LAB 3: PREPARATION AND STERILIZATION OF CULTURE MEDIA

Introduction

      A culture media or growth media is a liquid or gel substance which is used for the growth of microorganisms and cells. In the culture media contain nutrients to support the growth of microorganisms. Different types of cell and microorganisms require different types of culture media. Initially, the culture media is in powders state, the addition of water is required when we want to use it. For culturing microorganisms which are not nutritionally fastidious, nutrient medium is a general purpose preparation. The self-made nutrition agar broth contains:

1.5 g/L “Lab-lemco” powder (a beef extract)

1.5 g/L Yeast extract

5.0 g/L Peptone (nitrogen source)

5.0 g/L Sodium chloride

15.0 g/L Agar powder

The ready-made nutrient agar contains 15 g/L agar and the same contents as the manually-made nutrient medium. The final pH of both media has to be 7.4.

                   

                     (Figure 1: Different types of culture media)

      Autoclaving is the process of sterilizing supplies and equipment with the use of high pressure that is saturated at 250°F (121°C) for about 15 minutes. Autoclaves are machines that make use of pressurized steam in order to eliminate microorganisms. Autoclaving is considered the most reliable system for the sterilization and decontamination of reagents, laboratory glassware, waste and other media.

      We put in the materials into the autoclave for sterilization and seal the chamber. And then, all the air is removed from it either by a simple vacuum pump or by pumping in steam to force the air out of the way. Next, steam is pumped through the chamber until it reach 103kPa above atmospheric pressure, so it reaches a temperature of about 121–140°C (250–284°F). The steam pumping continues 15 minutes (large volumes item require longer than 15 minutes).After sterilisation, the steam pressure and temperature will be decreased slowly to the atmospheric pressure and room temperature. Then the sterilized materials can be taken out.

                

                                (Figure 2: Autoclave in laboratory)  

Objective

To prepare sterile nutrient agar for culturing microorganisms.

Materials and Reagents

Commercial Nutrient Agar

Brain Heart Infusion Broth (BHI)

Trypticase Soy Broth (TSAYE)

Peptone powder

Beef extract powder

Sodium chloride

Yeast extract

Electronic Weighing Balance

Distilled water

Scott bottles

Measuring cylinder

Glass rod

Beakers

Results

For the preparation of each type of agar, the appropriate amount of agar powder is weighed and dissolved in subsequent amount of distilled water in a beaker. The mixture is then mixed well by stirring it using a glass rod. The mixed solution is then transferred to a Scott bottle. After this, the cap of Scott bottle with the agar solution is slightly capped and is ready for the sterilisation. All media prepared is then sterilised with autoclaving machine at 121 °C, 103 kPa for 15 minutes. The media is allowed to cool after autoclaving and the caps of each Scott bottles is tighten.

After the lab, 4 culture media has been prepared by our group, which is:

a)     Commercial nutrient agar which is made from 11.2g of commercial nutrient agar powder dissolved in 400ml of distilled water.

b)    Brain Heart Infusion Agar (BHI) which is made from 5.2g of BHI agar powder dissolved in 100ml of distilled water.

c)     Trypticase Soy Agar (TSAYE) which is made from 4.0g of TSAYE agar powder dissolved in 100ml of distilled water.

d)    Self-made nutrient agar powder which is made from these ingredients:

i.                   0.6g of “Lab-lenco” powder

ii.                 0.6g of yeast extract

iii.              2.0g of peptone

iv.              2.0g of Sodium chloride (NaCl)

v.                 6.0g of agar powder

Where all the ingredients above dissolved in 400ml of distilled water.

(Photo 1: Bottles of ingredients powder)

(Photo 2: Weighing procedure of ingredient)

(Photo 3: Stirring the mixture of agar powder and distilled water)

(Photo 4: Transferring of mixed medium into Scott bottle)

(Photo 5: All Cultural Medium Prepared)

Discussions

There are some precautions that need to be followed during conduction of this lab work.

-  The pan of the balance is cleaned with a brush before we weigh culture medium powder to ensure that its surface is clean and more accurate during weighing.

-  The “tare” button is pressed every time after the empty beaker is put onto the balance to get the accurate reading and avoid zero error.

-  The distilled water was measured using measuring cylinder and the water should not pour all into beaker, because there should be some distilled water reserved for washing of leftover powder from the weighing beaker into beaker.

-  The correct amount of distilled water is added to make sure we produce the correct composition of culture media.

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

-  The entire medium has been poured into Scott bottle. The caps of the Scott bottles are slightly tightened to prevent the Scott bottle from breaking during autoclaving process.

Besides, before the Scott bottles with different medium placed in to the autoclaving machine for sterilization, these are the steps need to be following:

-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°C 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 room pressure before removing the basket with Scott bottles from the autoclave chamber.

-After autoclaving, the Scott bottle was removed from the autoclave machine and the cap of the bottle is tightened.

(Photo 6: Scott bottles placed inside the autoclaving machine)

Conclusion

The nutrients which can be found in each type of agar broth media are essential for the growth of microorganisms such as nitrogen source, carbon source, water are added. The pH of the media is also maintained to provide an adequate environment for microorganisms. The culture media are also sterilized to ensure there will be no other microorganism exist in it. All of the precaution steps should be carried out carefully to ensure there is no errors during the preparation of the cultural media. Autoclaving is an effective and efficient means of sterilization. In short, the proper ways to carry out the preparation and sterilization of culture media are very important to prevent contamination of the unwanted foreign microorganisms onto the agar medium.

References

http://en.wikipedia.org/wiki/Autoclave

http://wiki.answers.com/Q/Principle_of_autoclave

http://ibglabreportgroup22010.blogspot.my/p/lab-report-3.html

http://www.explainthatstuff.com/autoclaves.html

https://en.wikipedia.org/wiki/Growth_medium

http://labreport102.blogspot.my/2014/11/introduction-growthmedium-or-culture_24.html

http://elearning.usm.my/1516sem1/

http://ibg102.wordpress.com/