Lab 3: Preparation and sterilization of culture media

Lab 3: Preparation and sterilization of culture media

Introduction

Bacteria may be identified by studying the colony character, thus , a media culture has to be use. Mixed bacteria that is accumulate together can be separated when they grow in media culture. Solid media is used for the isolation of bacteria as pure culture. 'Agar' is most commonly used to prepare solid media. Agar is polysaccharide extract obtained from seaweed. Agar is an ideal solidifying agent as it is, bacteriologically inert, remains solid at 37°C, and transparent.There are mainly 2 types of culture media that we can prepare, they are commercial culture and own prepared culture. Commercial culture media nowadays is very convenient to use as the nutrient is already prepared in powder form. The addition of water is needed to prepare the media. While for the own prepared culture media, the amount of different kind of nutrient in powder form have to be added together and dissolve in water. For example, in this experiment , 3.0 g “Lab-lemco” powder (a beef extract), 2.0 g yeast extract, 5.0 g peptone (a nitrogen source), 5.0 g sodium chloride, and 2.0 g agar powder are dissolved in water.

        The culture media prepared is then undergo autoclave. Autoclave is a process that use moist heat and pressure so that all parts of the material to be sterilized reach 121oC for 15 minutes. An autoclave is, in essence, a large pressure cooker; a chamber which may be sealed off against surrounding air. Materials for sterilization are placed in the chamber, the door is sealed, and pressurized steam is forced into the chamber. The incoming steam displaces cooler air through an exhaust valve; this valve closes when the cell cooler air has been vented. Steam is continually forced into the chamber until the pressure reaches 103 kPa above atmospheric pressure; at sea level, this pushes the temperature in the chamber to 121oC. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121oC throughout. After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The sterilized objects can then be removed.

Objective

To prepare sterile nutrient agar for culturing microorganisms

To learn the aseptic technique

Materials and Reagents

Commercial nutrient agar

Balance

Distilled water

Scott bottles

Procedure

Preparation of commercial culture media

1. The appropriate amount of commercial agar powder was weight into Scott bottles and dissolved with distilled water. Mixed it well.

Figure 1: The amount of broth needed is weight.

Figure 2: The broth was stirred with amount of distilled water needed.

Figure 3: Poured it into a Scott bottle

2. Loosely recapped the bottles and set aside for sterilization.
3. All the media was sterilized at 121oC for 15 minutes.
4. After autoclaved the media, removed the media. Allow the broth preparation to cool then tighten the cap of each bottle.
5.  Repeat the method from “ Preparation of commercial culture media” step 1 to 4 to complete the procedure.

Result

For the own recipe of culture media, the color is lighter and it should contain

• 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

For the Commercial Culture Media, the color is more brownish and it should contain

• 11.2g commercial nutrient agar
• 5.2g BHI agar
• 4.0g TSAYE agar

Discussion

• The culture media must be in sterile condition and must ensure that is no contamination to the culture media. To ensure this, a distilled water must be used instead of tap water. 
• Next, the reading of weighing machine must be accurate in order to ensure the ingredient is enough to the amount needed. 
• The powder must be completely dissolved before pouring into a Scott bottle and must mix well. This is to ensure that is no solidification of agar at the bottom of Scott bottle.
•  Before the scott bottle is put into the autoclave machine, the cap is slightly loosen. This is to prevent the cracking of bottle in the autoclave machine. The high temperature and high pressure in the autoclave machine may increase the pressure in the Scott bottle. 
• For the own prepared culture media, make sure every nutrients are added together before pouring the water. This is to make sure there is no insufficient of nutrient in the media.