LAB REPORT 3: PREPARATION AND STERILIZATION OF CULTURE MEDIA

INTRODUCTION

Culture media are available commercially as powders; they require only the addition of water. Nutrient medium is a general purpose preparation for culturing microorganisms which are not nutritionally fastidious. 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

The agar has the same composition, except that it contains 15g/L agar. The final pH of both media is 7.4.

Autoclaving

Autoclaving is a process that use moist heat and pressure so that all parts of the material to be sterilized reach 121^oC 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 121^oC. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121^oC 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

MATERIALS AND REAGENTS

• Commercial nutrient agar
• Balance
• Distilled water
• Scott bottles
• Brain Heart Infusion Broth (BHI)
• Trypticase Soy Broth (TSAYE)
• Peptone powder
• Sodium chloride
• Yeast extract
•  Balance
• Distilled water
• Scott bottles
• Measuring cylinder
• Glass rod
• Beakers

PROCEDURE

1. Appropriate amount of broth (with agar) powder weighed into Scott bottles and dissolved with distilled water and it mixed well.
2. Loosely recap the bottles and set aside for sterilization.
3.  All media had sterilized at 121^oC for 15 minutes.
4. The media had removed after autoclaving process to allow the broth preparation to cool. Then the cap of each bottle is tighten up.

 

The Commercial Nutrient Agar

1. The commercial nutrient agar was weight about  34.0 mg.
2. The distilled water was measured 100mL by using measuring cylinder .
3. Both commercial nutrient agars and distilled water was poured together and mixed them well .
4. The solution was divided into three and poured them into different scott bottles that had been sterilized.
5. The distilled water was measured 60mL by using measuring cylinder.
6. The bottles was recapped loosely and moved aside for sterilization.
7. All media was sterilized at 121°C for 15 minutes.
8. After autoclaving , the media were removed . The commercial nutrient agar were allowed to cool then the cap of each bottles were tighten.

Own Prepared Nutrient Agar

1. 5.00 g of peptone, 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.

 

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

RESULT

3 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) , 100 mL of Brain-Heart Infusion (BHI) broth and 100 mL of Trypticase Soy Agar with 0.6% Yeast Extract (TSAYE). For the own-prepared NBA, the recipe is as stated below:

• Peptone                  5.00 g/L
• Sodium Chloride  5.00 g/L
• Yeast extract         1.50 g/L
• Agar                         15.00 g/L

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

DISCUSSION

There are a few precautions that need to be highlighted when conducting this lab work which are  the pan and the balance is cleaned with a small brush to ensure more accurate reading then  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. Next all of the apparatus are cleaned and rinsed with distilled water before used to avoid contamination and 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 is felt to prevent the Scott bottles from breaking during the autoclaving process.

CONCLUSION

As a conclusion, we able to learn correct methods to prepare sterile nutrient agar for culturing microorganisms. Culture media must be stored at the specified temperature, under specified conditions such as pH and humidity. Direct sunlight have to be avoided at all times from exposure of culture media and their component. To prevent humidity of laboratory, all plastic containers are sealed. There are specific temperature for sterilization of culture media. The culture media need to be sterilized to make sure all pathogen was damaged. Besides that we managed to know the sterilization method and also know how to use autoclave. 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. Other precautions is when preparing commercial media, we must read the label and instruction on the container before use. In the progress of experiment, use distilled water to clean all the apparatus. Then, measuring cylinder is used to measure the volume of distilled water required accurately and stir the mixture continuously to ensure that the nutrient powder dissolves completely. Last but not least, autoclaving is a good sterilization process.

References

1. www.neogen.com/Acumedia/pdf/ProdInfo/7146_PI.pdf
2. http://www.csudh.edu/oliver/demos/bal-use/bal-use.htm
3. http://www.ehow.com/info_8131230_types-agar-plates.html
4. http://www.studentsguide.in/animal-biotechnology/animal-cell-and-tissue-culture/preparation-and-sterilization-of-medium.html