MRS Broth Test- Principle, Procedure and Results

MRS Broth Test is a microbiological test that is used to identify and enumerate lactobacilli in various samples. Lactobacilli are gram-positive, rod-shaped bacteria that are widely distributed in nature and are involved in many fermentation processes. They are also part of the normal flora of the human gastrointestinal tract and vagina, and have beneficial effects on health and immunity. Some lactobacilli are also used as probiotics, starter cultures, or bio-preservatives in food and dairy industries.

The MRS Broth Test is based on the ability of lactobacilli to grow and ferment glucose in a selective medium that contains various nutrients and inhibitors. The medium, known as MRS Broth or De Man, Rogosa and Sharpe Broth, was developed in 1960 by De Man et al. to enhance the growth of fastidious lactobacilli that were difficult to cultivate on other media. The test can be performed on pure cultures or mixed samples, such as milk, cheese, yogurt, wine, beer, silage, feces, etc.

The objectives of the MRS Broth Test are:

• To cultivate and enumerate lactobacilli in a laboratory setting
• To determine the ability of lactobacilli to form gas during glucose fermentation
• To differentiate lactobacilli from other bacteria based on their growth and gas production patterns

The MRS Broth Test is a simple, reliable, and inexpensive method to identify and quantify lactobacilli in various samples. It can be used for quality control, research, or diagnostic purposes. However, it also has some limitations and requires confirmation by other biochemical or molecular tests. In this article, we will explain the principle, procedure, results, limitations, and quality controls of the MRS Broth Test in detail.

MRS Broth Test is based on the ability of lactobacilli to grow and ferment glucose in a selective medium that inhibits the growth of other bacteria. Lactobacilli are gram-positive, rod-shaped bacteria that produce lactic acid as the major end product of glucose fermentation. They are widely distributed in nature and are involved in various food fermentation processes, such as yogurt, cheese, and sourdough bread. They are also part of the normal flora of the human gastrointestinal and urogenital tracts, where they play a role in maintaining the balance of microbial ecology and preventing infections by pathogenic bacteria.

The selective medium used in MRS Broth Test is MRS Broth, which was developed by De Man, Rogosa and Sharpe in 1960. MRS Broth contains various ingredients that provide optimal conditions for the growth and metabolism of lactobacilli. These ingredients include:

• Enzymatic digest of animal tissue, beef extract, and yeast extract: These are the sources of carbon, nitrogen, vitamins, and other essential nutrients for lactobacilli.
• Dextrose: This is the fermentable carbohydrate that lactobacilli can utilize to produce lactic acid and energy.
• Sodium acetate: This is an inhibitory agent that suppresses the growth of most other bacteria by creating an acidic environment and interfering with their membrane function.
• Ammonium citrate: This is an energy source and a selective agent that prevents the overgrowth of contaminating organisms by chelating metal ions and reducing their availability for bacterial growth.
• Potassium phosphate: This is a buffering agent that maintains the pH of the medium at around 6.5, which is favorable for lactobacilli but not for most other bacteria.
• Magnesium sulfate and manganese sulfate: These are cations that are involved in various enzymatic reactions and metabolic pathways of lactobacilli.
• Polysorbate 80: This is a surfactant that facilitates the uptake of nutrients by lactobacilli and enhances their growth.

The principle of MRS Broth Test is simple: A sample containing lactobacilli is inoculated into MRS Broth and incubated at 35-37°C for 24-48 hours. During this time, lactobacilli will grow and multiply in the medium, producing turbidity (cloudiness) that indicates their presence. Some lactobacilli will also produce gas (mainly carbon dioxide) as a by-product of glucose fermentation. This gas will be trapped in a small inverted tube (called Durham tube) that is placed inside the broth tube. The presence of gas bubbles in the Durham tube indicates a positive test for gas production.

By observing the turbidity and gas production in MRS Broth Test, one can determine whether the sample contains lactobacilli or not. However, this test does not differentiate between different species or strains of lactobacilli, which may have different characteristics and applications. Therefore, further biochemical tests are required to confirm the identity and properties of lactobacilli isolates.

MRS Broth is a complex and selective medium that supports the growth of lactobacilli and inhibits the growth of other bacteria. The composition of MRS Broth is as follows:

• Enzymatic digest of animal tissue: This provides carbon, nitrogen, and amino acids for the growth of lactobacilli.
• Beef extract and yeast extract: These are rich sources of vitamins, minerals, and other growth factors for lactobacilli.
• Dextrose: This is the fermentable carbohydrate that lactobacilli can use to produce lactic acid and other organic acids.
• Sodium acetate: This is an inhibitory agent that suppresses the growth of non-lactobacilli by lowering the pH of the medium.
• Ammonium citrate: This acts as a source of energy and a selective agent for lactobacilli. It also helps to buffer the medium against acidification by lactobacilli.
• Potassium phosphate: This is a buffering agent that maintains the pH of the medium at 6.5, which is optimal for lactobacilli.
• Magnesium sulfate and manganese sulfate: These provide essential cations for the metabolism of lactobacilli.
• Polysorbate 80: This is a surfactant that facilitates the uptake of nutrients by lactobacilli and enhances their growth.

The medium also contains a Durham tube, which is a small inverted tube inside the larger test tube. The Durham tube traps any gas produced by lactobacilli during fermentation. The presence or absence of gas in the Durham tube indicates whether the organism can form gas from dextrose or not.

The final pH of MRS Broth is 6.5 +/- 0.3 at 25°C. The medium is sterilized by autoclaving at 121°C for 15 minutes. The medium should be clear and light amber in color after sterilization. Any turbidity or discoloration indicates contamination or deterioration of the medium.

The procedure of MRS Broth Test is simple and straightforward. It involves the following steps:

1. Prepare MRS broth medium by dissolving 55 g of dehydrated powder in 1 liter of distilled water. Mix well and dispense into tubes containing inverted Durham tubes. Sterilize by autoclaving at 121°C for 15 minutes.
2. Inoculate MRS broth tubes with a loopful of an 18 to 24 hour culture of the organism to be tested. Alternatively, you can use a sterile swab to collect a sample from a colony on an agar plate and transfer it to the broth tube.
3. Incubate the inoculated tubes at 35°C to 37°C for 24 to 48 hours in ambient air. Do not shake or disturb the tubes during incubation.
4. Examine the tubes for turbidity and gas production. Turbidity indicates growth of the organism, while gas production is indicated by a bubble in the Durham tube. Record your observations and compare them with the expected results.

The results of the MRS Broth Test can be interpreted by observing the turbidity and gas production in the broth medium after incubation. The turbidity indicates the growth of lactobacilli, while the gas production indicates the fermentation of glucose by some lactobacilli species.

• If the broth is turbid and there is no gas bubble in the Durham tube, it means that the organism is a lactobacillus that does not produce gas from glucose fermentation. An example of such an organism is Lactobacillus acidophilus.
• If the broth is turbid and there is a gas bubble in the Durham tube, it means that the organism is a lactobacillus that produces gas from glucose fermentation. An example of such an organism is Leuconostoc sp.
• If the broth is clear and there is no gas bubble in the Durham tube, it means that the organism is not a lactobacillus or it does not grow well in this medium. An example of such an organism is Pseudomonas aeruginosa.

The results of the MRS Broth Test should be confirmed by further biochemical tests to identify the lactobacilli species. Some examples of biochemical tests are catalase test, Gram stain, carbohydrate fermentation test, and bile esculin test.

• MRS Broth Test is a selective and differential medium for the cultivation and enumeration of lactobacilli, but it is not a definitive identification method. Lactobacilli are a diverse group of bacteria that share some common characteristics, such as gram-positive, non-spore-forming, rod-shaped, and acid-tolerant. However, they also differ in many aspects, such as morphology, physiology, biochemistry, genetics, and ecology. Therefore, isolates from MRS Broth Test should be further confirmed by other tests, such as Gram staining, catalase test, carbohydrate fermentation test, and molecular methods.
• MRS Broth Test may not support the growth of some lactobacilli strains that have specific nutritional or environmental requirements. For example, some lactobacilli are obligate anaerobes that cannot grow in the presence of oxygen, while others are microaerophilic or facultative anaerobes that need reduced oxygen tension for optimal growth. Some lactobacilli are thermophilic or psychrophilic that require high or low temperatures for growth, while others are mesophilic that grow best at moderate temperatures. Some lactobacilli are fastidious or heterofermentative that need complex media or additional supplements for growth, while others are non-fastidious or homofermentative that can grow on simple media. Therefore, MRS Broth Test may not be suitable for all types of lactobacilli and may result in false-negative results.
• MRS Broth Test may also allow the growth of some non-lactobacilli organisms that can tolerate the selective agents and produce similar results. For example, some enterococci, streptococci, pediococci, leuconostocs, bifidobacteria, and propionibacteria can grow in MRS Broth Test and produce acid and/or gas from glucose fermentation. These organisms may be confused with lactobacilli if only MRS Broth Test is used for identification. Therefore, MRS Broth Test may not be specific enough to differentiate lactobacilli from other related bacteria and may result in false-positive results.

Quality control is an essential step in any laboratory procedure to ensure the accuracy and reliability of the results. Quality control for MRS Broth Test involves using known positive and negative control strains to check the performance of the medium and the test procedure. The control strains should be inoculated and incubated under the same conditions as the test strains. The expected results for some common control strains are:

• Lactobacillus fermentum (ATCC 9338): Growth, gas in Durham tube
• Lactobacillus acidophilus (ATCC 4356): Growth, no gas in Durham tube
• Pseudomonas aeruginosa (ATCC 27853): No growth

If the control strains show the expected results, it indicates that the medium is prepared correctly, the inoculation and incubation methods are appropriate, and the test can be interpreted with confidence. If the control strains do not show the expected results, it indicates that there may be some problems with the medium, the inoculation, the incubation, or the interpretation of the test. In such cases, the test should be repeated with fresh medium and controls, and the source of error should be identified and corrected.

Quality control for MRS Broth Test should be performed regularly, preferably with each batch of medium prepared or received from a supplier. Quality control should also be performed whenever there is a change in the source or quality of the ingredients, or when there is a doubt about the validity of the test results.

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