DEHYDRATED CULTURE MEDIA 113 



Another method for isolation of Brucella by blood culture is the double 

 medium method as described by Castaneda.^ This method may also be used in 

 culturing other organisms from the blood. His method consisted of preparing a 

 medium containing 2 per cent Bacto-Tryptose, 0.5 per cent sodium chloride, 

 0.5 per cent sodium citrate and 3.0 per cent agar. This medium is sterilized in 

 15 ml. amounts in 100 ml. flat-sided rectangular bottles. The bottles are placed 

 on their side so that the agar medium solidifies on one of the narrow side walls 

 forming an even, transparent layer. To each bottle are then added under aseptic 

 conditions 10 ml. of sterile broth containing 2 per cent Bacto-Tryptose and 2 

 per cent sodium citrate. The air in the bottles is then replaced by the desired 

 mixtures of carbon dioxide and air by a suitable mechanical device. The double 

 medium is incubated at 35-3 7 °G. for 3 or 4 days to test sterility, during which 

 time the surface of the agar is wetted with the broth by tilting the bottle at 24 

 hour intervals. The sterile double medium is then inoculated with 10 ml. of the 

 patient's blood and the mixture of broth and blood washed over the surface of 

 the agar. Incubation is at 36°G. with the bottle in an upright position. The 

 medium is examined at daily intervals, and every other day the blood-broth 

 mixture is allowed to flow over the agar layer. Castaneda reported that if col- 

 onies developed in the agar layer in 24 to 48 hours it was likely that the culture 

 had been contaminated. When colonies appear 24 to 48 hours after the second 

 inoculation the cultivated organism was usually found to be a Salmonella, less 

 frequently a staphylococcus or a streptococcus. It may be a Brucella; however, 

 colonies of these organisms are more generally encountered after the sixth day 

 of incubation, that is after the third inoculation of the Tryptose Agar with the 

 blood-broth mixture. The culture is discarded as negative after 20 days incuba- 

 tion. There are many modifications of this method in routine practical use. The 

 quantity of media, size of bottle, amount of inoculum varying in the various 

 laboratories. Marvin^^ also described a blood culture bottle utilizing a Tryptose 

 Agar and a Tryptose Phosphate Broth combination as being a practical method 

 for blood culture work. 



Huddleson^^'^2 h^g established the differentiation of Brucella types by their 

 behavior in the presence of certain bacteriostatic dyes. Bacto-Tryptose Agar is 

 employed effectively as a base for the thionin and basic fuchsin media used by 

 Huddleson, but the dye content of these media must be less than that employed 

 for Liver Infusion Agar. Bacto-Thionin is employed in 1:100,000 dilution (1 ml. 

 1 per cent solution of Bacto-Thionin per liter), and basic fuchsin in 1:100,000 

 dilution (1 ml. 1 per cent solution of Bacto-Basic Fuchsin). The plates should 

 be inoculated within 24 hours after pouring, as the dyes become reduced in the 

 medium on standing. The bacteriostatic action of the dyes in these concentra- 

 tions in Tryptose Agar is in every way comparable with that previously de- 

 scribed by Huddleson. B. melitensis and B. suis will grow on Tryptose Agar 

 containing thionin, while B. abortus is inhibited; B. melitensis and B. abortus 

 develop on Tryptose Agar containing basic fuchsin and B. suis is inhibited. 



For the differentiation of the Brucella types on the basis of hydrogen sulfide 

 production it is recommended that Bacto-Tryptose Agar be dissolved in an 

 infusion prepared from Bacto-Liver (page 289). Differentiation of the three 

 Brucella species by means of their hydrogen sulfide production is not clearly de- 

 fined when distilled water alone is used in preparing the medium. 



When voluminous growth of organisms is desired, as in the preparation of 

 Brucella antigens, it is recommended that a seed culture be prepared first by 

 propagating the organisms in Bacto-Tryptose Broth. An incubation period of 

 24 hours at 35-3 7 °G. is generally sufficient to produce a heavy growth. The 

 seed culture is then spread upon the surface of the medium. A medium pre- 

 pared by dissolving Bacto-Tryptose Agar in an infusion of Bacto-Liver (page 



