196 DIFGO MANUAL 



thioglycollate. He noted the value of media containing thioglycollic acid and 

 agar particularly in the initiation of growth of anaerobes from catgut sutures. 

 Marshall, Gunnison and Luxen^^ reported that the Thioglycollate Medium of 

 Brewer was satisfactory for the cultivation of anaerobes, and permitted the 

 growth of organisms in the presence of mercurial preservative. Nungester, Hood 

 and Warren^2 ^nd Portwood^^ confirmed the neutralization of the bacteriostatic 

 effect of mercurial compounds by sodium thioglycollate. Malin and Finn^* re- 

 ported that it has been observed that the commonly used medium containing 

 thioglycollate is inhibitory to some organisms in the presence of carbohydrate. 



The National Institute of Health in 1941 ^^ specified the use of two Thiogly- 

 collate Media in sterility testing. The Brewer formula was prepared from infusion 

 of meat and 1 per cent peptone. The Linden formula contained 2 per cent 

 peptone and 0.2 per cent yeast extract. Each medium, in addition to 0.1 per 

 cent sodium thioglycollate, contained potassium phosphate, sodium chloride, 

 dextrose, agar and 1 : 500,000 methylene blue as an Eh indicator. In a study of 

 media used for sterility testing Christensen^^'^'^ reported that methylene blue as 

 used in the media was toxic for a number of organisms, and suggested the use 

 of resazurin as an Eh indicator. Sodium chloride and potassium phosphate were 

 toxic for some organisms encountered in sterility tests. Sodium thioglycollate, if 

 used in concentrations not greater than 0.05 per cent, was only slightly toxic 

 and in this concentration adequately neutralized the toxicit) of mercurial pre- 

 servatives used in biologicals. A reaction of pH 7.0 was suggested as being opti- 

 mum for the sterility test medium. Many suggestions on the media for sterility 

 testing leading to the present revised formula, as given by the National Institute 

 of Health,^ were carefully investigated and summarized by Pittman.^^ 



Bacto-Fluid Thioglycollate Medium is prepared according to the latest revision 

 as given by the "Compilation of Regulations for Tests and Methods of Assay and 

 Certification of Antibiotic Drugs," Federal Security Agency, Food and Drug 

 Administration, and conforms also to the specifications of the Federal Register 

 of the Food and Drug Administration,^ the U. S. Pharmacopeia^ and the Na- 

 tional Formulary.* This medium is carefully tested for its growth-promoting 

 abilities, oxidation-reduction qualities and its suitability to arrest mercurial stasis. 

 The medium supports the growth of aerobic as well as anaerobic organisms in a 

 cotton plugged tube without a special seal or other manipulation. Tubes 20 x 150 

 mm. containing 15 ml. of medium give the best ratio of surface exposed to depth 

 of medium for all types of organisms. Following autoclave sterilization the 

 medium should be cooled promptly to 25 °C. and kept at 20-30° C, preferably 

 in the dark, until ready for inoculation. If 30 per cent of the upper layer has a 

 pink color (oxidized resazurin) the medium should be heated in a boiling water 

 or steam bath to drive off absorbed oxygen. Do not reheat the medium more than 

 once, as continued reheating gives rise to toxicity. 



In testing the sterility of solutions containing one of the customary mercury 

 salts in 1:10,000 dilution as a preservative, 15 ml. of Bacto-Fluid Thioglycollate 

 Medium will inactivate the mercurial preservative contained in 3 ml. of the 

 solution. When testing preparations containing other preservatives, the amount 

 of Fluid Thioglycollate Medium used must be sufficient to dilute the inoculum 

 beyond the bacteriostatic limits of the preservative. Using 1 ml. of inoculum of 

 biologicals or other preparations containing various preservatives, the minimum 

 quantities of culture media to use are given in the accompanying table. 



Following inoculation the contents of the container must be thoroughly mixed 

 and mixed again when the first or 48 hour sterility test reading is made. 



To rehydrate the medium, suspend 29.5 grams of Bacto-Fluid Thioglycollate 

 Medium in 1000 ml. cold distilled water, and heat to boiling to dissolve the 

 medium completely. Distribute in tubes or flasks and sterilize in the autoclave 



