STERILIZATION AND MEDIA PREPARATION

 

STERILIZATION AND MEDIA PREPARATION

 

 Introduction:

 

Sterilization: Autoclaving is a process that use moist heat and pressure so that all parts of the material to be sterilized reach 121 degree Celsius 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 degree Celsius. The high pressure prevents solutions from boiling over at this temperature. Larger volumes require longer than 15 minutes to heat up to 121 degree Celsius throughout. After sterilization, the steam pressure is slowly decreased to atmospheric pressure. The sterilized objects can then be removed.

Media Preparation: Microorganisms depend on a number of factors such as nutrients, oxygen, moisture and temperature to grow and divide. In the laboratory, except for the above factors, the culture medium should be sterile and contamination of a culture with other organisms should be prevented.

The properties of agar which make it ideal in bacteriology are

1) Solid agar melts (dissolves) at 100 °C,

2) Remains solid at all incubation temperatures,

3) Transparent,

4) It is not heat-labile and therefore easily sterilized, and

 5) It is unaffected by almost all bacteria.

Liquid agar solidifies at 42-44 °C which is useful because sterile, heat-labile components such as antibiotics, blood, serum, carbohydrates and even bacterial cultures may be added before allowing the medium to solidify. Solid media generally contain agar at a concentration of 1.5%. Semi-solid media contain 0.05-0.3% agar and are useful in culturing anaerobic and microaerophilic organisms because such media form an oxygen gradient in test tubes, allowing all degrees of oxygen tension to exist in the culture vessels.

AIM:

            To understand the sterilization process using an autoclave and to learn the procedures used in preparing media needed for culturing microorganisms.

MATERIALS REQUIREMENT:

 

Commercial nutrient agar, Balance, Distilled water, Scott bottles, Measuring cylinder Beaker, Forceps, Universal bottles

PROCEDURE: STERILIZATION

The usual procedure for sterilization using the autoclave is as follows:

1. Open door, and place items to be sterilized into the autoclave chamber.

2. Close door. Push down door lock lever until door studs are completely in place.

3. Turn hatch wheel clockwise until it is secured tightly.

4. The temperature of the autoclave is set at 121°C.

5. Set timer by turning the large knob just below the hands to the desired setting.

6. Crank operating handle around to the Sterilize position till the red steam light goes on.

7. Wait and be sure the chamber reaches the proper temperature and pressure.

9. The Slow Exhaust and Fast Exhaust & Dry cycles both take 12-15 minutes longer than the time set to finish.

10. At the end of the run the white STERILE light will go on, and a loud, obnoxious buzzer will come on:

a. Rotate the operating handle all the way to OFF. Check that the chamber pressure is zero, and the temperature is below 100 °C.

b. Turn hatch wheel counterclockwise, push up door lock lever and slowly open door.

c. Use heatproof gloves to remove materials.

11. Allow liquid materials to cool before tightening caps.

PROCEDURE MEDIA PREPARATION:

 

1.      Measure approximately 250 ml of distilled water in a 1 L graduated cylinder and pour into a 1 L flask.

2.      Weigh out 1.5 g beef extract and 2.5 g peptone and add into the flask. Use approximately 100 ml of the water to rinse any powder stuck to the side of the flask down into the mixture.

3.      Stir over gentle heat from a bunsen burner to dissolve completely.

4.      Pour the mixture into the 1 L graduated cylinder and add warm water to the 500 ml mark. Pour back into the flask.

5.      Check the pH of the medium and adjust to pH 7.

6.      Autoclave the flask and the petriplates for 15 minutes at 121 °C and 15 lb/in2 pressure at the slow exhaust mode.

7.      After removing the media from the autoclave, allow the media to cool, and store for later use.

8.      Lay your petri dishes on the bench. The cover should be on top. Light your Bunsen burner, and then remove the NA flask from the water bath.

9.      Remove the tapes and cotton plug from the flask. Carefully flame the neck of the flask, open the plate cover about half way and pour the media in to petri plate about 30 ml.

10.  Flame the neck of the flask between each plate. Allow plates to solidify completely, which will take 15 minutes. Then invert, label and incubate at 37 °C overnight to dry off excess moisture and check for contamination.

 

CONCLUSION:

Different types of agar are needed for the cultivation of different types of microorganisms. Agar of the same composition with the commercial agar can be made by following the correct procedures. Preparation and sterilization of culture media should be done with great care to avoid contamination of unwanted microorganisms. We had learnt the preparation and sterilization of culture media via autoclaving process and the precaution that we need to take into consideration when handling this experiment.