Lab Report 3 - Preparation and sterilization of culture media
PREPARED BY:
GROUP 5
PREPARED FOR:
PROF. DR. LIONG MIN TZE
LECTURER OF IBA 104 – PRACTICAL FOR TECHNOLOGISTS
DATE: 24 MAY 2023
TITLE :PREPARATION AND STERILIZATION OF CULTURE MEDIA
1.0 Objectives
To prepare sterile nutrient agar for culturing microorganisms
2.0 Introduction
Culture media is a media that supplies important nutrients that are needed for microbes to grow under optimum condition. A gelatinous substance known as a culture medium contains vital nutrients needed to cultivate the desired microorganisms for future use. Nowaday, a wide variety of commercial cultural media are available in the market. Commercial culture media are usually in powder form with peptone, yeast extract, sodium chloride and agar powder as the ingredients. With the addition of water, the culture media formed.
Heat-sterilization, which is autoclaving, was selected as the method for sterilization of culture media as it is simple and more efficient in the laboratory compared to other methods. The culture media was sterilized in autoclave under the pressure of 103 kPa, temperature of 121°C for 15 minutes as the autoclave forced the moist, hot steam water into the chamber inside the autoclave to destroy the microorganism within the culture media. Steam was continuously forced into the chamber until the pressure inside the chamber achieved approximately 103 kPa, and cooled down after this pressure. Microbes within the culture media died due to the extreme condition.
There are two types of culture media prepared which are commercially nutrients agar solution which contain the typical used recipe for culture media and Luria-Bertani (LB) which contain tryptone, sodium chloride, yeast extract and agar powder. Both culture media were sterilized under the same method and condition.
3.0 Materials and Reagents
Commercial nutrient agar powder, yeast extract powder, tryptone(a nitrogen source), sodium chloride(NaCl) , agar powder, distilled water
4.0 Apparatus
Autoclave, measuring cylinder, Scott bottles, 500 ml beaker, spatula, electronic balance
5.0 Procedure
Part A : Preparation of commercial nutrient agar:
8.4 g of commercial nutrient agar powder was weighed using electronic balance and added in a beaker.
300ml of distilled water was added into the 500 ml beaker.
The solution was mixed in the beaker using a spatula.
Once the agar powder was dissolved, the agar solution was poured into a Scott bottle.
After Scott bottles are covered with a cap, it is prepared for sterilization.
Picture 1: Commercial nutrients agar solution in Scott bottle
Before initial preparation of the Luria-Bertani medium, the mass of each of the materials should be calculated.
Part B : Preparation of Luria-Bertani medium (LB)
The required amounts of each material below was weighed and measured by using electronic balance :
1.5 g of yeast extract
3.0 g of tryptone
3.0 g of sodium chloride (NaCl)
4.5 g of agar powder
The required amounts of material above were all added into a 500 ml beaker .
300ml of distilled water was added into the 500 ml beaker.
The agar solution was mixed well in the 500 ml beaker by using a spatula.
Once all the materials were dissolved, the agar solution was poured into the Scott bottle.
After Scott bottles are covered with caps, it is prepared for sterilization.
Picture 2: Luria-Bertani medium in Scott bottle
Part C : Sterilization of culture media
Both Scott bottles which were prepared in part A and part B were labelled and placed into an autoclave and subjected to high-pressure steam at 121°C (250°F) for 15 minutes.
Both media were sterilised at 121°C for 15 minutes.
After sterilization, the steam pressure was slowly decreased to atmospheric pressure, and the broth was allowed to cool to room temperature.
Tightened the cap of each bottle after the broth had cooled.
Picture 3: The Scott bottle was labelled
Picture 4: The Scott bottle was placed into the chamber of autoclave
Precaution :
1. Do remember to close the cap of the Scott bottle loosely or not too tight as the high pressure and temperature inside the autoclave may cause the Scott bottle to explode.
2. Aseptic technique may be applied to minimizing contamination.
6.0 Results
Calculation:
In order to prepare a culture media with 300 ml volume, mass of materials need to be calculate ：
Part A : Commercial nutrient powder
For commercial powder, the unit is 28 g / l which label on the material packaging
28g1000ml
x300ml
x=(28300)1000
= 8.4g
Hence, 8.4 g of commercial nutrient powder was needed.
Part B : Luria-Bertani medium (LB)
For yeast extract , the unit is 5 g / l which label on the material packaging
Yeast extract :
5g1000ml
q300ml
q=(5300)1000
q=1.5g
Hence, 1.5 g of yeast extract was needed.
For tryptone, the unit is 10 g / l which label on the material packaging
Tryptone :
10g1000ml
r300ml
r=(10300)1000
r=3.0g
Hence, 3.0 g of tryptone was needed.
For sodium chloride, the unit is 10 g / l which label on the material packaging
Sodium chloride ：
10g1000ml
p300ml
p=(10300)1000
p=3.0g
Hence, 3.0 g of sodium chloride was needed.
For agar powder, the unit is 15 g / l which label on the material packaging
Agar powder
15g1000ml
p300ml
p=(15300)1000
p=4.5g
Hence, 4.5 g of agar powder was needed.
7.0 Discussion
Sterilization is a method which is carried out to completely remove, destruct or eliminate all the viable organisms or inhibit the growth of all the living forms especially the microbes from any object or place. Sterilization of the culture media was needed before the culture media was utilised to prevent contamination of the culture media which may have caused the deviation of the results obtained. There were various sterilzation procedures such as heat sterilization, radiation sterilization and so on. In this experiment, heat sterilization which is autoclaving was selected as it is the most effective and efficient method. (Merck.K.D., 2023)
For preparation of culture media, two types of culture media were prepared which are commercially nutrient agar and Luria-Bertani medium (LB) . Both media should be prepared by using aseptic techniques to avoid contamination. Since the typical culture media recipe which is labelled on the material packaging only produces 1 litre culture media with g / l unit, the volume other than 1 litre needs to be recalculated to the required amount of values. (Mettler. T., 2023) . Therefore, the material required in part A and part B was calculated.
For commercially nutrients agar powder, it contains a lot of nutrients that are suitable for most microbes to grow. For the Luria- Bertani medium, it contains yeast extract, tryptone, sodium chloride and agar powder. Yeast extract was added for the optimum growth for bacterial multiplication. Tryptone was added as a source of nitrogen and carbon for microbes growth. Sodium chloride was also added for osmotic equilibrium and agar powder to favours the growth of aerobes as well as anaerobes. (Jain, A., Jain, R., & Jain, S., 2020)
For part A, the required amount of commercial nutrient agar powder was 8.4 g and added with 300 ml of distilled water to make a 300 ml culture media. For part B, 1.5 g of yeast extract, 3.0 g of tryptone, 3.0 g of sodium chloride (NaCl) and 4.5 g of agar powder was added with 300 ml of distilled water to make a 300 ml culture media. This result was calculated by using a ratio method which included the mass of material and volume of culture media. After adding the required amount of material for Luria- Bertani medium, continuously stirring was needed followed by the heating to dissolve the agar powder as it is difficult to dissolve. Media with agar should be soaked sufficiently with proper agitation before heating. (Merck.K.D., 2023)The colour of the culture media needs to be consistent, it should be golden colour which indicates that it contains agar powder. Any increase of colour may be caused by incorrect way of autoclaving or wrongly preparing for culture media. (Gamborg, O. L., & Phillips, G. C., 1995)
After the preparation, sterilization of culture media also was done to avoid contamination. Heat-sterilization which is autoclaving was selected. The Scott bottle which contains culture media was put into the autoclave for sterilization. Autoclave involves the heating of water to produce a high temperature steam inside the chamber so that the pressure inside the autoclave is able to achieve the 108 k Pa. The culture media was exposed under the condition, pressure of 108 kPa, temperature of 121°C for 15 minutes inside the autoclave. This is because most of the microorganisms will die under this extreme condition after 15 minutes. (Jackie.R., 2019)
8.0 Conclusion
Culture media are nutrient and mineral rich mediums that facilitate the development of microorganisms in the lab. It must be kept in the suitable place under optimum temperature, humidity and pH levels. To prevent contamination, culture media were also sterilised. Autoclaving, which is a method of heat sterilization, was chosen to destroy the undesired microbes. The autoclave was used to sterilise the Scott bottle containing the culture media. In order for the autoclave's internal pressure to reach 108 k Pa, water must be heated in order to create a high temperature steam inside the chamber.
The culture medium was subjected to a temperature of 121°C and a pressure of 108 kPa for 15 minutes. This is due to the fact that under these harsh conditions, the majority of microbes died within 15 minutes. Eventually, a sterilized and microbes-free culture media was obtained and may be used for the culturing of microorganisms Any precautionary measures should be properly followed to prevent unintended mistakes. For instance, before starting the preparation of culture media, sanitised the workplace to avoid contamination.
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