PURPOSE

The purpose of this experiment was to be demonstrated the techniques for transforming E. coli.

THEORY:

Bacterial growth on/in a medium is called a culture. Microbiology laboratories work with pure cultures. A pure culture is when there is onlyone microorganism growing in/on the medium. The transferring of a bacterium from a stock culture to either a solid or liquid medium is called inoculation. Inoculating a bacterium on/in a sterile medium ensures the purity of a culture. Properly transferring a bacterium without contamination is called an aseptic transfer. Many steps are taken to ensure that neither the bacterium nor the medium is contaminated. Aseptic Techniques are the precautionary measures taken to prevent contamination of pure cultures and sterile laboratory equipment. Treat all organisms as potential pathogens. Many of the organisms can be opportunistic in their abilities to cause infection. Microorganisms in the lab atmosphere may come to rest on the desktop between classes and overnight, so disinfect lab top thoroughly before and after each lab period. This is accomplished by spraying the lab top down with a commercial disinfectant or a 10% bleach solution and allowing this to stand for a minute. You may then wipe down the bench with the paper towel.

Disinfection is the destruction or removal of infectious or harmful microorganisms from nonliving objects by physical or chemical methods. Heating process developed by Pasteur to disinfect beer and wines is called Pasteurization. It is still used to eliminate microorganisms from milk and beer. Not all microbes are destroyed by pasteurization. Chemicals used to disinfect objects are calleddisinfectants. When this treatment is directed at a living tissue, it is called antisepsis and the chemical is called antiseptic. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics.

MATERIALS:

• Nutrient Agar plate

• Inoculating loop

• Nutrient Agar slant

• Inoculating needle

• Nutrient Agar deep

• Bunsen burner

• Nutrient Broth

• Test tubes and test tubes rack

• Nutrient Broth of culture of E. coli

• Etuv

• Nutrient Agar plate of E. coli                        

• lighter

PROCEDURE:

1- Microorganisms in environment;

a) Firstly; nutrient agar was taken, then the cover of a petri plate nutrient agar was removed and nutrient agar was exposed to air for5-10-20 minutes. Afterwards; cap of petri plate was replaced on the plate, and finally nutrient agar was incubated at 37 °C for 24 hours.

b) Again, another nutrient agar petri plate was taken petri plate was divided two parts underside by acetate pencil, and then we pressed slightly my finger on the nutrient agar medium, then it was incubated at 37 °C for 24 hours.

c) Again, another nutrient agar petri plate was taken and its cap was opened then one hair was put in the plate, after that; its cap was closed and it was incubated at 37 °C for 24 hours.

2- Transfer Techniques; 

i) Broth to Broth Transfer: Firstly; inoculating loop was taken and all of wire was heated to redness for sterilization. Afterwards, tube containing E. coli was taken my left hand and at the nozzle of tube, cotton was got out and the rim of tube was rendered sterile by flame. After that; inoculating loop was immersed to E. coli tube and E. coli was taken on the loop from nutrient broth. The rims of tube was held to re-flame, nozzle of tube was closed with cotton and preparing nutrient broth in a tube was taken and nozzle of the tube was rendered sterile with flame, and loop was immersed in the nutrient broth then nozzle of nutrient broth tube was held re-flame and was closed with cotton and nutrient broth was incubated at 37 °C for 24 hours.

ii) Broth to Slant Transfer: Firstly; inoculating loop was taken and all of wire was heated to redness for sterilization. Then, tube containing E. coli was taken my left hand and at the nozzle of tube, cotton was got out and the rim of tube was rendered sterile by flame. Next, inoculating loop was immersed to E. coli tube and E. coli was taken on the loop from nutrient broth containing E. coli. The nozzle of tube was held to re-flame, tube’s nozzle was closed by cotton again. Afterwards; our slant tube was taken and its cotton was removed, then nozzle of tube was held to flame in a nearly horizontal position. Next, loop containing E. coli was gone in a zigzag on the slant surface. E. coli was cultivated on the slant surface. Finally; nutrient slant tube was incubated at 37 °C for 24 hours.

iii) Broth to Deep Transfer: Firstly; inoculating needle was taken and all of wire was heated to redness for sterilization. Then, tube containing E. coli was taken my left hand and at the nozzle of tube, cotton was got out and the rim of tube was rendered sterile by flame. Next, inoculating needle was immersed to E. coli tube and E. coli was taken on the needle from nutrient broth containing E. coli. The nozzle of tube was held to re-flame, tube’s nozzle was closed by cotton again. Afterwards; our deep tube was taken and cotton was removed from tube and nozzle of tube was rendered sterile by flame. Needle containing E. coli was penetrated to deep nutrient agar tube, and then nutrient agar deep tube was incubated at 37 °C for 24 hours.

3- Agar Slant as source of inoculum; 

Slant to Broth Transfer: Firstly; inoculating loop was taken and was sterilized by flame as far as redness. Then tube containing E. coli was taken my left hand and cotton was got out at nozzle of tube. Next, the rim of tube was rendered sterile by flame. Then, inoculating loop was immersed to E. coli tube and E. coli was taken on the loop from nutrient slant containing E. coli and rim of tube was sterilized again, and cotton was closed the nozzle of tube again. Afterwards; our broth tube was taken, cotton was got out from nozzle of tube and nozzle of tube was rendered sterile by flame and loop containing E. coli was immersed to tube and was shaken, then nozzle of tube was held to re-flame and cotton was again closed , finally; nutrient broth tube was incubated at 37 °C for 24 hours.

4- Colony Selection;

In here; there was a nutrient agar petri plate containing E. coli and the cap of plate was opened carefully and E, coli was taken by sterilized loop in a short time. Afterwards; our nutrient broth was taken and cotton was got out, then nozzle of tube was rendered sterile by flame, and loop containing E. coli was immersed to nutrient broth, loop was shaken in the tube slightly. Next; nozzle of our nutrient broth tube was held to re-flame and was rendered sterile, cotton was closed. Finally; nutrient broth tube was incubated at 37 °C for 24 hours.

RESULTS:

Broth to Broth Transfer: There was turbidity and microorganisms were observed.

Broth to Slant Transfer: Propagation of microorganisms was observed on the surface.

Broth to Deep Transfer: There were microorganisms and they were observed toward top part more than toward bottom part.

Slant to Broth Transfer: There was turbidity, microorganisms were observed.

Plate to Broth Transfer: There was turbidity, microorganisms were observed.

Petri Plate (finger press): There were microorganisms, 11 small microorganisms were observed.

Petri Plate (air): 1 microorganism was observed at 5 minutes.

Petri Plate (one hair): no microorganisms.

DISCUSSION:

In this experiment, transfer of E. coli tried to learn with a certain transfer techniques, that these techniques are Broth to Broth, Broth to Slant, and Broth to Deep Transfer. Certain results were obtained from these transfers. For example; the formation of microorganisms were observed at broth to broth transfer and there was turbidity, at broth to slant transfer propagation of microorganisms were observed on the slant surface, at broth to deep transfer there were the formation of microorganisms but they were decreasing from above to deep, at slant to broth and at plate to broth transfers turbidity was observed and there were the formation of microorganisms. Meanwhile; petri plates were also examined against the formation of microorganisms, and at finger press; twelve small microorganisms were observed, another petri plate had been exposed to air for 5 minutes that one microorganism was observed, and finally; at another petri plate finding a hair there was no the formation of microorganisms.

In here; we understood that nutrient agar slant and deep media are more suitable for preservation of microorganisms in a long time. In addition; when the microorganisms live they produce toxic materials and these toxic materials spread out fast in broth media. However; slant and deep media slow down the expansion of toxic materials. Because of this some microorganisms are preserved in the slant and deep media in order to protect a long time.

As a result, we say that we learned transfer techniques and, inoculum and incubation of microorganisms. In addition; we learned that when the transfers of microorganisms are transferred sterilization of media, environment and using apparatus must be taken care sufficiently, and must be rendered sterile.

PURPOSE

The purpose of this experiment was to be demonstrated the techniques for transforming E. coli.

THEORY:

Bacterial growth on/in a medium is called a culture. Microbiology laboratories work with pure cultures. A pure culture is when there is only one microorganism growing in/on the medium. The transferring of a bacterium from a stock culture to either a solid or liquid medium is called inoculation.
Inoculating a bacterium on/in a sterile medium ensures the purity of a culture. Properly transferring a bacterium without contamination is called an aseptic transfer. Many steps are taken to ensure that neither the bacterium nor the medium is contaminated. Aseptic Techniques are the precautionary measures taken to prevent contamination of pure cultures and sterile laboratory equipment. Treat all organisms as potential pathogens. Many of the organisms can be opportunistic in their abilities to cause infection. Microorganisms in the lab atmosphere may come to rest on the desktop between classes and overnight, so disinfect lab top thoroughly before and after each lab period. This is accomplished by spraying the lab top down with a commercial disinfectant or a 10% bleach solution and allowing this to stand for a minute. You may then wipe down the bench with the paper towel.

Disinfection is the destruction or removal of infectious or harmful microorganisms from nonliving objects by physical or chemical methods. Heating process developed by Pasteur to disinfect beer and wines is called Pasteurization. It is still used to eliminate microorganisms from milk and beer. Not all microbes are destroyed by pasteurization. Chemicals used to disinfect objects are called disinfectants. When this treatment is directed at a living tissue, it is called antisepsis and the chemical is called antiseptic. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics. An antiseptic is a solution used to disinfect the skin or other living tissues. Disinfectants are strong chemical substances and are more destructive to living tissues than antiseptics.

MATERIALS:

• Nutrient Agar plate

• Inoculating loop

• Nutrient Agar slant

• Inoculating needle

• Nutrient Agar deep

• Bunsen burner

• Nutrient Broth

• Test tubes and test

  tubes rack

• Nutrient Broth of culture of E. coli

• Etuv

• Nutrient Agar plate of E. coli                        

• lighter

PROCEDURE:

1- Microorganisms in environment;

a) Firstly; nutrient agar was taken, then the cover of a petri plate nutrient agar was removed and nutrient agar was exposed to air for 5-10-20 minutes. Afterwards; cap of petri plate was replaced on the plate, and finally nutrient agar was incubated at 37 °C for 24 hours.

b) Again, another nutrient agar petri plate was taken petri plate was divided two parts underside by acetate pencil, and then we pressed slightly my finger on the nutrient agar medium, then it was incubated at 37 °C for 24 hours.

c) Again, another nutrient agar petri plate was taken and its cap was opened then one hair was put in the plate, after that; its cap was closed and it was incubated at 37 °C for 24 hours.

2- Transfer Techniques;

i) Broth to Broth Transfer:

Firstly; inoculating loop was taken and all of wire was heated to redness for sterilization. Afterwards, tube containing E. coli was taken my left hand and at the nozzle of tube, cotton was got out and the rim of tube was rendered sterile by flame. After that; inoculating loop was immersed to E. coli tube and E. coli was taken on the loop from nutrient broth. The rims of tube was held to re-flame, nozzle of tube was closed with cotton and preparing nutrient broth in a tube was taken and nozzle of the tube was rendered sterile with flame, and loop was immersed in the nutrient broth then nozzle of nutrient broth tube was held re-flame and was closed with cotton and nutrient broth was incubated at 37 °C for 24 hours.

ii) Broth to Slant Transfer:
Firstly; inoculating loop was taken and all of wire was heated to redness for sterilization. Then, tube containing E. coli was taken my left hand and at the nozzle of tube, cotton was got out and the rim of tube was rendered sterile by flame. Next, inoculating loop was immersed to E. coli tube and E. coli was taken on the loop from nutrient broth containing E. coli. The nozzle of tube was held to re-flame, tube’s nozzle was closed by cotton again. Afterwards; our slant tube was taken and its cotton was removed, then nozzle of tube was held to flame in a nearly horizontal position. Next, loop containing E. coli was gone in a zigzag on the slant surface. E. coli was cultivated on the slant surface. Finally; nutrient slant tube was incubated at 37 °C for 24 hours.

iii) Broth to Deep Transfer:
Firstly; inoculating needle was taken and all of wire was heated to redness for sterilization. Then, tube containing E. coli was taken my left hand and at the nozzle of tube, cotton was got out and the rim of tube was rendered sterile by flame. Next, inoculating needle was immersed to E. coli tube and E. coli was taken on the needle from nutrient broth containing E. coli. The nozzle of tube was held to re-flame, tube’s nozzle was closed by cotton again. Afterwards; our deep tube was taken and cotton was removed from tube and nozzle of tube was rendered sterile by flame. Needle containing E. coli was penetrated to deep nutrient agar tube, and then nutrient agar deep tube was incubated at 37 °C for 24 hours.

3- Agar Slant as source of inoculum;

Slant to Broth Transfer: Firstly; inoculating loop was taken and was sterilized by flame as far as redness. Then tube containing E. coli was taken my left hand and cotton was got out at nozzle of tube. Next, the rim of tube was rendered sterile by flame. Then, inoculating loop was immersed to E. coli tube and E. coli was taken on the loop from nutrient slant containing E. coli and rim of tube was sterilized again, and cotton was closed the nozzle of tube again. Afterwards; our broth tube was taken, cotton was got out from nozzle of tube and nozzle of tube was rendered sterile by flame and loop containing E. coli was immersed to tube and was shaken, then nozzle of tube was held to re-flame and cotton was again closed , finally; nutrient broth tube was incubated at 37 °C for 24 hours.

4- Colony Selection;

In here; there was a nutrient agar petri plate containing E. coli and the cap of plate was opened carefully and E, coli was taken by sterilized loop in a short time. Afterwards; our nutrient broth was taken and cotton was got out, then nozzle of tube was rendered sterile by flame, and loop containing E. coli was immersed to nutrient broth, loop was shaken in the tube slightly. Next; nozzle of our nutrient broth tube was held to re-flame and was rendered sterile, cotton was closed. Finally; nutrient broth tube was incubated at 37 °C for 24 hours.

RESULTS:

Broth to Broth Transfer: There was turbidity and microorganisms were observed.

Broth to Slant Transfer: Propagation of microorganisms was observed on the surface.

Broth to Deep Transfer: There were microorganisms and they were observed toward top part more than toward bottom part.

Slant to Broth Transfer: There was turbidity, microorganisms were observed.

Plate to Broth Transfer: There was turbidity, microorganisms were observed.

Petri Plate (finger press): There were microorganisms, 11 small microorganisms were observed.

Petri Plate (air): 1 microorganism was observed at 5 minutes.

Petri Plate (one hair): no microorganisms.

DISCUSSION:

In this experiment, transfer of E. coli tried to learn with a certain transfer techniques, that these techniques are Broth to Broth, Broth to Slant, and Broth to Deep Transfer. Certain results were obtained from these transfers. For example; the formation of microorganisms were observed at broth to broth transfer and there was turbidity, at broth to slant transfer propagation of microorganisms were observed on the slant surface, at broth to deep transfer there were the formation of microorganisms but they were decreasing from above to deep, at slant to broth and at plate to broth transfers turbidity was observed and there were the formation of microorganisms. Meanwhile; petri plates were also examined against the formation of microorganisms, and at finger press; twelve small microorganisms were observed, another petri plate had been exposed to air for 5 minutes that one microorganism was observed, and finally; at another petri plate finding a hair there was no the formation of microorganisms.

In here; we understood that nutrient agar slant and deep media are more suitable for preservation of microorganisms in a long time. In addition; when the microorganisms live they produce toxic materials and these toxic materials spread out fast in broth media. However; slant and deep media slow down the expansion of toxic materials. Because of this some microorganisms are preserved in the slant and deep media in order to protect a long time.

As a result, we say that we learned transfer techniques and, inoculum and incubation of microorganisms. In addition; we learned that when the transfers of microorganisms are transferred sterilization of media, environment and using apparatus must be taken care sufficiently, and must be rendered sterile.