CULTIVATION OF BACTERIA 49 



the varieties of dysentery bacilli, some fermenting mannite and others 

 not. In careful work the bouillon must be rendered sugar free. 



PEPTONE SOLUTION (Dunham's). This is a simple 1 to 2 per cent, 

 solution of peptone in tap or distilled water to which 5 per cent, of 

 sodium chloride is added. The peptone and sodium chloride are dis- 

 solved by heating. The fluid is filtered, placed in tubes, and sterilized. 

 The reaction is slightly alkaline to litmus and suitable for most pur- 

 poses. It can be altered or standardized if desired. 



(a) Sugar-peptone Solution, etc. The various sugars, mannite, inulin, 

 and glycerin are added to the peptone solution just as previously 

 described for bouillon. 



(b) Sugar-free Bouillon. A quantity of a culture of bacillus coli or 

 of bacillus lactis aerogenes is added to the meat extract and incubated 

 at 37 for twenty-four hours. The acidity is neutralized, peptone and 

 salt added, and treated as described under bouillon. 



GELATIN MEDIA. These are simply the foregoing bouillon and 

 peptone media to which gelatin is added as follows: To the bouillon 

 already prepared as described add 10 per cent, of sheet gelatin and 

 neutralize. Add the whites of two eggs for each litre, and boil for a few 

 minutes. Filter, place in tubes or flasks, and sterilize. Instead of add- 

 ing gelatin to bouillon already prepared, it may be added to the meat 

 infusion at the same time the peptone and salt were added in preparing 

 nutrient bouillon as just described. Different preparations of gelatin 

 differ greatly as to their melting point. Boiling lowers the melting 

 point, so that heat should not be applied any longer than necessary. 

 The melting point of different samples of nutrient gelatin varies between 

 20 to 27 C. The "gold-label" gelatin is employed. 



AGAR MEDIA. These are the various bouillon and peptone media 

 to which 1 to 2 per cent, of agar are added. In order to lessen the effect 

 of heat on sugars simple nutrient agar is first prepared and then the 

 sugar added. They are prepared by adding to stock bouillon 1 or 2 

 per cent., as desired, of thread agar, melting it by placing over a free 

 flame or in the autoclave or steam sterilizer. When the agar is brought 

 into solution over a free flame there may be considerable loss of fluid by 

 evaporation. This should be compensated for by adding additional 

 water before boiling. Agar may be added directly to the meat infusion 

 along with the peptone and salt. Indeed, this is an advantage, as agar- 

 agar is very difficult to bring into solution, and is not injured in the least 

 by prolonged boiling. The agar may be added to water alone in double 

 the amount finally desired. To this is added an equal quantity of nu- 

 trient broth, which is also double its usual strength. Glycerin agar is 

 simply nutrient agar plus 3 to 6 per cent, of glycerin. It is added to the 

 hot nutrient agar just previous to putting it in the flasks. Nutrient agar 

 begins to thicken at a fairly high temperature, and should be filtered as 

 hot as possible. When small amounts are made it is well to place the 

 filter and receiving flask in the sterilizer while filtering. 



MILK. This fluid is a good culture medium for most pathogenic 

 bacteria. It should be obtained as fresh as possible, so that but little 



