478 BACTERIOLOGIC TECHNIC. 



a later test with the same culture and technic stained very well. In 

 every case whenever the test is to be made, a fresh anthrax preparation 

 should be simultaneously stained, and all preparations should be sub- 

 jected to the action of alcohol for the same length of time (one or two 

 minutes) . Then we can judge very well whether a variety of bacte- 

 rium retains or gives up the stain. A separation of the individual 

 genera and varieties by Gram's stain now seems scarcely at all possi- 

 ble, since there are found within a single genus all stages, from those 

 organisms which stain well to those that stain poorly. 



The following may usually be stained by Gram's method: 



Micrococci (not the Micr. gonorrhoeae). 



Sporulating bacilli (symptomatic anthrax and malignant edema 

 are uncertain). 



Of the non-sporulating bacilli: Proteus, mouse septicemia, 

 swine erysipelas, lactic acid bacterium. (Uncertain: fluorescens, 

 some bacteria not unlike the coli Bact. ) 



Genus Corynebacterium, Mycobacterium, and Actinomyces. 



The following usually do not stain: 



Micr. gonorrhoeas, Bact. influenzae, Bact. coli and typhi, Fried- 

 lander's Bacillus pneumoniae, bacteria of pest and glanders, the 

 vibrios and spirilla. 



3. Demonstration of Capsules. — The following is Johne's method: 

 (1) Cover the preparation with 2% solution of gentian-violet and 

 warm until it steams; (2) wash in water; (3) moisten with 2% acetic 

 acid for six to ten seconds; (4) wash with water. 



By this method also in varieties which are not regarded as "cap- 

 sule-bacteria," a distinct membrane may often be demonstrated about 

 the intensely stained bacterial cell. The capsule is seen most beauti- 

 fully when examined in water. 



4. Staining of Flagella. — The flagella, which are almost always 

 invisible when unstained, are usually demonstrated according to 

 Lbffler's method: 



1. Making the preparation (rubbing up a trace of young agar 

 streak culture — not bouillon culture — in a very small drop of water, 

 spreading well, and drying rapidly). 



2. Heating the preparation with the mordant (p. 476) until it 

 steams (not boils!) for one-half to one minute. 



3. Wash with a vigorous stream of water. 



4. Wash in alcohol to remove the remains of the mordant adhering 

 at the edge. 



5. Cover with staining solution (a few crystals are dissolved in 

 10 c.c. of anilin water, and to this is added, drop by drop, 0.1% 

 sodium hydroxid solution until the clear fluid just begins to become 

 opaque — precipitation) and warm until it steams for one minute. 



6. Wash with water, dry, and mount in Canada balsam. 

 Extreme cleanliness is essential in the manipulations, especially 



very thorough cleaning of the cover-glasses with acid and alcohol, or, 

 still better, with soap. Also the cultures must be young, although it 

 is not essential, as some authors claim, to employ cultures which are 

 only twenty-four hours old. We have often obtained very good prepa- 



