Pure culture techniques
Aim
To learn the pure culture techniques and to isolate
pure culture of bacteria
Principle
The microbial population in our environment, air,
soil, water, etc is large and include many species of bacteria, fungi, algae,
etc. For studying these populations, it is necessary to isolate them as pure
cultures. Pure culture represents a population of organism of a single species
or more precisely growth derived from single cell or spore. There are various techniques for preparing
microbes as pure cultures from mixed population. Aseptic techniques or the sterile techniques
are the processes required for transferring a microbial culture from one
container to another without contaminating the culture or the environment. For successful aseptic technique, the work
area should be wiped with an antiseptic, the instruments, medium and containers
should be sterile and the procedure should be done quickly to minimize the time
of exposure.
The commonly used methods are streak plate, spread
plate and pour plate technique. These
techniques involve thinning of organisms and immobilization on or in a nutrient
medium.
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Isolation of pure culture by Streak
Plate technique
Streak plate technique is used for the isolation of
organisms, mostly bacteria, into pure culture from mixed population. The inoculum is streaked over the agar
surface in such a way that it “thins out” the bacteria and individual bacterial
cells are separated and well-spaced from each other. The sample is diluted by
streaking it through successive quadrants. While streaking in successive areas
of the plate, the inoculum is diluted to the point where there is only one
bacterial cell deposited every few millimeters on the surface of the agar
plate. When these bacterial cells divide
and give rise to thousands of new bacterial cells, an isolated colony is formed. Usually by the third or fourth quadrant
individual isolated colonies will be formed. Pure cultures can be obtained by
picking well isolated colonies and re-streaking these on fresh agar
plates.
Materials
required
Bacterial
culture, Inoculation loop, Bunsen burner, Nutrient Agar plates
Procedure
- The
inoculating loop was sterilized in the bunsen burner by putting the loop
into the flame until it is red hot and it was allowed to cool.
- Using
the loop, a loopful of culture was drawn from the culture and spreaded it
over the first quadrant (approximately 1/4 of the plate) and spread using
close parallel streaks.
- The
loop was flame sterilized, cooled down and streaked gently over a quarter
of the plate starting from the previous inoculated area using a back and
forth motion.
- The
loop was again flame sterilized, cooled down and the streaks from the step
3 are extended in to next quarter
- The
loop was again flamed and allowed to cool and the streaks from the step 4
are extended in to next quarter.
- The
loop was flamed again and allowed to cool. The streak area of step 5 are
extended into the center area of the plate. The loop was flamed loop once more.
- The
plates are incubated in an inverted position in the incubator at 37o
C for 24 hours.
Results
A
confluent growth was seen at the initial streak lines and the growth was less
dense at the later streak lines. Any
growth not present on the streak line is considered as a contamination. Well isolated colonies were observed towards
the last streak lines.
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Enumeration
of microbial cells by pour plate and spread plate technique
Aim
To
Enumerate of microbial cells by pour plate and spread plate technique
Principle
Pour-plate
method and Spread-plate method are pure culture techniques as well as are used
enumeration of bacterial sample. These techniques need the sample to be
serially diluted prior to plating. In serial dilution technique, the inoculum
from a mixed culture is subjected to serial transfer through a sterile liquid. The aim is to dilute the microbial suspension
so that the final tubes in the series contain lesser number and varieties of
microorganisms.
In
pour plate technique, diluted samples are mixed with melted agar medium and
then poured into sterile petri dishes. Here, 1 ml dilutions of bacteria is
mixed with melted agar medium maintained in the liquid state at a temperature
of 42-45°C (agar solidifies below 42°C). The bacteria and the melted medium are
mixed well in separate Petri plates, allowed to solidify, and then incubated.
The bacterial colonies develop, both within the agar medium (subsurface
colonies) and on the medium (surface colonies). Pour
plate method has certain disadvantages such as, the subsurface colonies needs
digging them out of the agar medium and the microbes must be able to withstand
temporary exposure to the 42-45° temperature of the liquid agar medium.
In spread
plate technique 0.1 ml of the serially diluted mixed culture is
placed on the center of an agar plate and spread evenly over the surface by means
of a sterilized bent-glass-rod and the medium is then incubated. The colonies
develop on the surface of the agar plates and the microorganisms are not
exposed to temperature of the melted agar medium.
Materials
required
Bacterial
culture, Inoculation loop, Bunsen burner, Nutrient Agar, sterile petri dishes,
9 ml sterile water blanks, sterile pipettes
Procedure
– Serial dilution
1 1 ml of
sample was aseptically added to first test tube containing sterile 9 ml water
and the tube was marked as 10-1.
2 1 ml
sample from the 10-1 tube was added aseptically to another 9 ml
sterile water blank marked as 10-2 and repeated until 10-9.
Procedure
–pour plate technique
1 sterile
petri dishes were inoculated with 1.0 mL of diluted sample from the tubes
marked 10-5, 106, 10-7, 10-8 and
10-9.
2 15-20 ml
sterile molten agar kept at water bath
at 45°C was poured into each Petri dish and the dish was rotated gently to mix
the culture and the medium thoroughly and to ensure that the medium covers the
plate evenly.
3 The agar
was allowed to solidify, and the plate was incubated in an inverted position at
37°C for 24-48 hours.
Procedure
– Spread plate technique
- sterile
petri dishes were poured with sterile nutrient agar and allowed to
solidify.
- The plates were inoculated with 0.1 mL of
diluted sample from the tubes marked 10-5, 106,
10-7, 10-8 and 10-9. The sample was pipetted onto the center
of the surface of an agar plate.
- The L-shaped glass spreader was dipped in
alcohol, and flamed over a bunsen burner.
- The sample was spread evenly over the surface
of agar using the sterile glass spreader.
- The plates were incubated in an inverted
position at 37°C for 24 -48 hours.
Observation
The
number of colonies and colony morphology was observed in the plates after
incubation. The colony forming unit
(CFU) was calculated.
CFU/ml = (no. of colonies x dilution factor) / volume plated.
Result
CFU from
pour plate was ……………
CFU from
spread plate was ……………
The
predominant colonies were found to be ……………………
……………………………………………………………………………………………….……………………………………………………………………………………………………………….………………….
in plates after pour plate technique and
……………………………………………………………………………………………………………………………………………………………………………………………………………………………………….
in plates after spread plate technique.
Serial dilution technique
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