Lab 3: Preparation and Sterilization of Culture Media

Introduction

     The study of microorganisms requires techniques for isolating cells from natural sources and growing them in the laboratory on synthetic media. Thus, development of synthetic culture media and culture techniques have played important roles in the advance of this field. Microbiologists use bacterial culture media for many purposes and applications. Media are used to isolate and identify bacteria, reveal their metabolic properties, and allow long-term storage of pure cultures. The most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or LB medium (Lysogeny Broth). Liquid media are often mixed with agar and poured via sterile media dispenser into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured.

The broth contains:

3.0 g/L "Lab-lemco" powder (a beef extract)
2.0 g/L yeast extract
5.0 g/L peptone (a nitrogen source)
5.0 g/L sodium chloride
2.0 g/L agar powder

An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C for around 15–20 minutes depending on the size of the load and the contents. Autoclaves use pressurized steam to destroy microorganisms, and are the most dependable systems available for the decontamination of laboratory waste and the sterilization of laboratory glassware, media, and reagents.

 

Materials

Commercial nutrient agar

Balance

Distilled water

Scott Bottles

Procedure

1.Weight appropriate amount of broth (with agar) powder into Scott bottles and dissolve 
   with distilled water. Mix well.

2. Loosely recap the bottles and set aside for sterilization.

3. Sterilized all media at 121°C for 15 minutes.

3. After autoclaving, remove the media. Allow the broth preparation to cool then tighten the 
    cap of each bottle 

Autoclave at 121 °C for 15 minutes

Result

The commercial and own recipe nutrient agar are prepared and ready to be sterilized.

The following week during our 4th lab, it can be seen that no contamination occurs in all of our media. So, it can be used perfectly ( Lab 4: Source of contamination and infection)

Discussion

1. The surface of the balance must to keep clean to avoid extra weight

2. During weighing, the door of the balance should be completely closed. 

3. The spatula must be completely dry.

4. The inside of the Scott bottles must be dry and clean before using it.

5. During autoclaving, the cap of Scott bottles should be loosened to prevent the bottles 
    from cracking due to pressured air. The cap also must not be too loose to avoid outflow of
    media inside the bottle

6. When mixing the ingredients to make the media, there should not be any clumps 
    present. 
7. Make sure the autoclave door is close tightly.

 Conclusion  

 In this experiment we have learned on how to prepare commercial and own recipe culture  
 media. The own made culture media are prepared based on the ingredient listed. We also 
 learn how to sterilize the culture media by autoclave. A few precaution step must be taken 
 during the preparation and sterilization of the culture media.

  Reference
  - http://generalbacteriology.weebly.com/culture-media.html
   -https://www.des.umd.edu/biosafety/auto/autoclave.html 
   -http://www.rapidmicrobiology.com/test-method/choosing-a-microbiology-laboratory-  
    autoclavesterilizer/
   -http://elte.prompt.hu/sites/default/files/tananyagok/microbiology/ch06s02.html