122 MANUAL OF MICROBIOLOGICAL METHODS 



Use of methylene blue as indicator of anaerobiosis. For all types of anaerobic jars 

 and containers, except individual-plating or tube-culture systems, it is convenient to 

 include an indicator tube which will serve as a check on the development of anaero- 

 biosis. The most commonly used system utilizes the change of methylene blue from 

 the colored (oxidized state) to the leuco form (reduced state). Using the solution 

 prepared as given below, any system which gives sufficient degree of removal of oxy- 

 gen from the atmosphere for anaerobic growth to develop will cause the blue color 

 of the solution to disappear or will maintain the colorless condition if the solution is 

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

 somewhat 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. 



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

 6.0 ml of O.IN NaOH diluted to 100 ml with distilled water, (2) 3.0 ml of 0.5 per cent 

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

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

 time the indicator solution is needed, mix 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 satis- 

 factorily 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 incu- 

 bation period.) 



Oxygen Removal by Combustion Using Laidlaw Principle 



For laboratories which are engaged in problems where anaerobic plating 

 is to be done frequently, it is advisable to plan for this and to purchase 

 equipment accordingly. Although the systems discussed above may be 

 adequate for this purpose, it is well to consider one of the jars which 

 utilize, on the Laidlaw (1915) principle, combustion as a means of securing 

 the anaerobic environment. These methods were designed especially for 

 incubation of plates, but other culture vessels (flasks, tubes, bottles, etc.) 

 may be used. Jars using this principle are those of Brewer (Brown and 

 Brewer, 1938) and Mcintosh and Fildes (Fildes and Mcintosh, 1921). 



Brewer Anaerobic Jar^ 



Materials for method of Evans, Carlquist, and Brewer (1948): (1) 

 Brewer jar complete with electric cord, (2) source of illuminating gas or 

 hydrogen, (3) plasticene (see footnote 2 on page 123), (4) vacuum pump 

 for evacuation. 



Method, Place plates in jar. Add tube of methylene blue solution. 

 Include a tube of soda lime in the jar to absorb excess CO2. Place roll 

 of (warmed) plasticene around rim of jar. Put on lid and press down 

 on plasticene to form seal. Add the lid clamp but tighten only slightly. 

 If used with illuminating gas, attach the jar by the rubber tubing to the 

 vacuum pump. Evacuate until the manometer or gauge reads approxi- 



1 Brewer jar. Baltimore Biological Laboratory, Baltimore, Maryland, and Fisher 

 Scientific Company, Pittsburgh, Pennsylvania. 



