Company, has been found to be more uniform in size and quality 

 than others tested. 



