AGf~ICULTURAL RESEARCH, PUSA.

METHODS OF ANAEROBIC CULTURE
Anaerobes are usually defmed as organisms that will
grow only in the absence of free oxygen (see p. 15).
It has been shown that it is not free oxygcn by itself
which is inimical to the growth of these organisms,
but that when molecular oxygen is present, a
peroxide is formed, probably hydrogen peroxide,
which prevents their multiplication. Anaerobes may
be cultivated, therefore, either by preventing the
admission of oxygen to cultures, or by destroying the
peroxide as fast as it is formed, by means of catalase
derived from fresh animal or vegetable tissue.
In the Smith - N ogueld method, for example, a
combination of these methods is used. Thc cultures
are sealed from the air by a vaseline plug; and any
peroxide that may be formed is at once destroyed by
the catalase present in a piece of fresh sterile rabbit
kidney. This method is described on p. lIS.
The method usually employed to establish anaerobic
.conditions is to remove the oxygen from the atmosphere
surrounding the culture, the oxygen being
sometimes replaced by an inert gas.
'fhe simplest method of securing anaerobiosis is by
growing the organisms in solid media. Deep agar tubes
are convenient and effwient for the pUl'pose. The
addition of 1 per cent. glucose to the medium is of
value, particularly when cultivating the saccharolytic
group of anaerobes. Glucose acts as a reducing
agent, thereby removing oxygen from the medium,
and fUl'ther serves as a suitable pabulum fOl' bacterial
growth. The agar may be inoculated when solid
by means of a long stmight wirc (vide p. 120). Thc
colonies develop best in the depth of the tube, becoming
fewer and smaller towards the surface. No
growth is usually noted in thc top half-inch of the
medium. An alternativc method is to melt the agar,
cool it to 50 0
C. and introduce nlC inOCllllll11 by means
of a capillary pipette. The contents of the tube are
mixed by rotation between thc palms of the hands.