Media Preparation and Autoclave, Plate Pouring

LAB 2 - MEDIA PREPARATION AND AUTOCLAVE, PLATE POURING

Introduction

Culture media are crucial in the controlled growth of bacteria in a laboratory. Two important requirements is that the culture media can nourish the bacteria and that it is fairly simple to make. These medias can be in the form of a liquid broth, or as a gelatinous agar. The use of each form of media will depend on whether you wish to inoculate the colony or want to visualize the bacterial colonies in a petri dish.

LB media is the most common culture media and ideal for pure conditions, made with ingredients such as tryptone, yeast, sodium chloride, and DI water. Due to the fact that LB is so rich in nutrients for bacteria, in order to prevent unwanted bacteria for eating all the nutrients, it is strongly recommended that the media is made just before autoclaving. R2A is another media that is also used for culturing bacteria, but allows the bacteria to grow slowly. R2A is the perfect media for mixed conditions, where there are slow- and fast-growing bacteria and both groups must be analyzed.

The purpose of this lab is to understand the importance of the culture media, understand and be able to operate an autoclave, and to properly and efficiently pour media into the petri dishes. Because this laboratory procedure is more oriented towards execution than analysis, there will a greater discussion on operation than on results. it is expected that some plates will not poured properly, and that some plates will be contaminated due to the amount of time they were open.

Materials and Methods

This lab was divided into tw section, with Part 1 discussing media preparation, autoclave operation and how culture medias work, while part 2 was focused on pouring premade culture media into petri dishes.

Part 1 was conducted verbally. The media to be used was premade. Media cultures LB and R2A were discussed, specifically the difference in nutrients that they each possess, as well as bacterial growth rate in each media. An analytical balance was used to measure 20g of LB broth, after which it was poured into a 2L flask along with 1 L of DI water. The water and brother were then mixed and the solution was distributed equally among 10 250mL beakers. Autoclave tape was then place on each beaker to indicate whether the mix was sterilized after autoclave operation or not. Autoclaving was also discussed, in terms of how it operates and how sterilization in an autoclave takes place. An autoclave was set to run for 20 minutes at 120° C. This operation can be already programmed onto the autoclave under a "LIQUIDS" setting. Water was added to the autoclave basin to create positive pressure in the chamber. Upon completion of cycle, the media was store at room temperature, while remaining closed off from the outside environment to prevent contamination.
Part 2 was conducted using 25 petri dishes and approximately 500mL of either LB or R2A. The culture media was poured into each dish as quickly and as effectively as possible. to provide a rough estimate, it was requested that 80% of the petri dish surface was to be covered by media and the dish be swirled around to make up for the remaining 20% of available surface area and create a thin film of culture media across the entire surface of the petri dish. A demonstration can be seen below:

Results and Discussion

To understand the importance of controlled bacterial growth in a laboratory setting, it is important to understand the vital role the culture media plays throughout the process. Important notes to be made are:

LB media allows for the rapid growth of microorganisms. This is due to the fact that it is extremely rich in nutrients meant to be metabolized by fast-growing bacteria. R2A is tailored to feed slower-growing microorganisms, as it uses more components and nutrients for metabolism than LB.
LB media is made exclusively of yeast, salt, and tryptone; R2A implements the use of yeast, peptone, dextrose, magnesium sulfate, starch, sodium pyruvate, and other components.
LB works really well for "pure" cultures, meaning that it is perfect for one one group of bacteria to be analyzed. R2A is better for mixed samples of bacteria, such as samples taken from the field.
Culture medias can be contaminated easily if left open for too long.

When it comes to the autoclave, one of the most important details is that although it is said that it is run for 20 minutes at 120° C, it is not totally true. What really occurs is that for the sterilization to occur, the autoclave has to build temperature and pressure up until it reaches the desired temperature (120° C), and then the 20 minute timer starts. Once the timer is finished, the autoclave has to be given time to depressurize and return to room temperature. Failing to allow the autoclave to properly build pressure will not allow the culture to be sterilized, and failing to allow the autoclave to lose pressure after the sterilization will cause the positive pressure to expand rapidly, or explode and hurt the experimenter as well as the culture medias. Figure 1 below shows an autoclave with the samples in the pressure chamber.

Figure 1: Autoclave with culture media preparing for sterilization

Pouring culture media into the petri dishes is important because that is where the culture media coagulates and becomes the gelatinous film upon which the microorganisms grow. A thin film of R2A in our case was needed to cover the bottom surface of the petri dish, and it was stressed that only a little was needed to promote bacteria growth. At the same time, it was important to not get any up on the sides or lid of the dish, as it would grow bacteria on those surface as well and skew the results we would get in an actual procedure. Because pouring was so important in microbiological testing, it was important for us to practice proper pouring technique, as can be appreciated in the video above. Figure 2 shows students pouring media into the dishes, which Figure 3 shows what the final petri dish should look like with media already poured into it. Additional pictures can be appreciated below Figure 3 for documentation, observation and recording purposes.