MODULE 7
ISOLATION OF BACTERIA INTO PURE CULTURE
Introduction
Isolation of bacteria into pure form is the best way to study and
characterize bacterial cultures. This is usually accomplished by spreading a
sample containing microorganisms on a solid medium so that a single cell
occupies a well-isolated portion of the agar surface. Cultures consisting of a
single species are considered to be pure cultures. There are three common
techniques used in isolating bacteria into pure cultures. These are streak
plating, spread plating, and pour plating. In streak plating, an inoculum is
spread over the surface of the medium by the inoculating loop. It is used in
isolating and purifying bacteria. In spread plating, it uses an L- shape rod to
spread the inoculum over the entire plate. Both streak and spread plating
can isolate aerobic microorganisms. In pour plate method, a medium is
poured into the plate with an inoculum and allowed to solidify. In this
method, anaerobic microorganisms can be isolated.
Learning Objectives
At the end of the activity, the students are expected to:
TLO17. Performs streak, pour and spread plate techniques for the isolation
of microorganisms.
TLO18. Describe the distribution of colonies from each plating method.
TLO19. Characterize types of isolates that can be grown from each
technique.
Materials
1.0-ml pipettor Inoculating loop
Sterile Pipette tips (blue) Alcohol lamp
Sterile petri dish disinfectant
L- rod beaker
24-hour plate culture 24-hour broth culture
Culture Media
Sterile Nutrient Agar (NA) in plates
Sterile Nutrient Agar (NA) in tubes (at least 15 ml each)
Procedures
A. Methods of Isolation
1. Streak Plating (See Figure 7.1; Video A on streak plating)
a. Prepare sterile NA plates by checking for contamination caused by
wrong storage or damaged agar surface. Once done, draw
quadrants at the base to guide you in your streaking. For this
procedure, we need five quadrants.
b. Get the 24-hr plate culture and follow aseptic technique in its use.
c. Heat the inoculating loop until it glows red. Let it cool for a few
seconds for the next step.
d. Pick out the desired colony from the bacterial culture with the
sterile inoculating loop, and then close the plate. Set aside.
e. Slightly open your sterile plate, gently touch the surface of the
first quadrant and streak lightly left to right, closing the plate
when done. To check, your streaks should have left light skid
marks on the first quadrant.
f. Before continuing with the second quadrant, flame the inoculating
loop again and let it cool for a few seconds. This time, make
parallel streaks starting from the end of the first quadrant marks
towards the entire second quadrant.
g. Repeat “f” until the fifth quadrant has been streaked.
h. Incubate the plates in an inverted position at 37˚C for 48 hrs.
i. Examine the plates for the presence of well-isolated colonies.
j. Answer items 1 and 2.
2. Pour Plating (See Figure 7.2; Video B on pour plating)
a. Retrieve the NA tubes and liquefy by boiling in water. Maintain the
tubes at 50˚C to keep the agar melted until ready for pouring.
b. Transfer 1 ml of the 24-hour broth culture into a tube with molten
NA then roll gently between your palms to mix.
c. Pour one mixture into a sterile plate, making sure of even
distribution throughout. Do the same with the rest of the tubes
with their assigned plates.
Note: An alternative technique is to pipette the inoculum into a
sterile plate, followed by the molten NA and swirling gently to
mixed both liquids.
d. Allow the agar to solidify and incubate the plates in an inverted
position at 37˚C for 48 hrs. Answer items 3 and 4 in the
worksheet.
3. Spread Plate (See Figure 7.3; Video C on spread plating)
a. Retrieve sterile NA plates and label for this procedure.
b. Using a pipette and sterile blue tips, draw about 0.1 ml of the 24-
hr broth culture and deposit into the designated NA plate. It’s
important that aseptic techniques are followed in every step.
c. Get a glass L- rod spreader and sterilize by dipping in alcohol and
flaming it until the alcohol has burned off. Alternatively, a
disposable sterile plastic spreader may be used (as in the video)
d. Use the L-rod to spread the inoculum evenly over the surface of
the agar by rotating it. Sterile once more the L-rod after use.
e. Incubate the plates in an inverted position at 37˚C for 48 hrs.
f. Answer items 5-8 in the worksheet.
Figure 7.1. Streak plating method
Figure 7.2. Pour plating method
Figure 7.3. Spread plating method
References:
1. Tabo NA & Tabo HAL. 2020. Laboratory Manual in General
Microbiology. Philippines: Siam Rein Publishing House.
