IBA104 PRACTICAL FOR TECHNOLOGIST - PREPARATION AND STERILIZATION OF CULTURE MEDIA
UNIVERSITI SAINS MALAYSIA
IBA104 - PRACTICAL FOR TECHNOLOGIST
LAB REPORT 3
GROUP 2
EXPERIMENT 3 : PREPARATION AND STERILIZATION OF CULTURE MEDIA
PREPARED BY :
LECTURER : PROF. DR. LIONG MIN TZE
LECTURER OF IBA104- PRACTICAL FOR TECHNOLOGIST
DATE : 25th MAY 2023
INTRODUCTION
Culture media is a solid, liquid, semi-solid that contains nutrients and is used to grow bacteria or microorganisms. It is also a nutrient framework for the growth and reproduction of microbes, plants, and animals that are made up of a combination of different nutrients. Carbohydrates, nitrogen, inorganic salts, trace elements, vitamins, and water are the most common ingredients. The culture media is not just the basic material that provides cell nourishment and promotes cell proliferation, but it is also the living environment in which cells develop and reproduce.
In this study, the preparation of culture media and nutrient mediums will be learned. Culture media are available commercially as powder. It can be prepared in either solid or liquid form. Nutrient medium purpose preparation for culturing microorganisms. This broth contains ‘Lab-lemco’ powder, yeast extract, peptone, sodium chloride and agar powder. The final pH of both media is 7.4.
Autoclaving is the most effective way to sterilize the dedicated laboratory equipment of liquid treatment products, which can kill harmful bacteria, viruses, fungi and spores. The phenomenon of the autoclaving process uses water or steam at high pressure to the boiling point.₁ All parts of the material to be sterilized reach 121⁰C for 15 minutes. An autoclave is a large pressure cooker and a chamber which may be sealed off against surrounding air.
In an autoclave, steam plays a critical role in the sterilization process. The heating process ensures that the steam reaches and maintains the desired sterilization temperature that is 121⁰C for the larger volume requires longer than 15 minutes. When the chamber temperature increases, the water vapor in the steam becomes superheated and reaches high temperatures. After the condensation process, a drying phase may follow the sterilization process.₂ The high temperature, pressure, and moisture of the steam work together to achieve successful autoclave sterilization.
MATERIALS AND REAGENTS
Commercial nutrient agar
Yeast extract
Tryptone
Sodium chloride
Agar
Balance
Distilled water
Scott bottles
PROCEDURE
Preparation of nutrient agar and Luria Bertani
Amount of broth (with agar) powder is weighed appropriately into Scott bottles and dissolved with distilled water. The broth is well mixed.
The bottles are recap loosely and set aside for sterilization.
The steps above are repeated to prepare 300 mL per group Luria Bertani agar.
1.5 g of yeast extract, 3.0 g tryptone, 3.0 g of sodium chloride and 4.5 g agar is weighed appropriately into Scott bottles and dissolved with distilled water.
The broth is well mixed.
Then, the bottles are recap loosely like a nutrient agar bottle and set aside for sterilization.
B. Preparation for autoclaved
All media is sterilized at 121°C for 15 minutes.
The media is removed after autoclaving. The broth preparation is allowed to cool then the cap of each bottle is tightened.
RESULTS
In this experiment, nutrient agar has been prepared by dissolving nutrient agar powder in 1000 mL of distilled water. Calculation has been used for the 300mL nutrient agar solution.
Weight of nutrient agar/mL = 28g/1000mL = 0.028 g
Weight of nutrient agar/300mL = 0.028g x 300 = 8.4 g
Thus, the weight of nutrient agar powder prepared for a 300mL solution of nutrient agar is 8.4g/L to be dissolved in 300mL of distilled water.
For the preparation of Luria Bertani agar is based on the calculation for 300mL solution :
5g of Yeast extract :
Weight of yeast extract = (5g x 300)/1000mL = 1.5 g
10g of Tryptone :
Weight of Tryptone = (10g x 300)/1000mL = 3.0 g
10g of NaCl :
Weight of NaCl = (10g x 300)/1000mL = 3.0 g
15g of agar :
Weight of agar powder = (15g x 300)/1000mL = 4.5 g
Thus, the calculations above have been used to prepare a 300mL solution of Luria Bertani agar.
DISCUSSION
General nutrition plates are available in a number of varieties. Luria BertaniLB agar is an ordinary nutrient for the normal evolution of bacteria, and does not specifically adapt to specific bacterial types. Miller's LB agar, which has variable amounts of the same substances in it, is a kind of LB. Trypticase Soy Agar (TSA) is another broadly applied medium made of casein, soybean meal used as an initial growth medium in order to evaluate bacterial morphology or stimulate bacterial growth for analysis and storage. PEA, which selectively inhibits Staphylococcus species and inhibits Gram negative bacteria by means of phenylethyl alcohol agar.
Brain Heart Infusion BHI agar is a general purpose medium, which can be cultivated for various organisms including bacteria, yeasts and moulds. The infusion of the brain's heart, peptone and dextrose components produces nutrients in BHI agar. Organic nitrogen, carbon dioxide, sulphur, vitamins and trace elements have been found in the peptone and infusion. The carbohydrates that bacteria use in their fermentation process are dextrose. Disodium phosphate has been added to buffer the medium.
Luria Bertani was the nutritional agar plates we'd prepared for this experiment. The initial measurements for the manufacture of Luria Bertani included 5g yeast extract, 10 g tryptone, 10 ml NaCl and 15 ml agar powder that was mixed into 1000 mL dilute water. A few modifications have been made to the 300 ml solution of luria Bertani agar, which contains 1.5mg yeast extract, 3 mg tryptone, 3 mg NaCl and 4.5g saran powder diluted in 300 ml distilled water. There are a number of ingredients in the media that have special purposes, with tryptone and yeast extract playing an important role in helping to create new organisms. They're providing a source of amino acid for the developing bacteria. NaCl is designed to maintain the equilibrium of otic pressure within the medium. Inflows and swelling are due to a decrease in the external otic pressure, whereas an increase is associated with water leakage and dehydration. When agar powder is added to Luria Bertani, it acts as an insulating agent and provides the bacteria with a firm surface of growth.
In this study, we focused on preparation of agar plate culture media. In the process of preparing culture media, there are many precautions steps to be taken. This is because to ensure the accuracy composition of nutrients so the bacteria could grow perfectly inside the culture media.
Before the culture agar powders could be weighed, the analytical balance must be cleaned with a small brush or tissue so that it is free from any dust or unwanted residue. The door of the balance machine should also be closed before weighing any substances and ‘tare’ button can only be pressed after the container is put onto the balance. Also, the balance should be allowed to stabilize for 1 minute after it has been on. These steps are important in order to obtain an accurate reading. Furthermore, the powdered components must be applied cautiously and gently using a spatula to avoid spillage. As a result, reliable weight readings of powdered materials are possible.
Furthermore, accurate measurements using the measurement cylinder shall be made of the volume of distilled water. It is for this reason that the special distillation volume necessary to produce a certain amount of agar powder must clearly be indicated on its label. Moreover, the measurement cylinder shall be placed on a flat solid surface in order to measure the volume of water for distillation. To obtain an accurate reading, the eyes must be at a level that is equal to the curvature of liquid meni
scus. It is not appropriate to fill the measuring bottle with distilled water completely, because there should be a surplus of distilled water that can be used for washing leftover powders out of the weighing vessel.
In the final step the culture medium will undergo the sterilizing process. Sterilization is defined as the complete destruction of all forms of microbial life, including bacterial spores. Steam sterilization generally refers to heating in an autoclave employing saturated steam. High pressures enable steam to reach high temperatures, thus increasing its heat content and killing power.
When all of the medium have been poured into the Scott bottles, we have to label the bottle properly for identification and loosen the cap of the bottles before putting them into the autoclave machine. Because the autoclave is operated in a high pressure environment. Loosening the cap will allow expansion of the bottle so that the bottles will not be broken or worst explode. In order to determine whether the bottle has been subjected to a suitable autoclave procedure, an autoclave tape was affixed to the bottles as well. At a temperature of 121℃, the autoclave will run for 15 minutes.
However, after the autoclaving process has ended, the Scott bottles will be remaining in the autoclave overnight in warm condition around 50℃. the culture medium can be removed from the machine and the cap of the bottles should be tightened. The bottles should also be turned over again for a few times to allow a balance distribution of agar in the bottle so that no agar will solidify at the bottom of the bottle. This step is to make sure that the culture agar can be poured inside the sterilize petri dish.
CONCLUSION
The manufacturer's directions are followed for preparing media from dehydrated commercial formulations. After being created, microbiological media still need to be sterilised due to microbial contamination from the air, glassware, and hands. The technique for preparing microbial culture media required that media containing agar be sufficiently saturated and well agitated before heating. The medium must be adjusted for pH before being poured into the proper containers for autoclave sterilisation using moist heat. For this experiment, the media must be autoclaved for at least 30 minutes at 121-123°C to achieve complete sterilisation. Saturated steam that has been used is under at least 15 psi of pressure. The caps are tightened after allowing the broth to cool. The media are available for culturing the cells.
REFERENCES
Microlit. (2021, February 24). What is autoclaving and how is it relevant to liquid handling instruments? Microlit. https://www.microlit.us/how-autoclaving-is-relevant-to-liquid-handling-instruments/
Scott, K. (1995). BIOTECHNOLOGY AND MEDICAL APPLICATIONS. In Handbook of Industrial Membranes (pp. 655–680). Elsevier.
Media preparation
Microbial culture media preparation
Hakobyan, L., Gabrielyan, L., & Trchounian, A. (2012). Yeast extract as an effective nitrogen source stimulating cell growth and enhancing hydrogen photoproduction by Rhodobacter sphaeroides strains from mineral springs. International Journal of Hydrogen Energy, 6519- 6526.
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