520
Bacterial Cell Culture Methods
reaction produced by oxidative organ-
isms is apparent frst at the surFace and
extends gradually downward into the
medium. Organisms that oxidize glu-
cosebu
tdono
tFe
rmen
ti
thaveneve
r
been observed to Ferment any other
carbohydrates and so the sealed tube
can be omitted when testing the other
sugars.
Production of aesculetin from aesculin.
Hy-
drolysis
oF
aesculin
is
shown
by
the
production oF blackening in the Follow-
ing medium. Peptone, 10.0 g; aesculin,
1.0 g; Ferric citrate, 0.5 g; water, 1 L; agar,
1.2 g.
Levan production.
Many bacteria, For ex-
ample, plant pathogens, are characterized
by an ability to produce levan polysaccha-
rides From sucrose. Such compounds may
also be produced by soil bacteria and help
in maintaining soil crumb structure. This
may be detected on a plate oF nutrient
agar containing 5% sucrose, poured and
a channel cut in the agar. This channel is
flled with the same medium with 0.1%
aniline blue in it. Polysaccharide produc-
ing organisms attract the dye toward them
while others remain colorless. The inocu-
lum is usually added in two ways; as streaks
to the leFt oF the channel and as individ-
ual spaced colonies to the right oF the
channel.
5.1.3
Biochemical Examination of
Anaerobic Bacteria
±or tests requiring plate cultures, anaer-
obic
jars
should
be
used
For
incu-
bation,
but
many
oF
the
tests
can
be
carried
out
without
this
appara-
tus, merely by slightly
modiFying the
media.
1. The media may be rendered semisolid
by adding 0.5% agar.
2. ±ermentative metabolism can be de-
monstrated by sealing cultures with
Vaseline.
5.1.4
Rapid Testing Methods
Analytical ProFle Index (API) test strips
.
API test strips, produced by Bio M´erieux
SA, ±rance, may be used to obtain test
results quickly. These consist oF a series
oF miniature cupules on a molded plastic
strip, each oF which contains a sterile dehy-
drated medium in powder Form. Addition
oF water containing a bacterial suspension
simultaneously rehydrates and inoculates
the medium. A rapid reaction is obtained
because oF the small volume oF medium
and the large inoculum used. Reactions
are evident From color changes oF indicator
chemicals either with or without addition
oF Further reagents. These changes occur
aFter incubation oF the strip in a small,
humidifed, plastic chamber. A range oF
strips is available containing between 20
and 50 tubes with cupules. These can be
used to identiFy specifc groups oF or-
ganisms, For example, members oF the
Enterobacteriaceae, staphylococci, anaer-
obes, lactic acid bacteria, streptococci, and
clinical yeasts.
Enterotube II system
. The basic philos-
ophy oF the Enterotube II system is the
same as the API system. The system pro-
vides ease, speed, and low cost in the
identifcation oF gram-negative Enterobac-
teriaceae. The system consists oF a single
tube containing 12 compartments, each
containing a diFFerent agar-solidifed cul-
ture medium. Compartments that require
aerobic conditions have small openings
that allow in air. Those requiring anaer-
obic conditions have a layer oF paraFfn
wax. There is a selF-enclosed inoculating
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