4o8 BIOLOGY OF THE LABORATORY MOUSE 



The carrier incidence in stock mice has not yet been adequately deter- 

 mined, but may be as high as 40 to 80 per cent. This state probably 

 develops after birth from contact with the mother and persists throughout 

 life. Organisms have been cultured from the conjunctiva, nasopharynx, 

 lungs, and brain, but not from the blood, liver, spleen, kidneys, or intestinal 

 contents (72, 230). Natural antibodies have not been demonstrated in 

 such animals. 



Etiology. — Pleuropneumonia-like organisms may be cultivated on special 

 agar media* directly from the lesions in mice, or from the conjunctiva and 

 nasopharynx, although they are more difficult to obtain from tissues of 

 normal animals. After 24 to 48 hours' incubation tiny colonies, 20 to 

 100 ju in diameter, may be seen under the microscope. Frequently they 

 have clear margins and dark centers and consist of granules, globules, and 

 fine filaments. Examination may most simply be made by staining the 

 colonies directly in the agar (48) with methylene blue or azure II, although 

 other special techniques have been employed (125, 126). 



Growth of the organisms ordinarily occurs in liquid media as a faint 

 opalescence appearing after 36 to 48 hours of incubation at 37°C. Meat 

 infusion or nutrient broth containing 30 per cent ascitic fluid or sterile 

 serum is satisfactory. Addition of 0.5 per cent glucose is apparently 

 advantageous with some strains. Dark field examination reveals tiny 

 granules and occasionally globules and small filaments. On subculture to 

 solid media, the characteristic microscopic colonies appear. The various 

 strains may be differentiated to some extent by culture, but more satisfac- 

 torily by immunological methods. Sabin (229) has stated that the members 

 of the pleuropneumonia group of organisms found in mice are immunologi- 

 cally and pathogenetically different from those found in the rat. 



The organisms are approximately 250 to 300 m/x in size, as determined 

 by filtration through gradocol membranes (225), and pass a Berkefeld V 

 filter. They are inactivated at 45°C. for 15 minutes but remain infective 

 for more than 30 days in 50 per cent buffered glycerin and for months if 

 frozen and dried by the Flosdorf-Mudd lyophile method. Toxin production 

 is thus far demonstrable with only one strain (Type A). The toxin appears 

 early during growth, lasts only about 2 days after its appearance, is inacti- 



* A satisfactory medium may be prepared as follows: 5 per cent defibrinated blood 

 is added to 2 per cent meat infusion agar (pH 7.6 to 8.0), the mixture brought to the 

 boiling point, immediately cooled to about 5o°C., and the clear supernatant removed 

 after the coagulated blood has settled out. To this is added about 30 to 40 per cent 

 ascitic fluid before pouring into Petri plates. 



