240 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
3. The Barber Method for the Isolation of a Single TV//.— It is occa- 
sionally necessary, in very refined bacteriological studies, to be abso- 
lutely certain that the starting-point of a pure culture is a single 
organism. Theoretically, single cells are the progenitors of the 
colonies observed in media inoculated by the plate or the streak 
method, and such is usually the case. Undoubtedly it may happen 
that a chain of streptococci may remain adherent and their descendants 
appear as a single colony, and it is equally certain that two alien 
bacteria may occasionally become adherent by intertwining of flagella 
or adhesion of viscid capsular substance and develop into a mixed 
colony. The apparatus of Barber,^ which consists essentially of a 
delicate capillary pipette mounted in the substage of the microscope, 
and capable of upward and downward motion in the optical axis of 
the instrument, is designed to circumvent this possibility. In prac- 
tice a very thin emulsion of bacteria in a fluid medium is placed on 
the surface of a sterile thin plate of glass in such a manner that a 
drop of the contaminated fluid hangs in the opening in the stage ordi- 
narily occupied by the condenser. The drop is manipulated by a 
mechanical stage, guided by direct observation with a \ inch lens until 
a single organism appears in the field of vision. The sterile capillary 
pipette is carefully brought upward until the tip engages the dependent 
drop; the organism will be seen to enter the pipette, which is then 
lowered and removed from its attachments to the microscope. The 
single cell is transferred to a suitable medium and incubated in the 
usual manner. 
For anaerobic bacteria, spores are preferably selected inasmuch as 
they appear to be less influenced by exposure to the air. The spore 
is "fished" in the usual manner, and the capillary tube containing 
the spore is filled with broth to displace air before incubation is 
practised. 
B. Anaerobic Bacteria. — 1. Plating Methods.— The cultivation of 
anaerobic bacteria which do not grow in the presence of atmospheric 
(free) oxygen requires special apparatus and technique. The simplest 
method, and one which is successful if gas-forming bacteria are absent, 
is to make dilutions in glucose agar precisely as described under 
Plating in the preceding paragraphs. The tubes should be filled to 
a depth of 10 cm. with the medium, and tubes of relatively large 
diameter— 2 to 3 cm.— are preferable. The tubes, previously heated 
to the boiling-point, and rapidly cooled to 43 to 45° C. to prevent 
reabsorption of oxygen, are inoculated by the dilution method, rotated 
between the hands to distribute the organisms uniformly, and cooled 
rapidly in an upright position. 
Colonies aj^pear within the depths of the media after incubation; 
in the thinly seeded tubes these colonies are discrete, and they may 
be removed without contamination, either in sterile capillary pipettes 
' Univ. Kansas Science Bull., 1907, vol. 4, No. 1; Jour. Kansas Med. Soc, November, 
1904. 
