PREPARATION OF MEDIA 39 



media selected arbitrarily or according to tradition may be less adequate for many 

 purposes than media whose composition has been determined according to the nutri- 

 tional rationale; indeed, the latter approach will more certainly lead to a clearer 

 understanding of the nature of the microorganism being cultivated. 



General Procedures in Media Preparation 



In selecting the components of a medium, it is desirable to employ sub- 

 stances of defined composition, purity, or mode of preparation. In the 

 preparation of synthetic media, known chemical compounds of chemically 

 pure (C.P.) or reagent grade should be used. It is less easy to define the 

 composition of some of the complex substances used in certain formulas. 



Peptones and gelatin should conform to the minimal standards given 

 in the United States Pharmacopeia XV. Gelatin, in addition, should 

 meet the additional specifications as set forth in the Military Medical 

 Purchase Description, ASIMPA 1-212-000. Tentative specifications for 

 bacteriological-grade agar have been proposed. The specifications 

 referred to above for agar, gelatin, and some peptones are included in the 

 appendix to this chapter. In addition to chemical analysis, the growth 

 response of selected test organisms is used in designating a product as 

 satisfactory for bacteriological use. Distilled water is used generall^^ to 

 dissolve the components of the medium, although it is obviously unneces- 

 sary when crude plant or animal extracts are employed. Many media, 

 especially those prepared from natural products, are turbid or become 

 turbid upon heating and require filtration through paper or other agents 

 prior to sterilization. 



The reaction of most bacteriological media is usually adjusted to a 

 hydrogen-ion concentration near neutrality. Since sterilization by wet 

 heat usually causes a drop of pH of about 0.2-0.4 unit, it is necessary to 

 adjust the pH of the medium before sterilization to a value higher by the 

 amount indicated. Although solutions of natural extracts often do not 

 require pH adjustment, each batch of medium should be tested to deter- 

 mine if the desired pH is obtained. Detailed instructions for testing and 

 adjusting the reaction of media are presented in Chap. IV. For ordinary 

 purposes, satisfactory results will be obtained by adjusting the medium 

 to slightly above (pH 7.2-7.4) the neutral point with bromthymol blue, 

 the aqueous solution of the sodium salt or the alcoholic (95 per cent 

 ethanol) solution of the unneutralized indicator (Table 2) being employed. 

 A comparator block containing either standard solutions of known pH 

 values and colors or a comparator consisting of colored glass standards for 

 the corresponding pH and indicator color values should be used to deter- 

 mine pH. For deeply colored media, a glass electrode pH meter should 

 be used. 



