188 DIFCO MANUAL 



Bacto-Phenol Red Agar Base and the complete carbohydrate* media described 

 below, are used in fermentation studies for the cultural identification of pure 

 cultures of microorganisms. While liquid media are generally employed in study- 

 ing the fermentation reactions of microorganisms, many bacteriologists prefer a 

 solid medium for this purpose. The solid media employed usually contain one 

 per cent of the selected carbohydrate and an indicator of reaction. They are 

 dispensed in tubes, slanted, and inoculated by smearing over the surface of the 

 slant and stabbing into the butt. 



The advantages claimed for a solid fermentation medium are that it permits 

 observ^ation of the fermentation reactions under both aerobic and anaerobic con- 

 ditions, that gas formation is indicated by splitting of the agar or accumulation of 

 gas bubbles in the base, and that deep tubes can provide sufficiently anaerobic 

 conditions for the development of the obligately anaerobic bacilli. 



Bacto-Phenol Red Agar Base is particularly well adapted to the study of 

 fermentation reactions of microorganisms. This medium supports excellent growth 

 of many fastidious bacteria. The basal medium is free from fermentable carbo- 

 hydrates which could give erroneous interpretation. With the exception of the 

 carbohydrate, which has been omitted, it is a complete medium prepared with 

 phenol red as an indicator of changes in reaction. Bacto-Phenol Red Agar Base 

 permits the user to prepare as much or as little medium as he requires, adding 

 to different portions any fermentable substance desired (usually one per cent of 

 the test carbohydrate being added). An entire series of carbohydrate agars may 

 thus be made up readily, conveniently and economically. 



Tubes of the sterile medium are inoculated by smearing over the surface of 

 the slant and stabbing into the butt. Obligately anaerobic bacteria may be inocu- 

 lated into the melted medium previously cooled to 45 °C. and subsequently allow- 

 ing it to solidify. After incubation, fermentation will be denoted by a change 

 in the color of the medium from red to canary yellow. Gas formation is indi- 

 cated by the collection of gas bubbles in the base, or by splitting of the agar. 



To rehydrate the medium, suspend 31 grams of Bacto-Phenol Red Agar Base 

 in 1000 ml. cold distilled water and heat to boiling to dissolve the medium com- 

 pletely. To this solution the selected carbohydrate is added. When the solution is 

 complete the medium is distributed in tubes which are stoppered with cotton 

 plugs or loosely fitting caps and sterilized in the autoclave for not more than 15 

 minutes at 15 pounds pressure (121°C.). The minimum amount of heat required 

 for sterilization is to be desired. By packing tubes loosely in the autoclave to 

 allow free circulation of the steam, the time required for sterilization may be ap- 

 preciably shortened, provided the temperature in the autoclave is actually 121 °C. 

 Some workers may prefer to add the carbohydrate in the form of a sterile solu- 

 tion to the melted sterile base, and then dispense the complete medium into 

 sterile tubes. This latter procedure is particularly recommended where only slow 

 operating autoclaves are available. In either case the sterile tubed medium con- 

 taining the carbohydrate is allowed to cool in a slanting position so as to provide 

 a slope and generous butt. The final reaction of the medium, without added 

 carbohydrate, will be pH 7.4. 



The addition of some carbohydrates may result in an acid reaction. In this 

 case, it is suggested that O.IN sodium hydroxide be added drop by drop to restore 

 the original color, taking care not to obtain too deep red or cerise color. 



One pound of Bacto-Phenol Red Agar Base will make 14.6 liters of medium. 



An extensive number of carbohydrates, polyhydric alcohols and glucosides, 

 Difco, is available (see page 291) for use in conjunction with Bacto-Phenol Red 

 Agar Base. There is also available a selected list of filter-sterilized ampuled solu- 



*The term carbohydrate, as used here, includes carbohydrates, polyhydric alcohols, glucosides and 

 other fermentable substances. 



