40 



MANUAL OF MICROBIOLOGICAL METHODS 

 Table 2. Acid-Base Indicators 



Indicator 



Bromphenol blue 



Bromcresol green 



Methyl red 



Bromphenol red 



Bromcresol purple 



Bromthymol blue 



Phenol red 



Cresol red 



Thymol blue (alk. range) . 

 Phenolphthalein 



Concentration 

 recommended, 



%* 



0.04 

 0.04 

 0.02 

 0.04 

 0.04 

 0.04 

 0.02 

 0.02 

 0.04 

 0.10 



Sensitive 

 pH range 



3.1-4.7 

 3.8-5.4 

 4.2-6.3 

 5.2-6.8 

 5.4-7.0 

 6.1-7.7 

 6.9-8.5 

 7.4-9.0 

 8.0-9.6 

 8.3-10.0 



Full 

 acid 

 color 



Yellow 



Yellow 



Red 



Yellow 



Yellow 



Yellow 



Yellow 



Yellow 



Yellow 



Colorless 



Full 



alkaline 



color 



Blue 



Blue 



Yellow 



Red 



Purple 



Blue 



Red 



Red 



Blue 



Red 



* Stock solutions in 95 per cent ethanol for the indicator acids or in water for the 

 indicator salts. See Chap. IV, Table 6, for details of preparation. 



Buffers are often required in medium to facilitate growth. This is par- 

 ticularly true of media composed of simple compounds or in which acid- 

 producing bacteria are cultivated. Mixtures of sodium and potassium 

 phosphates are generally employed (see Chap. IV), although dibasic 

 phosphate is also used singly. Buffer requirements will be indicated in 

 the media described below. 



To determine pH changes during growth, indicators may be included 

 in the medium. For this purpose, an indicator covering the desired 

 range (Table 2) is selected and 1-2 ml of a 1-2 per cent alcoholic solution 

 is added to each liter of medium before sterilization. Although litmus 

 is an insensitive pH indicator, it has the advantage of also indicating 

 major changes in oxidation-reduction potential and is thus useful to show 

 these changes, particularly in milk media. 



General directions for media sterilization will not be presented here, 

 since these are available in many bacteriological laboratory guides. A 

 few critical points will, however, be emphasized. Most tubed media are 

 safely sterilized in an autoclave, using 121°C (250°F) for 15 min, care 

 being taken not to pack containers tightly; media in large flasks or bottles 

 require a longer sterilization time (15-30 min). The autoclave should 

 reach this temperature rapidly (within 10 min) and should cool down just 

 as rapidly after interrupting the steam flow. It is to be noted that 

 temperature is the important factor in heat sterilization, pressure being 

 merely the means of obtaining water at elevated temperatures. Thus, 

 during sterilization the temperature must be observed (a pressure gauge 

 is not reliable as an indicator of temperature) and used as the criterion 

 of an adequate level of heat. It should be realized that overheating a 

 medium may lead to modifications of its composition. In general, main- 



