Bacteria Are Single Cell Microorganisms Biology Essay

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Bacteria are single-cell microorganisms that are able to live in living organisms or nonliving things. They are prokaryotic so they do not have any membrane-bound organelles and nucleus. Some types of bacteria can photosynthesize and produce its own food. However, there are types of bacteria which are not able to execute that function.

Bacteria are the type of most commonly used microorganisms in laboratories because they reproduce so fast. [1] The bacteria Escherichia coli were named for the Austrian doctor, Theodor von Escherich (1857-1911). E.coli was first isolated the genus of bacteria belonging to the family enterobacteriaceae, tribe Eschericheae by him. [2] E.coli is a large group of bacteria. Some strains of E.coli are harmless, however, some of them may cause diarrhea. When working with cell cultures, one can work with a 'pure culture' which is a well isolated colony or a culture that has been studied in the laboratory before and become known how the cells appear and react, it is called strain.

There are some microbiological techniques in order to carry out the research properly. Microbiological techniques are used in cell culture and for identifying microorganisms. While working with microorganisms, there is always a risk of contamination. To prevent and reduce the risk of contamination, aseptic techniques must be used. One of the important techniques is disinfection which helps to reduce the access of any living organisms. Mostly, flame is used for disinfection. Other technique which is used to prevent contamination is sterilization. It is required to destroy microorganisms that can contaminate cultures. There are 3 types of sterilization: Heating, using chemicals and irradiation. Using heat for sterilization in an autoclave causes the destruction of microorganisms by denaturation of enzymes and proteins. The recommendations for sterilization in an autoclave are 15 minutes at 121-124 °C. [3]

Bacteria can grow on any food that contains carbon and nitrogen. Solid or liquid nutrient that bacteria grow on is called medium. 2 types of medium were used in laboratory: LB broth which is liquid and LB agar which is solid.

2. MATERIALS

Petri dishes containing LB agar

E.coli

Automatic pipettor

Tips

1.5 ml Eppendorf tubes containing LB broth

15 ml Falcon tube

Plastic inoculation loop

Glass inoculation loop

Alcohol burner

Lighter

Paraffin

Incubator

Gloves

Ethanol

3. PROCEDURE

First, to prevent any contamination, gloves were worn. First step of the experiment was inoculation. Alcohol burner was burnt and plastic inoculation loop was heated for a second. Then the lid of falcon tube which is containing E.coli was opened and mouth of falcon tube was passed through the flame. After that, plastic loop was inserted in falcon tube and picked up a drop of liquid. Then lid of petri dish which is containing LB agar was opened and E.coli was spread over the agar. Finally, plastic loop was burned in order to kill bacteria.

Second step of the experiment was streaking. Plastic incubation loop was also used in this step. It was heated and cooled a little in order not to kill E.coli. Lid of petri dish was opened and a line was drawn to the centre by using loop. Then, the same loop was moved in a zigzag pattern until ¼ of the plate was covered. After that, petri was rotated about 45 degrees and loop was moved from the end of first zigzag to untouched area with the same pattern. Same technique was applied until all areas in petri dish were done. Finally, lid was closed and swathed with paraffin. Plastic loop was burned, again.

The third step was serial dilution. Five empty petri dishes were labeled as the dilution that will be used: 10-1, 10-2, 10-3, 10-4 and 10-5. In addition, 100 µl E.coli was added to 1.5 ml eppendorf tube which is containing 900 µl LB broth. Then, tip was attached to pipettor and 100 µl of liquid from eppendorf was taken. For almost 20 times, liquid was transferred between new eppendorf tube and pipettor in order to scatter bacteria equally. Consequently, 10-1 dilution of E.coli was obtained and transferred to eppendorf tube. After that, same method was applied for eppendorf which contains 10-1 E.coli. In this way, 10-2 E.coli was obtained and transferred to new eppendorf tube. This procedure was carried out until five eppendorf tubes which are containing 10-1, 10-2, 10-3, 10-4 and 10-5 dilution of E.coli were obtained. Later on, spreading method was applied. Petri dish which is labeled as 10-1 was taken and E.coli was transferred to petri from pipettor, then tips were burned and discarded. After that, glass inoculation loop was heated to sterilize and cooled not to kill bacteria in the petri dish. By the near of flame, E.coli was spread all over the agar by using glass loop at one time. Petri was rotated slowly while spreading. Same procedure was applied all the dilutions. Besides, all procedure was applied by the near of flame.

The final step was incubation. After the spreading, petri dishes containing E.coli were put into a machine called incubator which provides adequate temperature for bacteria. After 24 hours incubation, E.coli had been reproduced enough to see with naked eye.

4. RESULTS

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Figure 1: Streaking Method Figure 2: Petri dish that we took home

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Figure 3: Spreading 10-1 Figure 4: Spreading 10-2

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Figure 5: Spreading 10-3 Figure 6: Spreading 10-4

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Figure 7: Spreading 10-5

To calculate the total number of E.coli through spreading method, 10-5 dilution of E.coli was counted. Then, it is calculated that how many E.coli would be counted in 100 dilutions.

If 10-5 dilution has 8 E.coli, than 100 dilution has 8*105 E.coli.

10-5 8

100 ?

? = 8*105 E.coli in 100 µl

Afterward, it should be calculated that how many E.coli would be counted in 100 ml. This step is necessary because spreading method had been applied in 100 µl. (1 ml = 1000 µl)

If 100 µl have 8*105 E.coli, than in 100 ml, number of E.coli is 8*108 .

100 µl 8*105

100 ml ?

? = 8*108 E.Coli

As a result, streaking method was not applied accurately. However, spreading method was applied successfully because bacteria colonies were easily countable. In addition, various organisms were seen in petri dish that was taken home.

5. DISCUSSION

In this experiment, how microbiological techniques which are aseptic techniques, inoculation, streaking, serial dilution and incubation are applied has been learned. First of all, to prevent any contamination, sterilization has to be provided. For instance, table was cleaned with %70 ethanol before and after the experiment. Besides, filtered tips were used for the pipettors and gloves were worn to prevent any contamination. First procedure of this experiment was inoculation. Most important thing while carrying out inoculation was working by the near of flame. It is important because air stream which is produced by flame helps to keep any microorganisms or particular away. Thus, contamination risk was reduced. Also, it is necessary to burn the loop after work with it. It is because bacteria on that loop may transfer and contaminate the experiment. Streaking method is used for getting isolated colonies of bacteria, checking whether culture is pure or contaminated and able to grow. [4] Unfortunately, the streaking method was not accomplished accurately. It is understood from that colonies were not separately countable. The reason for this mistake may be the wrong application of plastic loop or not to start streaking where it is ended. Other reason for that mistake can be not to rotate petri enough to streak all over the surface. Next steps which are serial dilution and spreading were done successfully. Aim of serial dilution is to determine the number of bacteria in the original culture. [5] As always, serial dilution was applied by the near of flame in order to prevent any contamination. Using pipettor properly was crucial since wrong usage of pipettor such as not to stop pressing the plunger after first point may cause draw the liquid into the pipettor so contamination. It is also important to burn tips after using because of the bacteria contamination. The crucial part of spreading method was sterilizing glass loop and using it accurately. After holding glass loop through the flame, it is important to wait a while until it cooled because high temperature may kill bacteria. Furthermore, spreading must be applied while rotating petri dish in order to spread bacteria equally. Best result was taken with 10-5 dilution in spreading so calculation was made according to that dilution. In conclusion, it is identified that various microorganisms reproduced in petri dish and formed colonies.

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