LAB 3 : PREPARATION AND STERILIZATION OF CULTURE MEDIA

Laboratory's Information :

Laboratory's Number - LAB A203

Laboratory Title : PREPARATION AND STERILIZATION OF CULTURE MEDIA

Lecturer's Name : Associate Professor Dr. Liong Min Tze

Laboratory Assistant : Madam Najmah

Group Members : Charles Ng Wai Chun, Siti Hawa Binti Ramli, Nuramirah Binti Ramlan

Date of Laboratory : 13 October 2015

LAB 3 : PREPARATION AND STERILIZATION OF CULTURE MEDIA

Introduction

Microbes require nutrients to grow. These are supplied by either solid or liquid culture media. The standard solid medium is nutrient agar, a gelatinous substance derived from seaweed. The basic liquid medium is nutrient broth, typically a mix of water, meat extract peptone, and sodium chloride. The broth contains:

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

1.5 g/L yeast extract

5.0 g/L peptone (a nitrogen source)

5.0 g/L sodium chloride

15 g/L agar powder

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

An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C (249°F) for around 15–20 minutes depending on the size of the load and the contents. It was invented by Charles Chamberland in 1879, although a precursor known as the steam digester was created by Denis Papin in 1679. The name comes from Greek auto-, ultimately meaning self, and Latin clavis meaning key—a self-locking device.

Sterilization autoclaves are widely used in microbiology, medicine, podiatry, tattooing, body piercing, veterinary science, mycology, funeral homes, dentistry, and prosthetics fabrication. They vary in size and function depending on the media to be sterilized. Typical loads include laboratory glassware, other equipment and waste, surgical instruments and medical waste.

A notable growing application of autoclaves is the pre-disposal treatment and sterilization of waste material, such as pathogenic hospital waste. Machines in this category largely operate under the same principles as conventional autoclaves in that they are able to neutralize potentially infectious agents by utilizing pressurized steam and superheated water. A new generation of waste converters is capable of achieving the same effect without a pressure vessel to sterilize culture media, rubber material, gowns, dressing, gloves, etc. It is particularly useful for materials which cannot withstand the higher temperature of a hot air oven.

Autoclaves are also widely used to cure composites and in the vulcanization of rubber. The high heat and pressure that autoclaves allow help to ensure that the best possible physical properties are repeatably attainable. The aerospace industry and sparmakers (for sailboats in particular) have autoclaves well over 50 feet (15 m) long, some over 10 feet (3.0 m) wide.

Objective

To prepare sterile nutrient agar for culturing microorganisms.

Materials and Reagents

1) Commercial Nutrient Agar

2) Brain Heart Infusion Broth (BHI)

3) Trypticase Soy Broth (TSAYE)

4) Peptone powder

5) Beef extract powder

6) Sodium chloride

7) Yeast extract

8) Electronic Weighing Balance

9) Measuring cylinder

10) Scott bottles

11) Distilled water

12) Beakers

13) Glass rod

Procedure & Results

1) Agar powder is weighted approximately in the beaker the it is dissolved with distilled water. The broth is mixed well.

2) The bottles are loosely recapped and set aside for sterilization.

3) All media is sterilized at 121°C for 15 minutes.

4) After autoclaving, the media is removed. The broth preparation is allowed to cool then the cap of each bottle is tight.

For step 1),

4 different types of culture media were prepared which are

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

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

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

IV) 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.

             

One of the ingredients in Self - made nutrient agar,
peptone, is weighed using an Electronic Weighing Balance

All the ingredients were well prepared for the self-made nutrient agar culture media..

All the culture media were prepared

Discussion

To prepare a good culture media with accurate nutrient content and with no contamination,there are several precaution steps that we need to take when conducting the experiment.

1) Balance (Electronic Weighing Balance)

• The precise and appropriate amount of broth powder and agar powder is weighed using electronic analytical balance which has the precision of one hundredth of a gram, ±0.01 or one ten-thousandth of a gram, ±0.0001 g.
• The surrounding of the pan and the pan of the balance must be clean without any debris or dusts. Place the receiver on the center of the pan of the balance and close the balance door. Then, press the appropriate tare key on the balance to set the signal from the strain gauge to zero so that the weight of the receiver is no longer indicated. With careful handling, add the powdered materials using a spatula until the desired amount is reached. Always handle the spatula filled with powdered materials with care to avoid spilling.
• If solids are spilled, remove the receiver and sweep out all of the spilled material from the balance using a brush.The spilled material must be properly disposed.

2) Autoclave (Autoclaving process)

• Before using the autoclave, check the drain screen at the bottom of the chamber.
• In order to have a high effectiveness of the autoclaving process, the autoclave must reach and maintain a temperature of 121-123 degree Celcius for at least 30 minutes. This is achieved by using saturated steam under at least 15 psi of pressure.
• All the debris must be removed and cleaned 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 should between range of low and high. If there are too low water level, water should be added in.
• The cap of the Scott bottles must not be too tight to prevent breakage off the Scott bottles.                      
• However, the cap of the Scott bottles must not be too loose to prevent the outflow of media from inside of the Scott bottles.   

• Autoclave doors must be firmly closed and locked into place before it starts running.                                                   
• Do not stack or store combustible material next to an autoclave (cardboard, plastic, volatile or flammable liquids).  
• Heat resistant gloves should be used when removing materials after sterilization to prevent any injure from the heat inside the autoclave.       
• Avoid touching the inner chamber surfaces after sterilization.
• To be effective the autoclave must reach and maintain a temperature of 121-123 degree Celcius for at least 30 minutes. This is achieved by using saturated steam under at least 15 psi of pressure.

.Scott bottles are ready to be sterilized in a autoclave.

3) Agar (Culture media)

• The ingredients and the uses of the agar have to be clearly known before we use it as the culture media.
• Commercial Nutrient agar is one of the most commonly used nutrient agar as culture media.
• Self - made Nutrient agar contains ingredients like "Lab-lemco" power (a beef extract), yeast extract, peptone, sodium chloride and agar powder is another agar used as culture media.
• Trypticase Soy agar (TSAYE) is a medium made with casein and soybean meal and is used as initial growth medium to observe bacterial morphology or increase bacterial growth for analysis or storage.
• Brain Heart Infusion (BHI) agar is a general purpose medium suitable for the cultivation of a wide variety of organism types, including bacteria, yeasts and moulds. The BHI agar derives its nutrients from the brain heart infusion, peptone and dextrose components. The peptones and infusion are sources of organic nitrogen, carbon, sulfur, vitamins and trace substances. Dextrose is the carbohydrate source that microorganisms utilize by fermentation action. The medium is buffered through the use of disodium phosphate.

Other precautions:

• Read the labels and instructions on the container before using them in preparing the culture media.                              
• Use distilled water to clean all the apparatuses that needed to be used in the experiment.                                                          
• Measuring cylinder is used to measure the volume of distilled water required accurately (Avoid using beaker to measure the volume of distilled water).
• Stir the mixture evenly to ensure that the nutrient powder dissolves completely.

Conclusion

       As a conclusion, from the experiment that we conducted, we have learned the correct techniques in preparing commercial and own recipe culture media based on the ingredient listed. Culture media must be stored at the specified temperature, under specified conditions such as pH and humidity . Exposure of sunlight to the culture media and their components has to be avoided . Moreover, the culture media must be sterilized before we can use it. One of the sterilizing methods is autoclaving which we were able to learn how to operate a autoclave machine with proper manners.

Reference

1) American Society for Microbiology (2014), Culture Media. Retrieved from http://www.microbeworld.org/careers/tools-of-the-trade/culture-equipment/culture-media

2) Wikipedia, the free encyclopedia (18 October 2015). Retrieved from https://en.wikipedia.org/wiki/Autoclave

3) Sagar Aryal (15 April 2015), Nutrient agar: Composition, Preparation and Uses. Retrieved from http://www.microbiologyinfo.com/nutrient-agar-composition-preparation-and-uses/