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ISOLATION OF PURE CULTURES

Lao AGL
National Institute of Molecular Biology and Biotechnology
College of Science, University of the Philippines
Diliman, Quezon City 1101
ABSTRACT
This experiment was conducted in an attempt to isolate pure
bacterial cultures from water samples taken from various sources
around the UP Diliman campus. The collected water samples were
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serially diluted up to the 10 dilution and were then cultured using
the spread plating technique. The plates were incubated for a 24
hour growth period, after which three colonies were chosen and
used to make streak plates. The outcome of the spread plating
yielded no statistically valid results. A total of 23 colonies were
obtained from the spread plates, and three isolated colonies were
streaked. From the streak plates, three colonies were chosen and
were restreaked. The second streak plating resulted in the growth
of several pure colonies, therefore the isolation of pure colonies
from the water samples was successful.
I.

Introduction

Various bacteria can be found living basically anywhere in nature. They are abundant in bodies of water
such as ponds and lakes. The most common types of bacteria in fresh water are Proteobacteria,
Cyanobacteria, Actinobacteria, and Bacteroidestes. These bacteria contribute greatly to the carbon cycle,
by decomposing organic matter, and the nitrogen cycle, by performing nitrification and denitrification.
The goal of this experiment is to isolate pure bacterial cultures from water samples using the spread
plating technique. The water that will be used was collected from the AS pond and the stream near the
Institute of Math. This will be diluted and then cultured on nutrient agar.
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The serial dilution technique will be used to dilute the water samples seven times, up to the 10 dilution.
Each of the seven resulting dilutions will be used to make corresponding agar plates.
Two types of plating techniques will be used in this experiment: spread plating and streak plating. Spread
plating will be used to make the seven corresponding plates for the seven dilutions. After 24 hours of
growth, three colonies will be chosen to be streak plated for further purification and identification.
Inoculation of a liquid culture will also be employed.
II.

Materials and Methods

A stock bacterial solution was prepared by pipetting one ten-mL portion each of water collected from AS
pond and the stream near the Institute of Math into an Erlenmeyer flask containing 40 mL of distilled
water. After mixing, one mL of the stock bacterial solution was inoculated into a test tube containing nine
mL of 0.9% saline solution. The liquid in the test tube was mixed by pipetting in and out. A fastpette was
used, along with a 5-mL stripette. One mL of this mixture was then inoculated into the next test tube,
which also contained nine mL of 0.9% saline solution (7.2 g NaCl, 800 mL distilled water). The dilution
was repeated six times, for a total of seven dilutions.
A positive dilution test and a dilution blank were also performed. The positive test was done by performing
the same dilution method using an overnight E. coli suspension. The dilution blank was done by
inoculating one mL of the culture medium, which was incubated in the same conditions as the E. coli
suspension, into nine mL of saline solution.

One spread plate was done for each dilution. Each plate, except for the dilution blank, was made by
dropping 100 microlitres (using a micropipettor) of diluted suspension onto a Petri dish containing
hardened nutrient agar (5 g peptone, 3 g beef extract, 8 g NaCl, 15 g agar, 1 mL 1N NaOH). The blank
plate was done on a Luria-Bertani agar plate (10 g tryptone, 5.0 g yeast extract, 10 g NaCl, 1mL 1N
NaOH, 15 g agar). After dropping, a glass spreader was used to spread the suspension evenly on the
surface of the agar. The finished plates were then sealed with parafilm. The plating was performed under
the laminar flow hood, and the aseptic technique was maintained throughout the plating. The completed
plates were incubated inverted at 37C overnight.
Three colonies were chosen from the completed plates, and a streak plate was done for each in order to
isolate pure colonies. The plates and broth solutions were incubated at 37C and the microbial growth
was observed after 24 hours.
III.

Results

The spread plates were observed after being incubated at 37C for 24 hours. The colonies were counted,
and it was found that in the positive test, there were no statistically valid plates. The most valid plate
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(relatively) was the plate made from the 10 dilution, shown in Fig. 1.

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Figure 1. Spread plate of the 10 dilution of the positive test (E. coli suspension).
For the actual water sample, no statistically valid plates were found as well. The colony count jumped
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from too many to count (TMTC) in the 10 plate to too little to consider (TLTC) in the 10 plate (Fig. 2)
The colony forming unit (CFU) was calculated for each plate using equation 1. Sample calculations can

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Figure 2. Spread plates from the 10 dilution (left) and 10 dilution (right) of the bacterial stock
solution.

be found in the appendix, and the results of the calculations are found in table 1.

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Table 1. Colony count and CFU for positive test, negative blank, and bacterial solution spread plates.
Dilution
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10
-2
10
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10
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10
-5
10
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10
-7
10
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Blank Media (10

Positive Test, Blank


Colony Count
CFU, colony/mL
TMTC
TMTC
TMTC
TMTC
TMTC
TMTC
9
343
3.43 x 10
0
0

Bacterial Solution
Colony Count
CFU, colony/mL
TMTC
TMTC
4
13
1.3 x 10
5
10
1.0 x 10
0
0
0
0
0
0
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The weighted mean for the CFU should have been calculated, but since there were no statistically valid
plates, the mean weight was not computed. The equation for weighted mean can be found in the
appendix.
Three streak plates were also done for the three colonies chosen from the spread plates (Fig. 3).

Figure 3. Streak plates from the three isolated colonies obtained from the spread plates.

Finally, three colonies from these streak plates were restreaked (Fig.4).

Figure 4. Plates of the three restreaked colonies.


IV.

Discussion
A. Isolation and Plating Techniques

Bacteria colonies were isolated during the spread plating section of the experiment. However, isolation of
colonies does not immediately guarantee that those colonies are pure. Since there could potentially be
multiple types of bacteria in the stock bacterial solution, it is possible that colonies contain a mixture of
two or more different kinds of bacteria. There is no way to guarantee that different kinds of bacteria will
grow separately from each other. However, the purity of an isolated colony can be checked in several
ways. One way is to visually examine the colonial morphology. If there seem to be distinct morphologies
in the colony, then the colony is most likely impure. Another way is to apply Gram staining to the colony to
see if the entire colony reacts the same way. And another way is to use streak plating. This is the method
employed in this experiment. Three isolated colonies were chosen to be streak plated. The streaking
diluted the bacteria in the colony so that each bacteria would be able to form its own colony separate from
the others.
Spread plating, pour plating, and streak plating techniques each have their own advantages and
disadvantages with respect to colony culturing and isolation. Pour plating is advantageous in that the
bacteria cultured by this method are not exposed to air. This means that there is a lower risk of
contamination. Pour plating is also good for anaerobic bacteria, since they will not be exposed to oxygen.
However, this also makes it a less useful technique for aerobic bacteria. Another disadvantage is that the
medium needs to be molten in order to perform pour plating. The temperature needed to keep the
medium in liquid state could potentially kill the bacteria that is being cultured.
Spread plating, meanwhile, allows the bacteria to grow on the surface of the agar, making it easily
accessible for inoculation. There is also less chance of the colonies clumping together since they are
spread out over the medium. This technique is better for aerobic bacteria than the pour plating technique,
but the colonies are more prone to air contamination.

Streak plating, like spread plating, allows colonies to form on the surface of the medium, meaning that the
bacteria are not trapped in the medium. Due to the streaking technique, this method is more likely to
produce individual colonies than the other methods.
In streak plating, the bacteria to be cultured has to be grown on a solid medium. In spread plating and
pour plating, the bacteria needs to be in a liquid suspension. This means that dilution has to be performed
before spread and pour plating can be done. Also, more plates are needed for these two techniques than
for streak plating.
As shown in this experiment, streak plating seems to be the most effective technique for obtaining pure
cultures, because it spreads out the bacteria so that by the end of the streaking, the bacteria are virtually
separated cell by cell, free to form colonies far apart from each other. This means that each new colony
arises from a single bacteria, ensuring a pure culture. Spread and pour plating do not guarantee the same
degree of isolation, because there is no way to ensure that bacteria settle far from each other.
B. Serial Dilution Results
There were many microorganisms in the bacterial stock solution. If plated directly, the medium would
have too many colonies to count. Viable plates should have between 30 and 300 colonies, meaning that
plates made using stock solution would be invalid. Therefore, serial dilution was employed to reduce the
number of microorganisms per unit volume. The technique involves diluting the solution several times,
usually to a power of ten. The serial dilution technique was used instead of straight dilution to ensure that
the stock bacterial solution was well represented in each sample dilution. Serial dilution can be used
when there is an unknown count of microorganisms in a sample.
For this experiment, seven serial dilutions were performed. The final dilution was 1:10000000. The
method could also be performed for different dilutions, such as 1:1000 or 1:50. (Fig. 5).

Prepare
bacterial
stock solution

1 mL
9 mL diluent
(0.9% NaCl
solution)

9 mL diluent
(0.9% NaCl
solution)

1 mL

Finished
(1:1000)
9 mL diluent
(0.9% NaCl
solution)

1 mL

Prepare
bacterial
stock solution
1 mL

1 mL
4 mL diluent
(0.9% NaCl
solution)

9 mL diluent
(0.9% NaCl
solution)
Finished
(1:50)

Figure 5. Flowcharts for the dilution of bacterial stock solution to 1:1000 (top)
and 1:50 (bottom).

0.9% NaCl solution was used as the diluent for the water samples because that concentration is isotonic
to most bacteria. A lower concentration would cause bacteria to lyse due to the hypotonic condition, and
higher concentrations could cause bacteria to lose water due to hypertonic conditions.
A spread plate was made for each of the seven dilutions. To be considered valid, the spread plates
needed to have between 30 and 300 colonies after incubation. For this experiment, no statistically valid
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spread plates were obtained. As shown in the results (Table 1), the plates made from the 10 and 10
dilutions were found to statistically because there were too many colonies to count (TMTC, or lawn) The
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plates made from the 10 to 10 dilutions were found to be invalid because there were too little colonies
to consider (TLTC). This indicates that the stock bacterial solution did contain bacteria, since the first
three dilutions were TMTC. But since the subsequent dilutions were found to be TLTC, this indicates that
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few bacteria were transferred from the 10 dilution to the 10 . This could be caused by poor dilution
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technique. Another cause could be that the 10 dilution was insufficiently mixed, causing the bacteria to
either sink to the bottom of the tube or stay at the top of the solution. Still another possible cause is a
problem with the culture medium or the growth conditions. However, this cause is removed by the results
of the positive test and the negative test blank.
The positive test and a negative test blank were also performed along with the actual experiment. The
positive test was run by using the same procedure that was used for the stock bacterial solution, except
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that the stock solution was replaced by an E. coli suspension. The E. coli suspension was diluted to 10 ,
and spread plates were made. The positive test functioned as a control group. The results of the test
would determine the expected results if bacteria were present in the stock solution. The negative test
blank, meanwhile, was performed by inoculating plain culture medium onto an agar plate. The culture
medium was the same medium used for the E. coli suspension, and was incubated in the same
conditions as that suspension. This negative test blank served as the other control group, which would
determine the expected result if no bacteria were present in the stock solution. Both tests used the same
culture medium and growth conditions as the experimental setup, and both tests were successful, proving
that the medium and conditions were not the reason for the failure of the spread plates.
V.

Summary and Conclusion

This experiment was performed to determine the CFU of water samples taken from the AS pond and the
Institute of Math stream, as well as to isolate and purify bacteria from these water samples. The sample
was subjected to serial dilution and then inoculated using the spread plating technique. After 24 hours of
growth, three colonies were selected and streak plated for more isolation. Three colonies from these
streak plates were then restreaked to further ensure the new colonies purity.
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The plates made from the 10 and 10 dilutions were statistically invalid because there were too many
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colonies on the plates for them to be valid, while the plates made from the 10 to 10 dilutions were
invalid because there were too few. This could be caused by poor dilution technique or poor water
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representation from the 10 dilution to the 10 . Because of this, no conclusions can be made as to which
dilution made the best spread plate. But even with the unsuccessful dilution, the streak plating of selected
colonies was successful, meaning that a number of pure colonies were obtained after the streaking. It is
recommended that more care be taken while performing serial dilution.

VI.

References

Clark, J.A., El-Shaarawi, A.H. (1993). Evaluation of Commercial Presence-Absence Test Kits for
Detection of Total Coliforms, Escherichia coli, and Other Indicator Bacteria. Applied and Environmental
Microbiology, 59(2), 380-388.
Esteban, G., Finlay,B., and Clarke, K. 2012. "Priest Pot in the English Lake District: a showcase of
microbial diversity". Freshwater Biology. 57: 321-330.

Harris, R.F., Sommers, L.E. (1968). Plate-Dilution Frequency Technique for Assay of Microbial Ecology.
Applied and Environmental Microbiology, 16(2), 330-334.
Hoben, H.J., Somasegaran, P. (1982). Comparison of the Pour, Spread, and Drop Plate Methods for
Enumeration of Rhizobium spp. in Inoculants Made from Presterilized Peat. Applied and Environmental
Microbiology, 44(5), 1246-1247.
Madigan, M., Martinko, J., Stahl, D., & Clark. D. (2010). Brock Biology of Microorganisms, 128-131.
Prentice Hall.

VII.

Appendix

A. Equations and Calculations

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Sample Calculation (for 10 dilution plate, positive test):

where n = total colonies counted in all considered plates


fa = number of plates of lowest dilution with countable colonies
fb = number of plates in the next countable dilution, and so on
df = dilution factor based on reciprocal of lowest countable dilution

B. Pictures

Figure A1. Spread plate for negative blank.

Figure A2. Spread plates for positive test, decreasing concentrations (left to
right, top to bottom)

Figure A3. Spread plates for water sample, decreasing concentrations (left to
right, top to bottom).