614 ORDER IV. EUBACTERIALES 



A. Protoplasm of young cells grown on glucose agar vacuolated if lightly stained. 

 Diameter of vegetative rods is 0.9 micron or more. 



1. Acid from mannitol with ammonium salts as source of nitrogen. Acetylmethyl- 

 carbinol not produced. 



1. Bacillus megaterhtm. 



2. No acid from mannitol with ammonium salts as source of nitrogen. Acetyl- 

 methylcarbinol produced. 



a. Saprophytic, certain strains weakly pathogenic, 

 b. Growth on agar not rhizoid. Usually motile. 



2. Bacillus cereus. 

 bb. Growth on agar rhizoid. Usually non-motile. 



2a. Bacillus cereus var. mycoides. 

 aa. Pathogenic* 



b. Causative agent of anthrax. Non-motile. 



3. Bacillus anthracis. 



bb. Cause of disease in certain insects. Usually motile. 



4. Bacillus thuringiensis. 



B. Protoplasm of young cells grown on glucose agar not vacuolated if lightly stained. 

 Diameter of vegetative rods is less than 0.9 micron. 



1. Growth on glucose agar as good as or better than on agar. Good growth on 

 soybean agar, 

 a. Growth in 7 per cent NaCl broth. t 



b. Starch hydrolyzed. Nitrites produced from nitrates. 



c. Good growth under anaerobic conditions in glucose broth; pH 

 of cultures is 5.2 or below. Gas produced from nitrates under alka- 

 line, anaerobic conditions. 



5. Bacillus licheniformis. 



cc. Scant if any growth in glucose broth under anaerobic conditions; 

 pH of cultures is higher than 5.2. No gas produced from nitrates 

 under alkaline, anaerobic conditions. 



6. Bacillus subti'lis. 



d. Black pigment on carbohydrate media only. 



6a. Bacilhis suhtilis var. aterrimus. 

 dd. Black pigment on tyrosine media only. 



6b. Bacillus subtilis var. niger. 

 bb. Starch not hydrolyzed. Nitrites not produced from nitrates. 



7. Bacillus pumilus. 

 _ aa. No growth in 7 per cent NaCl broth. 



'^ --- b. Glucose utilized. Weak, if any, hydrolysis of gelatin. 



8. Bacillus coagulans. 



* Smith et al. (loc. cit.) pointed out that pathogenicity is a variable character in these 

 two species and that academically they should be classified as variants of Bacillus cereus, 

 the stable parent form. From the practical standpoint and to avoid complications that 

 would arise as to priority, it has been thought best to retain these as separate species in 

 the Manual until more work has been done. 



t Gordon and Smith (Jour. Bact., 58, 1949, 327) recommended the use of 5 per cent NaCl. 

 Ford et al. (Bact. Proc, 1952, 18), however, found that certain strains of Bacillus coagulans 

 would grow in 5 per cent broth, thus rendering this character useless as a means of separa- 

 ting Bacillus coagulans from Bacillus subtilis. Since then the writers have obtained growth 

 in 7 per cent NaCl broth by all strains of Bacillus licheniformis. Bacillus subtilis (one excep- 

 tion) and its varieties and Bacillus pumilus. The use of the higher percentage of NaCl is, 

 therefore, recommended to overcome the objection of Ford and his co-workers. (Also see 

 Smith, Gordon and Clark, op. cit., 1952.) 



