LAB 3: PREPARATION AND STERILIZATION OF CULTURE MEDIA

INTRODUCTION

Microbes require nutrients to grow. Therefore, for microorganisms culturing, we need to prepare a culture media to provide them enough nutrients to survive. Culturing organisms with specific nutritional (or even environmental) needs requires special purpose media. There are different types of culture media suitable for growing different types of cells, such as basal, enriched, selective, differential, transport and storage media. A standard solid culture media is nutrient agar or agar plates. Agar is polysaccharide extract obtained from seaweed. Agar is an ideal solidifying agent as it is bacteriologically inert, no influence on bacterial growth, it remains solid at 37°C and it is transparent. The broth contain:

5.0g/L peptone
1.5g/L beef extract

5.0g/L sodium chloride, NaCI

1.5g/L yeast extract

15.0g/L agar powder

Autoclaving is a method used in sterilization process. It is conducted by using of pressurized steam at specific condition: 121°C and 15 psi for 15 minutes, to kill infectious agents and denature protein thus causing thus causing destruction of all types of microorganisms, excluding some sporeformers. The pressurized steam produce a heat called ‘wet heat’ which has large amount of vaporisation heat capacity is sufficient and efficient to destroy microorganisms inside the autoclave chamber. Hence, it is commonly used to sterile laboratory glassware, other equipment and waste, surgical instruments and medical waste.

Autoclaving machine

OBJECTIVE

To prepare sterile nutrient agar for culturing microorganisms

Materials and reagents

Commercial nutrient agar

Balance

Distilled water

Scott bottles

Spatula

Brain Heart Infusion Broth agar (BHI)

Trypticase Soy Broth agar (TSAYE)

Beef extract

Yeast extract       

Peptone

Sodium chloride

Agar powder

Procedure

Commercial Nutrient Agar Preparation

1.       11.2g of commercial nutrient agar powder was weighed and poured into a 500ml beaker.

2.       400ml of distilled water was measured by using 1000ml measuring cylinder and poured into beaker which contained the commercial nutrient agar powder.

3.       The mixture was stirred by using spatula until the powder dissolved completely.

4.       The mixture was poured into a labelled scott bottle.

5.       The scott bottle was loosely recapped and autoclaved at 121°C/15psi for 15 minutes.

6.       The media was removed after autoclaving and was allowed to cool before tighten the cap of the scott bottle.

Self-Made Nutrient Agar Preparation

1)      6.0g of agar powder, 0.6g of beef extract, 0.6g of yeast extract, 2.0g of peptone and 2.0g of sodium chloride was weighed and poured into a 500ml beaker.

2)      400ml of distilled water was measured by using 1000ml measuring cylinder and poured into beaker which contained the mixture of powders.

3)      The mixture was stirred by using spatula until the powder dissolved completely.

4)      The mixture was poured into a labelled scott bottle.

5)      The scott bottle was loosely recapped and autoclaved at 121°C/15psi for 15 minutes.

6)      The media was removed after autoclaving and was allowed to cool before tighten the cap of the scott bottle.

Brain Heart Infusion Agar (BHI) and Trypticase Soy Agar (TSAYE) Preparation

1.       5.2g of Brain Heart Infusion Agar (BHI) powder was weighed and poured into a 250ml beaker.

2.       100ml of distilled water was measured by using measuring cylinder and poured into beaker which contained the powder.

3.       The mixture was stirred by using spatula until the powder dissolved completely.

4.       The mixture was poured into a labelled scott bottle.

5.       The scott bottle was loosely recapped and autoclaved at 121°C/15psi for 15 minutes.

6.       The media was removed after autoclaving and was allowed to cool before tighten the cap of the scott bottle.

These steps were repeated by using Trypticase Soy Agar (TSAYE) powder.

Result

The recipe for preparing the self-made nutrient agar is:

Beef extract 1.5g/L

Yeast extract 1.5g/L      

Peptone 5.0g/L

Sodium chloride 5.0g/L

Agar powder 15g/L

For preparing 400ml self made nutrient agar, 0.6g beef extract, 0.6g yeast extract, 2.0g peptone, 2.0g sodium chloride and 6.0g agar powder are mixed with 400ml distilled water.

The others culture media that have been prepared are 400 ml commercial nutrient agar (by mixing 11.2g agar powder with 400ml distilled water), 100ml BHI agar (by mixing 5.2g agar powder with 100ml distilled water) and 100ml TSAYE agar (by mixing 4g agar powder with 100ml distilled water).

Discussion

There are certain precautions that have to be taken during the experiment, including of:

1.     All the scott bottles have to be labelled first.

2.     The surface of balance must be cleaned before weighing any powder to ensure the accurate quantity of powder can be obtained.

3.     All scott bottles must rinsed with distilled water and be in dry condition before using.

4.     The media should be stirred evenly by spatula before pouring into the scott bottles to ensure the powders mix well with distilled water.

5.     Before autoclaving, the caps of the scott bottles should be loosen to prevent them from breaking due to the high pressure during autoclaving.

6.     The autoclaving machine has to be closed tightly the the media can be sterilised at 121 °C.

7.     After autoclaving, the media are cooled down and the caps of scott bottles are closed tightly.

Conclusion

In this experiment, we learned the importance of sterilization of culture media and the process of preparing these culture media. One of the ways to sterilize the culture media is by autoclaving.  Sterilization of culture media is important to prevent the contamination of other microorganisms.

Reference

http://www.microbeworld.org/careers/tools-of-the-trade/culture-equipment/culture-media

https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/culturing-microorganisms-6/culturing-bacteria-58/culture-media-364-5325/

http://medical-dictionary.thefreedictionary.com/autoclave