VI4.-4 MANUAL OF METHODS FOR PURE CULTURE STUDY 



particularly in the case of an intermediate product which is formed 

 within the cell. 



Relation to Free Oxygen 

 A section of Leaflet V having the same heading as this describes 

 methods for distinguishing roughly between aerobes and anaerobes. 

 For a careful physiological study of any organism one must realize, 

 however, that such determinations as those mentioned in Leaflet V 

 are quite incomplete. It is especially to be observed that the rough 

 methods given there do not distinguish between strict anaerobes and 

 microaerophilic organisms. For a more adequate study of the relation 

 of an organism to free oxygen, there are two points in particular 

 which require careful investigation: first, the optimum oxygen tension 

 (which may be considered in the case of anaerobes from the stand- 

 point of oxygen tolerance); second, the respiratory quotient. 



Optimum Oxygen Tension. Vessels large enough to furnish an ade- 

 quate oxygen supply must be used. Probably the best method is to 

 place the cultures growing on liquid or agar as desired, in a Novy jar, 

 to evacuate and to replace the air with a mixture of gases containing a 

 known percentage of oxygen. Such a method is well adapted to deter- 

 mining oxygen tolerance of microaerophilic organisms. It should be 

 particularly remarked that the 'absence' of oxygen in the gas space 

 over the bacterial culture should be tested for directly by employ- 

 ment of a suitable indicator (e.g., solution of reduced methylene blue 

 or indigo disulfonate) properly applied to the gas phase. Use of the 

 indicator within the medium is of uncertain value. 



Respiratory Quotient. A similar apparatus may be used if provided 

 with stop-cocks to allow the removal of samples of gas for analysis. A 

 manometer should always be present on such a system to show 

 changes in gas pressure. Inthe sample of gas removed, the carbon 

 dioxide may be determined by absorption with standard alkali, after 

 which the oxygen may be removed by alkaline pyrogallol. The respir- 

 atory quotient is obtained by dividing the volume of carbon dioxide 

 produced by that of the oxygen consumed. 



Details of these methods are not given here and must of necessity 

 be varied with the organisms under investigation. A useful set-up for 

 determining the points above mentioned is described by Soule (1928). 

 Attention should also be called to the manometric tcchnic for physio- 

 logical studies on microorganisms. This technic provides a powerful 

 method of attack and should find wide use in bacteriological research. 

 It offers the most convenient and accurate method available for fol- 

 lowing reactions in which gas is evolved or taken up, and has been 

 extended to include chemical determination of products. The 

 manometric technic has been used successfully in studies determining 

 rates of reaction (especially when CO2 is evolved, O2 taken up, or 

 acids formed), vitamin or growth factor requirements, CO2 utiliza- 

 tion, and efficacy of disinfectants. Manometric methods can be 

 adapted to a wide variety of uses. For general purposes in physio- 

 logical bacteriology, the Warburg type of manometer is used. This 



