EXERCISE

9
MICROBIOL
OGY
Pure Culture Techniques

Learning Outcomes
After completing this exercise, you should be able to
Obtain isolated colonies of a mixed culture using the
streak-plate method.
Obtain isolated colonies from a bacterial culture using the
loop dilution and pour-plate method.
Evaluate the purity of your isolated colonies
by transferring a single colony to an agar slant and
obtaining the growth of a single type of organism.

When we try to study the bacterial flora of the body,

soil, water, or just about any environment, we realize
quickly that bacteria exist in natural environments as
mixed populations. It is only in very rare instances
that they occur as a single species. Robert Koch, the
father of medical microbiology, was one of the first
to recognize that if he was going to prove that a
particu- lar bacterium causes a specific disease, it
would be necessary to isolate the agent from all
other bacteria and characterize the pathogen. From
his studies on pathogenic bacteria, his laboratory
contributed many techniques to the science of
microbiology, including the method for obtaining
pure cultures of bacteria. A pure culture contains
only a single kind of an organ- ism, whereas a mixed
culture contains more than one kind of organism. A
contaminated culture contains a desired organism
but also unwanted organisms. With a pure culture,
we can study the cultural, morphologi- cal, and
physiological characteristics of an individual
organism.
Several methods for obtaining pure cultures are
available to the microbiologist. Two commonly used
procedures are the streak plate and the pour plate.
Both procedures involve diluting the bacterial cells
in a sample to an end point where a single cell
divides, giving rise to a single pure colony. The
colony is therefore assumed to be the identical
progeny of the original cell and can be picked and
used for further study of the bacterium.
In this exercise, you will use both the streak-plate
and pour-plate methods to separate a mixed culture
of bacteria. The bacteria may be differentiated by the
characteristics of the colony, such as color, shape, and
Figure 9.1 A streak plate demonstrating well-isolated
colonies of three different bacteria.
other colony characteristics. Isolated colonies can then
be subcultured and stains prepared to check for purity.
Streak-Plate Method
The streak-plate method is the procedure most often
used by microbiologists to obtain pure cultures. It is
simple and allows for economy of materials. How-
ever, it requires a certain level of skill which is only
obtained through practice. Your instructor may want
you to try more than one method or only concen-
trate on one of the quadrant streak-plate procedures.
Figure 9.1 illustrates how colonies of a mixed bac-
terial culture should be spread out and separated
on a properly made quadrant streak plate shown
in method B of figure 9.2. Good spacing between
colonies on the plate is critical so that a single pure
colony can be aseptically isolated from quadrant
4 and used for further testing and study. This will
insure that you are not working with a mixed or con-
taminated culture.
Figure 9.2 shows three procedures for producing
a streak plate that will yield isolated colonies. By far
the most popular and most utilized procedure is the

1
EXERCISE 9 Pure Culture Techniques

Quadrant Streak
Quadrant Streak
(Method A)
(Method B)
1 s
3

2 s
1
4 2

4 s

s
3

(1) Streak one loopful of organisms over Area 1 near edge of

the plate. Apply the loop lightly. Don’t gouge into the
medium.
(2) Flame the loop, cool 5 seconds, and make 5 or 6 streaks from
Area 1 through Area 2. Momentarily touching the loop to a
sterile area of the medium before streaking insures a cool loop.
(3) Flame the loop again, cool it, and make 6 or 7 streaks from
Area 2 through Area 3.
(4) Flame the loop again, and make as many streaks as
possible from Area 3 through Area 4, using up the
remainder of the plate surface.
(5) Flame the loop before putting it aside.
(1) Streak one loopful of organisms back and forth over Area
1, starting at point designated by “s”. Apply loop lightly.
Don’t gouge into the medium.
(2) Flame the loop, cool 5 seconds, and touch the medium in a
sterile area momentarily to insure coolness.
(3) Rotate dish 90 degrees while keeping the dish closed.
Streak Area 2 with several back and forth strokes, hitting
the original streak a few times.
(4) Flame the loop again. Rotate the dish and streak Area
3 several times, hitting the last area several times.
(5) Flame the loop, cool it, and rotate the dish 90 degrees again.
Streak Area 4, contacting Area 3 several times and drag out
the culture as illustrated.
(6) Flame the loop before putting it aside.

Radiant Streak

(1) Spread a loopful of organisms in a small area near the

edge of the plate in Area 1. Apply the loop lightly. Don’t
gouge into the medium.
(2) Flame the loop and allow it to cool for 5 seconds. Touching
a sterile area will insure coolness.
(3) From the edge of Area 1 make 7 or 8 straight streaks to
the opposite side of the plate.
(4) Flame the loop again, cool it sufficiently, and cross streak
over the last streaks, starting near Area 1.
(5) Flame the loop before putting it in its receptacle.

Figure 9.2 Three different streak techniques.

quadrant streak plate, shown in methods A and B.

to master the streak plate as success in future
All of the methods depend upon the physical exercises will depend on using this technique to
dilution of cells over the plate surface until a single
obtain isolated cultures. This is especially true for
cell is deposited in an area and grows to produce an the exercises in Part 8 involving the identification of
isolated bacterial colony. It is important for
an unknown bacterium.
beginning students
2
 Pure Culture Techniques EXERCISE 9

Materials Agar-agar, the solidifying agent in this

• electric hot plate and beaker of water
• Bunsen burner or incinerator
• inoculating loop, thermometer, and marking pen
• 20 ml nutrient agar pour and 1 sterile petri plate
• mixed culture of Serratia marcescens or
Escherichia coli, and Micrococcus luteus or
Chromobacterium violaceum
1. Prepare your tabletop by disinfecting its surface
with the disinfectant that is available in the lab-
oratory (Roccal, Zephiran, Betadine, etc.). Use
paper towels to scrub it clean.
2. Label the bottom surface of a sterile petri plate
with your name and date. Use a marking pen
such as a Sharpie.
3. Liquefy a tube of nutrient agar, cool to
50°C,
and pour the medium into the bottom of the
plate,
following the procedure illustrated in figure 9.3.
Be sure to flame the neck of the tube prior to
pouring to destroy any bacteria around the end
of the tube.
After pouring the medium into the plate,
gen- tly rotate the plate so that it becomes evenly
dis- tributed, but do not splash any medium up
over the sides.
medium, becomes liquid when boiled and reso-
lidifies at around 42°C. Failure to cool it prior
to pouring into the plate will result in condensa-
tion of moisture on the cover. Any moisture on
the cover is undesirable because it can become
deposited on the agar surface, causing the
organ- isms to spread over the surface and
thereby defeating the entire isolation procedure.
4. Streak your plate using one of the methods
shown in figure 9.2. Method B is the most
commonly used procedure.
Caution
Be sure to follow the routine in figure 9.4 for obtaining
the organism from culture.
5. Incubate the plate in an inverted position for 24
to 48 hours. If the plate is not incubated in an
inverted position, condensate from the dish lid
will be deposited on the agar surface, allowing
organisms to swim and preventing the formation
of individual colonies.

(1) Liquefy a nutrient agar pour by (2) Cool down the nutrient agar pour to 50°C. Hold at 50°C for 5 minutes.
boiling for 5 minutes.

(3) Remove the cap from the tube (4) Pour the contents of the tube into the bottom of
and flame the open end of the tube. the petri plate and allow it to solidify.

Figure 9.3 Procedure for pouring an agar plate for streaking.

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EXERCISE 9 Pure Culture Techniques

(1) Shake the culture tube from side to

(2) Heat loop and wire to red-hot. Flame (3) Remove the cap and flame the neck
side to suspend organisms. Do not
the handle slightly also. of the tube. Do not place the cap
moisten cap on tube.
down on the table.

(4) After allowing the loop to cool for (5) Flame the mouth of the culture (6) Return the cap to the tube and place
at least 5 seconds, remove a loopful tube again. the tube in a test-tube rack.
of organisms. Avoid touching the
side of the tube.
continued
Figure 9.4 Routine for inoculating a petri plate.

76
 Pure Culture Techniques EXERCISE 9

(7) Streak the plate, holding it as shown. Do not gouge into the (8) Flame the loop before placing it
medium with the loop. down.

Figure 9.4 (continued)

Results for isolating colonies using the four pro- method is the most commonly used and gives consis-
cedures are shown in figure 9.5. The quadrant streak tent results.

(a) (b)

(c
)
Figure 9.5 Spread plates for the three procedures in figure 9.4 for producing isolated colonies: (a) quadrant streak
method A; (b) quadrant streak method B; (c) radiant streak.
©McGraw-Hill Education. Lisa Burgess, photographer.
77
EXERCISE 9 Pure Culture Techniques
Pour-Plate Method
(Loop Dilution)
This method of separating one species of bacteria
from another consists of diluting out one loopful
of organisms with three tubes of liquefied nutrient
agar in such a manner that one of the plates poured
will have an optimum number of organisms to pro-
vide good isolation. Figure 9.6 illustrates the general
procedure. One advantage of this method is that it
requires somewhat less skill than that required for a
good streak plate; a disadvantage, however, is that it
requires more media, tubes, and plates. Proceed as
follows to make three dilution pour plates, using the
same mixed culture you used for your streak plate.
Materials
• mixed culture of bacteria
• 3 nutrient agar pours
• 3 sterile petri plates
• electric hot plate
• beaker of water
• thermometer
• inoculating loop and marking pen
1. Label the three nutrient agar pours I, II, and III
with a marking pen and place them in a beaker
of water on an electric hot plate to be liquefied.
To save time, start with hot tap water if it is
available.
2. While the tubes of media are being heated, label
the bottoms of the three petri plates I, II, and
III.
one loopful one loopful
Mixed
culture I II
I II
Figure 9.6 Three steps in the loop dilution technique for separating out organisms.
78
3. Cool down the tubes of media to 50°C, using the
same method that was used for the streak plate.
4. Following the routine in figure 9.6, inoculate
tube I with one loopful of organisms from the
mixed culture. Note the sequence and manner of
han- dling the tubes in figure 9.7.
5. Inoculate tube II with one loopful from tube I
after thoroughly mixing the organisms in tube I
by shaking the tube from side to side or by
rolling the tube vigorously between the palms of
both hands. Do not splash any of the medium
up onto the tube closure. Return tube I to the
water bath.
6. Agitate tube II to completely disperse the organ-
isms and inoculate tube III with one loopful
from tube II. Return tube II to the water bath.
7. Agitate tube III, flame its neck, and pour its con-
tents into plate III.
8. Flame the necks of tubes I and II and pour their
contents into their respective plates.
9. After the medium has completely solidified,
incubate the inverted plates at 25°C for 24 to 48
hours.
Evaluation of the Two Methods
After 24 to 48 hours of incubation, examine all four
petri plates. Look for colonies that are well isolated
from the others. Note how crowded the colonies
appear on plate I as compared with plates II and III.
Plate I will be unusable. Either plate II or III will
have the most favorable isolation of colonies. Can
you pick out well-isolated colonies on your best pour
plate that are distinct from one another?
one loopful
III
III
 Pure Culture Techniques EXERCISE 9

Draw the appearance of your streak plate and
pour plates on the Laboratory Report.
Subculturing Techniques
The next step in the development of a pure culture is
the transfer of an isolated colony from the petri plate
to a tube of nutrient broth or a slant of nutrient agar.
Use your loop to carefully pick an isolated colony
and aseptically transfer a colony to the broth tube or
slant. To insure that the broth is inoculated, rotate
the loop
in the broth several times before withdrawing it from
the broth tube. For the slant, make an “S” motion by
drawing the loop from the bottom of the tube up the
surface of the slant. Use the following routine to
sub- culture the different organisms that you have
isolated.
Materials
• nutrient agar slants
• inoculating loops
• Bunsen burners

III II
I

I I

(1) Liquefy three nutrient agar pours, (2) After shaking the culture to disperse (3) Transfer one loopful of the culture
cool to 50ºC, and let stand for 10 the organisms, flame the loop and to tube I.
minutes. necks of the tubes.

I I

(4) Flame the loop and the necks of both

(5) Replace the caps on the tubes and (6) Disperse the organisms in tube I by
tubes.
return culture to the test-tube rack. shaking the tube or rolling it between
the palms.
continued
Figure 9.7 Tube-handling procedure in making inoculations for pour plates.
79
EXERCISE 9 Pure Culture Techniques

II
I

(7) Transfer one loopful from tube

(8) After shaking tube II and transferring (9) Pour the inoculated pours into their
I to tube II. Return tube I to the
one loopful to tube III, flame the respective petri plates.
water bath.
neck of each tube.

Figure 9.7 (continued)

1. Label one tube Micrococcus luteus and a second
Serratia marcescens.
2. Select a well-isolated red colony on either the
streak plate or the pour plate. Use your inoculating
loop to pick a well-isolated colony and transfer it
to the tube labeled S. marcescens.
3. Repeat this procedure for a yellow/cream-colored
colony and transfer the colony to the tube
labeled
M. luteus.
4. Incubate the tubes at 25°C for 24 to 48 hours.
Evaluation of Slants
higher temperatures inhibit the formation of the
organ- ism’s red pigment. Draw the appearance of the
slant. What color is the M. luteus culture? Draw the
slant.
You cannot be sure that your cultures are pure
until you have made a microscopic examination of
the respective cultures. It is entirely possible that the
S. marcescens culture harbors some contaminating
M. luteus and vice versa. Prepare smears for each
culture and Gram stain the smears (see Exercise 14).
S. marcescens is a gram-negative short rod, whereas
M. luteus is a gram-positive coccus. Draw the Gram-
stained smears on the Laboratory Report.

After incubation, examine the slants. Is the S. marc-

Laboratory Report
escens culture red? If the culture was incubated at
a Complete the Laboratory Report for this exercise.
temperature higher than 25°C it may not be red
because

80
Laboratory Report 9 Student:

Date: Section:

9 Pure Culture Techniques

A. Results
1. Evaluation of Streak Plate
Show within the circle the distribution of the colonies on your streak plate. To identify the colonies, use
red for Serratia marcescens, yellow for Micrococcus luteus, and purple for Chromobacterium
violaceum. If time permits, your instructor may inspect your plate and enter a grade where indicated.

Grade

2. Evaluation of Pour Plates

Show the distribution of colonies on plates II and III, using only the quadrant section for plate II. If
plate III has too many colonies, follow the same procedure. Use colors.

plate II plate III

81
Pure Culture Techniques (continued)

3. Subculture Evaluation
With colored pencils, sketch the appearance of the growth on the slant diagrams below. Also, draw a few
cells of each organism as revealed by Gram staining in the adjacent circle.

Serratia marcescens Micrococcus luteus Escherichia coli

or
Chromobacterium
violaceum

4. Compare the results of your streak and pour plates. Which method achieved the best separation of
species?

5. Do your slants contain pure cultures? How would you confirm their purities?

B. Short-Answer Questions
1. In regard to bacterial growth on solid media, define the term “colony.”
Bacteria colonize solid medium and develop as colonies. A colony is described as a visible mass of individuals of the same
kind of microorganisms, all of which originated from a single bacterium cell. Because colonies are generated from a single
cell that multiplies and expands outward, they are genetically similar.

2. What colony characteristics can be used for differentiation of bacterial species? As an example,
compare the properties of colonies of Serratia marcescens and Micrococcus luteus on your streak plate.

Size, color, form, texture, opacity, and odor are all useful colony features for differentiating bacterial
species. Serratia marcescens colonies, for example, are red, irregular in form, and have a pungent odor.
Colonies of Micrococcus luteus, on the other hand, are yellow, opaque, spherical, and dome-shaped.

3. Why is dilution a necessary part of pure culture preparation?

82
 When working with a culture containing millions of cells, dilution onto a solid medium is critical for separating
the cells and giving them enough space to form into separate colonies. Highly diluted solutions are also made using
serial dilutions. In the same way, a microbial culture can be diluted. To obtain microorganisms diluted enough to count
on a spread plate, the dilution of germs is critical.

83
Pure Culture Techniques (continued)

4. What advantage(s) does the streak-plate method have over the pour-plate method?
It is quick and requires fewer resources. There's bacterial growth in or on a nourishing broth, an agar
slant, and an agar plate. In microbiology, agar streak plates are a must-have instrument. They allow bacteria and
fungi to grow in separate colonies on a semi-solid surface. These colonies can be used to identify the organism,
purify the strain to remove contaminants and create a genetic clone that is 100% pure.

5. What advantage(s) does the pour-plate method have over the streak-plate method?
The pour plate method is simpler to apply, comes with built-in optimization, and is more likely to get the
desired result.

6. Why is the loop flamed before it is placed in a culture tube? Why is it flamed after completing the
inoculation?
Before entering a culture tube, the loop is flamed to guarantee that no contaminating germs are
introduced. No culture germs are introduced into the working environment since the loop is flamed
subsequently.

7. Before inoculating and pouring molten nutrient agar into a plate, why must the agar first be cooled to 50°C?
Agar should be chilled to 50 degrees Celsius to prevent bacteria from being destroyed by intense heat, but
not too cold since it will harden before the cells can be distributed and transferred into a new container. If the
liquid is poured while it is too hot, excess moisture will collect on the plate's cover.

8. Explain why plates should be inverted during incubation.

Plates are tilted during incubation to prevent moisture from collecting on the lid and falling onto the agar
surface. The diluting effect will be gone since the organisms will be spread. In addition, agar plates dehydrate
more quickly when kept upright.

83