Lab 3: Streak Plate, Culture Transfer Instruments and Techniques, Isolation and Maintenance of a Pure Cultur
Lab 3: Streak Plate, Culture Transfer Instruments and Techniques, Isolation and Maintenance of Pure Culture
Introduction
The purpose of this lab will ultimately be to grow a pure culture of Escherichia coli, place a colony inside LB broth and freeze a stock all to be used in later labs. However, this will be complicated by the fact that microorganisms do not grow in neat, pure populations. A pure culture must be isolated first from a mixed community. The first of such ways would be to grow a sample onto selective media so that the media's components only allow the growth of limited or one type of microorganisms. (A selective media is a specialized concoction of specific elements that provide for the growth and success of limited microorganisms. The list of ingredients of selective media differs from non-selective media in that it is much longer and more complex.) After this is done, a single, specific colony can be removed and either plated immediately or incubated in LB broth and frozen for future uses.
Methods and Materials
ETHANOL STERILIZATION As microbes are very delicate and sensitive entities, sterilization was key to this experiment. Using the squeezable bottles containing 70% ethanol, the entire bench top and all gloves were administered the solution and then dried using a paper towel. All instruments needed to be sterile as well. This is to protect the purity of the culture we aim to isolate.
STREAK PLATE FROM FROZEN STOCK A sterile toothpick was used to pick up a single E. coli colony from the already grown plate. (As opposed to the frozen stock solution). This toothpick with the colony was then plated as follows: A zig zag was traced on the media in the first quadrant of the dish. The tooth pick was then thrown away and another was opened. This toothpick zig zagged into the already plated quadrant once and then repeated in the second quadrant. This was then repeated for two more quadrants. The lid was then placed on the dish and it was placed in the incubator.
STREAK PLATE FROM FROZEN STOCK Another colony was then lifted using a sterile toothpick and put into a flask of sterile LB broth and gently swirled around. This flask was then placed in the incubator and allowed to incubate for 24 hours at 37*C.
LB BROTH TO FROZEN STOCK An previously incubated flask was then used for this next part. 1 mL of E.coli was pipetted out of the flask and placed in a cryogenic tube along with 1 mL of glycerol. This tube was then placed in the deep freeze at -80*C for later used of a pure culture.
Everything was then heavily sterilized because it would be bad if the entire class became infected with E. coli.
Discussion
Pinterest [Digital image]. Accessed online 2 October 2017 at <https://i.pinimg.com/474x/21/3b/88/213b88b2a7ea063a44d3b0743cba6ae6--medical-laboratory-lab-rats.jpg>.
Thomson, Ashley. "Lab #3: Streak Plate, Culture Transfer Instruments and Techniques, Isolation and Maintenance of a Pure Culture."
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| (Photo: Pinterest) |
The purpose of this lab will ultimately be to grow a pure culture of Escherichia coli, place a colony inside LB broth and freeze a stock all to be used in later labs. However, this will be complicated by the fact that microorganisms do not grow in neat, pure populations. A pure culture must be isolated first from a mixed community. The first of such ways would be to grow a sample onto selective media so that the media's components only allow the growth of limited or one type of microorganisms. (A selective media is a specialized concoction of specific elements that provide for the growth and success of limited microorganisms. The list of ingredients of selective media differs from non-selective media in that it is much longer and more complex.) After this is done, a single, specific colony can be removed and either plated immediately or incubated in LB broth and frozen for future uses.
ETHANOL STERILIZATION As microbes are very delicate and sensitive entities, sterilization was key to this experiment. Using the squeezable bottles containing 70% ethanol, the entire bench top and all gloves were administered the solution and then dried using a paper towel. All instruments needed to be sterile as well. This is to protect the purity of the culture we aim to isolate.
STREAK PLATE FROM FROZEN STOCK A sterile toothpick was used to pick up a single E. coli colony from the already grown plate. (As opposed to the frozen stock solution). This toothpick with the colony was then plated as follows: A zig zag was traced on the media in the first quadrant of the dish. The tooth pick was then thrown away and another was opened. This toothpick zig zagged into the already plated quadrant once and then repeated in the second quadrant. This was then repeated for two more quadrants. The lid was then placed on the dish and it was placed in the incubator.
STREAK PLATE FROM FROZEN STOCK Another colony was then lifted using a sterile toothpick and put into a flask of sterile LB broth and gently swirled around. This flask was then placed in the incubator and allowed to incubate for 24 hours at 37*C.
LB BROTH TO FROZEN STOCK An previously incubated flask was then used for this next part. 1 mL of E.coli was pipetted out of the flask and placed in a cryogenic tube along with 1 mL of glycerol. This tube was then placed in the deep freeze at -80*C for later used of a pure culture.
Everything was then heavily sterilized because it would be bad if the entire class became infected with E. coli.
 |
| (Photo: Amy He) |
Discussion
The intent of this entire lab was to familiarize ourselves with handling pure cultures of microorganisms. From the plating to the freezing, everything must be done with extreme care and caution so as to not spoil the culture or contaminate anything else.
The plating of the E. coli colony proved to be the hardest part as we did it incorrectly anyways using a colony from an already plated culture instead of the frozen stock, as well as dealing with the strange substance that is the growth media. If we pressed too hard, the jelly would break and if we didn't press hard enough, it was difficult to see if we plated anything. Furthermore, the streaking technique of zig zagging and such is to ultimately remove one colony from that plate and use that as the basis for the LB broth and such later on. A single colony is used for the entire plate to keep the future colonies consistent and the genetic mutations to a minimum. Instead of streaking 8 million different colonies at once and ending up with a mixture in the final quadrant, we can pretty much say the entire plate is uniform.
The other thing worth commenting on is the reason for the addition of glycerol to the cryogenic tube. While E. coli can survive at the extremely low temperature of -80 *C, they become brittle when frozen. The glycerol acts as a shield against the cracking or destroying while keeping the integrity of the culture the same.
Overall, we now have a pure culture to work with later on and can watch our pretty little E. coli grow on the plates we created. What a wonderful Circle of Life.
ReferencesThe plating of the E. coli colony proved to be the hardest part as we did it incorrectly anyways using a colony from an already plated culture instead of the frozen stock, as well as dealing with the strange substance that is the growth media. If we pressed too hard, the jelly would break and if we didn't press hard enough, it was difficult to see if we plated anything. Furthermore, the streaking technique of zig zagging and such is to ultimately remove one colony from that plate and use that as the basis for the LB broth and such later on. A single colony is used for the entire plate to keep the future colonies consistent and the genetic mutations to a minimum. Instead of streaking 8 million different colonies at once and ending up with a mixture in the final quadrant, we can pretty much say the entire plate is uniform.
The other thing worth commenting on is the reason for the addition of glycerol to the cryogenic tube. While E. coli can survive at the extremely low temperature of -80 *C, they become brittle when frozen. The glycerol acts as a shield against the cracking or destroying while keeping the integrity of the culture the same.
Overall, we now have a pure culture to work with later on and can watch our pretty little E. coli grow on the plates we created. What a wonderful Circle of Life.
Pinterest [Digital image]. Accessed online 2 October 2017 at <https://i.pinimg.com/474x/21/3b/88/213b88b2a7ea063a44d3b0743cba6ae6--medical-laboratory-lab-rats.jpg>.
Thomson, Ashley. "Lab #3: Streak Plate, Culture Transfer Instruments and Techniques, Isolation and Maintenance of a Pure Culture."
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