Lab.4
Isolation of bacteria in pure culture
To
identify a bacterial pathogens, it’s necessary to isolate the bacteria in pure
culture, many techniques can be used to isolate different microorganisms.
1. Streak plate technique:
Streak
method is routinely employed for isolation of bacteria in pure culture. This
technique involves the following steps:
Put
a sterile wire loop over the flame of the burner until be red-hot and allow to
cool.
Remove
very small amount of bacterial culture or clinical materials by sterile wire
loop.
Hold
the late of medium near to the burner by free hand and put the inoculums on
peripheral of the plate and spread over a small area.
Rotate
the plate to 90° and streak over agar surface by making a
parallel line vertical to the first one.
Sterile
the loop again and rotate the plate toward right angle and making another lines
of streak vertical toward quarter by touching the previous lines.
By
the same process, repeat the streaking for 3 to 4 times until the whole surface
of the agar plate is inoculated.
Streak
the center of plate in a zigzag motion.
Incubate
the plate in an inverted position at 37°C for
18-24hr.
2. Spread plate technique:
In
this method take 0.1ml of sample of bacterial culture by pipette place over the
surface of agar medium. Spread the bacteria culture over the surface by glass
spreader then incubate the plate at 37°C for
18-24hr.
This
technique is used for counting bacteria and also for swabbing from clinical
lesions such as throat swab, ear swab, vaginal swab and others.
3. Pouring plate technique:
Inoculate
a sterilized empty Petri dish with 0.1ml of bacterial culture or sample by
using sterile pipette.
Pour
the media into the Petri dish and allow to solidify.
Incubate
the plate at 37°C for 18-24hr.
To
differentiate between aerobic and anaerobic bacteria, aerobic bacteria grow and
reach to the surface of the agar medium.
Stabbing method:
This
technique is used to differentiate between motile and non-motile bacteria. The
procedure steps include:
Put
10 ml of semi-solid media in a test tube and leave to cool in vertical
position.
Inoculate
with a straight wire (needle) making a single stab down of center of the tube
to about half the depth of media.
Incubate
in 37°C for 1-2days.
Non
motile bacteria give growth that is restricted to the line of the stab, while
motile bacteria diffuse to media making a cloud of growth and the out-growth
may reach of the wall of the media.
Cultivation of anaerobic bacteria:
Obligate
anaerobic grow only in absent of O2, because the bacteria lack mechanism of
oxidation through respiratory enzymes (like cytochrome oxidase, catalase,
peroxidase) resulting in H2O2 accumulation. The H2O2 is toxic for Bacterial
growth.
The
methods described for achieving anaerobic conditions are:
Exclusion
of O2 by heating or vacuum.
Absorption
of O2 by chemical means.
Reduction
of O2 by candle or Gas Pak Jar.
Gas
Pak system is now the method of choice for isolation of anaerorbes. It generate
CO2 and H2 in moist condition. H2 combine with O2 to for H2O in present palladium
pellets, the pellets actas catalyst for combination.
,ethylene
blue strip act as indicator, is colorless in anaerobic conditions, and remain
blue in aerobically.
The procedure:
Place
inoculated agar plates media into the Jar in an inverted.
Open
the indicator strip and place it in the Jar.
Open
the Gas Pak envelop and introduce 10ml D.W. into the envelop and put it in the
Jar.
Place
the lid and tighten the screw clamp.
Incubate
the Jar at 37°C for 24-48hr.