GBE-203

MICROBIOLOGY LABORATORY

EXPERIMENT 1
CULTIVATION OF MICROORGANISMS

SUBMITTED BY
CEYLİN BAYKOÇ

SUBMITTED TO
MELİS RAHİME YILDIRIM

EXPERIMENT DATE: 19.10.2023

SUBMISSON DATE: 25.10.2023

Yeditepe University
Genetics and Bioengineering Department
Istanbul
 1. AIM

The main objective of the experiment, cultivation of microorganisms, is isolating a pure strain of
Escherichia coli bacteria with streaking method under aseptic techniques. In the meantime, to
obtain diluted colonies, serial dilution has performed with spreading technique to observe colonies
individually. To estimate the approximate number of colonies in the stock sample, the colony-
forming unit (CFU) has calculated.

2. INTRODUCTION

Escherichia coli (E. coli) is a gram-negative, rod-shaped, facultative anaerobic bacterium

belonging to Enterobacteriaceae bacterial family. Theodor Escherich identified this bacterium in
1885. Most E.coli bacteria are harmless for humans and animals. In fact, it lives in the
gastrointestinal systems of humans and warm-blooded animals. However, certain strains of E.coli
are pathogenic particularly to children who are not in sufficient hygienic conditions or who
consume contaminated foods.

E. coli can survive at temperatures of 4°C to 53°C up to 3 months on solid media. Under aerobic
conditions, Escherichia coli (E. coli) grows best at 37°C in a nutritious agar or broth medium at a
neutral pH of 7.0. For example, in Luria-Bertani broth E.coli can achieve an overnight cell density
of 109 CFU/mL of culture in stated conditions. LB Broth and LB Agar are classified as rich
mediums because they both provide the elemental nutrition to the microorganisms, such as C, H2O,
N, O, and S. Therefore, 1L Luria-Bertani broth medium consists of 10 grams of peptone, 5 grams
of yeast extract, and 10 grams of sodium chloride (NaCl). LB Agar has the same components as
LB Broth, with 12 g of Agar per liter.

For ease of use for biotechnological research, nonpathogenic K-12 and B strains of E. coli are
widely used as a model organism in laboratories. The cultivation of microorganisms provides
information about morphology and metabolism for identification, increasing and isolating several
colonies, testing antibiotics, and lastly producing antigens, proteins, or plasmids.

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To obtain clear results in this biotechnological research, the cell culture process should be
completed according to aseptic techniques. The aseptic technique is based on a variety of measures
aimed to reduce the possibility of contamination from the environment. Aseptic technique includes
a sterile work environment, good personal hygiene, and sterile reagents. For this reason, 70%
Ethanol (C2H5OH) is used to clean laboratory benches with Bunsen burners to create an air
circulation to upward direction.

Figure 2.1. Streaking method for formation single colonies.[1]

Figure 2.2. Spreading method for obtaining colonies as a specific amount.[2]

In this experiment three different techniques were applied: Streaking, spreading and serial dilution.
Streaking is a technique used to isolate a pure strain from a single species of microorganism. On
the other hand, spreading technique is used to obtain single colonies from diluted samples serially.
When the experiment has done, the colony-forming unit (CFU) has calculated for diluted sample
to determine the approximate number of cells in the stock sample. For long-term storage, E. coli
can be combined with glycerol and kept at -80°C continuously.

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑥 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟

𝐶𝐹𝑈 =
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝐿)

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 3. MATERIALS

• Escherichia coli culture

• Inoculation Loops
• LB Nutrient Agar
• LB Nutrient Broth
• P1000 Micropipette
• P200 Micropipette
• 200 µL Pipette Tips
• 1000 µL Pipette Tips
• 1.5 mL Microcentrifuge Tubes x 6
• 35 mm Polystyrene Petri Dishes x 5
• 100 mm Polystyrene Petri Dish
• Lighter
• Bunsen Burner
• 37°C Incubator
• Parafilm
• 70% Ethanol (CH₃CH₂OH)

4. METHOD

Streak Plating

1. Benches in the laboratory were disinfected with 70% ethanol.

2. Bunsen burner was lit with a lighter.
3. The petri dish to be used for streaking was labeled.
4. The inoculation loop was kept on the fire for 2-3 seconds.
5. The inoculation loop was cooled for approximately 5 seconds by slightly opening the lid
of the petri dish close to the fire.
6. Approximately 1 colony of E.coli bacteria was taken from the stock agar with the
inoculation loop.

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 7. Starting from the upper left side of the Petri dish and ending at the lower left side (1 quarter
of the area), bacteria were distributed in straight lines downwards continuously.
8. The inoculation loop was kept on fire for 1-2 seconds and cooled on the lid of the petri dish
close to the fire.
9. Starting from the last place left in step 7 (bottom left), the bacteria were distributed to the
other quarter of the petri dish, again ending at the bottom left.
10. The inoculation loop was kept on fire for 1-2 seconds and cooled on the lid of the petri dish
close to the fire.
11. Starting from the last place left in step 9 (bottom left), the bacteria were distributed to the
other quarter of the petri dish, again ending at the bottom left.
12. The inoculation loop was kept on the fire for 1-2 seconds and cooled close to the fire on
the lid of the petri dish.
13. Starting from the last place left in step 11 (bottom left), the bacteria were distributed to the
other quarter of the petri dish, this time towards the center of the petri dish, similar to the
letter 'S'.
14. The inoculation loop was kept on fire for 1-2 seconds.
15. The petri dish was covered with parafilm.
16. The Petri dish was kept in the incubator at 37° Celsius for 24 hours and the result was
recorded the next day.

Serial Dilution and Spread Plating

1. Five 1.5 mL microcentrifuge tubes, each containing 900 µL Nutrient Broth, were labeled
1 to 5.
2. 100 µL was taken from the 1.5 mL Stock microcentrifuge tube and added to
microcentrifuge tube 1 and mixed by pipetting.
3. 100 µL was taken from tube 1 and added to microcentrifuge tube 2 and mixed by pipetting.
4. 100 µL was taken from tube number 2 and added to microcentrifuge tube number 3 and
mixed by pipetting.
5. 100 µL was taken from tube number 3 and added to microcentrifuge tube number 4 and
mixed by pipetting.

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 6. 100 µL was taken from tube number 4 and added to microcentrifuge tube number 5 and
mixed by pipetting.
7. 5 Petri dishes were labeled as 101, 102, 103, 104 and 105.
8. 50 µL of each of the 5 microcentrifuge tubes (1, 2, 3, 4, 5) containing the diluted bacteria
were added to 35 mm petri dishes labeled 101, 102, 103, 104 and 105 respectively, following
aseptic techniques.
9. The inoculation loop for each petri dish was kept on fire for 1-2 seconds and cooled close
to the fire on the lid of each petri dish.
10. Using the inoculation loop for each petri dish, the bacteria were distributed to the petri
dishes by spreading technique.
11. The inoculation loop was kept on fire for 1-2 seconds.
12. Petri dishes were covered with parafilm.
13. Petri dishes were kept in an incubator at 37° Celsius for 24 hours and the results were
recorded the next day.

5. RESULTS

Petri dishes incubated in an incubator at 37° Celsius were obtained and photographed 24 hours
after both experiments were performed. The streak plating part of the experiment was performed
by one person. (Figure 5.1) The results obtained by the other members of the group are shown
below. (Figure 5.2) Spread plating part of the experiment was performed as a group and
photographed. (Figure 5.3) The colony-forming units per milliliter (CFU/mL) of the original
sample was determined using the following equation for 105 labelled petri dish as a spread plating
part of the experiment:

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑥 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟

𝐶𝐹𝑈 =
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝐿)

𝐶𝐹𝑈 = 270 𝑥 105 / 3

𝐶𝐹𝑈 = 9 𝑥 10! 𝐶𝐹𝑈/ 𝑚𝐿

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 Figure 5.1. The results of the experiment's Figure 5.2. The results of the experiment's
streak plating method after 24h, which was streak plating method after 24h, which was
carried out by a single person. carried out by other group members.

USSI
Figure 5.3. The results of the experiment's serial dilution and spread plating method after 24h,
which was carried out as a Group 5.

6. DISCUSSION

In this experiment, for both streaking and spreading methods, the aseptic technique was applied in
the beginning. The aseptic technique consists, of lighting a Bunsen burner and using 70% of
Ethanol for disinfecting areas. Bunsen burner is used to create an upward direction of air flow for
eliminating of any bacteria or particles that may be present on the surface of benches. Using 70%
Ethanol has a major effect on killing other contaminants. Because it can penetrate the cells with

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the help of water. The water in the 70% Ethanol helps to dissolve the proteins and other organic
compounds in the cell wall. Therefore the bacteria or any impurity can be eliminated easily. The
inoculation loop was kept on fire for 1-2 seconds for every new streaking. The main purpose of
flaming the inoculation loop is to sterilize it and prevent cross-contamination. When both two
experiments were done the Petri dishes were covered with parafilm to maintain a controlled
environment and retain moisture for bacteria to grow. Petri dishes were placed upside down in the
incubator to reduce the chance of any condensation or droplets forming and falling onto the agar
surface. The streaking experiment has been completed without any errors. Only, in the experiment,
when compared to the other group members, it was observed that only the first one or two lines in
Area 2 did not touch perpendicularly to Area 1 in any of the samples. In this part of the experiment,
streaking should made in the correct order. For instance, it is important that only the first one or
two streaks in area 2 should touch area 1 perpendicularly. This enables for more accurate growth
and viewing of individual colonies, which is useful for analyzing their metabolism and
characteristics. The spreading experiment has not been completed without any errors. When the
experiment was carried out in groups, 50 μl of each diluted sample should have been taken from
each sample using a micropipette in the range of 200-20 μl and placed in 35 mm Petri dishes, but
50 μl could not be taken from the samples because the micropipette in the range of 1000-100 μl
was used. Although the serial dilution was performed correctly, the accuracy of the results is not
correct because the volume of the sample placed in the Petri dishes is not accurate. As can be seen
in Figure 5.3, the inconsistency of the number of colonies on the petri dishes is visible. Therefore,
CFU was calculated according to an approximate value.

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REFERENCES

1. Allocati, N., Masulli , M., Alexeyev, M. A. F., & Di Ilio, C. (2013). Escherichia coli in
Europe: An Overview. International Journal of Environmental Research and Public
Health. https://doi.org/10.3390/ijerph10126235

2. World Health Organization (n.d.). E.Coli. https://www.who.int/news-room/fact-

sheets/detail/e-coli

3. (n.d.). Aseptic Technique. Thermo Fischer Scientific.

https://www.thermofisher.com/tr/en/home/references/gibco-cell-culture-basics/aseptic-
technique.html

4. (n.d.). LB Broth and LB Agar. Thermo Fisher Scientific.

https://www.thermofisher.com/tr/en/home/life-science/cell-culture/microbiological-
culture/bacterial-growth-media/lb-broth-and-lb-agar.html

5. Gallagher, S. R., & Wiley, E. A. (2012). Current Protocols Essential Laboratory

Techniques (2nd ed.). Wiley Blackwell.

6. Tortura, G. J., Funke, B. R., & Case, C. L. (1998). Microbiology: An Introduction (6th
ed.). Addison Wesley Longman Inc.

7. (n.d.). Streak Plate Method- Principle, Types, Methods, Uses. Microbe Notes.
https://microbenotes.com/streak-plate-method-principle-methods-significance-
limitations/ [1][2]