How to prepare MRS agar for Lactobacillus spp.

Introduction:

Welcome to the course on preparing MRS agar with dry CaCO3.

• This course will provide you with step-by-step instructions on how to prepare the medium, including sterilization, pH adjustment, and CaCO3 addition.
• MRS agar is a commonly used medium for cultivating lactic acid bacteria, and the addition of CaCO3 can help regulate the pH of the medium and improve bacterial growth.
• In this course, we will cover the materials needed, the steps involved, and some important tips to ensure that your MRS agar with CaCO3 is prepared in a scientific and reliable manner.

When it comes to plating techniques, lactobacilli can be grown on both pour and spread plates.

However, pour plates are typically used for enumerating bacterial populations, while spread plates are generally used for isolating individual colonies.

So the choice between pour or spread plating may depend on the specific experimental needs and objectives.

Materials:

The materials needed for preparing MRS agar with CaCO3 include:

• MRS agar powder
• Calcium carbonate (CaCO3)
• Distilled water
• Autoclave or pressure cooker
• Sterilized petri dishes
• Hot air oven
• Weighing scale
• Sterile spatula
• pH meter or indicator paper

Steps:

1. Weigh out the desired amount of MRS agar powder using a weighing scale, and add it to a flask containing the appropriate amount of distilled water according to the manufacturer's instructions.
2. The typical ratio is 50g of MRS agar powder in 1 liter of water.
3. Mix the agar powder and water thoroughly until it dissolves completely.
4. Autoclave or pressure cook the medium at 121°C and 15 psi for 15-20 minutes to sterilize it.
5. While the MRS agar is autoclaving, weigh out 1% of the total medium weight of CaCO3 powder (e.g. 10g CaCO3 powder for 1 liter of medium).
6. Preheat the dry CaCO3 powder in a hot air oven at 180°C for 2 hours to sterilize it.
7. After autoclaving, let the MRS agar cool down to around 50-60°C, so it is not solid but still liquid.
8. Add the preheated CaCO3 powder to the MRS agar using a sterile spatula.
9. Mix the agar thoroughly to distribute the CaCO3 evenly throughout the medium.
10. Caution: The medium may still be hot, so take necessary precautions to prevent burns.
11. Check the pH of the medium after adding the CaCO3 and adjust if necessary.
12. The typical pH range is 6.2-6.6.
13. Allow the MRS agar with CaCO3 to cool to room temperature.
14. Make sure it is still in a liquid state and not yet solidifying.
15. Pour the medium into sterile petri dishes using sterile techniques to minimize the risk of contamination.
16. Allow the medium to solidify in the petri dishes at room temperature or in a refrigerator at 4°C.

Conclusion:

• Congratulations, you have successfully completed the course on preparing MRS agar with dry CaCO3!
• By following these steps, you should be able to prepare the medium in a scientific and reliable manner.
• Remember to use proper sterile techniques and maintain a clean workspace to avoid contamination of the medium.
• With practice, you will be able to prepare MRS agar with CaCO3 efficiently and effectively.

Did you know?

• The addition of CaCO3 to MRS agar can help regulate the pH of the medium and improve bacterial growth.
• Lactic acid bacteria, which are commonly grown on MRS agar, produce lactic acid as a byproduct of their metabolism, which can lower the pH of the medium and inhibit bacterial growth.
• CaCO3 acts as a buffer, maintaining the pH of the medium at a desirable level for bacterial growth.
• CaCO3 also provides a source of carbon for the bacteria to utilize.
• Some strains of lactic acid bacteria have the ability to produce extracellular enzymes that can degrade CaCO3, releasing carbon dioxide and making the carbon available for bacterial metabolism.
• The addition of CaCO3 to MRS agar is not always necessary or desirable, depending on the specific strains of bacteria being grown and the experimental conditions.
• Therefore, it is important to consult the literature or a microbiology expert to determine whether or not to add CaCO3 to your MRS agar medium.
• Additionally, it is important to ensure that the CaCO3 is sterilized before adding it to the medium, as it can be a source of contamination if not properly treated.
• Preheating the dry CaCO3 powder in a hot air oven is an effective way to sterilize it before use.

Also,

• MRS agar is named after the three scientists who first developed the medium in 1960: de Man, Rogosa, and Sharpe.
• The medium was designed to support the growth of lactic acid bacteria, specifically those from the genus Lactobacillus, which are commonly found in the gastrointestinal tract of humans and animals.
• The medium is a selective and differential medium, which means that it contains ingredients that selectively promote the growth of certain types of bacteria while inhibiting others, and it also allows for differentiation between different types of bacteria based on their physical and biochemical properties.
• The addition of various nutrients and supplements to the base medium can modify its selectivity and allow for the growth of a broader range of bacteria.
• Today, MRS agar is widely used in microbiology research for the cultivation, isolation, and identification of lactic acid bacteria, including those used in the production of fermented foods and probiotics, as well as those associated with human and animal health.

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