MICROBIOLOGY OF HIBERNATION IN GROUND SQUIRRELS 



days were termed psychrophiles. In addition, pour plates were made 

 in TGE agar as above, except that these cultures were incubated at 

 37° C for 48 hours. The results from these latter cultures were re- 

 ferred to as the total count at 37° C. The number of fecal strepto- 

 cocci was determined using (Difco) azide dextrose agar with an in- 

 oculum of 1 ml and 37° C incubation temperature. All plate counts 

 were made using a Quebec colony counter and only those plates 

 having between 30 and 300 colonies were included. 



Bacterial Virus Study 



In order to gain some information concerning the normal occur- 

 rence of bacterial viruses in the animals, 44 adult ground squirrels 

 were examined repeatedly (154 individual samples) for the presence 

 of bacteriophage in their intestinal contents. The bacterial host cells 

 used were strains of Escherichia coli previously isolated from the 

 animals in the group. 



The method used for the detection of phage was as follows. Fresh 

 fecal material was collected in a sterile tarred 50 ml centrifuge 

 tube and the weight of the sample determined, A one to 10 dilution 

 (weight in volume) was prepared in sterile nutrientbroth and a uni- 

 form suspension made with the aid of a sterile glass rod. Following 

 centrifugation at 17,000 X g for 30 minat 5° C the clear supernatant 

 fluid was withdrawn and tested for the presence of virus by two 

 methods. In the first, 1 ml of the fluid and 1 ml of a 3 hr broth cul- 

 ture of the strain of E, coli being tested was added to 5 ml of tryp- 

 ticase soy broth. This suspension was incubated overnight at 37 C 

 and the supernatant fluid therefrom was assayed for the presence of 

 virus according to the following procedure. Serial 10-fold dilutions 

 (10"^ to 10~ ) of the fluid were prepared in sterile nutrient broth and 

 0.1 ml added to a tube containing 2 ml of melted (43° C) trypticase 

 soy agar (0.7 per cent) and 0.1 ml of an 18 hour broth culture of the 

 test strain of E, coli. This was carefully mixed and poured over the 

 surface of a layer of nutrient agar in a petri plate. These cultures 

 were incubated at 37° C and examined for the presence of virus as 

 indicated by the formation of plaques. In the second method the pre- 

 liminary overnight incubation was eliminated, and the original super- 

 natant fluid was tested for bacterial virus by the plaque method. 



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