Microbiology and Parasitology

Laboratory

Lab Lesson 4 Plate Techniques

of Isolating Pure Culture
In the natural setting microbes exist as one part of a complex community of differing
population of organisms. Isolation techniques are applied to separate certain type of bacteria
that enable one to study it morphological, cultural and physiological characteristic. After
isolating a certain colony, this can be grown as pure culture. Pure cultures are comprised of
one species of bacteria. This culture possible be kept or maintained in a culture collection.
Several methods are employed in obtaining pure culture. Each has certain advantages
and limitations. Any method used in isolating microbes must ensure that a single cell is
introduced into a sterile medium using appropriate culture glasswares.
Plating is the most common technique used in isolating pure culture. On a sterile
medium a sample is placed at one site and then dispersed on the growth medium. As the
sample is scattered, cells become isolated. Each isolated cell will produce a colony on the
culture medium. Three common plating methods are used routinely in the laboratory including
pour plate, streak plate, and spread plate.

Activity
A) Streak Plate Technique
A small amount of sample is transferred using either a loop or needle on the surface of a
suitable, agar medium either by loop or transfer needle. Successive streaks are made to
provide dilutions that eventually produce isolated colonies.

Objective
At the end of the exercise the students must be able to isolating pure culture using quadrant
and radiant streak plate method.

Materials
 2 agar plates  2 alcohol lamps
 2 inoculating loops  Mixed bacterial culture in broth tube

Procedure
1. Prepare your table by disinfecting its surface with disinfectant available.
2. Label the bottom surface of your agar plate with your name, section and date.
3. With a sterilized loop obtain mixed culture of bacteria
by dipping the loop into the culture tube.
4. Streak each plate with one of the methods described
below.

Quadrant Streak Method

a. Spread the sample over a small area near the
edge of the plate. This is area 1 (figure 4-1).
b. Apply the loop lightly to the medium to avoid
digging into it.
c. Flame the loop and cool it for 5 seconds.
d. Make five or six streaks from area 1 to area 2.

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e. Flame the loop again and allow it to cool. Figure 4-1. Quadrant streak method
of obtaining pure
culture.
f. Make five or six streaks from area 2 to area 3
g. Flame the loop again and make five or six streaks from area 3 to area 4.
h. Flame the loop again before putting it down.

Radiant Streak Method

a. Spread the sample over a small area near the
edge of the plate. This is area 1(figure 4-2).
b. Flame the loop and allow it to cool for 5 seconds.
c. From the edge of area 1 make seven or eight
streaks to opposite side of the plate.
d. Flame the loop again and cross streak over the last
group of streaks.
e. Flame the loop again before putting it down.

Figure 4-2. Radiant streak method.

5. Incubate plates at 35OC for 48 hours. Inoculated plates are always incubated in an
inverted position.
6. Look for isolated colonies. Draw the colonies.

Serial dilutions of sample for spread plate and pour plate techniques

Often times the number of microbes in a certain samples is so numerous such that dilution
is imperative. Serial dilution allows quantification of microbes in a given amount sample.

 Dissolve 1g of soil in 9ml of sterile distilled water. This dilution is 1:10. Using sterile
pipet, transfer another 1ml from the 1:10 dilution to further dilute the mixture until you will
obtain a 1:1,000,000 dilutions (Figure 4.3).

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 Microbiology and Parasitology
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Figure 4.3. Serial dilution of sample for quantitative plating.

B) Spread Plate Method

A known diluted sample is placed in the agar surface and is scattered evenly using a
bent glass rod. In this exercise you will use the soil sample previously prepared.

Objective
At the end of the exercise the students must be able to isolating pure culture using spread
plate technique.

Materials
 5 agar plates  test tube rack
 5 sterile broth tubes  weighing scale
 1 alcohol lamps  2 bent glass rods
 5 sterile 1ml pipets

Procedure
1. Place a 0.1 ml each of serially-diluted soil sample in a sterile agar plate. Use separate
sterile pipet for each dilution. Mark the bottom of the plates with the respective sample
dilution.
2. Use a bent glass rod as spreader. Dip the spreader in a beaker of alcohol and pass it in
a flame to ignite the alcohol. Remove the spreader to burn-off the alcohol. Repeat the
procedure 3 times.
3. Spread the sample by moving the rod back and forth. Rotate the dish and move the rod
again back and forth (figure 4-4).
4. Incubate the plates for 48 hours. Examine bacterial growth.

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 Microbiology and Parasitology
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Figure 4-4. Spread-plate method in isolating pure culture.

C) Pour Plate Method

One ml each of successively diluted samples is mixed with 9 ml of sterile, molten agar
maintained at 450C in a petri dish. The contents are carefully mixed and cooled to solidify.
The dishes are incubated at 350C.

Objective
At the end of the exercise the students must be able to isolating pure culture using pour
plate technique and quantify microbial population in a soil sample.

Materials
 5 petri dishes
 5 20ml sterile nutrient agar in test tubes maintain at 45OC water bath
 5 sterile 1ml pipets
 test tube rack
Procedure
1. Pipette 0.1 ml of serially-diluted sample in a sterile petri dish (figure 4-5). Again, use
separate sterile pipet for each dilution. Mark the bottom of the plates with the respective
sample dilution.
2. Add 20 ml of molten nutrient agar maintained at 450C in a water bath.
3. Swirl gently the mixture to disperse the sample evenly. Avoid splattering the mixture in
the cover.
4. Incubate the plate for 48 hours. Check the growth.
5. Examine the plate and look for dilution containing colonies less than 300.
6. Calculate the number of bacteria/gram of soil as follows:

Bacteria/gram = number of colonies x reciprocal of dilution

For example:
Number of colonies on the plate = 32
Dilution = 1:10,000

Bacteria/gram = 32 X 10,000
= 320,000

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 Microbiology and Parasitology
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Figure 4-5. Pour-plate method in isolating pure culture.

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Name: _____________________________ Score: ____________________

Lab Schedule: _______________________ Term/Date: ___________________

Aseptic Transfers
and Inoculation Procedures Lab Report 4
Results and Observation Attendance
prelab: _______________
Examine each of the streak plates, and make a sketch postlab: ______________
indicating the distribution of growth.

A) Streak Plate Method

Compare the two patterns.

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Radiant streak Quadrant streak

Questions
1. Why the loop being sterilized before transferring from one area to another area?

2. Why is serial dilution of inoculum important in obtaining pure culture?

3. Which area in the radiant streak obtained isolated colonies?

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….. in quadrant streak?

B) Spread Plate Method

Questions
1. What is the consequence of not spreading the inoculum adequately on the agar culture?

2. Where do you observe the growth (surface, subsurface or bottom)?

Why is this so?

C) Pour Plate Method

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Calculation

Questions
1. Where is the colony growth located?

2. What would happen to the growth if the original sample contains too many cells?

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