150 BIOLOGY AND TECHNIQUE 



The medium is then titrated accurately and brought to the desired reaction. 

 The medium usually gets slightly more acid on autoclaving, so that it is 

 better to adjust to about 2 points more alkaline on the hydrogen ion scale, 

 than the final reaction desired. It is important, in making hormone media, 

 never to filter in any way. Cotton, paper, and cheese cloth filters are equally 

 undesirable. If a firm clot is obtained after the first heating, there is usually 

 no difficulty in obtaining a clear medium. On the first autoclaving a second 

 precipitate usually forms, from which the clear medium can easily be decanted. 

 It is best to check up the reaction after the final sterilization. The Hormone 

 media can also be cleared with the Sharpless centrifuge. 



Sugar-free Broth. Make one liter of infusion broth 11 according to the 

 directions given. Inoculate with a young culture of B. coli comnmnis. 

 Incubate for twenty-four to forty-eight hours. The bacteria will ferment 

 and thus destroy any sugar (mono-saccharide) which may be present in the 

 broth, and thus render the medium sugar-free and acid. 



Arnold for one hour to kill the B. coli. 



Titrate and adjust. 



Arnold again for thirty minutes. 



Filter through paper until clear. 



This sugar-free medium is used as a basis for fermentation reactions. The 

 different sugars are added in 1 per cent concentrations and the medium is 

 then sterilized for three successive days in the Arnold, since the higher 

 temperatures of the autoclave tend to split the more complex sugars into 

 the simpler ones. 



Glycerin Broth. To ordinary, slightly acid or neutral meat infusion 

 broth, add six per cent of C. P. glycerin. Sterilize by fractional method. 



Calcium Carbonate Broth. This medium is designed for obtaining mass 

 cultures of pneumococcus or streptococcus for purposes of immunization or 

 agglutination. 



To 100 c.c. of meat infusion broth in small flasks, add one per cent 

 of powdered calcium carbonate, and one per cent of glucose. It is a wise 

 precaution to sterilize the dried calcium carbonate in the hot-air chamber 

 before using. Small pieces of marble may be used as suggested by Bolduan. 



METHOD FOR THE PREPARATION OF NUTRIENT BROTH 12 (Avery) (for 

 Streptococcus Agglutination). One pound of lean chopped beef allowed to 

 infuse in a liter of tap water over night in the ice-box. The unfiltered meat 

 infusion boiled for 30 minutes, filtered through paper, and the loss by evapora- 

 tion made up by the addition of water. One per 'cent peptone (Faircliild) 

 and 0.2 per cent sodium phosphate (Na.JIPOJ are now added. The mixture 



"It is not necessary to filter the infusion if it is to be used for making 

 sugar free broth. 



12 We may add that this method of growing streptococcus for agglutination 

 purposes does not always work out successfully but is the best we know of so far. 



