METHODS FOR MICROSCOPIC STUDY OF BACTERIA 217 
through the clear water and turbid medium containing indicator to 
the eye. On each side, in the front row, are placed tubes containing 
known solutions and indicator whose color by preliminary examina- 
tion most closely approaches that of the unknown. Back of each 
standard tube is a control tube containing the unknown solution, 
but with no indicator. The control tube furnishes the same color and 
turbidity factor as the medium tube containing indicator. Thus, the 
two disturbing factors of turbidity and color are nearly compensated 
for. The comparison between unknowm and known standards through 
tubes of practical identity of color and turbidity gives the pH value 
of the unknown solution. 
The adjustment of reaction from the pH value observed id that 
desired is made by determining the amount of standard alkali (or 
acid) of -^ strength required to change the color of 5 cc. of medium 
from the pH value observed to that required. 
Thus: 5 cc. of broth of pH 7.8 required 0.35 cc. of ^r acid to reduce 
the alkalinity to pii 7.3. One liter of this broth would require 3.5 cc. 
of normal acid to effect the theoretical reduction in alkalinity. F^xper- 
ience has shown that sterilization tends to increase the hydrogen-ion 
concentration somewhat; therefore, it is difficult to prepare media 
of the exact pH value desired. If, however, the reaction after adjust- 
ment and sterilization lies between pH 7.2 and pH 7.6, it is suitable for 
ordinary bacterial growth. 
Alternate Frocediire.— The reaction of media may be determined by 
direct titration, using phenolphthalein, neutral red or other suitable 
indicator, whose color cfiange is known with reference to its hydrogen- 
ion concentration. The procedure is as follows: 
Five cubic centimeters of media are added to 45 cc.of distilled water in 
a white porcelain evaporating dish and 1 cc. of phenolphthalein.^ 
Heat to expel CO 2, cool and titrate to the first appearance of a pale 
pink color. Inasmuch as cultural media are usually acid to phenol- 
phthalein, this procedure is a very useful one in determining the reac- 
tion. ^ XaOH is used in the titration. 
From the amount of ^ alkali required to bring the 5 cc. of medium 
to the desired end-point, the amount of normal alkali required to 
neutralize 1 liter of the medium is readily calculated. Thus: 
5 cc. of media are neutralized to phenolphthalein by 3 cc. of 
2^ XaOH. 
100 cc. of media would be neutralized to phenolphthalein bv 3 cc. 
of ¥ NaOH. 
1000 cc. of media would be neutralized to phenolphthalein bv 30 cc. 
of ? NaOH. 
It must be remembered that the first faint pink color which is the 
end-point in this determination with phenolphthalein represents a pH 
value of 8.3. This is somewhat too alkaline for the best development 
' Dissolve 0.5 gm. phenolphthalein in 100 cc. of 50 per cent alcohol. 
