U-^ 



DEHYDRATED CULTURE MEDIA 199 



Bacto-N.I.H. Agar Medium contains no thioglycoliate. If the medium is to be 

 used to test the sterility of a biological product containing a mercurial preserva- 

 tive, 0.05 per cent sodium thioglycoliate or 0.03 per cent thioglycoUic acid should 

 be added. 



To rehydrate the medium, suspend 38.5 grams of Bacto-N.I.H. Agar Medium 

 in 1000 ml. cold distilled water and heat to boiling to dissolve the medium com- 

 pletely. Distribute in tubes or flasks and sterilize in the autoclave for 18-20 

 minutes at 15-17 pounds pressure ( 121-123 °G.). The final reaction of the 

 medium will be pH 7.1. 



One pound of Bacto-N.I.H. Agar Medium will make 11.7 liters of medium. 



1 National Institute of Health Circular: Culture 

 Media for the Sterility Test, and Revision, 

 Feb. 5, 1946. 



BACTO 



THIOGLYGOLLATE MEDIUM (B363) 

 without Dextrose 



DEHYDRATED 



Bacto- Yeast Extract 5 g. 



Bacto-Gasitone 15 g. 



Sodium Chloride 2.5 g. 



/-Cystine, Difco 0.25 g. 



ThioglycoUic Acid 0.3 ml. 



Bacto-Agar 0.75 g. 



Bacto-Methylene Blue 0.002 g. ^- 



Bacto-Thioglycollate Medium without Dextrose is a sugar-free basal medium 

 containing thioglycoUic acid and 0.075 per cent Bacto-Agar and to which carbo- 

 hydrate can be added for studying the fermentation reactions of anaerobic micro- 

 organisms. The medium contains thioglycoUic acid and Bacto-Agar which gives 

 and maintains a low Eh, permitting the growth of the strictest anaerobes without 

 special seal, making the medium ideally suited for fermentation studies of anaer- 

 obes. Similar media prepared without Eh indicator and without Dextrose or Eh 

 Indicator are described below. 



The value of small amounts of agar in liquid media used for the cultivation 

 of anaerobes and microaerophiles as well as aerobes has been pointed out by 

 Kitchens^ and others. The use of alkaline sulfides and of sulfhydryl compounds 

 such as thioglycoliate, cysteine and glutathione for the reduction of the Eh poten- 

 tial of culture media for the propagation of anaerobes was first described by 

 Trenkmann^ and Quastel and Stephenson.^ Later Brewer"^ combined the use of 

 thioglycoliate with agar in liquid media for anaerobic culture. 



The presence of the thioglycoliate in the medium will also inactivate any 

 mercurial that might be carried over with the inoculum, as was demonstrated 

 by Marshall, Gunnison and Luxen.° Methylene blue serves as an indicator of Eh. 

 Most organisms, including anaerobes, will show earlier and more vigorous growth 

 in the presence of a carbohydrate. 



The sterile medium should not be stored in the refrigerator, especially if the 

 medium is to be used for the cultivation of anaerobes. The amount of oxidation 

 in the medium is shown by the depth of the color zone of the Eh indicator. Tubes 

 showing about one-third or more oxidized methylene blue are not satisfactory for 

 anaerobe cultivation and should be heated to the boiling point in steam or hot 

 water to drive off dissolved gases and rapidly cooled prior to inoculation. The 

 medium should be heated but once in this manner. 



To rehydrate the medium, suspend 24 grams Bacto-Thioglycollate Medium 



