6 General Bacteriology. 
A. 
1.) Titrate as follows: Pipette off 5 cc. of the fluid 
into a4-inch evaporating dish, add 45 ce. of distilled 
water, boil for three minutes, add 1 cc. of phenol- 
phthalein (0.5% substance in 50% alcohol), and then 
carefully run in, drop by drop, from a burette a twen- 
tieth normal *solution of sodium hydroxide (yNa 
OH) until the solution turns a faint pink color. Treat 
two other samples in the same way. If the amount 
of Na OH required is approximately the same in each 
case the average can be taken as the amount necessary 
to neutralize 5 cc. Calculate the amount necessary 
to neutralize the whole (1000-15 ec.). Since this 
amount would dilute the medium too much, a stronger 
solution (normal) is used, hence, 
2.) Neutralize by adding #yth of the volume eal- 
culated above of a normal solution of sodium hydrox- 
ide. Test the accuracy of the work at this point by 
the addition of a few drops of phenolphthalein to a 
ec. or so of the medium. If a faint pinkish tintis not 
obtained, titration and neutralization must be re- 
peated. 
B. 
Use a normal solution of so- 
dium hydroxide ({NaOH). Add 
to the hot solution a few ec. at a 
time, at first, later afew drops, 
stirring thoroughly with a glass 
rod. After each addition, test 
by placing a drop of the solu- 
tion by means of the glass rod 
on a strip of red litmus paper, 
and then moisten the paper with 
distilled water. The addition 
should continue until the red 
litmus paper is turned blue, but 
no change occurs on blue litmus 
paper.. 
If by mistake more alkali is added than is required, the reaction ean be corrected by 
the use of a normal solution of hydrochloric acid. 
h. Boil for 5 minutes and restore weight. 
1. Test reaction and adjust if necessary. 
j. Add 0.5 to 1.5% of a normal hydrochloric ania if neutralized by method A, oth- 
erwise omit. 
The amount of acid to be added varies with the purpose for which the 
medium is to be used, e. g., in water analyses +1.5 (acid) is preferable, with the path- 
ogenic bacteria a smaller amount of acid (+ 0.5) more nearly meets requirements. 
k. Filter through moistened filter paper (Abbott p. 96), or absorbent cotton, 
(VII. m). 
If the filtrate is not perfectly clear, cool to 60° C., 
add the white of an 
egg, thoroughly mix and boil for 5 minutes without stirring. 
The filtrate (bouillon) should be of a light straw color, perfectly clear, and should 
not give a precipitate on boiling. 
REFERENCES. 
SPECIAL DIRECTIONS. 
Secure and put to soak meat for VII. 
A. 90; M. & R. 48; McF. 124; N. 234; P. 212; 
P. B.C. 18-24. 
Prepare 1 liter of bouillon according to method C. 
EXERCISE V. FILLING TEST-TUBES AND FLASKS WITH CULTURE MEDIA. 
GENERAL DIRECTIONS. 
In filling tubes be careful not to allow the media to touch 
the neck of the vessels as this will cause the cotton to stick to the glass when the plugs 
are removed. Place the culture fluid to be tubed in a funnel arranged with a delivery 
* Normal solutions are prepared so that one liter at 16° C. shall contain the hydrogen equivalent 
of the active reagent weighed in grams (Sutton). 
hydrate is sufficiently accurate. 
For present purposes a 4 % solution of sodium 
