DIFCOMANUAL 21 



THE PREPARATION 



of Media from Dehydrated Culture Media, Difco 



The advantages of dehydrated media and their efficiency for the cultivation of a 

 large variety of saprophytic and pathogenic microorganisms have been recognized 

 by the authors of many bacteriological texts, including "Standard Methods for 

 the Examination of Water and Sewage," "Standard Methods for the Examination 

 of Dairy Products" and "Diagnostic Procedures and Reagents" of the American 

 Public Health Association. 



The preparation of media from Bacto Dehydrated Culture Media is a time- 

 saving and economical process by which large or small quantities of standard and 

 uniform culture media are made available with the minimal expenditure of effort. 



The composition of each medium is stated on the label of the bottle with the 

 quantities of the ingredients present in one liter of the finished medium. All that 

 is necessary to rehydrate the medium is to weigh accurately the dehydrated 

 medium, dissolve the powder in freshly distilled water or boiled distilled water 

 and sterilize the solution. For ease of preparation and for best results with Bacto 

 Dehydrated Media, a discussion of these methods is given in detail. 



Distilled water should be used in the rehydration of dehydrated culture media. 

 Distilled water that has been stored at room temperature for any length of 

 time is apt to absorb sufficient gases to actually alter the final reaction and com- 

 position of the medium. It is recommended that freshly distilled water or boiled 

 distilled water at room temperature be used in the preparation of all media. 



The quantity of dehydrated culture medium to use per liter is specified on each 

 package. Amounts for small or large quantities may be easily calculated, and the 

 desired amount weighed. Best results will be obtained by adding the powder to 

 a dry flask, and then adding the freshly distilled water a little at a time with con- 

 stant agitation to prevent the formation of lumps. A stirring rod may be used to 

 secure an even mixture. The entire amount of distilled water is added when all 

 of the powder is thoroughly wetted. Broth or liquid media are readily soluble in 

 v/ater at room temperature. Agar media must be heated to the boiling point for 

 complete solution. This may be accomplished in several ways. For small quanti- 

 ties (up to 300 ml.) the best procedure is to heat the flask over a free flame, 

 keeping the contents of the flask well agitated to prevent burning; for larger 

 quantities, solution of the agar media may be more easily effected in flowing 

 steam, stirring from time to time, or in the autoclave. Another satisfactory 

 method, which reduces the heating period of the medium is to boil three-quarters 

 of the distilled water over a free flame and suspend the dehydrated medium in the 

 remaining cold distilled water, taking care that all of the medium is thoroughly 

 vv^etted and evenly suspended. The suspension is then finally added to the boiling 

 water and boiling continued until solution is complete. Gelatin media are best 

 dissolved by heating to 50 °G. in a water bath. Agar or gelatin media must be in 

 complete solution before being dispensed into the containers in which they are 

 to be sterilized. 



Care should be exercised to avoid contamination of media during the rehydra- 

 tion process. Only chemically clean glassware should be used in dissolving the 

 media, and for distribution in the final container. It has been shown that deter- 

 gents used in washing glassware, if not satisfactorily removed by repeated rinsing, 

 may be responsible for growth inhibition and changes in reaction. Three highly 



