PREPARATION OF MEDIA 53 



associates described growth of this organism in liquid semisynthetic 

 medium, containing surface-active agents (esters of long-chain fatty acids 

 and polyhydric alcohols), which supported comparatively rapid cell 

 multiplication with a small inoculum and yielded cultures of uniform 

 turbidity without granule or pellicle formation (see Frobisher, 1949). 



MEDIA FOR DETERMINATION OF PHYSIOLOGICAL PROPERTIES 



In the study of pure cultures, the so-called cultural characters and 

 physiological reactions assume importance in the classification of many 

 groups of heterotrophic bacteria. The latter reactions include the dis- 

 similation of various carbohydrates and polyalcohols, the breakdown of 

 simple and complex nitrogenous compounds, and the production of 

 specific compounds as end products of the metabolism of the bacteria 

 under study. The media for some of the more commonly used reactions 

 will be included in this section. (See also Chap. VII, ''Routine Tests for 

 the Descriptive Chart.") 



Carbohydrate indicator media. The common procedure is to add the 

 carbohydrate or polyalcohol to be studied to a basal medium (either 

 liquid or agar) to which an indicator has been added to detect changes in 

 pH which develop during growth. Growth of some organisms, particu- 

 larly the sporeforming anaerobes, may result in a marked reduction of the 

 indicator, in which case the indicator must be added after rather than 

 before growth ; use of a spot plate or other methods of pH determination 

 is then made at the time of observation. Early observation of fermenta- 

 tion results generally eliminates the difficulty due to reduction of the indi- 

 cator, since acidity changes usually precede reduction. Production of 

 gas is detected in liquid media by placing Durham tubes (small inverted 

 vials which will fill with liquid during sterilization) in the tubes at the 

 time the medium is dispensed. The tubes are unnecessary if a solid or 

 semisolid medium is used. Semisolid agar is prepared by adding 0.3-0.5 

 per cent of agar to a satisfactory liquid medium and making stab inocula- 

 tions in the column of medium with a straight inoculating needle. In 

 such a medium (or in full-strength agar) gas production will be denoted 

 by the appearance of gas bubbles and cracks in the medium; the same 

 semisolid medium may also be used to determine motility. Full-strength 

 (1.5 per cent) agar should be cooled m slanting position and inoculated on 

 the surface of the slant. 



The basal medium to be employed for fermentation tests should pro- 

 vide the necessary nutrients for the organism to be studied and must be 

 free of fermentable carbohydrates. If good growth can be obtained on 

 2 per cent casein or gelatin-peptone solutions (or agar), these media are 

 preferred. According to Vera (1949), some samples of beef and yeast 



