1899 ] PRODUCTION OF FLUORESCENT PIGMENT at 
The addition of an oxidizing agent, as dilute potassium perman- 
ganate, has a similar etiolating effect upon the pigment. 
INFLUENCE OF THE REACTION OF THE MEDIUM. 
The presence of acid checks the production of pigment 
Even in slightly acid solutions where considerable multiplication 
may occur, pigment is not formed. The interference of the 
acid seems to be rather with the metabolic activities of the 
bacteria than with the pigment, as the following facts indicate. 
The pigment when it is once formed is not destroyed by acid, 
but simply rendered invisible. If a few drops of acid be added 
to a finely pigmented culture of any of the species employed, the 
color is completely discharged, but the addition of alkali brings 
it back as vividly as ever. On adding acid again and then alkali 
the same change again occurs, and this can be repeated for at 
least six times upon the same culture without any perceptible 
weakening of the intensity of the pigment. When, furthermore, 
a well-developed culture is made quite strongly acid with HCl 
and allowed to stand for twenty-four hours, the color reappears 
when alkali is added with the same intensity as before. The 
existence of an aphanochromatic substance, at one time conspicu- 
ously visible in the form of the fluorescent pigment, at another 
colorless and not apparent, is clearly demonstrated by these 
experiments. This substance, however, is not formed in acid 
solutions. 
The influence of acid upon the pigment is beautifully shown 
in the fermentation tube where the pigment acts as an indicator. 
In 3 per cent. glucose-broth (prepared from broth freed of 
muscle-sugar by the method suggested by Theobald Smith”), 
&. viridans for the first four to five days after inoculation develops 
no pigment although the broth becomes very turbid. No color 
appears when alkali is added to the broth at this stage, conclu- 
sively showing that the presence of acid interferes with the pro- 
duction of the pigment or its aphanochromatic double and does 
not simply mask the presence of this substance. After eight or 
* Journ. Expt. Med. 2: 543. 1897. 
