LAB REPORT 3: Preparation and Sterilization of Culture Media

Introduction

A growth medium, or culture medium is a liquid or agar designed to support the growth of microorganisms or cells. Today, there are many types of culture media available commercially but the two major types are agar plates and nutrient broth, used depending on the type of cell to be cultured.

Nutrient broth is in liquid form whereas an agar plate is a type of nutrient broth added with agar powder so that when the suspension cools down, it will coagulate as a semi-solid medium. The microorganisms will grow on the surface of the agar and this makes examination work so much easier as the colonies of microorganisms remain stationery and clearly visible.

The agar preparation is similar to the preparation of a nutrient broth. The composition of both types of growth media is as listed below:

Peptone                 5.00 g/L

Beef extract          1.50 g/L

Sodium Chloride  5.00 g/L

Yeast extract         1.50 g/L

The difference between the two is that the agar-nutrient broth has been added with 15.00g/L of agar powder. The nutrient broth and the agar-nutrient broth contained essential nutrients for the growth of the microorganisms, therefore it has to undergo the autoclaving process to be sterilized so that there won’t be any other unwanted microorganisms growing in the media.

Autoclaving, sometimes called steam sterilization, is the use of pressurized steam to kill infectious agents and denature proteins. This kind of "wet heat" is considered the most dependable method of sterilizing laboratory equipment and decontaminating biohazardous waste. Usually it utilises moist heat and pressure that reach 121°C or 15 psi in 15 minutes to disinfect medical supplies and laboratory equipment, extending their useful lifespan.

           

Objective

To prepare sterile culture media for microorganisms culturing

Materials and reagents

Commercial Nutrient Agar

Lactobacillus MRS Broth

Brain Heart Infusion Broth (BHI)

Trypticase Soy Broth (TSAYE)

Peptone powder

Beef extract powder

Sodium chloride

Yeast extract

 Balance

Distilled water

Scott bottles

Measuring cylinder

Glass rod

Beakers

Procedure

Commercial Nutrient Agar

1.)    13.00 g of the commercial nutrient agar and 15.00 g of the agar are weighed using the balance and are put into the beaker.

2.)    1000 ml of distilled water is measured by measuring cylinder. The nutrient media is mixed up with the distilled water. By using the glass rod, the solution is stirred until it mixes well.

3.)    The solution is then poured into the scott bottle that have been sterilized.

4.)    The bottle is loosely recapped and is set aside for the sterilization.

5.)    All media is sterilized at 121°C for 15 minutes.

6.)    The media is removed after autoclaving. The broth preparation is allowed to cool and then the cap of each bottle is tighten.

                         

Own Prepared Nutrient Agar

1.)    5.00 g of peptone,  1.50 g of beef extract, 5.00 g NaCI_2, 1.50 g of yeast extract and 15.00 g of the agar are weighed using the balance and are put into the beaker.

2.)    1000 ml of distilled water is measured by measuring cylinder. The nutrient media is mixed up with the distilled water. By using the glass rod, the solution is stirred until it mixes well.

3.)    The solution is then poured into the Scott bottle that have been sterilized.

4.)    The bottle is loosely recapped and is set aside for the sterilization.

5.)    All media is sterilized at 121°C for 15 minutes.

6.)    The media is removed after autoclaving. The broth preparation is allowed to cool and then the cap of each bottle is tighten.

                      

Lactobacillus MRS Broth

1.)    13.79 g of Lactobacillus MRS broth in powder form is weighed using the balance and is put into the beaker.

2.)    250 ml of distilled water is measured by measuring cylinder. The nutrient media is mixed up with the distilled water. By using the glass rod, the solution is stirred until it mixes well.

3.)    The solution is then poured into the scott bottle that have been sterilized.

4.)    The bottle is loosely recapped and is set aside for the sterilization.

5.)    The media is sterilized at 121°C for 15 minutes.

6.)    The media is removed after autoclaving. The broth preparation is allowed to cool and then the cap of the bottle is tighten.

Brain Heart Infusion Agar (BHI)

1.)    5.20 g of  BHI agar in powder form is weighed using the balance and is put into the beaker.

2.)    100 ml of distilled water is measured by measuring cylinder. The nutrient media is mixed up with the distilled water. By using the glass rod, the solution is stirred until it mixes well.

3.)    The solution is then poured into the scott bottle that have been sterilized.

4.)    The bottle is loosely recapped and is set aside for the sterilization.

5.)    The media is sterilized at 121°C for 15 minutes.

6.)    The media is removed after autoclaving. The broth preparation is allowed to cool and then the cap of the bottle is tighten.

Trypticase Soy Agar (TSAYE)

1.)    4.00 g of  TSAYE agar in powder form is weighed using the balance and is put into the beaker.

2.)    100 ml of distilled water is measured by measuring cylinder. The nutrient media is mixed up with the distilled water. By using the glass rod, the solution is stirred until it mixes well.

3.)    The solution is then poured into the scott bottle that have been sterilized.

4.)    The bottle is loosely recapped and is set aside for the sterilization.

5.)    The media is sterilized at 121°C for 15 minutes.

6.)    The media is removed after autoclaving. The broth preparation is allowed to cool and then the cap of the bottle is tighten.

Distilled water

1.)    500 ml of distilled water is measured using measuring cylinder.

2.)    The distilled water measured is then poured into the scott bottle that have been sterilized.

3.)    The bottle is loosely recapped and is set aside for the sterilization.

4.)    The media is sterilized at 121°C for 15 minutes.

5.)    The media is removed after autoclaving. The broth preparation is allowed to cool and then the cap of the bottle is tighten.

Results

1. 6 culture media has been prepared which are 1 L of commercial Nutrient Broth ( with 15 g/ L of Agar ), 1 L of own-prepared Nutrient Broth Agar (NBA) , 250 mL of Lactobacilli MRS broth, 100 mL of Brain-Heart Infusion (BHI) broth, 100 mL of Trypticase Soy Agar with 0.6% Yeast Extract (TSAYE) and 500 mL of water (H₂0).
2. For the own-prepared NBA, the recipe is as stated below:

          Peptone                5.00 g/L

          Beef extract         1.50 g/L

          Sodium Chloride 5.00 g/L

          Yeast extract       1.50 g/L

          Agar                    15.00 g/L

3. Each particular nutrient medium above are weighed appropriately and then dissolve in subsequent amount of distilled water. Those medium are then mixed well and thoroughly with water.

Discussion

1. There are a few precautions that need to be highlighted when conducting this lab work:

       ·         The pan and the balance is cleaned with a small brush to ensure more accurate reading.

       ·         All of the doors of the electronic balance are closed before weighing and the ‘tare’ button is                pressed every time after the beaker with substances is put into the balance to avoid zero                        error.

       ·         All of the apparatus are cleaned and rinsed with distilled water before used to avoid                              contamination.

       ·         All of the media are stirred well with a spatula or rod to ensure balance mixing with distilled              water and to maintain the concentration of the media.

       ·         The caps of the Scott bottles are tightened until a slightly tight feeling (but not over tight) is      felt to prevent the Scott bottles from breaking during the autoclaving process.

2. All of the Scott bottles which contain the different medium are then placed into a special basket then the whole basket with all the Scott bottles is put into the autoclave chamber for sterilization. The steps are as stated below:

•  The drain screen at the bottom of the chamber is checked  before using the autoclave.
• Any debris noticed is cleaned up for efficient heat transfer as steam must flush out of the autoclave chamber. If the drain screen is blocked with debris, a layer of air may form at the bottom of the autoclave and prevent proper operation.
• The water level is ensured to be higher than the bottles in the autoclave.      
• The cover of the autoclave chamber is tightened.
• The exhaust valve is tightened too to ensure the pressure goes up.
• The temperature is checked so it is always maintained at 121 ^oC and the pressure is ensured to reach 103 kPa above the atmospheric pressure, with steam is continuously forced into the chamber.
• The time for destruction of the most resistant bacterial spore is now reduced to about 15 minutes. For denser objects, up to 30 minutes of exposure may be required. The conditions must be carefully controlled or serious problems may occur.
• Then, the exhaust valve is opened to ensure the pressure drops to nearly 0 kPa before removing the basket with Scott bottles from the autoclave chamber.

Conclusion:

Preparation and sterilization of culture media are very important to prevent contamination of the unwanted microorganisms. Besides, different types of agar are needed for the cultivation of different types of microorganisms. Any of the precaution steps should be carried out carefully to ensure unwanted errors to occur.  Last but not least, autoclaving is a good sterilization process.

Reference

1.)   obtained from  http://ibg102labreports.wordpress.com/2013/04/15/lab-3-preparation-and-sterilization-of-culture-media/ on 21/11/14

2.)   obtained from  http://pasteristem.blogspot.com/2011/03/lab-3-preparation-and-sterilization-of.html on 21/11/14

3.)   obtained from  http://ibg102.wordpress.com/ on 21/11/14