Lab 2: Media Prep and Autoclave, Plate Pouring
Lab 2: Media Prep and Autoclave, Plate Pouring
Introduction
The purpose of this lab will ultimately be to prepare and pour plates of two different medias in preparation for later labs. Pouring the appropriate amount of media into each petri dish will be the biggest issue and it is predicted that the technique will get better as more plates are poured.
While there are many different types of media used in microbiology labs, one of the most common is Luria-Bertani (LB). Due to its richness in nutrients, it provides good food for bacteria and is simple to make. In addition to its prime conditions for a pure culture, it provides enough food so a culture may grow quickly.
While LB is typically used to grow Escherichia coli, it is a common media in every lab. The most common recipe is: combine 10g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1 liter of distilled water; adjust the pH to 7.0 with 1 N NaOH; and autoclave for 25 minutes at 120*C. However, for this lab, a premade mix will be used. Due to its heaven like conditions for baby microbes, LB should be made just before autoclaving to avoid bacterial contamination.
LB can take two forms: broth or agar. Broth is utilized for liquid cultures, such as injecting a liquid culture into a 250 mL flask. However, agar is used in petri dishes for solid cultures.
Another media is R2A. Unlike LB, R2A will grow cultures more slowly due to its limited nutrient media. It includes yeast extract, peptone, dextrose, magnesium sulfate, starch, sodium pyruvate and others. R2A is preferred in instances such as plating lake water, where there are both fast and slow growing bacteria. Because R2A is less nutrient rich, it will slow the growth of fast growing bacteria to give the slower growing bacteria a chance to come to fruition. This is to prevent the fast growing bacteria from eating up all the yummy, delicious nutrients that are all the rage these days.
Methods and Materials
MEDIA PREP & AUTOCLAVE In a 1 L autoclave bottle, 20 g LB broth powder and 500 mL ultrapure (DI) water was added. In another 1 L autoclave bottle, 9.05 g R2A agar powder and 500 mL ultra pure (DI) water was added. The top of each bottle was double layered with aluminum foil and labeled appropriately. A new piece of autoclave tape was placed on top and each bottle was placed on a mixing plate and allowed to mix. Each bottle was placed in the autoclave for 20 minutes at 121*C on the liquid cycle (making sure to add water to the autoclave basic before starting the cycle). When the autoclave finished, the bottles were carefully removed and allowed to cool to a temperature that was comfortable enough to handle for a few "mississippis" -Dr T.
PLATE POURING The bench top was first sterilized using ethyl alcohol. All dishes were labeled either 'LB' or 'R2A' according to what was being prepared. Quickly, the petri dish lid was opened, the foil was slightly bent back and just enough media was poured in the petri dish to completely cover the bottom of the dish and the lid was attached. The dish was then calmly swirled to ensure the entire bottom was covered.
Once all dishes were poured, they were all stacked on top of each other and the plastic sleeve was placed on top of them and later stored upside down in the cold room once they were set.
Results
Photo 1. Poured LB plates
References
Chester. Cute Chemistry Redbubble [Digital image]. Accessed online 5 September 2017 at <https://www.redbubble.com/people/chayground/works/10056504-youre-growing-on-me-petri-dish-cute-chemistry>.
Thomson, Ashley. "Lab #2: Media Prep and Autoclave, Plate Pouring."
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| (Photo: Chester) |
The purpose of this lab will ultimately be to prepare and pour plates of two different medias in preparation for later labs. Pouring the appropriate amount of media into each petri dish will be the biggest issue and it is predicted that the technique will get better as more plates are poured.
While there are many different types of media used in microbiology labs, one of the most common is Luria-Bertani (LB). Due to its richness in nutrients, it provides good food for bacteria and is simple to make. In addition to its prime conditions for a pure culture, it provides enough food so a culture may grow quickly.
While LB is typically used to grow Escherichia coli, it is a common media in every lab. The most common recipe is: combine 10g of tryptone, 5 g of yeast extract, 10 g of NaCl and 1 liter of distilled water; adjust the pH to 7.0 with 1 N NaOH; and autoclave for 25 minutes at 120*C. However, for this lab, a premade mix will be used. Due to its heaven like conditions for baby microbes, LB should be made just before autoclaving to avoid bacterial contamination.
LB can take two forms: broth or agar. Broth is utilized for liquid cultures, such as injecting a liquid culture into a 250 mL flask. However, agar is used in petri dishes for solid cultures.
Another media is R2A. Unlike LB, R2A will grow cultures more slowly due to its limited nutrient media. It includes yeast extract, peptone, dextrose, magnesium sulfate, starch, sodium pyruvate and others. R2A is preferred in instances such as plating lake water, where there are both fast and slow growing bacteria. Because R2A is less nutrient rich, it will slow the growth of fast growing bacteria to give the slower growing bacteria a chance to come to fruition. This is to prevent the fast growing bacteria from eating up all the yummy, delicious nutrients that are all the rage these days.
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| (Photo: Steve) |
MEDIA PREP & AUTOCLAVE In a 1 L autoclave bottle, 20 g LB broth powder and 500 mL ultrapure (DI) water was added. In another 1 L autoclave bottle, 9.05 g R2A agar powder and 500 mL ultra pure (DI) water was added. The top of each bottle was double layered with aluminum foil and labeled appropriately. A new piece of autoclave tape was placed on top and each bottle was placed on a mixing plate and allowed to mix. Each bottle was placed in the autoclave for 20 minutes at 121*C on the liquid cycle (making sure to add water to the autoclave basic before starting the cycle). When the autoclave finished, the bottles were carefully removed and allowed to cool to a temperature that was comfortable enough to handle for a few "mississippis" -Dr T.
PLATE POURING The bench top was first sterilized using ethyl alcohol. All dishes were labeled either 'LB' or 'R2A' according to what was being prepared. Quickly, the petri dish lid was opened, the foil was slightly bent back and just enough media was poured in the petri dish to completely cover the bottom of the dish and the lid was attached. The dish was then calmly swirled to ensure the entire bottom was covered.
Once all dishes were poured, they were all stacked on top of each other and the plastic sleeve was placed on top of them and later stored upside down in the cold room once they were set.
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| (Photo: Amy He) |
Photo 1. Poured LB plates
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| (Photo: Lauren Lukasik) |
Discussion
While the LB agar is used in plating petri dishes, LB broth is used for liquid cultures when a liquid culture must be injected. For this lab, we prepared dishes with both LB and R2A. LB will be used to quickly grow pure cultures due to its abundance of nutrients. On the other hand, R2A will later be used to grow mixed cultures, evening out the playing field for both fast and slow growing bacteria by restricting the nutrients available. "Leave no bacterias behind!" is definitely R2A's lifestyle motto. Furthermore, the pH may need to be adjusted to 7 on the media because the ideal environment for growing bacteria cannot be too acidic or too basic. Like Goldilocks, the pH must be juuuuust right in order to successfully grow a happy, healthy, wholesome set of cultures.
As hypothesized, plates got more appropriately portioned and uniform as more were prepared (see photo below). While I was trying to make the future homes of our bacteria stocked with nutrients (in preparation for the hurricane obviously), but was informed that they will be just fine with the bare minimum media to cover the dish. The uniformity can be seen in the plates at the top of our stack.
As hypothesized, plates got more appropriately portioned and uniform as more were prepared (see photo below). While I was trying to make the future homes of our bacteria stocked with nutrients (in preparation for the hurricane obviously), but was informed that they will be just fine with the bare minimum media to cover the dish. The uniformity can be seen in the plates at the top of our stack.
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| (Photo: Lauren Lukasik) |
Chester. Cute Chemistry Redbubble [Digital image]. Accessed online 5 September 2017 at <https://www.redbubble.com/people/chayground/works/10056504-youre-growing-on-me-petri-dish-cute-chemistry>.
Thomson, Ashley. "Lab #2: Media Prep and Autoclave, Plate Pouring."
Nicely done.
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