Lab #2: Media Prep and Autoclave, Plate Pouring

Lab #2: Media Prep and Autoclave, Plate Pouring

Introduction

We had made/ used one of many types of commonly used media. Luria-Bertani (LB) being the most common was my groups choice for this lab. It’s very rich in nutrients and food source for bacteria, but even better is that it’s very easy to create. Since its very rich in food for bacteria, it is used to grow pure cultures quickly.

                  LB broth or medium was a sample in the lab and usually always will be. It usually brings out Ecoli (Escherichia coli) and also many other bacteria. The main recipe used in this lab for the LB broth that we had used was a combination of 10 g 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. After the mixture was put together, you have to autoclave the mixture for 25 minutes at 120 degrees Celsius.

                  In this lab we used two different forms of medium for LB: we made a broth and an agar. The Broth is used for a liquid culture of bacteria. Agar then being for more of a solid culture like a petri-dish. R2A was the second media on the list for this lab. It is used as a media that allows more of a slow and controlled growth for bacteria compared to LB which is a very quick growth pattern. R2A however is much more complex, including yeast extract, peptone, dextrose, magnesium sulfate, starch, sodium pyruvate, and others. LB is used for cultures that are pure like Ecoli, compared to cultures that are mixed like water from a river. This is when R2A is handy. It allows you to see all types of bacteria instead of just the fast growing bacteria like LB would show.

Methods and Materials

In a 1L autoclave bottle we added ×20 g LB broth powder and 500 mL ultrapure (DI) water. We had to cover the top with double-layered aluminum foil. Placing a fresh piece of autoclave tape on the top to show sterilization. Place on mixing plate to mix. Autoclave (121 C, 20 minutes) on a liquid cycle, but being sure to add water to the autoclave basin before starting the cycle!. After cycle is complete, remove and let it cool to about 50 C. This is to allow handling of the flask so you don’t burn yourself, caution this is still hot. Pour out the media into petri dishes. First, remove the lid to a dish and remove the foil cover to the flask. Then pour just enough LB agar into the petri dish to completely cover the bottom of the dish. Quickly put the lid back on and swirl the dish gently enough to make sure the entire bottom covered. Do not get any on the sides of the dish. The agar will harden so try and be a little speedy with this process. To end, store plates back in the plastic sleeve the petri dishes originally came in, should be labeled. Make sure dishes are stored upside down in the cold room.

Pics: 

Results:

                  This was a set up lab for another lab to come so hang around for the results later J

Discussion

Why did we make LB broth and agar?

                  We want a comparison between the results of each and how it would vary. We know that LB broth is made to allow growth in a liquid and agar is made to allow growth in more of a solid.

Why did we make LB and R2A?

                  This was to see how different growth patterns are formed in media. LB is a rapid and allows very fast growing. This then allows only the fast growing bacteria to accumulate on the dish which then generally only shows up as one type of bacteria. R2A on the other hand allows for slow growth thus allowing all different types of bacteria to accumulate.

How do your teams’ plates look? Do they look uniform?

                  We moved pretty quickly at first so the pours were more giving than the later pours therefor we definitely didn’t pour evenly. However, we did always cover the bottom completely and I believe our dishes were better than the rest due to me being the one doing them (Too good of a pourer)

Why might we need to adjust the pH to 7 on a broth media?

                  Most of these bacteria are found in living animals or people’s intestines which generally sits around pH of 7. They also like it here because if a bacterium is in too basic of an environment or even too acidic then they tend to die. Neutral is where they thrive.