6 North Carolina Agricultural Experiment Station 
of nitrogen assimilating bacteria, will illustrate the possibilities which may 
come in similar studies from the use of very acid or very alkaline solid media. 
In routine work it will be found to be advantageous to flask and sterilize the 
media in 200 cc. quantities for the reason that the addition of 1 or 2 drops of 
strong acid or alkali to this quantity will bring about a change in concentration 
of about pH 0.2. When acid is added to agar in flasks at 50° to 60°C. it may 
be thoroughly agitated by whirling, 10 cc. portions may be removed for com¬ 
parison in reaction with the color standards of Clark and Lubs (1917) and 
when the usual precautions against contamination are observed the material 
in the flasks may be kept sterile, while the adjustment to the desired pH con¬ 
centration is being made. The agar may then, before it has had time to solidify, 
be poured into sterile test tubes or sterile Petri dishes, whereupon it is ready 
for use. The danger of contamination from this procedure, as judged by ex¬ 
perience in making several thousand tubes of media, is no greater than when 
the tubes are sterilized after the media have been placed in them, as is usually 
done. In summary, this procedure removes the necessity of sterilization after 
adjustment of reaction,* eliminates the chances of change of reaction or of 
other chemical changes which may be hastened by a rise in temperature and 
does not, within a wide range, destroy the jellifying powers of the agar or 
gelatin. 
*Experience gained in later studies has led to the adjustment of reaction of 
cooled sterile media and to the avoidance of subsequent heating as a routine 
practice. Either strong acid or strong alkali or sterile solutions of dilute acid 
or alkali have been employed. This procedure is by no means novel and has 
much to commend it. As long ago as 1897, Deeleman (1) corrected his media 
by adding sterile acid or sterile alkali after the media were sterilized because 
he noted that titrable acidity is increased by sterilization. Much of the data 
obtained in these early investigations before accurate methods were elabo¬ 
rated for determining true acidity rather than titrable acidity are now only of 
historic interest. The necessity of avoiding sterilization subsequent to adjust¬ 
ment of pH is emphasized, however, in a number ‘of recent studies as illus¬ 
trated by that of Wyeth in 1919 (2). 
The temperature of the medium at time of adjustment is another factor 
whose importance is not generally recognized although its significance, in a 
manner quite different from that shown in the present paper, was strikingly 
demonstrated by Clark in 1915(3). He employed a medium consisting of 10 per 
cent gelatin, 1 per cent peptone and 0.5 per cent meat extract which, when 
titrated hot before final sterilization was reported to have a titrable acidity of 
+ 10. After sterilization, the acidity should have been greater. Instead, it 
showed a titrable acidity of +5, with pH=8.5 as the neutral point. At room 
temperature the titre was +6, at the boiling point with undiluted medium 
+ 10, and with 5 cc. diluted to 50 cc. with conductivity water, +19. In con¬ 
clusion, Clark states that no procedure deserves more severe criticism than hot 
titrations and that they are irrational since titres thus obtained cannot be 
translated into hydrogen ion concentrations, in consequence of which a large 
body of available data is ruined. An increasing mass of data, based on the 
classic works of Sorensen and Michaelis, must be regarded as proving beyond 
question that the determining of titrable acidity of media, no matter what 
its temperature, is an illogical procedure which cannot yield values that meas¬ 
ure true acidity and should, therefore, be universally abandoned in bacterio- 
logic technic. 
References : 
(1) Deeleman, M. Der Einflusz der Reaktion des Nahrbodens auf das Bak- 
terienwachstum. Arb. a. d. k. Gsndhtsamte 13: 374, 1897. 
(2) Wyeth, F. J. S. The effects of acids, alkalis and sugars on the growth 
and indole formation by Bacillus coli. Biocliem, Jour. 13: No. 1, 19-24, 
1919. 
(3) Clark, W. M. The reaction of bacteriologic culture media. Jour. Infect, 
Dis. 17: No. 1, 109-136, 1915. F. A. W. 
