182 Medical Bacteriology. 
BACTERIUM TUBERCULOSIS. Place the sputum in a Petri dish over a black surface 
and select one of the little cheesy masses, if these are present, and smear it on a cover- 
glass. Where these particles are not present a loop or two of the thick portion is used. 
The cover-glass preparations are to be stained by one of the following methods: 
1) Gabbett, see Part 1, p. 38. 
2) Ziehl-Neelson: 
a. Carbol-fuchsin ten times through the flame. 
b. Nitric acid (30%) momentarily. 
c. Water. 
d. Aleohol (60%) until red color disappears. It may be necessary to immerse 
preparation in acid a second time, but the greatest care must be exercised to prevent 
extraction of dye from tubercle bacterium. 
é. Loeffler’s methylen blue, 1 minute. 
f. Mount and examine: 
‘While the tubercle bacteria may be detected when present in considerable numbers 
with a } in. objective when there are few present a 7s in. oil immersion will be neces- 
sary, and this ought to be used to search all slides where the tubercle germ has not been 
_ found with a lower power. A mechanical stage is a great convenience in a systematic 
search. 
At least two preparations should be stained and thoroughly examined before a meg 
ative result is pronounced. 
The viscosity of sputa may be overcome and the bacteria concentrated where the num- 
ber is very small by 1) Ribbert’s method which consists in the addition of a 2% solution 
of caustic potash and boiling. This dissolves the mucus and the bacteria are then depos- 
ited with the sediment. This sediment can be obtained by allowing the mixture to 
stand in a conical glass vessel or more quickly by the use of a centrifuge. 2) Ham- 
mond’s method: 
1. Add 5% of crystallized carbolie acid (in the case of sputum add 5 times its bulk 
of a 5% solution of carbolic acid). 
2. Place 15 ec. in the tubes of a centrifuge and whirl for 15 minutes. 
3. Pour off supernatant fluid and treat precipitate with 8 cc. of a 5% KOH solution. 
Mix thoroughly and allow to stand 2 minutes. 
4. Fill to 15 ce. mark with distilled water and whirl 20 minutes. 
5. Make cover-glass preparation of sediment (or purify same by repeated washings 
and centrifugalizations with distilled water). 
A centrifugal machine should be able to make at least 2,500 revolutions per minute. 
This speed ought to be maintained for 15 minutes. Sputum may be preserved by ad- 
dition of small quantity of carbolic acid (5%). 
Negative results are of positive diagnostic value only when repeated examinations 
are made of different samples taken at different times. 
BACTERIUM INFLUENZAE. This micro-organism is frequently present in enormous 
numbers (100 or more) and sometimes in almost pure cultures in the greenish purulent 
masses in the sputum. It stains readily with the ordinary dyes, and when lightly stained 
presents the bipolar stain. Carbol-fuchsin diluted 10 times is one of the best stains. 
Gram’s stain is negative. 
Sputum from suspected cases should be collected either by means of a probang or 
in a bottle and examined: 
