Bacterial Cell Culture Methods
507
2
Methods of Bacterial Isolation
2.1
Sterilization
Media are usually sterilized after distribu-
tion into bottles, tubes, or flasks. Sterile
media may be transferred aseptically into
previously sterilized containers. Table 1
lists the methods commonly used for the
sterilization of media.
2.2
PuriFcation of Bacteria from Contaminated
Cultures
Progressive dilution is the basis of isolation
techniques. This can be applied as follows:
Poured plate method
Melt and cool to 45
Cth
reetube
so
f
an agar medium. Inoculate one tube
with one loopful of the suspension and
mix well by rotating it between the
hands.
Flame the loop thoroughly and transfer
one loopful of the mixture from the ±rst
tube to the second. Mix again.
Repeat for the third tube. Pour the
contents of all the tubes into the sterile
petri dishes and allow the contents
to set.
The
second
or
third
plate
should
show well-separated colonies after in-
cubation. Distinct colonies can then
be picked off and examined. If the
suspension is very heavy, initial dilu-
tion in sterile saline may have to be
made.
The streak plate method.
This is a quicker,
though less reliable method.
Prepare a plate of solid medium. Dry the
plates for 30 min before use by placing
them open with the open sides facing
downwards in a 45
Cincubator
.
Mark the plate on the underside into
four sectors. With a sterile loop, place
a small drop of suspension on the
agar at one side of a marked sector
n
e
a
rt
h
ee
d
g
eo
ft
h
ed
i
s
h
.D
r
a
w
this out in a single broad tangential
streak.
Flame the loop, cool it by touching
the agar at the end of the dish away
from the inoculum and draw a set of
Tab. 1
Common methods of sterilization.
Treatment
Comments
Autoclave
Most media can be sterilized by treatment in an autoclave, usually a 15–20 min
treatment at a pressure of 1 kg cm
2
.Thisra
isesthesteamtemperatureto
121
C
Steaming
Media that cannot be autoclaved (e.g. sugar media) may be sterilized by
intermittent steaming. Samples are heated over boiling water in a steamer
(85–95
C) for 15 min on each of 3 successive days. Between treatments the
material must be left at a suitable temperature for the growth of endospores
Filter
sterilization
It is necessary to sterilize some ingredients separately before use. Heat-labile
ingredients (some antibiotics, sugars, and vitamins) are sterilized by ±ltration
through a membrane ±lter (pore size 0.2–0.45
µ
m), which excludes bacteria
Dry heat
Dry glassware such as empty flasks, e.g. pipettes cannot be satisfactorily
autoclaved and must be sterilized in a hot air oven for a minimum of 2 h at a
temperature of 160
C. All material should be allowed to cool to room
temperature before removal from the oven
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